THIRD EDITION

Fetal
Medicine
BASIC SCIENCE AND CLINICAL PRACTICE
Pranav P. Pandya, BSc, MBBS, MD, FRCOG
Consultant and Director of Fetal Medicine
University College London Hospitals
London, England, UK
Dick Oepkes, MD, PhD, FRCOG
Professor of Obstetrics and Fetal Therapy
Department of Obstetrics
Leiden University Medical Center
Leiden, The Netherlands
Neil J. Sebire, MBBS, BClinSci, MD, FRCOG, FRCPath, FFCI
Professor of Paediatric and Developmental Pathology
Department of Histopathology
Great Ormond Street Hospital
London, England, UK
Ronald J. Wapner, MD
Professor of Obstetrics and Gynecology
Vice Chair of Research
Director of Reproductive Genetics
Columbia University Irving Medical Center
New York, NY, USA
© 2020, Elsevier Limited. All rights reserved.

First edition Harcourt Brace and Company 1999

Second edition Elsevier Limited 2009

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Foreword
It seems difficult to believe that 20 years have passed since the first
edition of Fetal Medicine was published in 1999. Over that time,
many things have changed. In fact, in the Foreword to the first
edition, John Hobbins charted the preceding 20 years to indicate
the progress that had been made in the speciality up to 1999. John
Queenan, writing the Foreword for the second edition in 2009,
emphasised how an improved understanding of the basic science
of fetal medicine together with the development of technologies
with which to treat fetuses had brought us to the point at which
we could reasonably talk of the ‘fetal patient’.
Now the four new editors of this, the third edition, have taken
things to a new level and are to be congratulated both in encouraging
previous experts to make further updated contributions and recruit-
ing some outstanding ‘new blood’ from experts all over the world.
It is clear from the excellent chapters that in the past 10 years,
there have been a consolidation of knowledge in many areas and
viii
some spectacular progress in subjects such as noninvasive genetic
diagnosis and fetal imaging and in the use of randomised studies
in the extremely difficult area of open fetal surgery.
This beautifully produced and illustrated edition provides read-
ers with a cornucopia of up-to-date knowledge in the field of fetal
medicine which should be a close companion for anyone involved
in the speciality.
Professor Charles H. Rodeck, MB, BS, BSc, DSc, FRCOG,
FRCPath, FMedSci
Emeritus Professor, Institute of Women’s Health, Obstetrics and
Gynaecology, University College London, London, UK
Professor Martin J. Whittle, MD, FRCOG, FRCP(Glas)
Emeritus Professor of Fetal Medicine, University of Birmingham,
Birmingham, UK
Preface
The two previous editions of this book are established as authorita-
tive textbooks in fetal medicine, with the last edition published in
2009. I was therefore delighted to be asked by Professors Rodeck
and Whittle to be an editor for the third edition. There have been
so many significant advances in fetal medicine during the past
decade that I thought it would only be possible to do justice to
a new edition with an expanded team of international expert co-
editors. This would not have been possible without Professors
Dick Oepkes, Neil Sebire and Ron Wapner and their monumental
efforts in preparing this new edition.
We were clear from the start that this edition should continue
and build on the same premise as the previous, namely combin-
ing basic science with contemporary clinical practice. At the same
time, we wished to thoroughly revise the content and style to reflect
the rapid advances in fetal medicine and science. We have changed
the appearance of chapters with a focus on major topics in fetal
medicine, providing scientific evidence on recent advances, struc-
tured text, key points at the beginning of each chapter, concise
chapter summaries, new images and new videos online. The aim is
to allow the readers to both rapidly access relevant information to
aid management and to provide a comprehensive understanding
of the topic. With these aims, there are numerous new chapters
and extensive revision of existing chapters to keep pace with the
dramatic innovation in fetal medicine.
We are deeply indebted to the multidisciplinary international
authors who are recognised experts in their field; they have gener-
ously contributed their time, knowledge and experience to this
book. They have delivered up-to-date information that is practi-
cal and accessible and provides deeper understanding of complex
issues within fetal medicine and basic science.
We also thank all the team at Elsevier for their constant sup-
port and help, in particular Sharon Nash, Beula Christopher,
Sarah Barth and Kate Dimock.
Finally, we would like to thank again Charles Rodeck and Martin
Whittle for trusting us to produce the third edition of this book.
Pranav P. Pandya
Dick Oepkes
Neil J. Sebire
Ronald J. Wapner
ix
List of Contributors

The editors would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, without whom this
new edition would not have been possible.

Ganesh Acharya, MD, PhD Guillaume Benoist, MD, PhD

Professor of Obstetrics and Gynecology Department of Obstetrics and Gynecology
Department of Clinical Sciences, Intervention and Technology University Hospital
Karolinska Institute Caen, France
Stockholm, Sweden University of Normandy
Normandy, France
Michael Aertsen, MD
Radiologist Colleen G. Bilancia, PhD, DABMGG
Department of Radiology Clinical Cytogeneticist and Molecular Geneticist
University Hospitals Leuven Lineagen, Inc.
Leuven, Belgium Salt Lake City, UT, USA

Yalda Afshar, MD, PhD Caterina M. Bilardo, MD, PhD

Maternal Fetal Medicine Fellow Professor in Fetal Medicine
Division of Maternal Fetal Medicine Department of Obstetrics and Gynaecology
Department of Obstetrics and Gynecology Amsterdam University Medical Centers
University of California VUmc
Los Angeles, CA, USA Amsterdam, The Netherlands;
University Medical Centre Groningen
Cande V. Ananth, PhD, MPH University of Groningen
Professor and Vice-Chair for Academic Affairs Groningen, The Netherlands
Chief, Division of Epidemiology and Biostatistics
Department of Obstetrics, Gynecology, and Reproductive Louise D. Bryant, BSc (Hon), PhD
Sciences Associate Professor of Medical Psychology
Rutgers Robert Wood Johnson Medical School Leeds Institute of Health Sciences
New Brunswick, NJ, USA University of Leeds
Leeds, England, UK
Michael Ashworth, MD, FRCPath
Consultant in Paediatric Pathology Colin R. Butler, BSc, MBBS, MRCS
Department of Histopathology Tracheal Research Fellow
Great Ormond St Hospital for Children Great Ormond Street Hospital for Children
London, England, UK London, England, UK

Patrick Au, MPhil, MSc Frank Van Calenbergh, MD, PhD

Scientific Officer (Medical) Professor of Neurosurgery
Prenatal Diagnostic Laboratory Academic Department of Neurosciences
Tsan Yuk Hospital Biomedical Sciences
Hong Kong SAR, China Faculty of Medicine;
Department of Neurosurgery
Spyros Bakalis, BSc, MBBS, MRCOG, MD University Hospital Gasthuisberg UZ Leuven
Consultant in Obstetrics and Fetal Medicine Leuven, Belgium
St Thomas’ Hospital
London, England, UK

x
 List of Contributors xi

Steve N. Caritis, MD Elisabeth de Jong-Pleij, MD, PhD

Professor Physician-Sonographer
Department of Obstetrics, Gynecology and Reproductive Department of Obstetrics and Gynaecology
Sciences, School of Medicine St. Antonius Hospital
Magee Women’s Hospital of UPMC Nieuwegein, The Netherlands;
University of Pittsburgh Department of Obstetrics and Gynaecology
Pittsburgh, PA, USA University Medical Centre Utrecht
Utrecht, The Netherlands
Lyn S. Chitty, BSc, PhD, MBBS, MRCOG
Professor of Fetal Medicine and Genetics Bart De Keersmaecker, MD
UCL Great Ormond Street Institute of Child Health Feto-Maternal Specialist
NE Thames Regional Genetics Service Fetal Medicine
Great Ormond Street Hospital for Children NHS Foundation Department of Obstetrics and Gynecology
Trust University Hospitals Leuven
London, England, UK Leuven, Belgium;
Department of Obstetrics and Gynecology
Patricia Collins, BSc(Hon), PhD Kortrijk, Belgium
Professor of Anatomy
AECC University College Jan Deprest, MD, PhD, FRCOG
Bournemouth, England, UK Professor
Head of the Department of Development and Regeneration
James Cook, MBBS, MSc, MRCPCH Department of Obstetrics and Gynecology
Subspecialty Trainee in Paediatric Respiratory Medicine University Hospital of Leuven Gasthuisberg
Department of Paediatric Respiratory Medicine Leuven, Belgium;
Great Ormond Street Hospital for Children NHS Foundation Institute for Women’s Health
Trust University College London
London, England, UK London, England, UK

Howard Cuckle, BA, MSc, DPhil Roland Devlieger, MD, PhD

Adjunct Professor Professor of Obstetrics and Gynaecology
Department of Obstetrics and Gynecology Academic Department of Development and Regeneration
Columbia University Medical Center Cluster Woman and Child, Biomedical Sciences
New York, NY, USA Faculty of Medicine, KU Leuven;
Centre for Surgical Technologies, Faculty of Medicine, KU
Anna L. David, PhD, FRCOG, MB, ChB Leuven;
Professor and Consultant in Obstetrics and Maternal Fetal Fetal Medicine Unit, Division of Woman and Child,
Medicine Department of Obstetrics and Gynaecology
Institute for Women’s Health University Hospital Gasthuisberg, UZ Leuven
University College London Leuven, Belgium
London, England, UK
Guido M. de Wert, PhD
Luc De Catte, MD, PhD Professor of Biomedical Ethics
Feto-maternal Specialist Department of Health, Ethics and Society
Fetal Medicine Research Schools CAPHRI and GROW
Department of Obstetrics and Gynecology University of Maastricht
University Hospitals Leuven Maastricht, The Netherlands
Leuven, Belgium
Jan E. Dickinson, MD, FRANZCOG, DDU, CMFM
Paolo De Coppi, MD, PhD Professor Maternal Fetal Medicine
NIHR Professor of Paediatric Surgery Division of Obstetrics and Gynaecology
Head of Stem Cells and Regenerative Medicine Section Faculty of Health and Medical Sciences
Developmental Biology and Cancer Programme The University of Western Australia
UCL Institute of Child Health Perth, Western Australia, Australia
Consultant Paediatric Surgeon
Great Ormond Street Hospital Mark Dilworth, PhD
London, England, UK MRC Career Development Award Research Fellow
Maternal and Fetal Health Research Centre, Faculty of Biology,
Medicine and Health
University of Manchester;
St. Mary’s Hospital
Manchester University NHS Foundation Trust
Manchester Academic Health Science Centre
Manchester, England, UK
xii List of Contributors

Wybo J. Dondorp, PhD Jenny Hewison, BA (Hon), MSc, PhD

Associate Professor of Biomedical Ethics Professor of the Psychology of Healthcare
Department of Health Leeds Institute of Health Sciences
Ethics and Society University of Leeds
Research Schools CAPHRI and GROW Leeds, England, UK
University of Maastricht
Maastricht, The Netherlands Richard J. Hewitt, BSc, DOHNS, FRCS (HNS-ORL)
Consultant Paediatric ENT
Caroline E. Dunk, PhD Head and Neck and Tracheal Surgeon
Research Associate Director of the National Service for Severe Tracheal Diseases
Research Centre for Womens and Infants Health Great Ormond Street Hospital for Children
Lunenfeld Tanenbaum Research Institute London, England, UK
Mount Sinai Hospital
Toronto, Ontario, Canada Liran Hiersch, MD
Staff Physician
Thomas R. Everett, BSc MBChB, MD, MRCOG Department of Obstetrics and Gynecology
Consultant in Fetal Medicine Lis Maternity Hospital
Leeds General Infirmary Tel Aviv Sourasky Medical Center
Leeds, England, UK Sackler Faculty of Medicine
Tel Aviv University
Jane Fisher, BA (Hon), MA Tel Aviv, Israel
Director
Antenatal Results and Choices Melissa Hill, BSc, PhD
London, England, UK Senior Social Scientist
Genetics and Genomic Medicine
Henry L. Galan, MD UCL Great Ormond Street Institute of Child Health
Professor NE Thames Regional Genetics Service
Department of Obstetrics and Gynecology Great Ormond Street Hospital for Children NHS Foundation
Colorado Fetal Care Center Trust
University of Colorado School of Medicine London, England, UK
Aurora, CO, USA
Sara L. Hillman, BSc, MBBS, PhD, MRCOG
Mythily Ganapathi, PhD, FACMG NIHR Academic Clinical Lecturer
Assistant Professor of Pathology and Cell Biology at CUMC Subspecialty Trainee in Maternal Fetal Medicine
College of Physicians and Surgeons University College London
Columbia University Medical Center and the New York London, United Kingdom
Presbyterian Hospital
New York, NY, USA An Hindryckx, MD
Consultant in Obstetrics and Fetal Medicine
Helena M. Gardiner, MD, PhD University Hospitals Leuven
Director of the Fetal Echocardiography Fellowship and Training Leuven, Belgium
Program
The Fetal Center Stuart B. Hooper, PhD
UT Health McGovern Medical School Centre Head
Houston, TX, USA The Ritchie Centre
The Hudson Institute for Medical Research
Cecilia Gotherstrom, PhD Department of Obstetrics and Gynaecology
Associate Professor Monash University
Department of Clinical Science Intervention and Technology Melbourne, Australia
Division of Obstetrics and Gynecology
Karolinska Institutet Berthold Huppertz, PhD
Stockholm, Sweden Chair
Division of Cell Biology, Histology and Embryology
Richard Harding, PhD, DSc Gottfried Schatz Research Center
Emeritus Professor Medical University of Graz
Department of Anatomy and Developmental Biology Graz, Austria
Monash University
Melbourne, Australia J. Ciaran Hutchinson, MRes, MBBS, DipFMS
Clinical Research Fellow
Department of Histopathology
Great Ormond Street Hospital
London, England, UK
 List of Contributors xiii

Jon Hyett, MBBS, BSc, MD, MRCOG, FRANZCOG Y.W. Loke, MD, FRCOG
Clinical Professor and Head of High Risk Obstetrics Emeritus Professor of Reproductive Immunology
RPA Women and Babies Kings College
Royal Prince Alfred Hospital University of Cambridge
University of Sydney Cambridge, England, UK
Camperdown, NSW, Australia
Enrico Lopriore, MD, PhD
Luc Joyeux, MD, MSc Professor and Head of the Neonatology Division
General Paediatric Surgeon and PhD Candidate in Fetal Surgery Division of Neonatology
Academic Department of Development and Regeneration Department of Pediatrics
Cluster Woman and Child, Biomedical Sciences Leiden University Medical Center
Faculty of Medicine Leiden, The Netherlands
Katholieke Universiteit;
Centre for Surgical Technologies George A. Macones, MD
Faculty of Medicine Professor and Chair
KU Leuven, Belgium Department of Obstetrics and Gynecology
Leuven, Belgium Washington University in St. Louis
St. Louis, MO, USA
Davor Jurkovic, FRCOG, MD
Consultant Gynaecologist Fergal D. Malone, MD, FACOG, FRCOG, FRCPI
Department of Obstetrics and Gynaecology Master of the Rotunda Hospital, Dublin
University College Hospital Chair and Professor of Obstetrics and Gynecology
London, England, UK Royal College of Surgeons in Ireland;
Consultant Obstetrician and Gynecologist and Maternal-Fetal
John C. Kingdom, MD Medicine Specialist
Professor of Maternal-Fetal Medicine Rotunda Hospital
Department of Obstetrics and Gynecology Dublin, Ireland
University of Toronto
Toronto, Ontario, Canada Anahit Martirosian, RDMS
Sonographer
Sylvie Langlois, MD Center for Fetal Medicine and Women’s Ultrasound
Professor and Clinical Geneticist Los Angeles, CA, USA
Department of Medical Genetics
University of British Columbia Fionnuala McAuliffe, MD, FRCOG, FRCPI
Vancouver, British Columbia, Canada Chair and Professor of Obstetrics and Gynecology
Head, Women’s and Children’s Health
Lara S. Lemon, PhD, PharmD University College Dublin
Research Assistant Professor Dublin, Ireland;
Department of Obstetrics, Gynecology and Reproductive Sciences Consultant Obstetrician and Gynecologist and Maternal-Fetal
University of Pittsburgh School of Medicine; Medicine Specialist
Data Scientist National Maternity Hospital
Department of Clinical Analytics Dublin, Ireland;
University of Pittsburgh Medical Center Council Member, Royal College of Obstetricians and
Pittsburgh, PA, USA Gynecologists
London, England, UK
Marianne Leruez-Ville, MD, PhD
Department of Virology Annie R.A. McDougall, PhD
Necker Enfants Malades Hospital Research Officer
University of Paris Descartes The Ritchie Centre
Paris, France The Hudson Institute of Medical Research
Melbourne, Australia
Liesbeth Lewi, MD, PhD
Professor and Staff Member Kenneth J. Moise Jr., MD
Department of Obstetrics and Gynecology Professor
University Hospital of Leuven Gasthuisberg Division of Maternal-Fetal Medicine
Leuven, Belgium Department of Obstetrics
Gynecology and Reproductive Sciences
Brynn Levy, MSc (Med), PhD, FACMG UT Health McGovern School of Medicine;
Professor of Pathology and Cell Biology at CUMC Co-Director
College of Physicians and Surgeons The Fetal Center
Columbia University Medical Center and the New York Children’s Memorial Hermann Hospital
Presbyterian Hospital Houston, TX, USA
New York, NY, USA
xiv List of Contributors

Ashley Moffett, MD, FRCOG Kuhan Rajah, MRCOG

Emeritus Professor of Reproductive Immunology Subspecialty Trainee in Reproductive Medicine
Department of Pathology Department of Obstetrics and Gynaecology
University of Cambridge University College Hospital
Cambridge, England, UK London, England, UK

Sieglinde M. Müllers, PhD Rashmi Rao, MD

Specialist Registrar in Obstetrics and Gynecology Assistant Professor
Royal College of Surgeons in Ireland Division of Maternal Fetal Medicine
Rotunda Hospital, Dublin, Ireland Department of Obstetrics and Gynecology
University of California
Ran Neiger, MD Los Angeles, CA, USA
Director
Maternal-Fetal Medicine Unit Jute Richter, MD, PhD
Ma’ayanei Hayeshua Hospital Professor and Staff Member
Bnei Brak, Israel Department of Obstetrics and Gynecology
University Hospital of Leuven Gasthuisberg
John P. Newnham, AM, MD, FRANZCOG, CMFM, DDU Leuven, Belgium
Professor of Maternal Fetal Medicine and Head
Division of Obstetrics and Gynaecology Joshua I. Rosenbloom, MD
The University of Western Australia Fellow
Perth, Western Australia, Australia Division of Maternal-Fetal Medicine
Department of Obstetrics and Gynecology
Sarah G. Obican, MD Washington University in St. Louis
Assistant Professor
University of South Florida Francesca Maria Russo, MD
Division of Maternal Fetal Medicine Research Fellow
Department of Obstetrics and Gynecology Department of Obstetrics and Gynecology
Tampa, FL, USA University Hospital of Leuven Gasthuisberg
Leuven, Belgium
Anthony O. Odibo, MD, MSCE
Professor of Obstetrics and Gynecology Anthony R. Scialli, MD
Director of Ultrasound and Fetal Therapy Director
University of South Florida Reproductive Toxicology Center
Morsani College of Medicine Washington, DC, USA
Tampa, FL, USA
Neil J. Sebire, MBBS, BClinSCi, MD, FRCOG, FRCPath, FFCI
Dick Oepkes, MD, PhD, FRCOG Professor of Paediatric and Developmental Pathology
Professor of Obstetrics and Fetal Therapy Department of Histopathology
Department of Obstetrics Great Ormond Street Hospital
Leiden University Medical Center London, England, UK
Leiden, The Netherlands
Andrew Sharkey, BA, PhD
Pranav P. Pandya, BSc, MBBS, MD, FRCOG Associate Lecturer
Consultant and Director of Fetal Medicine Department of Pathology
University College London Hospitals University of Cambridge, UK
London, England, UK Cambridge, England, UK

Lawrence D. Platt, MD Susan C. Shelmerdine, MBBS, BSc, MRCS, FRCR

Professor Clinical Research Fellow
Center for Fetal Medicine and Women’s Ultrasound Department of Radiology
Division of Maternal Fetal Medicine Great Ormond Street Hospital
Department of Obstetrics and Gynecology London, England, UK
University of California
Los Angeles, CA, USA Colin Sibley, PhD, DSc
Professor of Child Health and Physiology
Rosalind Pratt, MBChB, Bsc Maternal and Fetal Health Research Centre
Clinical Research Fellow Faculty of Biology, Medicine and Health
University College London University of Manchester;
London, England, UK St. Mary’s Hospital
Manchester University NHS Foundation Trust
Manchester Academic Health Science Centre
Manchester, England, UK
 List of Contributors xv

Saul Snowise, MD Raman Venkataramanan, PhD

Minnesota Perinatal Physicians and the Midwest Fetal Care Professor
Center Department of Pharmaceutical Science, School of Pharmacy
Children’s Hospital Department of Pathology, School of Medicine
Minneapolis, MN, USA University of Pittsburgh
Pittsburgh, PA, USA
Sylke Steggerda, MD, PhD
Neonatologist Yves Ville, MD, FRCOG
Division of Neonatology Professor
Department of Pediatrics Department of Obstetrics and Fetal Medicine
Leiden University Medical Center Necker Enfants Malades Hospital
Leiden, The Netherlands University of Paris Descartes
Paris, France
Emily J. Su, MD, MSCI
Associate Professor Magdalena Walkiewicz, PhD
Department of Obstetrics and Gynecology Assistant Professor
Colorado Fetal Care Center Department of Molecular and Human Genetics
University of Colorado School of Medicine Baylor college of Medicine
Aurora, CO, USA Baylor Genetics Laboratories
Houston, TX, USA
Mary Tang, FRCOG
Clinical Associate Professor Colin Wallis, FRCPCH, MD
Prenatal Diagnostic and Counselling Division Consultant Respiratory Paediatrician
Department of Obstetrics and Gynaecology Respiratory Unit
University of Hong Kong Great Ormond Street Hospital
Pokfulam, Hong Kong SAR, China London, England, UK

Arjan B. Te Pas, MD, PhD Lilian Walther-Jallow, PhD

Neonatologist Department of Clinical Science Intervention and Technology
Division of Neonatology Division of Obstetrics and Gynecology
Department of Pediatrics Karolinska Institutet
Leiden University Medical Center Stockholm, Sweden
Leiden, The Netherlands
Ronald J. Wapner, MD
Alan T. Tita, MD, PhD Professor of Obstetrics and Gynecology
Professor and Director Vice Chair of Research
Center for Women’s Reproductive Health Director of Reproductive Genetics
Department of Obstetrics and Gynecology Columbia University Irving Medical Center
University of Alabama New York, NY, USA
Birmingham, AL, USA
Magnus Westgren, MD, PhD
Frederick Ushakov, MD Professor
Specialist in Fetal Medicine Department of Clinical Science Intervention and Technology
Fetal Medicine Unit Division of Obstetrics and Gynecology
University College London Hospital Center for Fetal Medicine
London, England, UK Karolinska University Hospital
Stockholm, Sweden
Ignatia B. Van den Veyver, MD
Professor Scott W. White, MBBS, PhD, FRANZCOG, CMFM
Departments of Obstetrics and Gynecology and Molecular and Clinical Senior Lecturer in Maternal Fetal Medicine
Human Genetics Division of Obstetrics and Gynaecology
Baylor college of Medicine The University of Western Australia
Houston, TX, USA Perth, Western Australia, Australia

Jeanine M. van Klink, PhD Louise C. Wilson, MB, BS, FRCP

Clinical Psychologist Consultant in Clinical Genetics
Division of Neonatology Great Ormond Street Hospital NHS Foundation Trust
Department of Pediatrics London, England, UK
Leiden University Medical Center
Leiden, The Netherlands
xvi List of Contributors

R. Douglas Wilson, MD, MSc Karen Wou, MD

Professor and Head Clinical Genetics Fellow
Department of Obstetrics and Gynecology Department of Pediatrics, Division of Clinical Genetics
Cumming School of Medicine Columbia University Irving Medical Center
University of Calgary New York, NY, USA
Calgary, Alberta, Canada
Yuval Yaron, MD
Dian Winkelhorst, MD Director, Prenatal Genetic Diagnosis Unit
PhD Student and Clinical Researcher Genetic Institute, Tel Aviv Sourasky Medical Center
Divison of Fetal Therapy Associate Professor, Department of Obstetrics and Gynecology
Department of Obstetrics Sackler Faculty of Medicine, Tel Aviv University
Leiden University Medical Center Tel Aviv, Israel
Leiden, The Netherlands
Kwok Yin Leung, MBBS, MD, FRCOG
Paul J.D. Winyard, BM, BCh, MA, PhD, FRCPCH Chief of Service and Consultant
Professor of Paediatric Education and Honorary Consultant in Department of Obstetrics and Gynaecology, Queen Elizabeth
Paediatric Nephrology Hospital
Developmental Biology and Cancer Programme Hong Kong SAR, China
UCL Great Ormond Street Institute of Child Health
London, United Kingdom Angela Yulia, MRCOG, PhD, PGCert, Med Ed
Subspecialty Trainee in Maternal and Fetal Medicine
Christoph Wohlmuth, MD, PhD, Priv. Doz. Fetal Medicine Unit
Department of Obstetrics and Gynecology University College London Hospitals NHS Foundation Trust
Paracelsus Medical University London, England, UK
Salzburg, Austria
1
Early Concepts and Terminology
PATRICIA COLLINS
KEY POINTS
• T his chapter considers the language used within embryologi-
cal research and how it is evolving.
• The terms used to describe embryos, cells and tissues derive
from the social constructs of science during the time they
were created.
• Newer terms have been added as scientific methods have
increased, although there may not be a consensus on the
definition of some terms.
• The application of computer sciences to development has
produced its own terminology.
• The use of computer ontologies may impact development
and the evolution of embryological concepts and terminology
with which to explain the observed processes.
The Changing Concepts and Language of
Embryology
Embryological terminology used today is a strange and diverse
mixture of terms accrued over the course of two centuries and
used as a vernacular language with different dialect depending on
the topic and techniques of study. The accumulated terms include:
• The very old concepts generated between 1830 and 1900
• The newer understanding of cell phenotype generated in the
20th century from in vitro and in vivo experimentation and cell
culture
• The very modern and rapidly changing 21st-century terms
which describe gene expression and metabolism within embry-
onic tissues
The language of the latter group is driven by computer ontol-
ogies: hierarchies of embryological terms and key words pro-
grammed as algorithms, which are used to mine databases and
publications. The spur for this interest is a future ability to unlock
embryological pathways as a method for treating adult pathology
and harnessing the regenerative potential of stem cells.  a different story. Richardson3 noted that drawings by contem-
Origin of the Early Embryological Terms
Embryological terms are a product of their time and a reflec-
tion of how developmental science was explained. At a time
when the theory of evolution was being formulated in the
mid-1800s, Ernst Haeckel promoted a concept which stated
that embryos would pass through all the previous evolutionary
2
stages, resembling a series of extant or extinct adult animals
as they recapitulated evolution during development.1 Thus
Haeckel designated a blastula stage of development when a
sphere or bilaminar layer of embryonic cells was present and a
later gastrula stage achieved after the blastula cells had invagi-
nated to produce more than one or two layers. He also inaugu-
rated the term gastrulation to describe the process where cells
initially on the embryonic surface move inside the embryo to
produce intraembryonic cell populations.
At this time the instruments for examining embryos were rudi-
mentary, and cell theory, being formulated also in the mid-1800s,
was still relatively young. Embryologists of the day saw layers of
tissue rather than the individual cells composing the layers and
did not link the morphology of the earliest cells with differences in
function. In studies in which it is clear from the publications that
cells could be seen, distinctions among early embryonic cell types
probably could not be made with the instruments available. The
concepts thus generated by these early embryologists were prod-
ucts of their time, dependent on the methods of experiment and
observation customary when they were formulated.
For most of the 20th century, textbooks supported the notion
that the tissues of the developed body were derived from one of the
‘three germ layers’. Whilst this is not untrue in simplistic terms,
the accent on three layers (ignoring the cell phenotypes) moved
attention away from and limited interrogation of the dynamic dif-
ferentiation processes occurring in embryos. Without a full range
of words with which to think about developmental processes the
reflections on, and explanations of, what is seen histologically
becomes obfuscated.
A similar interpretive process driven by evolution theory
occurred with the description of external embryonic form. In
1828, Von Baer2 noted that all vertebrate embryos pass through
externally similar stages, and Haeckel published a series of draw-
ings demonstrating remarkable similarity between embryos
which go on to become very dissimilar adults. This latter concept
remained unchallenged for more than a century. Recent examina-
tion of Haeckel’s pictures, together with a clear analysis of the
developmental stages of various organs in each embryo, revealed
poraries of Haeckel show much more accurate interpretations
of mammalian embryos of the same developmental stage with
clear differences among them. He noted that Haeckel’s drawings
had given a misleading view of embryonic development. Thus
the idea of one stage of development, during which all vertebrate
embryos are the same, promoted extensively at the turn of the
20th century and repeated unchallenged, hampered investigation
into what really occurs in a number of vertebrate embryos. This

again obfuscated the search for what is actually present in embryos
by limiting the embryological concepts taught, the language used
and consequently the expectations and explanations of the pro-
cesses observed.
Embryology was advanced in the middle and later years of
the 20th century by in vitro studies of developing reptile, avian
and mammalian embryos, particularly using chimeric embryos in
which specific cells lines could be followed.4 These experiments
provided information about the similarities and differences among
species. Also at this time, the genes expressed within developing
tissues were studied, and the range of genes used in basic cell func-
tioning and at particular points of development were elucidated.
The functions of many genes were studied by the experimental
production of animals in which specific genes were knocked out
or knocked in, and the effects of the homozygous were compared
with the wild type. These experiments demonstrated the impor-
tance of some genes, with knock-out causing lethality; in other
cases, the actual effect of taking one gene out within an embryo
was confounded by the catch-up mechanisms in built into devel-
opment: the change in one part of the system causing compensa-
tory change in the remainder.  derm and mesoderm. The terms are still
Early Embryonic Cell Interactions
Expansion of in  vitro fertilisation techniques and the selection
of healthy embryos for implantation have demonstrated that
the secondary oocyte has a range of genes ready for expression
to ensure cleavage, morula and blastocyst formation. When
the dividing cells are an appropriate size, polarity is expressed,
junctions are formed and embryonic cell–cell interaction com-
mences.5 After hatching from the zona pellucida, the blastocyst
is able to interact with maternal tissue. There is no time when
the genome is not being read and epigenetic consequences,
because of local environmental conditions, are not part of the
next process.
Three cell lineages are now identified in the blastocyst, leading
to extraembryonic and embryonic cell populations (Table 1.1). All
of these lineages express polarity genes and form epithelia.
The process of gastrulation produces cells which do not have
an epithelial phenotype (i.e., mesoblast and mesenchymal cells).
Recovery of embryonic cells has led to the development of embry-
onic stem cells which can be immortalised in two-dimensional
culture conditions.  cells. Need specific culture conditions; grow
Embryonic Cells in Culture
Historically, the definitions of the terms used to describe the
putative abilities of embryonic cells were easily found. Today
recent papers note the difficulty of accurately defining these terms
(Table 1.2). Adult cells can be induced to grow in culture and
now so can embryonic cells.
Early in vitro culture techniques mainly concerned the growth
of adult cells within a two-dimensional physical environment.
Cells are grown to confluence and then split into subcultures (pas-
saging) and are regrown many times. The passages and new media
promote expansion of the numbers of cells in the culture and pro-
long the life of the cells beyond that of the original donor. This
methodology is still utilised.
Three-dimensional environments, the norm for all body tis-
sues, are being explored in in  vitro culture. It has been noted
that cells in three-dimensional culture systems form organ-
oids, in which the epithelial cells form spheres surrounded by
Chapter 1  Early Concepts and Terminology 3
TABLE
1.1 Lineage Concepts
Term Meaning
Three cell lineages • T rophectoderm—will become placenta and
identified as extraembryonic membranes
zygote under- • Hypoblast—sometimes called primitive
goes cleavage endoderm; maintains the primitive streak
• Inner cell mass—sometimes called primi-
tive ectoderm; usually termed epiblast; all
embryonic cell lines are derived from this
lineage
Polarity genes Cells within an early embryo form epithelia and
mesenchyme. The epithelial cells exhibit
polarity genes which specify the apical, basal
and lateral surfaces; the position of junctional
complexes; and the direction of the mitotic
spindle during cell division. Not observed
before compaction in morula.6
Germ layers These were historically the ectoderm, endo­
widely used regardless of cell phenotype.
They all derive from the epiblast of the early
blastocyst.
TABLE Definitions of the Terms Used to Describe
1.2 Zygotes and Stem Cells
Term Meaning
Totipotent The ability of a single cell to develop into an adult
organism and generate offspring. In humans,
the zygote is totipotent. The loss of totipotency
is now seen as a process.
Pluripotent The ability of a single cell to develop into cells
from one of the ‘three germ layers’ and ‘germ’
cells in vitro and in vivo. EpiSCs are postim-
plantation epiblast stem cells.
Embryonic stem Human embryonic stem cells (hESCs). Origin not
cells clear. Not quite the same as inner cell mass
in two dimensions. Now have been adapted to
long-term in vitro culture.
Human-induced Cells derived from adult cells (e.g., fibroblasts) which
pluripotent have been cultured with specific transcription
stem cells factors. They undergo transition to epithelial cells
(hiPSCs) and express epithelial genes, becoming polarised.
They also change their metabolism.
Human spheroids When hiPSCs are grown in three-dimensional cul-
or organoids ture and encouraged along a particular devel-
opmental pathway, they form spheres of inner
epithelial and outer mesenchymal phenotypes.
Organoids have been created from hiPSCs
specified as endoderm which differentiate into
airway or gut phenotypes with appropriate
epithelial and supporting mesenchymal cells.
Self-organisation into layered tissues has also
been seen in three-dimensional cultured brain
cortical cells and retinal tissue.
  
4 SE C T I O N 1     Early Fetal Development
mesenchymal cells.7,8 These self-organising cell lines have been
implanted into animals and will continue growing.9 The ulti-
mate aim of these studies is to grow replacement portions of gut,
respiratory conducting airways or kidney which ultimately can be
used for transplant.
Cultured cells have also been purposefully arranged in specified
three-dimensional shapes by bioprinting methods and encouraged
to grow and differentiate along specific lines by the addition of tar-
geted growth factors to the media.10 The complexity of setting up
all three-dimensional systems and recording cell growth, move-
ment and interaction are particularly challenging.
Further experimental methods have attempted to immortalise
embryonic cell lines. By specifying the growth factors used in the
culture media and the oxygen levels supplied and by forcing cells
to change their phenotype (mesenchyme to epithelial), the cells
have been driven along particular developmental pathways to
form specific cell lines, e.g., cardiac myocytes, hepatocytes and
neurons.11,12 Such cultures are used for further optimisation of
culture conditions, to gain knowledge of the genes the cells are
utilising, and for testing drugs on cells in culture rather than on
laboratory animal species.  WormBase
Interpretation of the Genome
The latter years of the 20th century and the beginning of the
21st saw an explosion of interest in embryological pathways,
first because of the identification of the human genome and the
genomes of the commonly used laboratory animals (Table 1.3)
and second because it was thought that re-expression of embry-
onic genes and pathways in an adult could lead to treatment of
many pathological conditions.
The success of the Human Genome Project led to elucidation
of the genomes of laboratory species and greater understanding
of the shared genes upregulated in development. Information
on the temporal and spatial regions of gene expression during
development superimposed on internal and external embryonic
form has been shared via the internet. Such websites also have the
methodologies for demonstrating specific genes and transcrip-
tion factors.  • Transgenic lines of chickens and quail
Computing Sciences and Embryological
Terminology
Embryology has now become a domain of computer sciences as
well as laboratory-based sciences. Powerful computing was neces-
sary for the collation of the genomes and the proteins encoded.
Two interrelated lines of research can now be noted: (1) the rela-
tionships among cells, tissues, organs and time within developing
embryos and (2) the relationship between genes and the mol-
ecules they encode. Ontologies, hierarchical structures of speci-
fied vocabulary, have been created for developmental processes;
embryonic cells and tissues; for genes, transcription factors and
proteins. New genes, or the spatial and temporal expression of
known genes, are added to specific ontologies either by a curator
or by the computer ontology algorithm itself (an inferred elec-
tronic annotation). Predictions of future gene function can be
gained from these methods.
The laboratory techniques of mass spectrometry and microar-
ray methods can identify proteins and metabolites in very small
samples of culture media. Information concerning what genes the
cells are expressing and what proteins they are making at each time
TABLE Internet Sources of Information on a Range of
1.3 Genomes
Genome and
proteins Internet site
Human National Human Genome Research Institute
Genome Has timeline for the Human Genome Project
Human • Human Proteome Project; Human Proteome Map
­Proteins • Human Protein Atlas
Human Developmental Studies Network (HUDSEN)
• Electronic atlas of a developing human brain
• Human spatial gene expression database
• Virtual Human Embryo
• Multidimensional Human Embryo
Both use Carnegie staging for human embryos
Brainspan Atlas of the developing human brain,
developmental transcription factors
FlyBase A database of the genome and genes expressed in
Drosophila throughout development
A database of the genome and genes expressed
Caenorhabditis elegans and other species
Mouse eMAP • E dinburgh Mouse Atlas Project
• eMA: three-dimensional mouse anatomy atlas
• eHistology, with serial sections of mouse
embryos throughout development
• eMAGE: gene expression database for mouse
embryos
DBTMEE
• Database of transcriptome in mouse early
embryos
echickatlas • T hree-dimensional anatomical atlas of chick
embryo development
• e-Chick Atlas of gene expression
Geisha Gallus Expression In Situ Hybridisation Analysis
• Anatomical atlases
• Chick development stage series
• Bird genomes
point of development has been shown to provide, for example,
objective data in the assessment of preimplantation embryos from
in vitro fertilisation.13
These methods have also been used to analyse the supernatant
of embryo cultures at specific time points or to analyse the super-
natant of induced pluripotential stem cells culture as the cells are
induced to follow specific phenotypic pathways (Table 1.4). The
techniques of high-resolution mass spectrometry are now able to
identify and quantify protein in single embryonic cells.16 The lan-
guage used to describe these techniques is now part of the embryo-
logical vocabulary.
The use of powerful computing has also enabled three-
dimensional images of animal and human embryos of all
stages to be made available, showing external form and serial
histological sections. The spatial and temporal location of spe-
cific gene expression may also be added to these images (see
Table 1.3).

TABLE
1.4 Common Bioinformatics Terms
Data-driven
technology term Meaning
Gene ontology The terms entered into a computer in a relational
hierarchy. It provides a structured language
to describe gene function and tools to predict
gene function.
Metabolome A range of small molecules (amino acids,
adenosine triphosphate, hormones, signalling
molecules) which can be measured in the
culture media in which embryos are grown.
These measurements give insights into the
biochemical and metabolic pathways operating
within the embryo at particular times of
development. They can be used to assess
viability of preimplantation embryos.14
Proteomics Study of all the proteins of a cell type and their
interactions15
Protein interaction A scheme which shows interactions between gene
network products of differentially expressed genes
Secretome The collection of transmembrane proteins and
proteins secreted into the extracellular space,
e.g., cell–cell signalling molecules within a
culture
Transcriptome The examination of the global transcription factors
expressed in cell or embryo culture by microarray
methods at specified a particular time of or after
cell perturbation
  
Much of the painstaking work of capturing and interpret-
ing photographs of cells in culture is now being completed by
computer software programmes and their ontological algorithms
(e.g., spatial expression of genes),17 and fluorescence of cyto-
skeletal elements.18 Metadata analyses now provide information
on comparative functional genomic studies and on systems-
level analyses integrating epigenetic and functional data in devel-
opment.19 
A Note of Caution
For many years, the painstaking work of those who examined
serial sections of embryos, elucidating and correlating the mor-
phological changes within embryos at specified stages (e.g., Matt
Kaufmann20 for mouse embryos and Ronan O’Rahilly and Fabiola
Müller21,22 for their extensive and fundamental work on the Carn-
egie collection of human embryos) was underrated. Yet an under-
standing of the four-dimensional changes and the interactions
between cells and tissues within an embryo is necessary for the
accurate interpretation of the newer developmental techniques.
The significance of the newer findings and how they relate to
human development, from the embryo to adult senescence, still
needs to be considered and theories and concepts re-examined.
Findings from techniques in which cell lines experience very dif-
ferent epigenetic processes to in vivo development must be treated
with care, just as differences in the timing of particular gene
expression between animal species are treated.
Chapter 1  Early Concepts and Terminology 5
In stem cell culture, changes in cell behaviour (from what
might be considered ‘normal’) caused by the culture conditions
may lead to the misinterpretation of experimental results and
should be considered with caution. Human-induced pluripo-
tent stem cells (hiPSCs) are generated by resetting their epigen-
etic landscapes.23 Although they are used as undifferentiated cells
which can be pushed into developmental pathways by the culture
media, undifferentiated embryonic cells are only transiently seen
in a developing embryo.24
In a similar construct, the more computers use specified
gene ontologies and make predictions on the results of their
algorithms on the basis of inferred electronic annotation, the
more distant the generated results become from the fundamen-
tal questions they may have been used to answer. Attempts are
being made to make the output of mining proteomic databases
easier to interpret,25 so those not familiar with embryological
development or cell interactions may promote the use of these
complex techniques, but they also separate further the under-
standing of the answer from the original question. Hutter and
Moerman26 urge caution when interpreting outcomes from
the use of ‘big data’ in biological sciences. If the input data
used to solve a problem becomes convoluted and obfuscated,
then the output may not be the answer to the question being
investigated.
This is an unprecedented time for embryological research.
The advances in knowledge are enormous; however, what each
finding means within the whole, beyond an increase in com-
plexity that could not have been imagined in the past century,
is not yet clear. The excitement of gathering an unprecedented
amount of data which computers analyse does not explain
which of the processes are operating in a human embryo at
a particular time, even more so now that the outcomes must
be genome, environmental and epigenetic specific. Although
embryological language may have evolved in its origin and
usage, this evolution will dwindle now that terms are fixed in
computer ontologies. As the complexity of research outcomes
increases and not all may be clear of the meaning ascribed to a
term, it may be important that researchers define the particular
meanings they attribute to concepts, cells and tissues within
their studies. 
Conclusion
This chapter has presented and considered the changing ter-
minology used in the study and description of embryological
development. Terms coined by researchers reflect the tools, sci-
entific constructs and societal beliefs at the time of the study
of developmental processes. Some older terms and language
have been retained regardless of their limited specificity and
utility to described recent advances in embryology. The use of
computers to analyse the extensive data gathered on genomes
and their products has contributed its own terminology. Com-
puter algorithms used in the analysis of vast data arrays may
now themselves be limiting the development of new evolution-
ary and conceptual terms. Careful reflection may be needed
concerning the outcomes of computer algorithms and analy-
ses in embryological research and the ways in which contin-
ued evolution of embryological concepts and language can be
achieved.
Access the complete reference list online at ExpertConsult.com
Self-assessment questions available at ExpertConsult.com

References
1. H  aeckel E. The gastrea theory, phylogenetic
classification of the animal kingdom and the
homology of the germ lamellae (trans. E.O.
Wright). Q J Micr Sci. 1874;14:142–165,
223–247.
2. Von Baer KE. Entwicklungsgeschichte der Tiere:
Beobachtung und Reflexion. Königsberg: Bon-
träger; 1828.
3. Richardson MK. Heterochrony and the phylo-
typic period. Dev Biol. 1995;172:412–421.
4.  Le Douarin NM. The Nogent Institute—50
years of embryology. Int J Dev Biol. 2005;49:
85–103.
5. Tao H, Inoue K, Kiyonart H, et  al. Nuclear
location of Prickle2 is required to establish cell
polarity during early mouse embryogenesis.
Dev Biol. 2012;364:138–148.
6. Gray RS, Roszko I, Solnica-Kresel L. Planar
cell polarity: coordinating morphogenetic cell
behaviours with embryonic polarity. Dev Cell.
2012;21:120–133.
7. Wells JM, Spence JR. How to make an intes-
tine. Development. 2014;141:752–760.
8. Eiraku M, Sasai Y. Self-formation of layered
neural structures in three dimensional culture
of ES cells. Cur Opin Neurobiol. 2012;22:
768–777.
9. Lei NY, Jabaji Z, Wang J, et al. Intestinal sub-
epithelial myofibroblasts support the growth
of intestinal epithelial stem cells. PLoS One.
2014;9(1):e84651.
10. K olesky DB, Homan KA, Skylar-Scott MA,
Lewis JA. Three-dimensional bioprinting of
thick vascularized tissues. Proc Natl Acad Sci
U S A. 2016;113:3179–3184.
11. Takahashi K, Yamanaka S. Induction of plu-
ripotent stem cells from mouse embryonic and
adult fibroblast cultures by defined factors.
Cell. 2006;126:663–676.
12. Takahashi K, Yamanaka S. A developmental
framework for induced pluripotency. Develop-
ment. 2015;142:3274–3285.
13. Katz-Jaffe MG, McReynolds S, et al. The role
of proteomics in defining the human embry-
onic secretome. Mol Hum Reprod. 2009;15:
271–277.
14. Botros L, Sakkas D, Seli E. Metabolomics and
its application for non-invasive embryo assess-
ment in IVF. Mol Hum Reprod. 2008;14:679–
690.
15. Graves PR, Haystead AJ. Molecular biologist’s
guide to proteomics. Microbiol Mol Biol Rev.
2002;66:39–63.
16. Lombard-Banek C, Moody SA, Nemes P.
Single-cell mass spectrometry for discovery pro-
teomics: quantifying translational cell hetero-
geneity in the 16-cell frog (Xenopus) embryo.
Angev Chem In Ed. 2016;55:2454–2458.
17. Visel A, Thaller C, Eichele G. GenePaint.org:
an atlas of gene expression patterns in the
mouse embryo. Nucleic Acids Res. 2004;32:
D552–D556.
18. Pavie B, Rajaram S, Ouyang A, et  al. Rapid
analysis and exploration of fluorescence micros-
copy images. J Vis Exp. 2014;(85): e51280.
19. N  ord AS, Pattabiraman K, Visel A, Rubenstein
JLR. Genomic perspectives of transcriptional
regulation in forebrain development. Neuron.
2015;85:27–47.
20. Kaufmann MH. The Atlas of Mouse Development.
St. Louis: Elsevier; 1992.
21. O’Rahilly R, Müller F. Developmental Stages
In Human Embryos. Publication 637. Carnegie
Institution of Washington; 1987.
22. O’Rahilly R, Müller F. Developmental stages
in human embryos: revised and new measure-
ments. Cells Tisues Organs. 2010;192:73–84.
23. Liang G, Zhang Y. Genetic and epigen-
etic variations in iPSCs: potential causes and
implications for application. Cell Stem Cell.
2013;13:149–159.
24. De Paepe C, Krivega M, Cauffman G, et  al.
Totipotency and lineage separation in the
human embryo. Mol Hum Reprod. 2014;20:
599–618.
25. Boyle J, Kreisberg R, Bressler R, Killcoyne
S. Methods for visual mining of genomic
and proteomic data atlases. Bioinformatics.
2012;13:58–68.
26. Hutter H, Moerman D. Big data in Cae-
norhabditis elegans: quo vadis? Mol Biol Cell.
2015;26:3909–3914.
5.e1
2
Cellular Mechanisms and Embryonic
Tissues
PATRICIA COLLINS
KEY POINTS
• T his chapter describes the tissue types present in early em-
bryos and the interactions between these tissues.
• The membrane systems and cytoskeletal elements within a
typical cell are reviewed.
• Early embryos contain only epithelial and mesenchymal
populations.
• Each tissue type produces specialised extracellular matrix
molecules and proteins which permit and encourage tissue
interactions.
• Specific interactions between developing epithelia and
mesenchyme are presented, and the common cytokines and
growth factors are discussed.
This chapter provides a brief overview of concepts of cells and
tissues and the terminology by which developmental processes
are described. It should be noted that the genetic programme of
the early conceptus is now being studied by analysis of substances
secreted1 and metabolites produced,2 some of the very early cell
interactions between the fertilised oocyte and the maternal endo-
thelium have been revealed,3 and the ways in which cell types
interact before and during organogenesis is now being explored
in human embryonic stem cell and human-induced pluripotent
stem cell culture.4
General Characteristics of all Cells
All cell types have a plasma membrane and internal organelles,
and all are supported by a range of cytoskeletal structures. The
details and arrangement of proteins within cells and within the
matrices they synthesise encompass a vast region of research
beyond the scope of this text. Only the main proteins and struc-
tures necessary for the appreciation of developmental processes
are presented. Readers are recommended to consult other texts for
further details.5
Plasma Membrane
The plasma membrane of cells is composed of phospholipids mol-
ecules arranged in a bilayer. Within this layer are vast numbers
of proteins which may reside entirely within the bilipid layer or
6
protrude intracellularly, extracellularly, or both. Proteins which
project exteriorly are covered with carbohydrates as glycoproteins or
proteoglycans and contribute to the glycocalyx of the cell (Fig. 2.1).
The glycocalyx also contains glycoproteins and proteoglycans
which have been secreted into the extracellular space around the
cells and then absorbed onto the cell surface. Thus it is difficult
to specify exactly where a cell plasma membrane ends and the
surrounding extracellular matrix (ECM) begins. The glycocalyx
is fundamental in cell–cell and cell–matrix communication. Cells
place specific protein and carbohydrate groups into the glycocalyx
when touching and adhering to other cells, when displaying cell
markers to other cells, in blood clotting cascades and in inflam-
matory responses.
The membrane systems (organelles) within the embryonic
cells (i.e., nucleus, mitochondria, endoplasmic reticulum, Golgi
apparatus, lysosomes and secretory vesicles) are also made of the
plasma membrane; secretory vesicles can become incorporated
into the external plasma membrane to release contained contents.
Intracellular membrane systems are supported within the cyto-
plasm by a range of cytoskeletal elements which also allow cells to
maintain surface specialisations (microvilli), to change shape (as in
the movements of endocytosis and exocytosis), to move in specific
directions (with the glycocalyx molecules) and to adhere strongly
to a substrate when movement ceases. 
The Cytoskeleton
The cytoskeleton is a highly dynamic network of protein filaments
that extends throughout the cell. As it also allows the cell to move
or move portions of its plasma membrane, the cytoskeleton is less
like a bony framework and more like a moveable muscular sys-
tem. Three types of protein filaments produce a diverse range of
cytoskeletal elements, including actin filaments, microtubules and
intermediate filaments (Fig. 2.2); these are synthesised from actin,
tubulin and a range of fibrous proteins (e.g., vimentin, laminin,
respectively).
Actin filaments. Actin filaments are polar structures com-
posed of globular molecules of actin arranged as a helix. They
work in networks and bundles, often found just beneath the
plasma membrane, where they crosslink to form the cell cor-
tex. Actin filaments are used to change the shape of the plasma
membrane, moving it outwards in projections or inwards in
invaginations. Whereas discrete bundles of actin, anchored into
the cortex, can produce thin spiky protrusions of the plasma
 CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 7

LUMEN

Transmembrane
= Sugar residue Adsorbed glycoprotein proteoglycan

Glycocalyx

Glycolipid

Bilipid layer

Transmembrane
CYTOSOL glycoprotein

• Fig. 2.1  The glycocalyx is synthesised by epithelial cells. It is a feltwork of glycoproteins on the luminal
aspect of an epithelium. It is the interface for communication between the lumen of a tube or body cavity
and the cells.

Actin
supporting
microvilli

Cell cortex Centrosome

Actin filaments Microtubules Intermediate filaments

• Fig. 2.2  Three types of cytoskeletal elements within a cell allow the cell to maintain a shape or move it.

membrane, microvilli, sheet-like extensions of the membrane
(lamellipodia) are supported by continuous flattened bundles
of actin similarly anchored. Conversely, actin filaments can pull
portions of the membrane inwards in the formation of endo-
cytotic vesicles or in cell division. Here, contractile bundles of
actin associated with the motor protein myosin form. Although
myosin is most familiar in muscle fibres, nonmuscle cells contain
various myosin proteins. Contractile bundles of actin filaments
and myosin filaments are synthesised for specific functions and
then disassembled (e.g., during cell division after chromosomal
separation, the plasma membrane constricts to the middle of the
cell, allowing two daughter cells to separate). Such assemblies of
actin and myosin are also found in development near the apical
surface of epithelial cells where they play a role in folding of
epithelial sheets and in mesenchyme where they can form stress
fibres which allow the cells to exert tension on the ECM. 
8 SE C T I O N 1     Early Fetal Development
Microtubules. Microtubules are long, hollow cylinders
composed of the globular protein tubulin. They are much
more rigid than actin filaments. Microtubules emanate from
the centre of the cell in a region termed the centrosome. They
lengthen by adding tubulin to the proximal end of each micro-
tubule while subunits are lost from the distal end. The centro-
some offers a focus and region of stabilisation for the proximal
ends of the hundreds of microtubules in a cell; it also contains
the two centrioles which are used by the cell when dividing.
Vast numbers of microtubules extend in all directions to the
plasma membrane and seem to ensure that the centrosome is
at the centre of the cell. From this position, the microtubule
array sites the other cellular organelles and holds them in place
using a range of contact proteins. If a cell touches another cell,
there may be internal movements of the organelles driven by
the microtubules, resulting in repositioning of the centrosome.
Microtubules display a dynamic instability, with new subgroups
being added or subtracted very rapidly. The turnover of distal
units can be slowed by contact with proteins close to the plasma
membrane; this allows cells to maintain a particular shape and
polarity. Microtubules are also used in cell-surface specialisa-
tions, where they form the basis of cilia in the familiar 9+2
arrangement of nine microtubule doublets around a pair of
single microtubules.  Embryonic basal laminae are composed chiefly of laminin.
Intermediate filaments. Intermediate filaments are made of
a variety of proteins all formed from highly elongated fibrous
molecules. They are arranged as rope-like fibres which span
each cell often from one cell junction to another. They are
termed intermediate filaments because their apparent diam-
eter on electron microscopy is between that of actin filaments
and thick myosin filaments. Specific varieties of intermedi-
ate filaments are present in epithelial and mesenchymal cells.
Whereas epithelial cells contain keratin filaments, mesenchy-
mal cells have vimentin and vimentin-related filaments, and
in the cells which will develop a myogenic lineage, desmin
filaments are seen. Neuroepithelial cells develop neurofila-
ments and glial fibrillary acidic protein filaments are seen in
astrocytes.  membrane.
Embryonic Tissues
Two early tissue arrangements can be seen in embryos – epithelia
and mesenchyme. Individual cells within each arrangement secrete
extracellular proteins which form the ECM. This structures the
space around and within cell populations and provides the appro-
priate conditions for development.
Epithelia
Cells composing epithelia are polarised with apical and basal sur-
faces. Whereas the apical surface commonly displays specialised
features such as microvilli, the basal surface is the site of extracellu-
lar protein deposition in the form of a basal lamina. Laterally, the
cells contact their neighbours via varieties of juxtaluminal junc-
tional complexes which bridge the narrow intercellular clefts. Epi-
thelial cell polarity factors regulate the relative size of the surfaces
or domains and the internal organisation of the cytoskeleton.6 The
transmembrane protein Crumbs specifies the apical domain, Baz/
PAR-3 controls the position and extent of the junctions, Scribble
restricts the size of the junctional domains, an internal contractile
actomyosin network which produces the planar polarity of epi-
thelia is contiguous across the lateral borders of the cells through
E-cadherin adhesion,7 and the basal region displays integrins
which link to the basal lamina.
Basal lamina. Basal laminae are thin, flexible sheets of ECM
which are made by and underlie epithelial cells (Fig. 2.3). Basal
laminae are also found surrounding individual skeletal muscle
fibres, fat cells and Schwann cells. The presence or absence of a
basal lamina beneath an epithelium during development is of
consequence. Basal laminae organise the proteins in adjacent cell
membranes, induce cell differentiation and cell metabolism, serve
as routes for cell migration and can influence cell polarity in those
cells that touch them; they can change with time during develop-
ment and thus can maintain a developmental impetus.
The basal lamina is described in electron microscopic studies as
having an electron lucent layer, the lamina lucida or rara, closest to
the basal surface of the cell and an electron-dense layer, the lamina
densa below. If the epithelial layer rests on underlying mesen-
chyme, a layer of collagen fibrils connects the basal lamina to the
underlying tissue, sometimes with specialised anchoring collagen
fibrils. The strength of this connection is important for develop-
ment and growth. Many textbooks do not distinguish between the
basal lamina as described and the basement membrane, a thicker
layer which includes the basal lamina and extracellular compo-
nents of the underlying connective-tissue matrix.
Later, other extracellular molecules such as type IV collagen, per-
lecan and entactin (see below) contribute to the feltwork of the
layer (see Fig. 2.3). In some regions of the body, basal laminae
form specialised structures or units which have a specific function
during development or in adult life. An example of this arrange-
ment is seen in tooth development, where initially ameloblasts
and odontoblasts are separated by a basal lamina. The ameloblasts
deposit enamel directly onto one side of this basal lamina, and
the odontoblasts deposit dentine onto the other (see Fig. 2.12); in
this way, the tooth is formed. In both the kidneys and the lungs,
the basal lamina from the specialised cells of the organ abuts
directly onto the endothelial basal lamina, producing a selectively
permeable barrier. In the kidneys, this is the glomerular basement
In development, the basal lamina acts as a selective barrier to
the movement of cells, and migrating cells will move along basal
laminae but not through them. Cells beneath an epithelial layer see
only the basal lamina which the overlying cells produce. Changes
in the local basal laminal composition is one way by which the
epithelial cells can communicate with the cells migrating beneath
them. In adult tissues, the basal lamina permits the movement
of macrophages, lymphocytes and nerve processes and plays an
important part in tissue regeneration after injury. 
Cell–Cell junctions. Juxtaposed cells usually do not touch.
For contact to be established, the cells produce specific mol-
ecules which promote the development of a cell–cell junction
between them (Fig. 2.4). Junctional complexes allow sheets
of epithelial cells to act in concert in maintaining a barrier
or in producing alterations in the overall epithelial morphol-
ogy; they also permit cell–cell communication and are in this
respect especially important in development. Cell junctions
are classified into three main groups: (i) tight junctions, which
prevent leakage of molecules between cells from one side of
a sheet of cells to the other8; (ii) anchoring junctions, where
the neighbouring cell membranes attach and are supported by
cytoskeletal elements within the cells, either actin or interme-
diate filaments (this type of junction also anchors epithelial
cells to the ECM); and (iii) communicating junctions, which
 CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 9

Key:

Entactin

Perlecan

Laminin

Type IV collagen

• Fig. 2.3  A basal lamina is synthesised by epithelial cells. It is a feltwork of proteins which attach to
the epithelial cells and provide an attachment for underlying cells. It is the interface for communication
between the extracellular space and the cells.

mediate the passage of electrical or chemical signals from one
cell to another.
The formation of these junctional complexes is dependent on
a range of cell adhesion molecules (CAMs) (see Fig. 2.4). In cell–
cell anchoring junctions (adhesion belts and desmosomes), the
CAMs involved are termed cadherins; they are attached intracel-
lularly to intermediate or actin filaments in the cell cortex.9 The
latter run parallel to the plasma membrane; thus the actin bundles
of adjacent cells are linked. Concomitant contraction of the actin
bundles results in narrowing of the apices of the epithelial cells
and rolling of the epithelial layer into a deep groove or a tube.
Epithelial cells contact the underlying basal lamina they syn-
thesise by different types of anchoring junctions (hemidesmo-
somes and focal contacts). In these cases, the transmembrane
linker proteins belong to the integrin family of ECM receptors.10
Cytoskeletal filaments support the connection of the integrin
within the cell membrane to the ECM.
Communication between adjacent epithelial cells is mediated
by gap junctions. In forming a gap junction, each cell contributes
six identical protein subunits (called connexins) which form a
structure, similar to an old-fashioned cotton reel, termed a con-
nexon. This is situated across the bilaminar membrane with the
thicker rims extending into the extracellular and intracellular
spaces. Each connexon is capable of opening and closing, thus
controlling the gap. When two connexons from adjacent cells are
aligned, a tubular connection is made between the cells. Each gap
junction is really a cluster of apposed connexons which each per-
mit molecules smaller than 1000 daltons to pass through them. In
early embryos, most cells are electrically coupled to one another
by gap junctions. Later in development, epithelial cells synthesise
gap junctions at particular stages when it is inferred that informa-
tion is passing from cell to cell. When gap junctions are removed,
there is often a difference in differentiation in the cellular progeny.
Gap junctions are seen in adult tissues (e.g., connecting cardiac
10 SE C T I O N 1     Early Fetal Development

Actin supporting
microvilli

Key to cell junctions:

Tight junction Adhesion

molecules:

Junctional Cadherins
Adhesion belt
complex
Actin
Intermediate
filaments
Desmosome Cadherins

Gap junction Connexins

Hemidesmosome
Integrins

Basal lamina
• Fig. 2.4  Junctional complexes form between epithelial cells, preventing passage between cells, permit-
ting communication between cells, and joining cells to each other and to the basal lamina. Junctions are
supported by adhesion molecules and the cytoskeletal elements within the cells.

myocytes to permit transmission of the electrical signals of the car-

diac cycle). (For further information on CAMs, see Alberts et al.5) 

Mesenchymal Cells
Mesenchymal cells, in contrast to epithelial cells, have no polarity
and thus no directional surface specialisations. They have junc-
tional complexes which are not juxtaluminal, and they produce
extensive ECM molecules and fibres from the whole cell surface
(Fig. 2.5). As development proceeds, proliferating mesenchymal
populations begin to differentiate. This is often first seen by the
upregulation of specific mRNA in the cell or in the production of
different ECM molecules by selected progeny.
Extracellular matrix. A substantial part of the developing
embryo is made up of ECM. This name is given to the vast array
of complex molecules which are secreted locally by mesenchy-
mal cells and assembled into networks which structure the spaces
between the embryonic cells. In early development, mesenchymal
populations are composed of migrating epithelial cells and mesen-
chymal cells generated from germinal (proliferative) epithelia. It
is the latter group which will give rise to the range of connective
tissues seen in the adult. Connective tissues form the architectural • Fig. 2.5  Scanning electron microscope view of mesenchyme cells. Mes-
framework of the body, and the matrices determine the tissue’s enchyme cells have no polarity. They control the space around them by
physical properties (i.e., in bone, cartilage or fascia). Variation in their synthesis of extracellular matrix. (Photograph by Dr P. Collins.)

• Fig. 2.6  Mesenchyme cells and extracellular matrix (ECM), forming a lamina propria beneath an epithe-
lium. The mesenchyme cells synthesise collagens, proteoglycans and glycosaminoglycans as a frame-
work and add other proteins (e.g., fibronectin) as necessary. ECM is attached to a basal lamina, forming
a basement membrane.
the constituents and amount of the matrix molecules gives the
diversity of connective tissues (Fig. 2.6).
There are two main classes of ECM molecules, glycosaminogly-
cans (GAGs) which may be linked covalently to proteins as proteo-
glycans, and fibrous proteins, such as collagen, elastin, laminin and
fibronectin. The GAG and proteoglycan molecules form a highly
hydrated gel-like ground substance into which the fibrous pro-
teins are embedded.
Four main groups of GAGs have been described: (i) hyaluronic
acid, (ii) chondroitin sulphate and dermatan sulphate, (iii) hepa-
ran sulphate and heparin and (iv) keratan sulphate. Hyaluronic
acid is the simplest GAG. It is especially abundant in embryos,
where it fills the spaces between cells and because of its level of
hydration becomes turgid and generally resists compression. By
synthesising hyaluronic acid, cells can open up migration path-
ways or support epithelia which are undergoing morphological
change. The other GAGs, which are more complex than hyal-
uronic acid, have much shorter disaccharide chains, contain sul-
phated sugars and are usually bound to a protein core forming
proteoglycans. Aggregates of hyaluronate and proteoglycans can
make huge molecules which occupy a volume equivalent to a
bacterium.
Within tissues, GAGs can form gels of varying pore size and
thus act as filters regulating the movement of molecules accord-
ing to their size or charge. The heparan sulphate proteoglycan
perlecan is found in the basal lamina of the kidney glomerulus
and functions as a filter in the glomerular basement membrane.
CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 11
Basal lamina
Basement Proteoglycans
membrane hooking into
basal lamina
Collagen
Proteoglycan
links
Lamina
propria
Hyaluronic acid
filling in gaps
Mesenchyme
cell
Proteoglycans which bind various growth factors act as reservoirs
for messages which can be positioned in the matrix by cells at one
stage to be read by cells developing later. GAGs and proteoglycans
associate with the fibrous proteins in the matrix and provide sup-
port between the fibres.
Collagens form the major fibrous proteins in the matrix. Sev-
eral families or groups of collagens are described, each made from
a range of basic α-chains and each encoded by a separate gene.
Each collagen molecule is made from three α-chains wound
around one another like a rope. The main types of fibrillar col-
lagen found in connective tissue are types I, II, III, V and XI.
These types aggregate into the huge hawser-like bundles which
can be seen on electron microscopy. Collagen types IX and
XII are smaller and link the larger fibres to one another and to
other matrix molecules. Types IV and VII are found in the basal
laminae, where type IV forms the feltwork of the mature basal
lamina, and VII forms the anchoring structures which attach
the basal lamina to the underlying matrix. Collagen is used to
provide the initial matrices for cartilage and bone and is particu-
larly seen in tendons and ligaments. In these cases, the amount
of GAGs is reduced, and the collagen fibres are aligned by the
fibroblasts in response to the direction of the stresses acting on
the collagen. In this way, the connective tissues of the body are
responsive to the physical demands placed upon them. Anoma-
lies of collagen synthesis can give rise to diseases in life (e.g., the
condition osteogenesis imperfecta is caused by mutations in type
I collagen production, leading to bones which are brittle and
12 SE C T I O N 1     Early Fetal Development
fracture with little stress; mutations in type II collagen lead to
disorders of cartilage).
Elastin is composed of short elastin proteins which are cross-
linked so that when relaxed, the fibres are randomly coiled but
when stretched each elastin molecule can expand so contribut-
ing to the overall effect. Elastic fibres are at least five times more
extensible than a rubber band of the same cross-sectional area.
Usually collagen fibres are interwoven with elastic fibres to prevent
overstretching and damage.
Laminin is one of the first extracellular proteins synthesised in
the embryo, forming most of the early basal laminae. Each mol-
ecule is shaped like an asymmetric cross. Molecules join together
to form a feltwork often supported by smaller entactin molecules.
Fibronectin is a high-molecular-weight glycoprotein found in
extracellular matrices and in blood plasma. Within the ECM,
fibronectins promote cells adhesion. Generally, contact with
fibronectin causes cells to move. It has been shown in culture
that neural crest cells preferentially migrate along fibronectin-
rich substrates, and within three-dimensional cultures, migrating
cells can achieve their greatest speeds migrating along fibronec-
tin pathways. It is interesting to note that bonding with fibro-
nectin does not necessarily fix a cell to one position within the
matrix, so contacts with this protein are made and then released
with ease.
Biomechanical properties of the ECM. It is now appreciated
that mechanical characteristics and movements of the ECM are
fundamental to developmental processes and that mesenchymal
cells are sensitive to the tension and stiffness of the matrix itself.
The application of shear forces to matrix molecules results in fibre
alignment along the axis of force and the development of ten-
sion: an individual mesenchymal cell is able to sense parts of the a
matrix as soft or stiff depending on whether the tension is gener-
ated perpendicular (soft) or parallel (stiff) to a fibre.11 ECM move-
ments occur across tissue-level scales exhibiting both vortical and
convergent extension motion patterns. The physical-chemical and
biochemical reactions which drive matrix fibre assembly collec-
tively exert compression and stretching forces on embryonic cell
arrangements.12,13  the regulation of the genes within the cell and the interaction of
Cell–Matrix junctions. Cells interact with the ECM molecules
via protein receptors or co-receptors in the plasma membrane.
Syndecans are proteoglycan co-receptors which span the plasma
membrane. The extracellular part carries chondroitin sulphate
and heparan sulphate chains while the intracellular domain inter-
acts with the cell cortex actin filaments. Integrins are receptor
proteins which bind to and respond to the ECM information.
An extracellular receptor site binds with matrix molecules, espe-
cially fibronectin and laminin; intracellularly, the integrin binds
via vinculin to the actin cytoskeletal network or intermediate fila-
ments. When the integrins in the cell membrane contact a matrix
molecule (e.g., fibronectin), the orientation of the fibronectin
will cause alignment of the actin cytoskeleton and a reorienta-
tion of the cell itself. Later, when the cells are depositing ECM
molecules, the actin cytoskeleton will exert forces to orient the
matrix molecules in a similar configuration. Thus the interaction
between matrix molecules and cells and then cells and matrix can
drive development and propagate order from cell to cell. The cell–
matrix junctions permit communications within the embryo just
as gap junctions permit communications between cells. However,
the cell–matrix mechanisms allow messages to be left in the matrix
which may indicate migration routes or halt migrating cells and
suggest a differentiation pathway. The matrix information system
can thus control the temporal pattern of development.  differentiation. Similar cultures of cells taken from embryos at
Transformation from Epithelium to
Mesenchyme
The two embryonic states of epithelial tissue and mesenchyme
are not necessarily immutable, and transition from epithelia to
mesenchyme and vice versa occurs during development. How-
ever, such a change requires a temporal or external inductive
agent and causes dramatic upregulation and synthesis of a whole
variety of special intra- and extracellular molecules as previously
described. Generally, in an embryo, epithelial cells seem to derive
from existing epithelial populations, but mesenchymal cells are
produced initially from proliferative (germinal) epithelia and then
later by amplifying mitoses within the mesenchymal population.
Changes from one cell state to another are considered important
in development.
The early migrating cells derived from the primitive streak
have a mesenchymal morphology yet become epithelial when they
reach their destinations forming the somites, the somatopleuric
and splanchnopleuric epithelia lining the intraembryonic coelom,
including the lining of the pericardial cavity. All of these epithe-
lia become germinal centres which produce further mesenchymal
populations. However, an additional group of mesenchymal cells
derived from the neural crest never reverts to epithelia. Angiogenic
mesenchyme, which is believed to arise from extraembryonic sources
initially, is especially proliferative and capable of great migration
within the embryo; it differentiates into endothelium throughout
early development. A small subset of endothelial cells in the heart
are induced to transform back to mesenchyme at the atrioventric-
ular canal and the proximal outflow tract. This may be the only
example of a mesenchymal population derived from an endothelial
lineage14; the cells retain expression of an endothelial marker. 
Embryonic Induction and Cell Division
The earliest cells of the embryo are described as ‘totipotent’, indi-
cating that they have the capacity to differentiate into any cell type
in the body. The pathways cells take to differentiation depend on
the cell with its environment and neighbours. Information from
these other sources causes shifts in the differentiative fate of the
cell’s progeny.
All embryonic cell populations are initially receptive to induc-
tive signals. They respond by becoming committed to a particu-
lar pathway of development which thus restricts their ability
to respond to further inductive influences (i.e., they become
restricted). After a series of restrictions, cells are said to become
determined. Determined cells are programmed to complete a pro-
cess of development which will lead to differentiation. The deter-
mined state is a heritable characteristic of cells and can be passed
on to progeny; it is stable and not dependent on environmental
factors. The differentiated state is usually not heritable; it is often
dependent on environmental conditions, and it may prevent fur-
ther cell division.
The state of determination or differentiation may be assessed
by studying cells in culture. For example, melanocytes display the
black pigment melanin. If melanocytes are cultured without the
tyrosine needed to synthesise melanin, the cells will become pale
and no longer appear differentiated. When the tyrosine is replaced
into the culture medium, the cells will again synthesise melanin
and return to their differentiated state, illustrating that they main-
tained their determination despite not being able to display their
 CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 13

different stages of development will reveal which types of protein

Neurons
the cells are capable of synthesising and thus their level of deter-
mination or differentiation. Cell proteins have been classified as

(number fixed near birth)

Skeletal muscle fibres

Determinate tissues
‘basic’, or ‘housekeeping proteins’, if they are considered essential
Seminiferous tubules
for cellular metabolism and are termed ‘primary’, as are the genes
which regulate them. As cells become determined, they synthe- Renal nephrons
sise proteins appropriate to their cell group (e.g., liver and kidney
Heart muscle fibres
cells, but not myocytes, produce arginase); this class of protein is
termed ‘secondary’. Finally, at the most differentiated state, cells Pulmonary alveoli
produce ‘tertiary’ proteins (also called ‘luxury proteins’) specific to
Intestinal villi
their needs (e.g., haemoglobin in erythrocytes). This range of
proteins – primary, secondary and tertiary – is an expression of
Ovarian follicles
stages of determination and differentiation, and they are coded by

(number not fixed near birth)

a range of genes.

Indeterminate tissues
Thyroid follicles

Proliferative and Quantal Mitoses
Within a mesenchymal population, cell divisions will both increase
the total numbers of cells but also provide the foci for changes in
determination. In normal cell division, sometimes described as
proliferative, transient amplifying or multiplicative mitoses, prog-
eny similar to the parents are produced. In some situations (e.g.,
as a response to a local inductive influence), the cells will enter a
quantal cell cycle in which the outcome is a quantal mitosis which
increases the restriction of their progeny. The progeny then con-
tinues in amplifying mitoses at the progressive level of determina-
tion. It is at these quantal mitoses that binary choices are made in
the embryo.  (After Gilbert SF. Developmental Biology, Sinauer Associates, MA, 1992.
Stem Cells and Progenitor Cells
Within a proliferating cell population, instead of a division pro-
ducing two progenies with increased determination, mitosis may
produce one determined progeny and another cell with the same
state of determination as the parent. This latter cell can reproduce
again, passing on an increased state of determination to only one
offspring. This type of division has been termed ‘asymmetric’ in
contrast to the ‘symmetrical’ proliferating mitoses. Stem cells are
seen in development and in adult life (e.g., in the bone marrow or
in the gut epithelium).
Progenitor cells are those which are already determined to some
extent. They may individually follow their differentiation pathway
or may proliferate producing more similarly determined progeni-
tor cells. In this case, no stem cell can be identified; all cells seem
capable of either differentiation of continued mitosis. An example
is populations of migrating myoblasts in the embryonic limb bud.
It should be noted that the ability of stem cells to continue cell
division is intricately linked to the metabolic activity of these cells.
Stem cells use glycolytic pathways to generate energy, a method
related to the low oxygen tension prevalent during early develop-
ment and organogenesis and a necessary requirement of human
stem cells grown in culture.15 Consideration of mitochondrial
structure and metabolism in early embryos suggests a dynamic
interplay between developmental and differentiation processes
informed by cell signalling and epigenetic regulation.16  cell is presented to another and as a consequence the behaviour of
Terminal Differentiation
The differentiative fates of cells within embryos may be to become
long-living lymphoblasts or neuroblasts but may just as well be to
undergo ‘apoptosis’, also called ‘programmed cell death’. Apop-
tosis is a mechanism whereby cells bequeath their organelles to
Hepatic chords
Exocrine acini
Endocrine cells
Blood cells
Birth Maturity
1 3 6 9 1 3 6 9 10 30 6090
Prenatal months Postnatal years
Duration of hyperplasia
• Fig. 2.7  The duration of multiplicative growth for various human tissues,
By permission of Oxford University Press, USA.)
neighbouring cells without releasing any cellular fragments which
might stimulate an inflammatory response. Apoptosis occurs in
the adult state as well as in embryos. In organogenesis, apopto-
sis allows for some slack in the system. More cells than may be
needed are produced by amplifying mitosis; later the cells are
supported by the local ECM or by innervation or blood supply.
Those cells which are in excess and cannot be supported by these
means undergo apoptosis.17 As different tissues might be expected
to produce different sets of survival factors, a cell in an abnormal
location deprived of its specific signals required for survival would
also die.
The times at which cells cease proliferative mitoses and become
differentiated is different for different tissues (Fig. 2.7). Although
some tissues will all enter determined pathways and differentiation
before birth, other cells retain the ability to divide (e.g., as stem
cells) or if environmental conditions changes, as in a wound, are
able to revert temporarily to the determined state, affect a repair
and then differentiate once again 
The Cell Cycle
Determination, differentiation and development, in general, all
result from a series of interactions in which information from one
one or both cells are changed. Earlier embryonic studies described
the changes in shape in individual cells and cell populations which
were seen in development and then how these might be modi-
fied by experimentation. Studies of genes and proteins within
embryonic cells and tissues have elucidated some of the drivers
and checks of development, including when they appear within
the cell cycle.
14 SE C T I O N 1     Early Fetal Development
An increase in cell restriction begins with a quantal cell divi-
sion; thus the mechanisms of mitosis need to be examined. Cells
which are undergoing amplifying mitoses pass through a cell cycle
which lasts for 12 hours or more. The cell cycle is traditionally
divided into four phases, of which the most dramatic is mitosis
and cell division (Fig. 2.8). The phases of the cell cycle are noted
by the letters M for mitosis and G1, S and G2 for the interphase.
During mitosis, the cell packs up most of its organelles, the centri-
oles duplicate and move to each end of the cell and begin synthesis
of microtubules which are arranged as the mitotic spindle, and the
chromatin in the nucleus condenses into the chromosomes. As
the cell has already replicated its DNA, the chromosomes which
become visible at metaphase are duplicated but held together at
the centromere. The nuclear membrane breaks down, and the
chromosomes gather at the equator of the cell and are drawn apart
towards the poles of the spindle, where they decondense and re-
form intact nuclei. The cell cytoplasm divides by cytokinesis, an
event which is viewed as the end of the M phase.
G1 is the time period when the cell grows until it is large enough
to begin DNA synthesis. It may move along to S phase (synthesis)
when it has reached an appropriate size and if the environmental
conditions are favourable. If the cell is not yet committed to DNA
replication or is going to follow a differentiation pathway, the cell
can step out of the cycle into G0 for days, weeks or longer before
resuming proliferation. The majority of cells in adults are in G0.
The S phase marks replication of the nuclear DNA and is followed
by G2, which is the time taken for further growth. At the end of
G2, the cell must be of sufficient size, have replicated all its DNA
and be in a suitable environment before it can continue into mito-
sis. Signals from the cells or from the environment can prevent the
cell moving from one phase to another.
The cycle itself is driven by complexes of cyclins, so called
because they undergo synthesis and degradation in each division
cycle of the cell. The cyclins bind to cyclin-dependent protein
kinases to trigger mitosis and to trigger DNA replication. They
thus provide a checkpoint for exit from G1 and entry into S phase
and exit from G2 and entry into M phase.
In early embryonic cell divisions, the zygote and blastomeres
are very large. Growth is not required at this time, and the cleav-
age divisions operate to restore the nuclear-to-cytoplasm ratio,
s is G2
he
nt
Sy
Mitosis
+
G1
• Fig. 2.8  The four stages of the cell cycle. After cell division (mitosis), the
cell grows continuously until the next mitosis. The phases G1, G2 and S are
parts of interphase. (If the cell is not dividing, it enters G0.)
producing cells of typical size. Under these conditions, cells pass
through M phase and S phase in quick succession. After each divi-
sion, the cell progeny is half the size of the parent. Later, the cycle
lengthens, and various control systems come into operation. It is
important that the rate of passage through the S phase allows com-
pletion of DNA replication. If a cell enters M phase before replica-
tion is complete, it will die. The cell cycle control system receives
a feedback signal from incompletely replicated DNA, which will
prevent movement to the G2 phase. Similar protective mecha-
nisms ensure that DNA is only replicated once during S phase. 
Tissue Interactions
For the information carried in the genome of an embryo to be
expressed, the cells produced by mitotic division must be able to
contact and respond to each other; this process occurs locally by
the construction of gap junctions. However, as an embryo grows,
it is composed of more and more cell populations, which become
differentiated into tissues and separated by the matrix molecules
they produce. In these more complex situations, the tissues have a
repertoire of communication methods which require both epithe-
lial and mesenchymal arrangements and their matrices working in
concert. From a starting point of an early embryo at the body plan
stage, all of the basic organs arise through interactions between
close epithelial populations (plus their basal laminae), epithelia
and mesenchyme (with the ECM) and between differentiating
mesenchymal populations. The basic interactions are the same.
Such sequential spatial and temporal reciprocal interactions have
been termed epigenetic cascades.18
Permissive Interactions
It has been shown by experimental study that neither epithelia nor
mesenchyme will grow alone; each needs the other for DNA syn-
thesis and mitosis to occur. However, in many cases, it is not the
cell bodies that are required. Epithelial cells will grow in culture as
long as there is some sort of mesenchymal extract in the medium,
and mesenchymal cells will grow in culture as long as they can
contact a basal lamina. Thus in both cases, factors in the matrix
which have arisen from the cell population are enough to support
growth. These basic requirements of development are termed per-
missive interactions. The supporting tissue may not be that usually
present in development, and indeed much research time has been
spent in investigating which mesenchymal tissues would support
growth of specific epithelia and vice versa. However, in many cases,
although growth was maintained in these experiments, develop-
ment would not proceed at all or in the normal manner. For such
development, more information is needed from the reciprocating
tissues, and such information could enable both tissues to change
in a manner that neither of them could do without the informa-
tion. This is the basis of instructive interactions. 
Instructive Interactions
Wessells19 proposed four general principles which can be seen in
most instructive interactions:
• In the presence of tissue A, responding tissue B develops in a
certain way.
• In the absence of tissue A, responding tissue B does not develop
in that way.
• In the absence of tissue A but in the presence of tissue C, tissue
B does not develop in that way.
 CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 15

Collagen
Narrow cleft Hyaluronidase produced
by mesenchyme cells
Collagen fibrils

Mesenchyme cells
Hyaluronidase

Epithelial cells

Collagen fibrils

A B
• Fig. 2.9  Branching of a tubular duct may occur as a result of an interaction between the proliferating
epithelium of the duct and its surrounding mesenchyme and extracellular matrix. A, Mesenchymal cells ini-
tiate cleft formation by producing collagen III fibrils locally within the development clefts and hyaluronidase
over other parts of the epithelium. Collagen III prevents local degradation of the epithelial basal lamina by
hyaluronidase and slows the rate of mitosis of the overlying epithelial cells. B, In regions where no collagen
III is produced, hyaluronidase breaks down the epithelial basal lamina and locally increases epithelial mito-
ses, forming an expanded acinus (arrows). (After Gilbert SF. Developmental Biology, Sinauer Associates,
MA, 1992. By permission of Oxford University Press, USA.)

• I n the presence of tissue A, a tissue D, which would normally
develop differently, is changed to develop like B.
Thus in an instructive interaction, one tissue induces another
to respond in a specific way. If the target tissue can respond to the
inductive signal, it is called competent. If the target tissue does not
respond, it is described as non-competent. Non-competence may
be because the tissue has previously responded to an earlier induc-
tive signal which has restricted its repertoire of possible responses.
As development proceeds, more and more cell populations will
become non-competent as they differentiate.
Inductive interactions may be more or less complicated: only
the induced tissue may change or both tissues may change and par-
ticipate, as in morphogenesis, or, more commonly, several recipro-
cal inductive interactions may be required over a prolonged period
of developmental time before a specific organ or tissue will form.  Interactions
Epithelial–Mesenchymal Interactions
The instructive interactions between epithelia and mesenchyme
produce the general morphological changes which are seen in
every system throughout embryogenesis. They are a subset of
embryonic tissue interactions occurring between epithelial tissue
and specific subdivisions of mesenchymal tissue which arise dur-
ing differentiation. These interactions provide a mechanism for
coordinating and fine tuning the mitotic rates and differentiative
abilities of the two tissues. A range of interactions occurring in
different systems of the embryo is described.
Branching Morphogenesis
Most organs, from the lungs to the kidneys, initially develop a
main duct from which branches arise often dichotomously; later
these mature into typical patterns seen in the fully formed organ.
The mechanism of branching morphogenesis is therefore similar
in a variety of systems. Although the early descriptions of this pro-
cess described only the morphogenesis, now the specific matrix
molecules involved during the interaction have been identified
(Fig. 2.9).
At the tip of a proliferating duct, the epithelium and its basal
lamina is in contact with the underlying mesenchymal cells and
their ECM molecules. Local production of hyaluronidase by the
mesenchyme breaks down the basal lamina and promotes prolif-
eration of the epithelium. Cleft production is initiated by the mes-
enchyme, which produces type III collagen fibrils within putative
clefts. The collagen acts to protect the basal lamina from the effects
of the hyaluronidase and the overlying epithelia have a locally
reduced mitotic rate. The region of rapid mitoses at the tip of the
acinus is thus split into two, and two branches develop from this
point. If the type III collagen is removed from the cleft, branching
does not occur; if excess collagen is not removed, supernumerary
clefts appear. Note that this interaction may occur with any epi-
thelial type and any subpopulation of mesenchyme.20 
Neural Ectoderm and Surface Ectoderm
The interactions described are clearly seen in formation of the lens
of the eye. The optic cup is an outgrowth of the diencephalon of
the brain. As the cup approaches the overlying surface ectoderm, it
induces a local change. The ectoderm cells become narrower at the
apical region, causing the sheet of cells to curve and move towards
the optic cup. Ultimately, a small lens vesicle invaginates, and the
unaffected surface ectoderm becomes confluent over the structure.
If the optic cup is removed, no lens vesicle develops. If the optic
cup is removed and placed beneath a different portion of surface
ectoderm, a similar lens vesicle is induced (Fig. 2.10). 
Neural Ectoderm and Neural Crest Mesenchyme
Interactions: The ‘Fly-Paper Model’ of Skull
Development
The subtle interplay between the mesenchyme and the epithelial
basal lamina is demonstrated in the ‘fly-paper model’ of skull
development.21 Here, an interaction occurs between the neuroec-
toderm of the neural tube and the surrounding neural crest mesen-
chyme. The neuroepithelium displays fibronectin amongst other
fibrous proteins in the basal lamina. This ensures the migration
of the neural crest over the neural tube and into the developing
face. As development proceeds, the neuroepithelium transiently
expresses type II collagen in the basal lamina on the basal aspect
of the neural tube, around the olfactory regions, around the optic
16 SE C T I O N 1     Early Fetal Development

(iii) (iv)
Tissue other than optic vesicle Isolated optic cup can still
implanted—no induction induce lens vesicle

(ii)
No optic vesicle (i)
No lens induced Lens vesicle induced by
underlying optic cup
• Fig. 2.10  Induction of the lens vesicle by the optic cup. This illustrates clearly the four general principles
of an instructive interaction. (Wessells,Tissue Interactions and Development, 1st ed., ©1977. Reprinted
by permission of Pearson Education, Inc., New York.)

Chondrocranial elements
Transient collagen
contributing to base of the skull
type II distribution
seen in
Transient collagen type neuroectoderm of:
II distribution seen in
neuroectoderm of: Olfactory capsule Olfactory region

Trabecula
Ventrolateral

– Diencephalon
surfaces

– Mesencephalon Optic capsule Optic vesicles

– Rhombencephalon
Parachordal

Otic capsule Otic vesicles

Notochord
First vertebra

• Fig. 2.11  Transient expression of type II collagen in the basal lamina of the neuroepithelium causes mesen-
chyme cells which touch it to upregulate their own synthesis of type II collagen and differentiate along a chon-
drogenic pathway. The pattern of expression in the neuroectoderm determines the form of the chondrocranium.
(From Thorogood, P., Bee, J. & Von Der Mark, K. (1986). Transient expression of collagen type II at epithelio-
mesenchymal interfaces during morphogenesis of the cartilaginous neurocranium. Devi Biol. 116, 497-509.)

cups before and during lens invagination, around the otic vesicles
which will form the inner ear, and on the basal and lateral sur-
faces of the diencephalon, mesencephalon and rhombencephalon.
The notochord also expresses type II collagen in its basal lamina.
Some time after the type II collagen has been removed from the
basal laminae, neural crest mesenchyme adjacent to the regions
listed commences synthesis of type II collagen and ultimately dif-
ferentiates along a chondrogenic pathway. It seems as if the type
II collagen in the basal lamina affects the cells which contact it
and causes their upregulation of type II collagen. The pattern of
the expression of type II collagen in the basal laminae determines
the form of the chondrocranium (Fig. 2.11). Slight alterations in

ORAL ECTODERM Dental epithelium
Epithelial
cell–tissue
transformations
Epithelium
Epithelium causes
Mesenchyme mesenchyme
to condense
Epithelial–
mesenchymal
interactions
Dental mesenchyme
induces oral
epithelium to become
dental epithelium Forming dental papilla
JAW MESENCHYME Dental mesenchyme
Mesenchymal
cell–tissue
transformations
• Fig. 2.12 A summary of the epithelial–mesenchymal interactions during tooth development. (From
Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)
the pattern of expression could have profound effects on the shape
and form of the chondrocranium, perhaps producing the diversity
of skull shapes seen in vertebrates.  case, the neural crest mesenchyme has already been induced along
Surface Ectoderm and Neural Crest Interactions
A further example of a reciprocal tissue interactions can be seen in
mammalian tooth development summarised in Fig. 2.12.22 Tooth
development begins along the dental lamina of the premaxilla,
maxillae and mandible. The epithelium proliferates to form an
enamel organ under the influence of the neural crest mesenchyme,
which forms a dental papilla; together this unit is a tooth bud or
germ. The enamel organ induces the dental papilla mesenchyme
to become odontoblasts; these cells then induce the epithelial cells
to differentiate into ameloblasts. The tooth is formed by matrix
deposition each side of the epithelial basal lamina, enamel on one
side and dentine on the other.
The neural crest mesenchyme is responsible for patterning
development of the pharyngeal arches, including tooth forma-
tion. Dental papilla mesenchyme is able to induce the formation
of teeth in nonoral epithelium and can specify the type of tooth
produced (e.g., incisor or molar). If cranial neural crest is cultured
alone, it will differentiate into cartilage. When it is recombined
with limb epithelium, then cartilage and bone will form. How-
ever, if cranial neural crest is recombined with mandibular epithe-
lium, salivary islands, hair and teeth form as well as cartilage and
bone, indicating that the mandibular epithelium is essential and
specific for the development of teeth.
Early recombination (9–11.5 days of development) of mouse
mandibular epithelium (first arch) and hyoid mesenchyme (sec-
ond arch) results in teeth in 90% of cases, showing that the
dental lamina epithelium can induce tooth development in the
head neural crest mesenchyme. The reverse recombination – early
second arch epithelium and mandibular arch mesenchyme – does
not produce teeth, indicating that it is only the first arch epi-
thelium which has this property. However, later recombination
CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 17
Enamel organ Pre-ameloblasts
Ameloblasts
ENAMEL
Enamel organ epithelium
induces dental papilla
mesenchyme to become
Odontoblastic mesenchyme
Dental papilla induces pre-odontoblasts
induces epithelial
epithelium to form an and odontoblasts
pre-ameloblasts to
enamel organ
become ameloblasts
Dental papilla Pre-odontoblasts Odontoblasts
Predentine
DENTINE
experiments (11.5–12 days of development), with second arch
epithelium and first arch mesenchyme, will produce teeth. In this
a dental lineage.23
The local specification of particular teeth can be changed
experimentally. If presumptive incisor region of the mandibular
epithelium is recombined with predetermined molar papillae
from post-day 12 tooth germs, the shape of the teeth can be rede-
fined by the epithelium and incisiform teeth develop. 
Surface Ectoderm and Somatopleuric
Mesenchymal Interactions in the Limb
The tissues involved in limb development arise from a ridge along
the flank of the embryo. Interaction of specialised regions of the
somatopleuric mesenchyme with the overlying ectoderm gives rise
to local, thickened regions of surface ectoderm and proliferation
of the underlying mesenchyme. At these sites, the ectoderm forms
a longitudinal ridge of high columnar cells, the apical ectodermal
ridge (AER) and the specialised somatopleuric mesenchyme main-
tains its growth. The AER and underlying mesenchyme together are
termed the progress zone, and the limb grows meristematically from
this point (i.e., proliferation produces the next distal portion of
the limb). These two populations require each other. Only the api-
cal ectodermal ridge can promote limb outgrowth, and only limb
mesenchyme will result in limb development. Positional values are
assigned to the proliferating epithelial and mesenchymal popula-
tions by the progress zone; thus first the humerus is developed, then
the radius and ulna, then the carpals and so on (Fig. 2.13).
Within the portion of the limb which has received its positional
value, the mesenchyme instructs the overlying ectoderm about the
appropriate epidermal structure to develop. Parts of a chick hind
limb develop different characteristics with feathers on the thigh
and scales on the leg. If mesenchyme from the thigh is transplanted
beneath the leg, feathers will develop instead of the normal scales.
This type of experiment has been repeated by recombining neck
18 SE C T I O N 1     Early Fetal Development

Specification of axes of the limb

Proximal

Pr
e-
ax
ia
l

Ventral

Dorsal
Po
Distal st
ax
ia
l
1. Proximodistalyaxis 2. Craniocaudalyaxis 3. Dorsoventralyaxis

1. Proximodistalyaxis

Progress zone

Extra AER Duplicated distal limb

Apical ectodermal
ridge (AER)

Somatopleuric
limb mesenchyme

Leg Leg features grow

mesenchyme distally on wing

2. Craniocaudalyaxis
III
Extra ZPA
IV II

III
IV

Zone of polarising activity (ZPA) Extra ZPA grafted Postaxial border with
specifies the post axial border on other side digit IV duplicated

3. Dorsoventral axis

No pre-axial or postaxial specification

ZPA dispersed in mesenchyme – replaced into ectoderm sleeve
Direction of joints still indicates dorsoventral axis
• Fig. 2.13  The three axes of the limb are specified by different interactions. The proximodistal axis is
specified by the progress zone, the craniocaudal axis by the zone of polarising activity, and the dorso-
ventral axis by the ectoderm of the limb. The pattern of development within the limb and the ectodermal
specialisations are controlled by the limb mesenchyme.

ectoderm, which will not normally develop feathers, with thigh
mesenchyme; in this case, the epidermis will develop feathers.
If thigh mesenchyme is inserted beneath the apical ectoder-
mal ridge of a developing wing which is at the stage to assign
radius and ulna, two things occur. First, information from the
apical ectodermal ridge about the age of the limb will override the
‘thighness’ of the mesenchyme, and it will express leg characteris-
tics appropriate to the developmental time of the wing. Thus the
proximal limb characteristics are replaced by distal ones. Second,
the wing epithelium is reassigned to develop leg characteristics by

the mesenchyme. Thus the limb develops a fibula and tibia and
the epidermis displays scales. This latter experiment shows the
reciprocal nature of inductive interactions with both tissues giving
and responding to information.
Positional information which causes the development of the
axes of the limb is controlled by both mesenchyme and epithe-
lium. A subset of mesenchyme situated at the postaxial border of
the limb bud termed the zone of polarising activity (ZPA) controls
the cranio-caudal axis of the limb (i.e., where the thumb devel-
ops). Active substances released at this region cause a number five
digit to form, the little finger (Fig. 2.13). The reducing influence
of this substance allows development of digits four, three, two
and then the thumb to develop. However, even if this system is
disrupted by mixing the mesenchyme up within the limb pro-
ducing five equally structured digits, the limb still displays dorsal
and ventral surfaces. The inductive influence for dorsal and ventral
patterning is specified by the ectodermal epithelium.  lium. The advance of the endodermal epithelial cells promotes the
Endoderm and Splanchnopleuric Mesenchyme
Interactions in the Lung
The respiratory tree derives from interactions between endodermal
epithelium and surrounding splanchnopleuric mesenchyme. The
trachea arises from the pharynx as a midline, ventral diverticulum
which grows causally then bifurcates into the primary bronchi,
which expand dorsally on each side of the oesophagus. Originally,
splanchnopleuric mesenchyme surrounding the pharynx enve-
lopes both the oesophagus and the trachea; however, the proxim-
ity of the lung buds to the pericardio-peritoneal canals, which will
later give rise to the pleural cavities provides a different mesenchy-
mal population. Proliferation of the adjacent splanchnopleuric coe-
lomic epithelium (of the primary pleural cavities) provides investing
mesenchyme around the developing trachea and lung buds from
stage 13 of development. The proliferative activity decreases in
stage 14, and the mesenchyme becomes arranged in zones around
the developing endoderm. This investing mesenchyme contains a
mixed population of cells, that which will pattern the endodermal
epithelium, a subpopulation of angiogenic mesenchyme which
may migrate in to form the endothelial networks surrounding the
air sacs, and splanchnopleuric mesenchyme which will give rise to
the smooth muscle cells which surround both the respiratory tubes
and the blood vessels. After its proliferative phase, the splanchno-
pleuric coelomic epithelium gives rise to the visceral pleura.
The control of the branching pattern of the respiratory tree
resides with the splanchnopleuric mesenchyme and particularly
Fgf10 expression.24 Whereas recombination of tracheal mesen-
chyme with bronchial respiratory endoderm causes an inhibition
of bronchial branching, recombination of bronchial mesenchyme
with tracheal epithelium induces bronchial outgrowths from the
trachea.19,25 Initially, the tracheal mesenchyme is continuous with
that surrounding the ventral wall of the oesophagus, but with
lengthening and division of the tracheal bud and deviation of the
lung buds dorsally, each bud becomes surrounded by its own spe-
cific mesenchyme, thus permitting regional differences between
the lungs (i.e., the number of lobes or the degree of growth and
maturity of a particular lung). Each lung develops by a process of
dichotomous branching as described in branching morphogen-
esis. Tenascin, an ECM molecule (also known as hexabrachion or
cytotactin), is present in the budding and distal tip regions but
absent in the clefts. Conversely, fibronectin is found in the clefts
and along the sides of the developing bronchi but not on the bud-
ding and distal tips.26  densation of mesenchyme termed the metanephric blastema; and
CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 19
Endoderm and Splanchnopleuric Mesenchyme
Interactions in the Gastrointestinal Tract
Liver. The liver is a very precocious embryonic organ, func-
tioning as the main centre for haemopoiesis in fetuses. It devel-
ops from an endodermal evagination of the foregut and from the
septum transversum mesenchyme, a region of unsplit lateral plate
mesenchyme at the very rostral edge of the disc before head fold-
ing. The development of the liver is intimately related to the devel-
opment of the heart as the vitelline, followed by the umbilical
veins, are disrupted by the septum transversum to form a hepatic
plexus the forerunner of the hepatic sinusoids.
Endodermal epithelial cells from the foregut proliferate and
extend as lines of epithelial cells into the septum transversum
mesenchyme. Contact of endodermal epithelium with the mes-
enchymal cells induces them to form blood islands and endothe-
conversion of more and more septum transversum mesenchyme
into endothelium and blood cells with only a little remaining to
form the scanty (human) liver capsule and interlobular connective
tissue.
The morphogenesis of the liver lobes is patterned by the sep-
tum transversum mesenchyme, and both endoderm and septum
transversum mesenchyme are required for normal liver develop-
ment.27 If a mechanical barrier is inserted across the mesenchymal
hepatic area just caudal to the endodermal outgrowth, liver tissue
will develop normally cranial to the barrier where it is in contact
with the endoderm. However, caudal to the barrier, the mesen-
chyme will form endothelial cells and hepatic lobes, but there will
be no hepatocytes present.
Experiments in which either the epithelium or the mes-
enchyme is changed will not result in liver development (e.g.,
cephalic and somitic mesenchyme do not promote the differ-
entiation of hepatic endoderm, and intestinal endoderm cells
combined with hepatic mesenchyme will not differentiate into
hepatocytes). However, all derivatives of the lateral plate mesen-
chyme, both somatopleuric and splanchnopleuric mesenchyme,
can promote the differentiation of hepatic endoderm, although
not so strongly as hepatic mesenchyme, and lateral plate mes-
enchyme will form blood sinusoids under these conditions. It
is inferred that matrix or cell surface properties are common
throughout the lateral plate mesenchyme but are different from
axial mesenchymal cells.28 
Gastric mucosa. Within the gastrointestinal tract, the local
organisation of the mucosa and smooth muscle layers is under
the control of the local splanchnopleuric mesenchyme. Recom-
bination experiments of chick gut epithelium and mouse mesen-
chyme and vice versa show that the patterning of the intestinal
villi is determined by the underlying mesenchyme. However, the
epithelial cells produce enzymes associated with the relevant spe-
cies (e.g., mouse epithelium produces lactase and chick epithelium
sucrase, regardless of the origin of the underlying mesenchyme).29
Indeed, culture of human pluripotent stem cells has resulted in the
development of human small intestinal organoids, which can be
transplanted into mice to mature.30,31 
Intraembryonic Mesoderm and Intermediate
Mesenchyme Interactions
The metanephric kidney develops from three sources, an evagi-
nation of the mesonephric duct, the ureteric bud; a local con-
20 SE C T I O N 1     Early Fetal Development
angiogenic mesenchyme, which migrates into the metanephric blas-
tema slightly later to produce the glomeruli and vasa recta.32-34 It
may also be the case that innervation is necessary for metanephric
kidney induction.
During embryonic development, functional mesonephric
kidneys develop but are remodelled in male embryos as parts
of the reproductive tracts. The mesonephric kidneys develop
on the posterior abdominal wall and extend ultimately from
the pleural region to the lumbar, with both development and
regression proceeding in a craniocaudal progression. However,
in the metanephric kidney, a proportion of the mesenchyme
remains as stem cells, which continue to divide and enter
the nephrogenic pathway later as individual collecting ducts
lengthen. Thus the temporal development of the metanephric
kidney is patterned radially with the outer cortex being the last
part to be formed.
In each kidney, a ureteric bud arises as a diverticulum from
the mesonephric duct and grows dorsally to enter the meta-
nephric mesenchymal population. The bud bifurcates when it
comes into contact with the metanephric blastema as a result
of local ECM molecule synthesis by the mesenchyme.35 Both
chondroitin sulphate proteoglycan synthesis and chondroitin
sulphate GAG processing are necessary for this and consequent
branching of the ureteric bud.36 Subsequent divisions of the
ureteric bud and the mesenchyme form the gross structure of
the kidney with major and minor calyces; the distal branches
of the ureteric ducts form the collecting ducts of the kidney.
As the collecting ducts elongate, the metanephric mesenchyme
condenses around them. The ureteric bud undergoes a further
series of bifurcations within the surrounding metanephric
mesenchyme, forming smaller ureteric ducts. The metanephric
mesenchyme condenses around the dividing ducts into smaller
condensations, which then undergo a mesenchyme to epithe-
lium transformation forming vesicles. To initiate this trans-
formation, the cells cease production of mesenchymal matrix
molecules and commence production of epithelial ones. This
has been demonstrated in tissue culture.
Initially, syndecan can be detected between the mesenchymal
cells in the condensate. The cells cease expression of the cadherin
N-CAM, fibronectin and collagen I and commence production
of the cadherin L-CAM (also called ‘uvomorulin’) and the basal
laminal constituents laminin and collagen IV. The mesenchymal
clusters thus convert to small groups of epithelial cells which
undergo complex morphogenetic changes (Fig. 2.14). Each epi-
thelial group elongates and forms a comma-shaped and then an
S-shaped body, which elongates further. It then fuses to a branch
of the ureteric duct at its distal end while expanding as a dilated
sac at the proximal end. The sac involutes with local cellular dif-
ferentiation such that the outer cells become the parietal glomeru-
lar cells, while the inner ones become visceral epithelial podocytes.
The podocytes develop in close proximity to invading capillaries,
which derive from local angiogenic mesenchyme37; this third
source of mesenchyme produces the endothelial and mesangial
cells within the glomerulus. Both the metanephric-derived podo-
cytes and the angiogenic mesenchyme produce fibronectin and
other components of the glomerular basal lamina. The isoforms of
type IV collagen within this layer follow a specific programme of
maturation, which occurs as the filtration of macromolecules from
the plasma becomes restricted.38
Interestingly, although the interactions in kidney development
were usually focused on the induction of metanephric mesen-
chyme by the ureteric bud epithelium, it had been noted that when
cultured across a filter, the inductive stimulus was quite weak. In
contrast, fragments of spinal cord were found to be very potent
inducers of metanephric mesenchyme, initiating epithelialisa-
tion. This suggested that perhaps nerves arriving at the interaction
site during development were of some importance. Indeed, it has
been shown that blocking nerve growth factor receptor mRNA
in the developing kidney not only prevents receptor synthesis
but also that nephrogenesis is also completely halted.39 The use
of human pluripotent stem cells provide additional study routes
of embryonic kidney cell populations; however, it is not yet clear
how well these in vitro populations reflect normal human kidney
development.40 
Other Cells Types Affecting or Affected by Local
Interactions
It seems that the basic epithelial–mesenchymal interaction
may not be so basic or simple after all. Whereas initial observa-
tions were made on cultured embryos, later studies were made
on cultured embryonic explants which could be perturbed in
some manner. The range of inductive tissues was ascertained
using other mesenchymes or epithelia from the same embryo
from different embryos of the same species and even from
different species. Now it seems that the homogeneity of the
mesenchymes under examination may have been assumed.
The presence of angiogenic mesenchyme may be fundamental
for some interactions. Early in development, it arises extra-
embryonically from the parietal hypoblast. Later, angiogenic
mesenchyme is seen close to endodermal epithelia but not
ectodermal. It is not clear whether the majority of angiogenic
mesenchyme arises close to endodermal populations as it has
now been shown to arise from somite derived mesenchyme.41
Early embryonic angioblasts are highly invasive, moving in
every direction throughout embryonic mesenchymal tissue.
It is likely that these cells contribute to interactive processes,
especially in those organs where close proximity of endothelia
to specialised cells is a feature.
Similarly, the innervation of blood vessels, glandular cells
and myoepithelial cells may need to be achieved early in devel-
opment, at the time of the early interactions. Indeed, the con-
dition Hirschsprung disease, which results in a failure of neural
crest cells to colonise the gut appropriately and form local con-
stituents of the enteric nervous system, is thought to occur
because of disordered basal laminal molecules. The enteric
nervous system arises from neural crest cells at somite levels
1 to 7 and from 28 onwards. Normally, the crest cells invade
the splanchnopleuric mesenchyme around the endoderm and
site themselves in the putative submucosal and myogenic lay-
ers. The splanchnopleuric mesenchyme will develop into both
the lamina propria connective tissue lineages and the smooth
muscle cells of the muscularis mucosa and the muscularis pro-
pria. Hirschsprung disease is characterised by a dilated segment
of colon proximally and lack of peristalsis in the segment distal
to the dilation. Infants with this condition show delay in the
passage of meconium, constipation, vomiting and abdominal
distension. A mouse model has been investigated which dem-
onstrates the same pathology and symptoms.42 In normal mice,
laminin and type IV collagen are found beneath the mucosal
and serosal epithelia and around the blood vessels. In mutant
mice, there is a broad zone of these basal laminal proteins
around the entire outer gut mesenchyme with an increase in
the amount of laminin, type IV collagen and heparan sulphate,
 CHAPTER 2  Cellular Mechanisms and Embryonic Tissues 21

Epithelial ureteric bud and Metanephric mesenchyme transforms

metanephric mesenchyme to form comma-shaped epithelial vesicle

Comma-shaped
vesicles become
S-shaped

Distal

Renal
corpuscle
Capsule
Proximal (simple squamous)

Tubule simple Podocyte

S-shaped vesicles elongate, cuboidal
forming parts of nephron
The proximal end invaginates and, Loop
with blood vessels, forms the
renal corpuscle

• Fig. 2.14  In the developing kidney there is a mesenchyme-to-epithelial transformation. The epithelial ure-
teric bud forms the collecting ducts in the kidney. It induces the metanephric mesenchyme to transform
into local epithelial vesicles, which develop into the nephrons.

specifically in the aganglionic portion of bowel. It is suggested
that the overabundance of basal laminal components prevents
the neural crest cells from penetrating the gut wall; their new
position outside the gut does not confer on them the environ-
mental stimuli for enteric nerve differentiation. Consequently,
these crest cells differentiate into autonomic ganglia and nerves
similar to the parasympathetic nerves which normally modu-
late enteric nervous system activity. This system demonstrates
the importance of local mesenchymal and ECM activity for the
normal induction of neurons as well as epithelial cells. 
22 SE C T I O N 1     Early Fetal Development
Cytokines and Growth Factors
As a result of experiments to find the most appropriate media for
maintaining the proliferation of cells in culture, two families of
‘factors’ were identified, arising initially from lymphocytes and
from platelets. Culture of lymphocytes and macrophages revealed
a family of factors within the supernatant which was able to facili-
tate cell–cell communication and proliferation. These soluble fac-
tors were isolated and collectively termed cytokines. The cytokines
include subtypes of interleukin (IL), which cause the main T- and
B-cell proliferation; types of interferon (IFN), which are mainly
antiviral in nature; tumour necrosis factor (TNF) α and β, which
are cytotoxic; transforming growth factor β (TGFβ), which inhib-
its T- and B-cell proliferation; and granulocyte-macrophage colony-
stimulating factor (GM-CSF) with factors for granulocytes and
macrophages individually, which promote growth. The prolifera-
tive actions of cytokines are mediated by specific cell receptors on
the surface of target cells.
Culture of mammalian cells was found to be more success-
ful with blood serum added to the medium rather than plasma.
Whereas serum is the fluid which remains after blood has clot-
ted, plasma is obtained by removing the cells without permitting
clotting to occur. The difference between these two fluids is the
presence of factors released from platelets as the blood clotted.
Experiment showed that an extract of platelets alone added to the
medium would support cell-culture proliferation, and the extract
was termed a growth factor. The particular growth factor from the
platelets was shown to be a protein and named platelet-derived
growth factor (PDGF). For PDGF to have an effect on a target cell,
the cell must display an appropriate receptor protein on its surface
as in the action of cytokines. Receptor proteins for cytokines and
growth factors are part of the cell glycocalyx along with inter alia
CAMs and integrins.
Large families of growth factors have now been identified, not
all of them proteins; steroid hormones which act on intracellular
receptor protein are an example. Growth factors have been divided
into broad- and narrow-specificity classes or families. PDGF is a
broad-specificity factor as is epidermal growth factor (EGF). PDGF
acts on fibroblasts, smooth-muscle cells and neuroglial cells, and
EGF acts on epidermal cells and on many embryonic epithelia.
Other broad-specificity growth factors are the insulin-like growth
factors (IGFI, IGFII) (previously termed ‘somatomedins’), stimu-
lating cell metabolism and with other growth factors stimulating
cell proliferation; fibroblast growth factor (FGF) with subgroups,
again being inductive in embryos; and transforming growth factor β
(TGFβ), having been identified in lymphocytes and also grouped
as a cytokine yet being produced widely by many cell types. The
TGFβ family also includes activins and bone morphogenetic pro-
teins (BMPs), which similar to TGFβ, may suppress growth as
well as stimulate it.
Of the narrow-specificity factors, nerve growth factor (NGF)
and related brain-derived neurotrophic factor (BDNF), and neu-
rotrophins 3 and 4 promote survival of neurons; erythropoietin
promotes proliferation and differentiation of erythrocytes; and
interleukin-3 (IL-3) and related haemopoietic colony-stimulating
factors (CSFs), which are also classed as cytokines, stimulate prolif-
eration of blood cell precursors.
Cytokines and growth factors can signal a wide variety of cel-
lular effects, including stimulation or inhibition of growth, differ-
entiation, migration and so on.43 Because each family is so large
and because there are a similarly extensive number of receptors,
the possible signalling combinations are considerable. Often, a
single growth factor can bind with varying affinities to individual
receptor family members. Whereas some of these receptors are
monogamous, recognising only one isoform of the growth factor,
others are polygamous, recognising all isoforms. The effects of any
individual growth factor may therefore depend on which recep-
tor isoform, or ratio of isoform receptors, is displayed on the cell
surface. It is now clear that developmental information resides not
in any single molecule but rather in the combination of molecules,
to which a cell is exposed as development progresses. Thus varying
combinations of growth factors, in varying concentrations, can
elicit quite different effects on similar cells.44
As well as an expansion in the range of growth factors identi-
fied within development, knowledge of the genes which code for
them and ways of demonstrating them has also contributed to
the complexity of understanding embryological processes. Many
genes have been shown to be ubiquitous throughout embryos
and conserved through evolution, making viable transfer of tis-
sues between animal groups possible. Responses to deletion of
specific gene action, or application of cytokines and growth fac-
tors at inappropriate times and places in the embryo add to the
body of knowledge, but because interactions are driven by com-
plex cascades of many growth factors, each study can only add
a small piece to the jigsaw. Stern45 noted that embryos generate
complexity with only a few extracellular signals, each of which has
multiple roles at different developmental times. Thus each time
we appear to understand a developmental process and declare a
‘default’ pathway of development that is understood, more com-
plexity is uncovered. 
Conclusion
This chapter gives an overview of the morphology of the cells within
an embryo and how they join to form tissues. The cell membrane
and the intercellular organelles which affect it are considered along
with the cytoskeletal elements which support apical specialisations
and cell membrane movements and give the three-dimensional
shape to a range of body cells. Epithelia and mesenchyme are pre-
sented, and the importance of the transition of one cell pheno-
type to the other is considered. The main epithelial–mesenchymal
interactions, including examples of branching morphogenesis, are
outlined for a range of developing systems. The terminology for
cell–cell interactions and the cell cycle is presented. An overview of
the main families of cytokines and growth factors is given.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 16. Folmes CDL, Terzic A. Metabolic determi-
nants of embryonic development and stem cell
31. Finkbeiner SR, Freeman JJ, Wieck MM,
et  al. Generation of tissue-engineered small
fate. Reprod Fertil Dev. 2013;27:82–88. intestine using embryonic stem cell-derived
1. Katz-Jaffe MG, McReynolds S, Gardner DK,
17. Zakeri Z, Penaloza CG, Smith K, et al. What human intestinal organoids. Biol Open.
Schoolcraft WB. The role of proteomics in
cell death does in development. Int J Dev Boil. 2015;12:1462–1472.
defining the human embryonic secretome. Mol
2015;59:11–22. 32. Grobstein C. Inductive interaction in the
Hum Repro. 2009;15:271–277.
18. Hall BK. Evolutionary Developmental Biology. development of the mouse metanephros. J Exp
2. Syggelou A, Iacovidou N, Atzori L, et  al.
London: Chapman and Hall; 1992. Zool. 1955;130:319–340.
Metabolomics in the developing human being.
19. Wessells NK. Tissue Interactions and Development. 33. Saxen L, Koskimies O, Lahti A, et  al. Dif-
Pediatr Clin North Am. 2012;59:1039–1058.
Menlo Park, CA: Benjamin/Cummings; 1977. ferentiation of kidney mesenchyme in an
3. Macklon NS, Bronsens JJ. The human endo-
20. Harunaga JS, Doyle AD, Yamada KM. Local experimental model system. J Adv Morphogen.
metrium as a sensor of embryo quality. Biol
and global dynamics of the basement mem- 1968;7:251–293.
Reprod. 2014;91(98):1–8.
brane during branching morphogenesis require 34. Saxen L. Failure to demonstrate tubule induc-
4. Takasato M, Little MH. The origin of the
protease activity and actomyosin contractility. tion in a heterologous mesenchyme. Dev Biol.
mammalian kidney: implications for rec-
Dev Biol. 2014;394:197–205. 1970;23:511–523.
reating the kidney in  vitro. Development.
21. Thorogood P. The developmental specifica- 35. O’Brien LL, McMahon AP. Induction and
2015;142:1937–1947.
tion of the vertebrate skull. Development. patterning of the metanephric nephron. Semin
5. Alberts B, Johnson A, Lewis J, et al. Molecular
1988;103(suppl):141–153. Cell Dev Biol. 2014;36:31–38.
Biology of the Cell. 6th ed. New York: Garland
22. Lumsden AGS. Neural crest contribution to 36. Fouser L, Avner ED. Normal and abnormal
Science; 2014.
tooth development in the mammalian embryo. nephrogenesis. Am J Kidney Dis. 1993;21:64–
6.  St Jonston D, Sanson B. Epithelial polar-
In: Maderson PFA, ed. Developmental and Evo- 70.
ity and morphogenesis. Curr Opin Cell Biol.
lutionary Aspects of the Neural Crest. New York: 37. Ekblom P, Sariola H, Karkinen-Jaaskelainen
2011;23:540–546.
Wiley; 1987:261–300. M, Saxen L. The origin of the glomerular endo-
7. Gorfinkiel N, Blanchard GB. Dynamics of
23. Jernvall J, Thesleff I. Reiterative signaling and thelium. Cell Differentiation. 1982;11:35–39.
actomyosin contractile activity during epi-
patterning during mammalian tooth morpho- 38. Bard JBL, Woolf AS. Nephrogenesis and the
thelial morphogenesis. Curr Opin Cell Biol.
genesis. Mech Dev. 2000;92:19–29. development of renal disease. Neurol Dial
2011;23:531–539.
24. Agha EE, Bellusci S. Walking along the fibro- Transplant. 1992;7:563–572.
8. Rajasekaran SA, Beyenbach KW, Rajasekaran
blast growth factor 10 route: a key pathway 39. Sariola H, Saarma M, Sainio K, et  al. Depen-
AK. Interactions of tight junctions with mem-
to understand the control and regulation of dence of kidney morphogenesis on the expres-
brane channels and transporters. Biochimica et
epithelial and mesenchymal cell-lineage forma- sion of nerve growth factor receptor. Science.
Biophysica Acta. 2008;1778:757–769.
tion during lung development and repair after 1991;254:571–573.
9. Garrod D, Chidgey M. Desmosome structure,
injury. Scientifica (Cairo). 2014;2014:538379. 40. Takasato M, Little MH. The origin of the
composition and function. Biochimica Bio-
25. Hilfer SR, Rayner RM, Brown JW. Mesenchy- mammalian kidney: implications for rec-
physica Acta. 2008;1778:572–587.
mal control of branching pattern in the fetal reating the kidney in  vitro. Development.
10. Walko G, Castañón MJ, Wiche G. Molecular
mouse lung. Tissue Cell. 1985;127:523–538. 2015;142:1937–1947.
architecture and function of hemidesmosomes.
26. Abbott LA, Lester SM, Erickson CA. Changes 41. Christ B, Huang R, Scaal M. Formation and
Cell Tissue Res. 2015;360:529–544.
in mesenchymal cell-shape, matrix collagen and differentiation of the avian sclerotome. Anat
11. Doyle AD, Yamada KM. Mechanosensing via
tenascin accompany bud formation in the early Embryol. 2004;208:333–350.
cell-matrix adhesions in 3D microenviron-
chick embryo. Anat Embryol. 1992;183:299–311. 42. Gershon MD. Phenotypic expression by neural
ments. Exp Cell Res. 2016;343(1):60–66.
27. Asahina K, Zhou B, Pu WT, Tsukamoto H. crest-derived precursors of enteric neurons and
12. Zamir EA, Rongish BJ, Little CD. The ECM
Septum transversum-derived mesothelium gives glia. In: Maderson PFA, ed. Developmental and
moves during primitive streak formation—
rise to hepatic stellate cells and perivascular Evolutionary Aspects of the Neural Crest. New
computation of ECM versus cellular motion.
mesenchymal cell in developing mouse liver. York: John Wiley; 1987.
PLoS Biol. 2008;6(10):e247.
Hepatology. 2011;53:983–995. 43. Sporn MB, Roberts AS. Peptide growth fac-
13. Atherton P, Stutchbury B, Jethwa D, Balles-
28. Le Douarin N. An experimental analysis of liver tors and their receptors. Vols. 1 and 2. Berlin:
trem C. Mechanosensitive components of inte-
development. Med Biol. 1975;53:427–455. Springer-Verlag; 1990.
grin adhesions: role of vinculin. Ext Cell Res.
29. Haffen K, Kedinger M, Simon-Assmann P. 44. Jessell TM, Melton DA. Diffusable factors
2016;343(1):21–27.
Cell contact dependent regulation of entero- in vertebrate embryonic induction. Cell.
14. Snarr BS, Kern CB, Wessels A. Origin and fate
cyte differentiation. In: Lebenthal E, ed. 1992;68:257–270.
of cardiac mesenchyme. Dev Dyn. 2008;237:
Human Gastrointestinal Development. New York: 45. Stern C. Neural induction: old problem, new
2804–2819.
Raven Press; 1989. findings, yet more questions. Development.
15. Rafalski VA, Mancini E, Brunet A. Energy
30. Watson CL, Mahe MM, Múnera J, et  al. An 2005;132:2007–2021.
metabolism and energy-sensing pathways in
in vivo model of human small intestine using plu-
mammalian embryonic and adult stem cell
ripotent stem cells. Nat Med. 2014;20:1310–1314.
fate. J Cell Sci. 2012;125:5597–5608.

22.e1
3
Staging Embryos in Development and
the Embryonic Body Plan
PATRICIA COLLINS
KEY POINTS
• T his chapter presents the concepts and timing of embryonic
development.
• The problems of using staging systems to describe a continu-
ous process are discussed.
• The method behind staging of animal development is pre-
sented.
• A revision of the timing of early human development is pre-
sented.
• Embryonic and obstetric stages of development are pre-
sented.
• The embryonic body plan, main embryological stages and
their approximate times are presented.
A variety of staging systems for human embryos were devised in
the early years of the past century. To enhance this information,
studies on other animals were undertaken and externally similar
embryos compared. However, devising a staging system is very
different from describing a day-by-day alteration of external
characteristics.
Chick and Mouse Embryo Staging Series
In recent years, the external characteristics of laboratory animal
species have been available and shown within staging schemes.
Computing power now permits the manipulation of external
images, sectional information and three-dimensional (3D) repre-
sentations of internal structures in embryos. Databases of devel-
opmental information of laboratory animals have been collated in
collaborative projects by those involved in experimental embryol-
ogy. Chick development was described by Hamburger and Ham-
ilton as a series of 46 stages over the 20-day incubation period.1ab-3
Photographs and movies of all stages of chick development are
available on an online database, e-Chick Atlas.
For the mouse similar staging systems have been developed
by Theiler4 based on the Streeter staging of human embryos (see
later) and continued by Kaufman.5 Kaufman noted the care with
which specimens needed to be prepared and sectioned and the
level of experience necessary for the interpretation of serial sec-
tions in order to understand the complexity of spatial and tem-
poral development of body systems and internal organs. The
use of magnetic resonance imaging (MRI) to obtain images of
both external features and sectional anatomy is available online
(emouseatlas.org) based on Kaufman’s original images. MRI stud-
ies of mouse embryos are also providing a further way of seeing
digital images through any plane.6
Both chick and mouse databases now display the expression of
a range of genes within the developing organs and tissues linked
to sections and 3D reconstructions of embryos. 
Human Stage Series
Staging of human embryos has always started from a different
standpoint to those used in animal series; stages are not a serialisa-
tion of external features. Human embryos were first placed in a
stage series by Mall,7 founder of the Department of Embryology
of the Carnegie Institution of Washington. His work was contin-
ued by George Streeter8-11 in the 1940s and O’Rahilly and Mül-
ler since that time.12-15 The monograph Developmental Stages in
Human Embryos12 has been a mainstay of embryonic develop-
mental stages.
Human embryos are assigned to a stage based on the develop-
mental status of many body systems in concert, not on any one
parameter alone. Development from fertilisation averages 266
days, or 9.5 months; the embryonic period, extending from fertili-
sation to about 58 days, is divided into 23 stages. The embryonic
period ends when bone marrow is seen replacing cartilage in the
humerus, a time defined by Streeter.
The original studies on human staging were based on 600
embryos within the Carnegie Collection obtained from hysterec-
tomies, and specimens were formalin fixed. It is not clear if all of
these embryos were normal. The time at which an embryo enters
and leaves a particular stage varies because of a number of fac-
tors, including placental health, genetic factors and individual
growth rate. In vivo imaging techniques of very early development
prompted the revision of some of the ages previously assigned to
early embryonic stages. O’Rahilly and Müller15 note that greatest
length, the length of an embryo, exclusive of the lower limbs, is
a valuable parameter which can be measured antenatally. By add-
ing the number 29 to the greatest length, within the range 3 to
33 mm, an age in days can be broadly estimated. The revision of
stage, the age and the specific measurement of greatest length are
taking time to percolate through newer embryological studies. It
would be of benefit to all if the references for the staging system
used were given and terms used were specified. The main features
23
24 SE C T I O N 1     Early Fetal Development

of the stages of human development are given before a consider- are becoming defined and result in neurulation and the begin-
ation of obstetric staging of the first trimester of pregnancy.  ning of somite formation, more clearly seen in stage 10, in which
embryos typically have 7 to 12 pairs of somites. The formation of
Main Stages in the Embryonic Period the neural plate and the beginnings of its rostral fusion contrib-
ute to the morphological movements of head folding, when the
Embryonic development is not apparent before stage 6. Stages 1 cardiac area, which was rostral to the neural epithelium, becomes
to 5 are concerned with setting up the cell populations for implan- ventral and forms a boundary of the cranial intestinal portal.
tation; most of the cell lines generated are extraembryonic and Stages 6b to 10 are concerned with embryogenesis when mor-
involved in establishing the placenta and fetal membranes. In stage phogenetic movements affecting the whole embryo move widely
6a, the primordial germ cells are sequestered into the extraembry- dispersed cell populations closer and into their relevant positions
onic mesoblast, and in stage 6b, the primitive streak appears. From for local interactive processes to commence. A stage 11 embryo is
this time, intraembryonic cell populations are generated, and the at the gateway of organogenesis, and all body systems can be seen
morphological movements of these populations produce a recog- to originate from this point. 
nisable embryo. Fig. 3.1 shows the formation of cell populations
during stages 1 to 11 matched to estimated age. The proliferation The Stage 11 Embryo, Body Plan Stage
of cells at the primitive streak occurs through stages 6b, 7 and 8,
when the notochord is first evident, and provides cell populations The criterion for stage 11 is the presence of 13 to 20 pairs of
which pass within the embryo. By stage 9, the neural populations somites (Fig. 3.2); during this stage, the rostral neuropore closes.
5a

10
4

11
7
3

6b
6a
2

5b

5c
1
Description of stage

First cleavage

After folding
Before folding

Intraemb. coelom

Neural tube

Neural crest
Implantation

Notochord

Neurenteric canal

Neural groove
Secondary yolk sac

Primitive streak

Somites
Preimplantation

Compaction
Free blastocyst
hatched from zona

Implanted
previllous
Fertilisation

Syncytio- Lacunae Intervillous

trophoblast spaces
Tropho-
blast
Oocyte Cyto-
Chorion Villi
trophoblast

Ootid
Visceral Extraemb. mesenchyme Haemopoiesis
hypoblast
Hypoblast Primary yolk Secondary
Zygote Parietal sac yolk sac
hypoblast
Inner Allantois
cell
mass

Extraemb.
Connecting stalk
mesoblast
Epiblast

Amnion

Primordial germ cells

Primitive streak
Notochordal
process/plate
Embryonic
endoderm
Mesoblast Mesenchyme
Somatopleuric and
splanchnopleuric coelomic epithelium
Epithelial somites

Caudal eminence
Neural
ectoderm
Neural
crest

Ectodermal placodes
Surface
ectoderm

Approx. 1 2–3 4–5 6 16–18 18–21 24–27 28–30

age in days
7–12 21–25 26–29

• Fig. 3.1 Developmental processes occurring during the first 10 stages of development. In the early
stages, a series of binary choices determines the cell lineages. Generally, the earliest stages are con-
cerned with formation of the extraembryonic tissues, and the later stages are concerned with the forma-
tion of embryonic tissues. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)
 CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan 25

Fusion occurs from the rhombencephalon rostrally towards the
mesencephalon and from the region of the future optic chiasma
towards the mesencephalic roof. The optic primordia have begun
evagination towards the surface ectoderm. The otic vesicle which
will invaginate and give rise to the inner ear (cochlea and semi-
circular ducts) has not yet formed but the surface epithelium
has thickened and begun to invaginate. Around the developing
pharynx, the mandibular processes are present, but the maxil-
lary processes have yet to arise. The second pharyngeal arch,
the hyoid, is present but not the third. Ventral to the foregut
is the heart, this develops very precociously and is seen in stage
9 embryos as tube-like with a united ventricular portion but
still separate atrial components. The specialised cells of the coe-
lom, which will give rise to the myocardium, can be identified
as can the matrix produced locally between the endocardium
and the myocardium. Cardiac contractions commence at the
beginning of stage 10 when the heart has a recognisable ven-
tricle, bulbus cordis and arterial trunk, and a cardiac loop can
be distinguished; the organ is already asymmetrical. By stage 11,
the sinus venosus, atria, left and right ventricles, truncus arterio-
sus and substantial dorsal aortae can be identified. The heart is
connected to a range of endothelial vessels and plexuses which
are most mature cranially and still forming caudally. The fluid
in the vascular system contains relatively few cells. It ebbs and
flows because of the pulsations of the myocardium, which also
cause movement of fluid in the intraembryonic coelom. These
combined circulations are sufficient to provide nutrient supply
to the embryonic tissues.
The intraembryonic coelom is especially important at this
stage of development. The coelom arises from confluent spaces
which appear in the embryo from stage 9. As head folding occurs,
these spaces coalesce to form a horseshoe-shaped cavity within the
embryonic body, which passes between the endoderm of the gut
and the ectoderm of the body wall on each side of the developing
fore- and midgut, and meets in the midline beneath the rostral
neuropore as the future pericardial cavity. The ends of the horse-
shoe are in wide communication with the extraembryonic coelom
around the embryo and within the chorion. The walls of the coe-
lom are composed of germinal epithelia, which provide mesenchy-
mal cell populations for the connective tissues and smooth muscle
of the respiratory and gastrointestinal tracts, the body wall and
especially the heart. The myocardium arises directly from the dor-
sal pericardial wall in the folded embryo. The ventral pericardial
wall will give rise to the serous, parietal pericardial layer.
The foregut develops as the head fold elevates and the pericar-
dial cavity swings ventrally. It is flattened dorsoventrally, extending
laterally to form the pharyngeal pouches. The buccopharyngeal
membrane is present at this stage and may begin to rupture. There
is only small indication of the future respiratory primordium. The
liver is represented by the septum transversum mesenchyme and
underlying endoderm, but the latter is still widely connected to
the yolk sac. The nephric system consists of a solid nephrogenic

Neural tube

Pharynx

Pericardial
cavity
Pericardial
Amnion cavity

Pericardioperitoneal canal
Foregut

Yolk sac
Entrance to pericardioperitoneal canal
wall
Neural tube
Somite

Midgut
Yolk sac
wall
Left umbilical vein

Peritoneal
cavity

Umbilical cord Hindgut

A B
• Fig. 3.2  A, The embryo at stage 11, showing the position of the intraembryonic coelom (contained by
the walls coloured blue). B, The three major epithelial populations within a stage 11 embryo, viewed from
a ventrolateral position. The neural tube lies dorsal to the gut. Ventrally, the intraembryonic coelom crosses
the midline at the level of the foregut and hindgut but is lateral to the midgut and a portion of the foregut.
(From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)
26 SE C T I O N 1     Early Fetal Development

cord lateral to somites 8 to 13. The cloacal membrane is situated
ventrally after tail folding just caudal to the connecting stalk which
passes to the developing placenta. The primordial germ cells can
be identified in the embryo at stage 11. They are initially in the
mesenchyme around the yolk sac and allantoic walls. With tail
folding, they are brought into the body cavity with the hindgut
epithelium and surrounding mesenchyme, and then by amoeboid
movement and by growth displacement, they migrate dorsocrani-
ally. They do not reach the gonads until stage 15, some 9 days
later, when the local cell populations are developed sufficiently to
receive them.
During stage 11, the most important epithelial layers attain
their position (i.e., surface epithelium, notochord, neural epi-
thelium, somites, gut epithelium and the lining of the coelo-
mic cavity; see Fig. 3.2). The germinal epithelia of the somites
and the coelomic cavity then generate extensive mesenchymal
populations at the same time that the neural crest mesenchyme
cells are migrating within the head and neck. Finally, each epi-
thelium is surrounded and supported by different mesenchymal
populations (i.e., the neural tube and notochord are surrounded
by neural crest mesenchyme in the head and somatopleuric
mesenchyme in the trunk). The gut epithelium is surrounded
by splanchnopleuric mesenchyme, which becomes specialised,
particularly around the respiratory diverticulum. The surface
ectoderm is supported by somatopleuric mesenchyme in the
trunk and by neural crest mesenchyme in the head. The coelo-
mic epithelia themselves produce specialised epithelial popula-
tions which give rise to the gonads, adrenal medulla and the
lining of the uterine tubes; these epithelia are all supported by
local mesenchyme. Angiogenic mesenchyme is found through-
out the embryo from stage 9. It is capable of extensive migration
and differentiates into endothelium or blood cells throughout
the embryo. Angiogenic mesenchyme is found within the endo-
dermal and splanchnopleuric layers but never within ectoderm
derived tissues.
Stage 11 may be considered to be the body plan stage. To
achieve it, genes functioning across the whole embryo are in oper-
ation. As stage 11 fades into stage 12, organogenesis is underway,
and the epithelial–mesenchymal interactions which result in all
development are now operating along local lines. Diversity of dif-
ferentiative outcomes is now possible by upregulation of specific
genes in specific regions of the embryo.
Between stage 11 and stage 23, a period of about 30 days, the
embryo grows in length from 3 mm to 30 mm. It passes from a
general vertebrate embryo to a fully formed but immature human.
Fig. 3.3 illustrates the dramatic increase in size and developmental
status during this time, and Fig. 3.4 shows the relative time at
which individual systems progress in development. 

First pharyngeal arch Mandibular Maxillary Pinna Eyelids

Neurulation
process process
Optic
Pericardial vesicle
Otic vesicle
cavity
and heart

Somites Cerebral
hemisphere
Yolk
Hand
sac

Upper limb Connecting Connecting Umbilical

Lower limb stalk
Connecting bud stalk cord
bud
stalk
Stage 10 Stage 13 Stage 16 Stage 18

Reflection of amnion

Epiblast population

Primitive node

Primitive streak

Connecting stalk

Stage 6

Embryonic stage 6 10 13 16 18 20 23

Size (mm) 0.4 1.5–3 4–6 8–11 13–17 21–23 28–30

Approximate age 16–18 26–29 30–33 35–40 41–45 46–50 53–58

(days)

• Fig. 3.3  The external appearance and size of embryos between stages 6 and 23. Early in development,
external features are used to describe the stage (e.g., somites, pharyngeal arches or limb buds). (From
Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015. Adapted with permission from Rodeck CH, Whittle
MJ. Fetal Medicine. London: Churchill Livingstone, 1999.)
 CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan 27

Embryonic stages 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Weeks post-ovulation 1 2 3 4 5 6 7 8 9 10 11 12

Head and tail folding Upper lip Digits on hand Eyelids fuse
External appearance
Pharyngeal arches Palate External ear

Neurulation Anterior lobe Posterior lobe

pituitary pituitary
Nervous Otic vesicle
First neural
crest cells Optic cup Membranous labyrinth

Trachea
Respiratory Lung buds Further division of bronchi
Primary bronchi

Fore-, mid-, Thyroid Pharyngeal pouches Midgut loop

hindgut Liver dorsal and ventral Midgut loop rotating returns to
Gastrointestinal abdomen
Pancreas
Urorectal septum Rotation of stomach

Mesonephros
Urinary Mesonephric duct Metanephric nephrons Kidneys ascend
Ureteric bud Major calyces Minor calyces

Germ cells in allantois wall Müllerian ducts Uterus and uterine tubes Testis at inguinal canal
Reproductive Indifferent gonad Testis differentiating Vagina Prostate
External genitalia indifferent External genitalia differentiating

Primitive Septum primum Septation of ventricles

vascular
Cardiovascular Heart beats Spleen Septum secundum
system
Heart tube

Somite period Cartilaginous Membranous

20 days .................................. 30 days
part of skull part of skull
Musculoskeletal Forelimb bud Forelimb digit rays
Hindlimb bud

• Fig. 3.4  A timetable of development of the body systems. The development of individual systems can
be seen progressing from left to right. Embryonic stages and weeks of development are shown. Embry-
onic stages are associated with external and internal morphological features rather than embryonic length.
To identify the systems and organs at risk at any time of development, follow a vertical progression from
top to bottom. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

Obstetric Timing and Staging of Embryos
and Fetuses
The obstetric timescale is involved in estimating a day of delivery
and then assessing the fetus to see if it seems appropriately aged
to deliver at that time. The commencement of gestation is deter-
mined clinically by counting from the date of the last menstrual
period. Estimated in this manner, a pregnancy averages 280 days,
or 10 lunar months (40 weeks). Fig. 3.5 shows age in weeks; the
embryonic, fetal and perinatal periods; and their relationship to
trimesters of pregnancy. The 2-week discrepancy between these
scales can be seen. Generally, books written for obstetricians or
fetal physicians use the lower, obstetric scale, and embryology
books use the upper, embryological scale. Fig. 3.6 shows details of
the estimated length of embryological stages and how they relate
to the obstetric timescale. It is recommended that sonographically
determined ages of embryos and fetuses, usually expressed in the
obstetric timescale, should be specific, giving weeks and days.16 A
reported age of 5 weeks and 2 days is two stages earlier than an age
of 5 weeks and 6 days. As improvements in imaging of the first
trimester using high resolution 3D transvaginal sonography are
made, awareness of both timescales is important, and interpreta-
tion of embryonic stage should be clear.
The terms ‘gestational’, ‘gestational week’, and ‘gestational age’
are considered ambiguous by O’Rahilly and Müller13; however,
the terms are used widely and colloquially in the obstetric lit-
erature. These authors also recommend the greatest length (GL)
exclusive of the lower limbs as the best prenatal measurement of
embryos directly or derived ultrasonically rather than the older
measurement of crown–rump length (CRL).15 Consideration
and caution of the shared measurements used to generate predic-
tive data against which to correlate fetal health is very pertinent.
Unless the most appropriate measurements are collected, the out-
comes of any correlations or predictions will not provide mean-
ingful information.
The successful delivery and survival of preterm infants at ages
equivalent to 19 or 20 embryonic weeks of development has illus-
trated that estimations of fetal age have become less important
than estimations of fetal maturity, which depend on aspects of
maternal health and placental growth.
A number of biometric indices used to determine fetal age
in utero have been evaluated in ultrasound studies for accuracy.
Charts of first-trimester growth based on biparietal diameter,
head circumference and abdominal circumference of normal
singleton fetuses, correlated against CRL (from 45–84 mm) are
considered to be more accurate than gestational age by menstrual
dates.17 It is suggested that the femur length:head circumference
ratio may be a more robust ratio to characterise fetal proportions
than femur length:biparietal diameter,18,19 and combining kid-
ney length, biparietal diameter, head circumference and femur
28 SE C T I O N 1     Early Fetal Development

Late neonatal period (7–28 days)

Implantation period Early neonatal period (birth–7 days)

Embryonic
stages Fetal period Perinatal period

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Age of embryo (weeks)

1 2 3 4 5 6 7 8 9 10 Age of embryo (months)

Implantation
Fertilisation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Pregnancy (weeks)

1 2 3 4 5 6 7 8 9 10 Pregnancy (lunar months)

Last menstruation Estimated date of delivery

First trimester Second trimester Third trimester (of pregnancy)

• Fig. 3.5 The two timescales used to depict human development. Embryonic development, in the
upper scale, is counted from fertilisation (or from ovulation, i.e., in postovulatory days; see O’Rahilly and
Müller12). Times given for development are based on this scale. The clinical estimation of pregnancy is
counted from the last menstrual period and is shown on the lower scale; throughout this book, fetal ages
relating to neonatal anatomy and growth will have been derived from the lower scale. Note that there is a
2-week discrepancy between these scales. The perinatal period is very long because it includes all pre-
term deliveries. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

Embryo Obstetric
week week

Days week week

Last
1
menstruation

Endometrial growth 2

Fertilisation, cleavage 0 1 2 3 4 5 6 1 3
stages 1–4 Days post-fertilisation

Implantation, placentation, 7 8 9 10 11 12 13 2 4
stages 5–6

Primitive streak 14 15 16 17 18 19 20 3 5
gastrulation, stages 7–8
21 22 23 24 25 26 27 4 6
Embryo folding, stage 9

28 29 30 31 32 33 34 5 7
Body plan 10 13
11 14
12
35
15
36 37 38 39 40 41 6 8
16 17
Stage and range
42
18
43 44 45 46 47 48 7 9
shown in colours
19
20
49 50 51
21
52 53 54 55 8 10
22
56 57 58 11
23
• Fig. 3.6  Details of the estimated times of embryonic stages alongside first trimester weeks of pregnancy.
 CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan 29

length also increases the precision of dating.20 Johnsen et  al18
reported that analysis of measurements of biparietal diameter
and head circumference at 10 to 24 weeks’ gestation gave a ges-
tational age assessment of 3 to 8 days greater than charts in use
at that time.
A multicentre study of fetal growth INTERGROWTH-21st
aims to standardise the collection of anthropometric measure-
ments of fetal and childhood growth,21 and The World Health
Organization similarly is promoting consideration of ethnicity
and social factors when collecting such data.22 These data will
contribute to local and population specific growth charts against
which normal development can be viewed.  stages and the obstetric timescale is given. It is hoped that the
Conclusion
This chapter outlines a commonly used staging system for human
embryonic development. Human embryos in the Carnegie Col-
lection were staged according to the system initiated by Streeter
and continued by O’Rahilly and Müller. It is based on internal
and external criteria and is not just a sequence of size or exter-
nal features. The staging system has been revised using the results
of ultrasound examination of pregnancies of known commence-
ment. The internal and external features of a stage 11 embryo are
given.
The duration of pregnancy in obstetric terms is based on the
date of the last menstrual period; thus the obstetric ‘age’ of an
early embryo is different to the Carnegie stage. Care must be taken
in recording the age of an embryo or fetus so that these differ-
ent timescales can be reconciled. A chart of estimated embryonic
anthropometric measurements of fetuses in a range of countries
and cultures will help develop accurate biometric indices which
can be used to estimate fetal age and health.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 9. Streeter GL. Developmental horizons in
human embryos. Description of age groups
abnormal fetal growth. In: Callan PW, ed.
Ultrasonography in Obstetrics and Gynecology.
XV, XVI, XVII, and XVIII, being the third St. Louis: Saunders; 2008:225–265.
1a. Hamburger V, Hamilton HL. A series of nor-
issue of a survey of the Carnegie Collection. 17. Salomon LJ, Bernard JP, Duyme M, et  al.
mal stages in the development of the chick
Carnegie Institution of Washington Publica- Revisiting first-trimester fetal biometry. Ultra-
embryo. J Morphol. 1951;88:49–92.
tion 575. Contrib Embryol. 1948;32:133–203. sound Obstet Gynecol. 2003;22:63–66.
1b. Hamburger V, Hamilton HL. A series of nor-
10. Streeter GL. Developmental horizons in human 18. Johnsen SL, Rasmussen S, Sollien R, Kiserud
mal stages in the development of the chick
embryos. Description of age groups XIX, XX, T. Fetal age assessment based on ultrasound
embryo. Dev Dyn. 1992;195:231–272.
XXI, XXII, and XXIII, being the fifth issue of a head biometry and the effect of maternal
2. Sanes JR. On the republication of the Ham-
survey of the Carnegie Collection (prepared for and fetal factors. Acta Obstet Gynecol Scand.
burger–Hamilton stage series. Dev Dyn. 1992;
publication by CH. Heuser & GW Corner). 2004;83:716–723.
195:229–230.
Carnegie Institution of Washington Publica- 19. Johnsen SL, Rasmussen S, Sollien R, Kise-
3. Hamilton V. Afterword: the stage series in the
tion 592. Contrib Embryol. 1951;34:165–196. rud T. Fetal age assessment based on femur
chick embryo. Dev Dyn. 1992;195:273–275.
11. Streeter GL. Developmental horizons in length at 10-25 weeks of gestation, and ref-
4. Theiler K. The House Mouse. Development and
human embryos. Description of age group erence ranges for femur length to head cir-
Normal Stages From Fertilization to 4 Weeks of
XI, 13 to 20 somites, and age group XII, 21 cumference ratios. Acta Obstet Gynecol Scand.
Age. 2nd ed. Berlin: Springer-Verlag; 1989.
to 29 somites. Carnegie Institution of Wash- 2005;84:725–733.
5. Kaufman MH. The Atlas of Mouse Development.
ington. Publication 541. Contrib Embryol. 20. Konje JC, Abrams KR, Bell SC, Taylor DJ.
London: Elsevier; 1992.
1942;30:211–245. Determination of gestational age after the 24th
6. Petiet AE, Kaufman MH, Goddeeris MM,
12. O’Rahilly R, Müller F. Developmental Stages week of gestation from fetal kidney length
et al. High-resolution magnetic resonance his-
in Human Embryos. Publication 637. Carnegie measurements. Ultrasound Obstet Gynecol.
tology of the embryonic and neonatal mouse: a
Institution of Washington; 1987. 2002;19:592–597.
4D atlas and morphologic database. Proc Natl
13. O’Rahilly R, Müller F. Mini-review: prenatal 21. Uauy R, Casanello P, Krause B, et al. Interna-
Acad Sci USA. 2008;105:12331–12336.
ages and stages—measures and errors. Teratol- tional Fetal and Newborn Growth Consortium
7. Mall FP. On stages in the development of
ogy. 2000;61:382–384. for the 21st Century. 2013 Conceptual basis
human embryos from 2 to 25 mm long. Anat
14. O’Rahilly R, Müller F. The Embryonic Human for prescriptive growth standards from concep-
Anz. 1914;46:78–84.
Brain. An Atlas of Developmental Stages. 2nd ed. tion to early childhood: present and future.
8. Streeter GL. Developmental horizons in human
Wiley-Liss; 1999. BJOG. 2013;120(suppl 2):3–8, v.
embryos. Description of age group XIII,
15. O’Rahilly R, Müller F. Developmental stages 22. Merialdi M, Widmer M, Gülmezoglu AM,
embryos about 4 or 5 millimeters long, and age
in human embryos: revised and new measure- et  al. WHO multicentre study for the devel-
group XIV, period of indentation of the lens ves-
ments. Cells Tissues Organs. 2010;192:73–84. opment of growth standards from fetal life to
icle. Carnegie Institution of Washington Publi-
16. Galan HL, Pandipati S, Filly RA. Ultrasound childhood: the fetal component. BMC Preg-
cation 557. Contrib Embryol. 1945;31:27–63.
evaluation of fetal biometry and normal and nancy Childbirth. 2014;4:157.

29.e1
4
Teratology
SARAH G. OBICAN AND ANTHONY R. SCIALLI
KEY POINTS
• A t baseline, each pregnancy has a 2% to 4% risk for a con-
genital anomaly diagnosed at birth.
• The adverse effects of exposures on embryo-fetal develop-
ment depend on the agent, dose, and timing of exposure.
• Resources are available for up-to-date information on specific
exposures during pregnancy.
Introduction
A teratogenic exposure is one that has the ability to interfere with
normal development of the fetus. Some exposures increase the
risk for structural anomalies, others may interfere with fetal organ
development, and others may increase the risk for other adverse
pregnancy outcomes, including intrauterine growth restriction,
preterm birth and intrauterine fetal demise.
This chapter discusses some of the exposures that might
increase the risk for abnormal development in human pregnancy.
At baseline, each pregnancy has a 2% to 4% probability of a struc-
tural anomaly diagnosed at birth.1 Chemically induced anomalies,
including those caused by medication exposure, are thought to
occur in fewer than 1% of these cases.2  more difficult to produce malformations with experimental dam-
Historical Perspective
In the 19th century, experimental teratology focused on frogs
and birds in which, for example, hypoxia could cause devel-
opmental aberrations. However, the mammalian uterus was
thought to be impervious to external hazards, and genetic
abnormalities were blamed for the occurrence of malforma-
tions. The notion that human embryos might be harmed by
environmental factors was a revolutionary concept until Nor-
man Gregg, an Australian physician, noted in his practice an
increase in children with congenital cataracts after a rubella
epidemic. He identified what came to be called the ‘congenital
rubella syndrome’ with a combination of additional findings,
including heart defects, hearing loss, thrombocytopenia and
poor growth.3
In response to the increased interest in studying birth defects,
the Teratology Society was formed in 1960. Shortly thereafter,
the field was changed by the unexpected tragedy of thalidomide.
Thalidomide, a sedative–hypnotic, given in the usual (medicinal)
doses caused fetal malformations in the absence of toxicity to
the mother. Since this discovery of what is now called selective
30
embryotoxicity, drug testing regulations have changed. For exam-
ple, in 1962, the Food, Drug and Cosmetic Act was expanded
to include the Kefauver-Harris Amendment, which requires drug
manufacturers to provide proof of effectiveness and safety before
approval as well as provide information regarding side effects. By
1966, the US Food and Drug Administration (FDA) instituted
standard test protocols for drug testing in pregnant laboratory
animals. 
Mechanisms of Teratogenicity
Experience in experimental teratology led to the development of
potential mechanisms for abnormal development, articulated by
James Wilson4 (Table 4.1). Wilson’s mechanisms included the
idea that impairment of survival and function in differentiated
cells in key locations in an embryo could perturb development.
This concept gave rise to the ‘all-or-nothing’ principle in which it
was believed that before gastrulation (approximately postconcep-
tion day 14), cells in the embryo were largely undifferentiated
and could substitute for one another, decreasing the ability of
an exposure to cause selective malformations without destroying
the entire embryo. This all-or-nothing principle has not proved
invariable in experimental teratology, but it remains true that it is
age to undifferentiated compared with differentiated embryonic
tissues.
Mechanisms of congenital malformations more recently have
been understood in terms of developmental pathways that might
be inhibited by a specific exposure. For example, DiGeorge syn-
drome, which is often associated with a deletion in the long arm of
chromosome 22, includes disruption of Tbx1, a gene that plays a
role in development of cells in the secondary heart field. As a con-
sequence, conotruncal heart defects can be seen in affected chil-
dren.5 It has not been identified, however, whether isotretinoin
therapy, which also has been associated with conotruncal heart
defects, works by way of interference with Tbx1.
There are basic cell behaviours during embryo development
that may serve as targets for exposures. Cell populations in the
embryo migrate to targeted locations, and interference with
migration may cause abnormal development. Neural crest cells
migrate into the branchial arches, for example, and interfer-
ence results in a group of disorders of the jaw, ears, and zygo-
matic arch, among other defects. Neurons migrate from their
birthplace in the centre of the brain towards the periphery, and
inhibition of migration can cause microcephaly. Cells also can
induce behaviours in neighbouring cells. The tips of the ureteric

TABLE
4.1 Mechanisms of Teratogenesis
Mutation
Chromosomal aberrations
Mitotic interference
Altered nucleic acid synthesis and function
Lack of precursors, substrates and coenzymes for biosynthesis
Altered energy sources
Enzyme inhibition
Osmolar imbalance
Changed membrane characteristics
From Wilson JG. Mechanisms of teratogenesis. Am J Anat 136(2):129–131, 1973.
  
buds induce the mesenchyme of the developing kidney to form
functioning nephrons. Cells produce substances that act at a dis-
tance to modify cell differentiation. For example, cells in the
medial (postaxial) limb bud secrete Sonic Hedgehog protein,
which diffuses across the limb paddle to help determine the
identity of the individual digits.
The greater understanding of cellular and molecular events
during embryo development has given rise to an opportunity
for understanding mechanisms of abnormal development in
more detail. Although the mechanistic details of malformations
in human beings associated with exposures are incompletely
understood, we expect that developments in the genetic basis
of malformations and in cell biology will lead to our improved
understanding of exposure-associated malformations.  dose.
Underlying Principles
The mechanisms of abnormal development were the basis of
one of James Wilson’s principles. The full set of principles is
listed in Table 4.2 as developed in the 1950s and 1960s.1 Of
note is Wilson’s fourth principle, which tells us that malforma-
tions are not the only kind of developmental toxicity of impor-
tance. An exposure that causes an organ, say the brain, not
to function correctly can be devastating even if there are no
recognisable structural malformations in the child. We com-
monly call these adverse effects developmental toxicity, which
is a more helpful term than teratogenicity in not requiring a
definition of what exactly counts as a malformation. An expo-
sure producing nonmalforming developmental effects does not
necessarily also produce malformations. Indeed, an exposure
that produces one kind of malformation does not of neces-
sity produce any other kind of malformation. Thalidomide, for
example, produces only certain kinds of limb defects, not all
kinds of limb defects.6 Effects of developmentally toxic expo-
sures are specific, producing a finite grouping of adverse effects.
Dr Wilson captured this idea of specificity in his third princi-
ple, and the Public Affairs Committee of the Teratology Society
made it explicit in 2005.7
Wilson’s sixth principle is among the most important for
practitioners because it reminds us that for all medications,
other chemicals, and physical agents, there are an exposure level
that produces no harm, an exposure level that produces death
and a range of exposures in between that produces a gradation
of effects. It is not useful to talk about agents (drugs, chemicals,
radiations) as teratogenic or nonteratogenic. It is preferable to
CHAPTER 4 Teratology 31
TABLE
4.2 Wilson’s Principles
1. Susceptibility to teratogenesis depends on the genotype of the con-
ceptus and the manner in which this interacts with environmental
factors.
2. Susceptibility to teratogenic agents varies with the developmental
stage at the time of exposure.
3. Teratogenic agents act in specific ways (mechanisms) on developing
cells and tissues to initiate abnormal embryogenesis (pathogenesis).
4. The final manifestations of abnormal development are death, malfor-
mation, growth retardation and functional disorder.
5. The access of adverse environmental influences to developing tis-
sues depends on the nature of the influences (agent).
6. Manifestations of deviant development increase in degree as dosage
increases from the no-effect to the totally lethal level.
From Wilson JG. Current status of teratology. General principles and mechanisms derived
from animal studies. In Handbook of Teratology, vol 1. General Principles and Etiology, pp.
47–74, JG Wilson, FC Fraser (eds.), New York: Plenum Press, 1977.
  
talk about teratogenic exposures, an exposure including the iden-
tity of the agent and the dose level at which it is encountered.
X-irradiation during pregnancy was associated with microceph-
aly and mental retardation when exposure levels were about
50 cGy from the atomic bombings of Japan.8 Flying across the
United States is associated with estimated radiation exposures
about 8000 times lower9 and would not be expected to have the
same effects. We do not, therefore, characterise x-ray as terato-
genic or nonteratogenic. It depends, among other things, on
How much evidence is needed before it is worthwhile warning
patients and health care providers about possible adverse effects
of exposures in reproducing men and women? There is some con-
troversy in this area because there are potential adverse effects of
excessive warning, namely anxiety, the interruption of otherwise
wanted pregnancies, and the discontinuation of important, even
essential, medical therapy.
It has been fashionable from time to time to claim that most
human teratogenic exposures were first identified by ‘astute clini-
cians.’ Thalidomide is often cited as evidence of this claim because
the first publications on thalidomide birth defects came from one
paediatrician in Germany and another in Australia who described
clusters of children with phocomelia and wondered whether there
was a pregnancy exposure as the cause. The astute clinician model
has been described in mathematical terms,10 but the astute clini-
cian model produces only a hypothesis that must be confirmed by
other evidence. The association between rubella and congenital
cataracts and between thalidomide and phocomelia were, in fact,
doubted by some teratologists until additional evidence was forth-
coming. Perhaps clinicians whose hypotheses are confirmed may
be as lucky as they are astute.
For a determination of causation in teratology, methods have
been advanced that are based on the Hill criteria.11 These criteria
(Table 4.3) were most famously applied in the consideration by
the US Surgeon General of the evidence for a causal relationship
between cigarette smoking and lung cancer.12 To entertain a con-
clusion of causation, you need not satisfy each of the Hill criteria,
but the more you satisfy, the more confident you can be that an
association is causal.
In this chapter, we discuss some exposures that have been asso-
ciated with developmental toxicity in human pregnancy. In some
32 SE C T I O N 1     Early Fetal Development
TABLE
4.3 Bradford Hill Criteria
1. Strength of the association (the likelihood that the association is not
due to chance, bias, or confounding); strength of the association
refers to findings in human epidemiological studies
2. Consistency of the association (the association is reproduced in dif-
ferent populations); consistency of the association refers to findings
in human epidemiological studies
3. Specificity (uniqueness of the association both with respect to the
exposure and with respect to outcome). In teratology, specificity
results in a distinctive pattern of birth defects that appear repeatedly
and consistently.
4. Temporal relationship (the putative cause comes before the effect)
5. Coherence (the association is compatible with related knowledge)
6. Biologic gradient (there is a dose–response effect)
7. Biologic plausibility (the association does not violate known prin-
ciples)
8. Experiment (reducing the putative cause reduces the effect)
9. Analogy (evidence is similar to that for similar cause-effect relation-
ships)
From Hill AB. The environment and disease: association or causation? Proc R Soc Med
58:295–300, 1965.
  
instances, there is evidence that the association is causal, but in
other cases, a causal association cannot be concluded. We recog-
nise, however, that giving patients advice about exposures during
pregnancy does not require conviction that causation criteria have
been satisfied. For example, we recommend that women who have
taken lithium during pregnancy consider fetal echocardiography,
even though causation criteria are not satisfied that lithium causes
Ebstein anomaly or any other heart defect. In the end, counsel-
ling about exposures relies on the same kinds of judgments about
adverse outcomes that we use every day as clinicians considering
possible risks and benefits of medication therapy.  exposure during pregnancy.
Personalised Risk Assessment and Resources
Several excellent books are available as resources. However, soon
after publication, books in this ever-changing field have the
potential to be outdated. Online databases offer summaries of
pregnancy exposures written and frequently updated by teratology
experts include TERIS (http://depts.washington.edu/terisdb/teris
web/index.html), REPROTOX (Reprotox.org), and Briggs (avail-
able through http://wolterskluwer.com). Lactmed (https://toxnet.
nlm.nih.gov/newtoxnet/lactmed.htm) is a free online resource for
medication exposure and lactation.
There are two networks of teratology information services, one
in North America called the Organization of Teratology Informa-
tion Specialists (OTIS; http://www.mothertobaby.org) and one
serving Europe, the European Network of Teratology Information
Services (ENTIS; http://www.entis-org.eu). Both networks are
staffed with physicians, genetic counsellors and teratology experts
who offer individualised risk assessments and up-to-date informa-
tion for any drug or environmental exposure during pregnancy and
lactation. These complimentary services are available to both health
care providers and patients. These networks also conduct prospec-
tive studies and patient follow-up after pregnancy exposures.
Pregnancy registries open to enrolment may be located from
the FDA’s Office of Women’s Health (www.fda.gov/ScienceRese
arch/SpecialTopics/WomensHealthResearch/ucm251314.htm).  pled epiphysis.22,23 Embryotoxicity is associated with exposure
Selected Human Exposures
Medication
A survey of 1000 pregnancies in southwest France found that
99% of the women received a prescription for at least one
drug during pregnancy with a mean of 13.6 medications per
woman.13 In other studies, approximately two thirds of preg-
nant women took at least one medication during pregnancy, and
about 60% of patients used a prescription medication.14 In the
past few decades, the overall use of medication and the number
of women using 4+ prescription medications anytime in preg-
nancy more than doubled, and for the first trimester, it more
than tripled.15
Thalidomide. No other therapeutic agent has had a greater
impact on how we think about teratogenic potential as thalido-
mide. In 1957, thalidomide was marketed as a sedative and anti-
emetic. Thalidomide did not cause acute toxicity in adults, making
it one of the few agents that are selectively embryotoxic. In the late
1950s and 1960, there were few case reports of phocomelia, an
unusual limb reduction defect in which the hand and foot arise
from the shoulder or hip. By 1961, Dr Lenz in Germany and
Dr McBride in Australia independently noted an association
between phocomelia and exposure to thalidomide. Thalidomide
exposure was associated with specific limb reduction defects,
oesophageal and duodenal atresia, congenital heart defects (tetral-
ogy of Fallot), external ear and cranial nerve abnormalities, and
renal agenesis. The sensitive time period for the limb defects was
21 to 36 days postconception.
Although thalidomide was removed from the market, it was
found later to be effective for erythema nodosum leprosum. In
1998, the US FDA approved Thalomid for this use. In 2006, the
medication was approved for multiple myeloma. Prescription and
dispensing of thalidomide is strictly controlled in the US through
a Risk Evaluation and Mitigation Strategy (REMS)16 to prevent
 
Isotretinoin. Isotretinoin (13-cis-retinoic acid), a derivative
of vitamin A (retinol), is an effective treatment of cystic acne
vulgaris. Use of the medication increases the risk for spontane-
ous abortion and the development of a specific set of anomalies,
including neural crest-related facial and palate defects, micro-
gnathia, external and internal ear anomalies (microtia or anotia),
conotruncal heart defects, thymic abnormalities and deficits in
intelligence.17-20 Effects on cognition can occur in the absence of
structural anomalies.20
There is no associated risk for poor fetal outcome in cases in
which isotretinoin was discontinued before pregnancy. The cur-
rent recommendation is to discontinue isotretinoin 1 month
before conception, but considering the half-life of 29 hours, after
1 week off therapy, maternal blood concentrations should be
negligible.
Use of topical retinoids does not increase the risk for structural
malformations or developmental delay due to decreased availabil-
ity of the medication and its metabolites in maternal plasma.21 
Warfarin. In 1948, warfarin was marketed as a potent roden-
ticide because of its capability of inducing internal haemor-
rhage.16 Warfarin prevents vitamin K from acting as a cofactor in
hepatic synthesis of factors II, VII, IX and X and was adapted for
human clinical use because of its oral bioavailability and revers-
ibility by vitamin K. Warfarin was associated with embryo-fetal
growth restriction, nasal hypoplasia, fibula hypoplasia and stip-

between 6 and 9 weeks of gestation,23 although one report did
not note a warfarin embryopathy in those exposed before 8
weeks of gestation.24
Other poor perinatal outcomes associated with warfarin expo-
sure include an increased risk for stillbirth, spontaneous abortion,
preterm birth and low birth weight.24-26 The underlying maternal
disease may contribute to the increased risk for poor pregnancy
outcomes.
Warfarin is still used by some practitioners in pregnancy,
especially in women with mechanical heart valves. Heparin and
low-molecular-weight heparin (LMWH) are the mainstays of
anticoagulant therapy during pregnancy, but they may not be
effective enough, especially in women with mechanical heart
valves. More favourable maternal and fetal outcomes may be
associated with the use of low-dose warfarin (<5 mg/day) fol-
lowed by LMWH close to the time of anticipated delivery,27
but low-dose warfarin therapy has also been associated with
warfarin embryopathy.28 Second and third trimester expo-
sure may increase the risk for central nervous system (CNS)
defects, possibly associated with microhaemorrhages in neuro-
nal tissues.23,29  Case reports and case series report a possible misoprostol
Methotrexate and aminopterin. Folic acid is a cofactor in the
synthesis of thymidylate, a rate limiting step in DNA synthesis.
Methotrexate (MTX), a folic acid analog, has been used for the
treatment of malignancy, rheumatic disorders, psoriasis and ecto-
pic pregnancy. Aminopterin is also a folic acid antagonist that is a
close structural analog of MTX. Aminopterin interferes with early
human fetal development and was used as an abortifacient in the
1940s and 1950s. Successful and failed abortion attempts using
this agent were associated in case reports with fetal malforma-
tions, including hydrocephalus, meningomyelocele, anencephaly,
limb malformations, cleft lip with cleft palate and developmental
delay.30-34
Similar human malformations were noted after pregnancy
exposure to MTX. Feldkamp and Cary (1993) presented a
review of case reports. Based on six malformed infants, they
concluded that 6 to 8 weeks after conception is the sensitive
period for malformations induced by MTX exposure. They
suggested that a MTX dose of more than 10 mg/week was
necessary to produce anomalies such as clover-leaf skull with a
large head, swept back hair, low-set ears, prominent eyes and
a wide nasal bridge.35 A disproportionality analysis study of
MTX and aminopterin case reports and case series provided
support for pulmonary atresia, craniosynostosis and limb defi-
ciencies, which were reported more often than expected in
MTX-exposed children.36
Because MTX-associated birth defects have only been
described in case reports, the incidence of malformations after
MTX exposure is not known. Of clinical pertinence is the inci-
dence of malformations after MTX exposure for a misdiagnosed
ectopic pregnancy when an intrauterine pregnancy exists. The
dose of MTX used for this purpose (75–100 mg) is higher than
the antirheumatic doses seen in relatively large series of MTX
exposure, and the medication is given earlier in gestation than the
proposed critical window, usually before 6 weeks postconception.
However, case reports have described malformations in these cases
and raised the question of a distinct syndrome caused by such
early exposures.37-39
Evidence is still not clear as to how long a woman should wait
to conceive after she has been successfully treated for a previous
ectopic pregnancy. MTX can persist in the maternal liver for
months. In 2009, a study showed no difference in outcome in
CHAPTER 4 Teratology 33
pregnancies conceived less than 6 months compared with more
than 6 months after MTX therapy.40 
Misoprostol. Misoprostol (Cytotec) is a synthetic prostaglan-
din E1 analog indicated for the prevention of gastric ulcers but
also used to empty the uterus after an incomplete abortion, to
ripen the cervix in preparation for labour and in the treatment of
a postpartum haemorrhage. In combination with other agents, the
use of oral misoprostol was approved by FDA for the termination
of pregnancy of less than 49 days’ duration. With the use of 200
μg of misoprostol, the uterine artery resistance indices increase,
supporting the theory of reduced uteroplacental perfusion as one
mechanism for abnormal development.41 Other mechanisms pro-
pose an increase in intrauterine pressure or interruption of normal
embryonic vascular development.42 Most misoprostol exposure in
pregnancy occurs between 5 and 8 weeks of gestation.43 Miso-
prostol as an abortifacient fails in 10% of cases, and the risk for
failure is higher when it is used as a single agent instead of in
combination with mifepristone or MTX.44,45 Birth defects after
misoprostol use are reported most often in countries where abor-
tion is illegal and not widely available.
association with terminal transverse limb reduction defects,46-48
abdominal wall defects,47,49 palsies of the sixth and other cranial
nerves (Möbius syndrome),46,50,51 bladder exstrophy52 and autism
spectrum disorder with Möbius syndrome.53 A review of four
studies that included 4899 cases of congenital anomalies and 5742
normal control participants reported an association with Möbius
syndrome (odds ratio (OR), 25.31; 95% confidence interval (CI),
11.11–57.66) and terminal transverse limb defects (OR, 11.86;
95% CI, 4.86–28.90).54 
Angiotensin-converting enzyme inhibitors and angiotensin
II receptor antagonists. Angiotensin-converting enzyme (ACE)
inhibitors reduce the activity of ACE and lower blood pressure.
These medications have been used in the treatment of hyper-
tension, cardiac failure and diabetic nephropathy. ACE inhibi-
tor exposure in the second and third trimesters can reduce fetal
blood pressure and renal function and has been associated with
oligohydramnios, growth restriction, hypocalvaria, and renal
failure.55
In one study, first trimester exposure to ACE inhibitors showed
an increased risk for cardiovascular defects (relative risk (RR),
3.72; 95% CI, 1.89–7.30) and CNS defects (RR, 4.39, 95% CI,
1.37–14.02) in offspring of women who filled a prescription for
an ACE inhibitor. These findings might have been due to mater-
nal diabetes, for which adequate adjustments did not appear in the
analysis. These findings were not replicated by subsequent studies
that might have been more successful in adjusting for maternal
diabetes.56-58
Angiotensin II receptor antagonist exposure in the first trimes-
ter was not associated with an increase in congenital anomlies.56,59
After the first trimester, these drugs have been associated with oli-
gohydramnios, limb contractures, pulmonary hypoplasia and fetal
renal dysfunction.60,61 
Lithium. Lithium has been used for the treatment of bipolar
disorder. Based on reports from the 1970s, lithium was thought
to increase the risk for Ebstein anomaly in which the tricuspid
valve is severely displaced toward the right ventricular apex, caus-
ing significant tricuspid regurgitation. Although very few cases of
Ebstein anomaly were noted in a lithium exposure registry, the
prevalence was higher than the expected rate of 1 in 20,00062;
however, this registry included cases reported after the diagnosis
of a birth defect had been made. According to one author, lithium
34 SE C T I O N 1     Early Fetal Development
might increase the risk for Ebstein anomaly to 1 in 1000 exposed
children.63 There has been disagreement with the conclusion that
lithium exposure causes Ebstein anomaly.64 If the structural mal-
formation risk exists, it is likely to be on the order of 0.1%. Fetal
echocardiography can be considered.  Malformation risk
Mycophenolate. Mycophenolate mofetil and mycophenolate
sodium are used as immunosuppressants after organ transplanta-
tion or in treatment of autoimmune disease. The US National
Transplant Pregnancy Registry (NTPR) reported 26 mycophe-
nolate exposed pregnancies born to 18 women.65 There were 15
liveborn children, 4 of whom had malformations. Three children
had microtia, and of those, two also had cleft lip and palate. The
fourth child had hypoplastic nails and shortened fifth fingers.
Many case reports emerged showing different malformations;
however, it was unclear if all of these malformations represented a
mycophenolate embryopathy. The most characteristic abnormali-
ties appeared to be ear abnormalities, facial clefts, and perhaps
conotruncal heart defects.10
The existence of a mycophenolate embryopathy was veri-
fied by epidemiology studies. The National Transplantation
Pregnancy Registry reported an increase in malformations and
other adverse outcomes among pregnancies of women exposed
to mycophenolate compared with other transplant patients
on different regimens.66 ENTIS reported 57 prospectively
ascertained pregnancies after maternal therapy with myco-
phenolate. There were 29 liveborn children, 16 spontaneous
abortions and 12 elective terminations. There were two late
terminations because of multiple malformations consistent
with mycophenolate exposure. Of the liveborn infants, six
had major congenital defects: two with external auditory canal
atresia, one with tracheoesophageal atresia, one with severe
hydronephrosis, one with an atrial septal defect and one with
a myelomeningocele. The malformation rate is thought to be
greater than 20%. In this study, there were also increases in
preterm birth (62%) and low birth weight (31%). A 2014
British study followed nine pregnant women exposed to myco-
phenolate. There were no malformations noted, but there was
an increase in composite poor pregnancy outcome (OR, 5.31,
95% CI, 1.05–26.96).67  Index.81,82 Preconceptional maternal exposure to lead can result
Anticonvulsants. Most anticonvulsants have been associated
with an increase in structural malformations, although it has
not always been easy to separate medication use from a possible
genetic or other contribution of maternal epilepsy. Pregnancy reg-
istries currently are collecting and evaluating outcome data with
which to address the possible risks of anticonvulsant use with
more precision. One such registry estimated malformation risks
of common anticonvulsants compared with that of lamotrigine
as shown in Table 4.4.68 In this study, valproic acid was associated
with increases in spina bifida, hypospadias, oral clefts and some
heart defects; phenobarbital was associated with heart defects and
oral clefts; and topiramate was associated with oral clefts. Val-
proic acid has also been associated with cognitive impairment and
autism.69,70
The information on anticonvulsant medications is updated
frequently because of the ongoing studies in this area. For more
information and to enrol exposed patients, clinicians can con­
tact one of the active registries. In the United States, the North
American Antiepileptic Drug (AED) Pregnancy Registry can be
accessed at http://www.massgeneral.org/aed, and in Europe and
other continents, the International Registry of Antiepileptic
Drugs and Pregnancy can be contacted at http://www.eurapinte
rnational.org.  dysarthria, visual field and auditory disturbances, and tremor.85
TABLE Malformation Risks Estimated by the North
4.4 American Anti-Epileptic Drug in Pregnancy
Registry68
95% confidence
Drug estimate interval
Valproic acid 5.1 3.0–8.5
Phenobarbital 2.9 1.4–5.8
Topiramate 2.2 1.2–4.0
Lamotrigine 1.0 Reference
Data from Hernández-Díaz S, Smith CR, Shen A, et al. Comparative safety of antiepileptic
drugs during pregnancy. Neurology. 2012;78(21):1692–1699.
https://doi.org/10.1212/WNL.0b013e3182574f39.
Environmental Agents
Lead. Human exposure to lead occurs from lead pipes or solder,
batteries, paint, dyes, gasoline, contaminated soil, pottery glazes,
wood preservatives and some traditional Asian or Mexican medi-
cations. Experimental animal and human studies show that lead
can be transported across the placenta to the fetus, and in the
human fetuses, transfer may occur as early as the 12th week of
gestation.71,72 Mid-20th century reports suggested that women
occupationally exposed to lead had a higher risk for spontane-
ous miscarriage and preterm rupture of membranes.73,74 However,
lead exposure in these reports was not well quantified and might
have exceeded the current occupational limits. In some but not all
studies, lead exposure was associated with fetuses that were small
for gestational age.75,76 More recent studies also have reported an
increased risk for preterm birth with lead exposure.76-79
In school-aged children, blood concentrations greater than
20 μg/dL are associated with a 7-point IQ decrease compared
with concentrations less than 20 μg/dL.80 Umbilical cord
blood lead concentrations greater than 10 μg/dL are associated
with lower infant scores on the Bayley Mental Developmental
in subsequent fetal exposure caused by mobilisation of lead from
maternal bone. This risk is reduced with appropriate maternal
intake of calcium.83 Although it is appropriate to monitor blood
lead concentrations during pregnancy in chronically exposed
women and women with a history of lead intoxication, there is
no consensus on the management of elevated blood lead dur-
ing pregnancy. Identifying and eliminating exposure sources has
been recommended if the maternal venous blood lead is above
5 μg/dL. Chelation can be considered if maternal blood concen-
trations exceed 45 μg/dL.84 
Mercury. There are two broad types of mercury: inorganic and
organic. Inorganic mercury includes that used in thermometers
and dental amalgams. Organic mercury, such as methylmercury
(MeHg), can be found in fatty fish, especially large predator fish,
including swordfish, king mackerel, shark, and tile fish.
High exposure to MeHg causes CNS impairment known as
Minamata disease, named for a city in the southwest of Japan’s
Kyushu Island where the water was contaminated with MeHg
from a chemical plant. Pregnant women ate fish and shellfish from
the contaminated water, resulting in infants with a phenotype sim-
ilar to cerebral palsy. Typical symptoms of congenital Minamata
disease included developmental delay, ataxia, sensory disturbances,

It has been more difficult to document neurologic impairment
from the intake of fish in typical diets. The downside to avoiding
fish is inadequate intake of omega-3 fatty acids, which are involved
in nervous system development. We recommend that pregnant
women eat two or three servings of fish or shellfish (about 8–12
oz) per week. However, pregnant or breastfeeding women should
avoid fish known to have higher mercury content.86,87  shielding. The high-dose studies include pelvic computed tomog-
Ionising radiation. Electromagnetic radiation includes wave-
lengths ranging from very long (radio waves) to very short (gamma
rays). Somewhere near the logarithmic middle of the range are
visible light wavelengths of about 1 μm. At shorter wavelengths,
about 10 nm, the radiation energy is sufficient to knock electrons
out of their orbits, resulting in ionisation. Ionising radiation with
a wavelength of about 10 to 0.01 nm is called x-ray, the kind of
radiation used in many diagnostic procedures in medicine.
There are four units commonly used to describe the amount
of radiation encountered by patients. The radiation absorbed dose
(rad) is a measure of the effects of radiation on matter, whether
biological or otherwise. The amount of radiation resulting in
absorption of 100 erg by 1 g of matter is 1 rad. If the matter
is human tissue, the amount of radiation is measured in rem
(roentgen equivalent man). In medicine, rem and rad are used
interchangeably, although they are not strictly the same. The cor-
responding International System of Units (SI) are the Gray (Gy),
which represents 100 rad, and the Sievert (Sv), which represents
100 rem.
We counsel pregnant women that exposures less than 5 rad
(∼50 mSv) do not increase the risk for congenital malformations.
This figure is extrapolated from the association of microcephaly
and cognitive impairment in offspring of pregnant women with
exposure to 50 rad (∼500 mSv) or more during the atomic bomb-
ings of Hiroshima and Nagasaki,8 which is then reduced by of an
order of magnitude to account for imprecision in the assessment
of exposure. The sensitive time period for these effects is 8 to 15
weeks’ gestation, the time when the most rapid proliferation of
neuronal elements is occurring and when most, if not all, neuro-
blast migration to the cerebral cortex from the proliferative zones
occurs.
The original investigators did not believe there was a threshold
dose of radiation below which there was no risk; they estimated a
0.4% increase in risk for microcephaly and cognitive impairment
for every rad of exposure. Based on other studies, it is currently
believed that there is a threshold dose of about 6 rad for intellec-
tual impairment.88
The risk for childhood cancer after intrauterine radiation
sometimes is considered not to exhibit a threshold dose below
which there is no increase; it was estimated that 1 in 2000
children exposed during pregnancy to x-ray pelvimetry would
develop leukaemia compared with a baseline risk for 1 in 3000
children.89 A review in 2014 estimated that an increase in cancer
risk could not be documented at fetal exposures less than 50 rad
(∼500 mSv).90
Natural sources of ionising radiation include stars such as the
sun and nuclides in soil, water, air and human tissue. Background
radiation from these sources results in absorption of about 1 mSv
by a human embryo and fetus over the course of pregnancy.91
Air travel entails additional exposure to cosmic radiation sources.
The US Federal Aviation Administration maintains an online cal-
culator that provides a detailed estimate of radiation dose dur-
ing travel, depending on the date of travel, the altitude achieved
and the amount of time at altitude.92 One-way trips within the
United States are generally associated with radiation absorption
CHAPTER 4 Teratology 35
well under 0.1 mSv. It would take about 10 round trips between
Toronto and Frankfurt to produce a fetal-absorbed radiation dose
of 1 mSv.93
Embryo-fetal radiation doses from diagnostic imagining range
from as little as a few mrad to about 5 rad (∼50 mSv).88 The low-
dose procedures include chest and dental x-ray with appropriate
raphy and whole-body positron emission tomography. Radiation
therapy for malignancy can result in much higher doses to the
conceptus, depending on the procedure, the radiation source,
and the technique. Most tumours remote from the pelvis can be
adequately treated with radiation doses to the embryo or fetus of
1 rad (∼10 mSv) or less, and there are case reports of normal out-
comes after such therapy.94 
Selected Infections
Rubella. Rubella is an RNA virus that causes what in the
prevaccine era was a common childhood illness called German
measles. Abnormalities in the offspring of women who contracted
rubella during pregnancy were described by the Australian oph-
thalmologist Norman Gregg.95 Dr Gregg was impressed by the
number of children with congenital cataracts whom he saw in his
practice. He conferred with colleagues and decided that the epi-
demic of cataracts was associated with maternal rubella contracted
from returning soldiers who introduced this infection to Austra-
lia during the Second World War. The association was confirmed
when Wesselhoeft published on 573 rubella-infected pregnancies
among which there were 521 abnormal children.96 Since the use
of rubella vaccination in developed countries, congenital rubella
has become unusual.97
The most common defect in congenital rubella is deafness,
associated with maternal infection during the first 16 weeks of
pregnancy. Other features include congenital cataract, patent duc-
tus arteriosus, pulmonary artery stenosis, growth and cognitive
impairment, and encephalitis. It has been estimated that 80% of
fetuses are affected after maternal infection in the first trimester,
and 50% are affected after maternal infection between 12 and
16 weeks.95 Adverse effects after 16 weeks’ gestation are unusual,
although deafness has been reported. 
Varicella-Zoster. Varicella-zoster virus is a DNA virus in the
herpesvirus family. The virus causes chickenpox and shingles. Pri-
mary varicella-zoster infection during pregnancy has been associ-
ated with severe maternal illness, including varicella pneumonia.
The first case report of congenital abnormalities after maternal
varicella-zoster virus infection appeared in 1947.98 The mother
had a febrile illness and rash during the eighth week of pregnancy,
which was interpreted as a classic case of varicella. At birth, there
was muscular atrophy of the infant’s right leg and underdevelop-
ment of the toes with clubfoot. The infant had optic atrophy and
may have had hydrocephalus and cortical atrophy.
Based on case reports, the fetal abnormalities associated with
maternal varicella infection included scarring with a dermato-
mal distribution, muscle or bone hypoplasia in a limb associated
with the scarring, cognitive impairment, chorioretinitis, cataracts,
microcephaly and abnormal laxity of rectal and bladder sphinc-
ters. A prospective study of 44 women with varicella with exami-
nation of 41 liveborn children at 1 to 2 years of age was reported
in 1986.99 There were 11 children whose mothers had varicella
during the first trimester, one of whom had skin abnormalities
of the right leg with atrophy and bony defects, chorioretinitis,
cortical atrophy, unilateral hydronephrosis and hydrocephalus.
36 SE C T I O N 1     Early Fetal Development
Another infant had hydrocephalus. There were no abnormalities
among infants whose mothers had second or third trimester vari-
cella or among infants whose mothers had zoster. From a series of
106 women with a clinical diagnosis of varicella during the first 20
weeks of pregnancy and a review of the literature, the incidence of
congenital varicella syndrome after first-trimester maternal infec-
tion was estimated at 2.2% (95% CI, 0%–4.6%).100  it occurred late. Because the relationship between gestational
Cytomegalovirus. Cytomegalovirus (CMV) is a DNA herpes-
virus that can cause primary infection during pregnancy or can be
reactivated in a pregnant woman with a history of previous infec-
tion. Congenital CMV infection has been estimated to occur in
up to 3% of births, with 90% of infants asymptomatic at birth.101
Symptomatic infants may have enlarged livers and spleens, micro-
cephaly, growth restriction and chorioretinitis. Follow-up of 34
symptomatic infants showed death in 10. Among the 23 survi-
vors, microcephaly was present in 16, cognitive impairment in 14,
hearing loss in 7 and chorioretinitis or optic atrophy in 5.102 There
were only 2 survivors who did not have neurological or auditory
handicaps. Sensorineural hearing loss was estimated to occur in
about half of children with congenital CMV infection.101  Nicotine is absorbed through the oral mucosa and the respira-
Zika virus. Zika virus is an RNA virus that belongs to the flavi-
virus family, which includes agents causing other arthropod-medi-
ated infections (West Nile, dengue, tick-borne encephalitis, yellow
fever). Zika virus was first described in 2015 and 2016 as a possible
cause of an epidemic of microcephaly in Brazil,103 and a retrospec-
tive evaluation of a 2013 to 2015 Zika outbreak in French Polynesia
estimated a 1% incidence of microcephaly associated with infec-
tion during pregnancy.104 Based on several data sets, a microcephaly
incidence of 1% to 14% was estimated after Zika virus infection.105
A report from Colombia did not identify abnormalities in the off-
spring if maternal infection occurred in the third trimester.106 Other
abnormalities reported in children born to women with Zika virus
infection include chorioretinal and optic nerve abnormalities, cutis
gyrata, hypertonia or spasticity, hyperreflexia, irritability, cerebral
calcifications, ventriculomegaly and lissencephaly.  molecules used in the detoxification of cigarette constituents.115
Parvovirus B19. Parvovirus B19 is a small DNA virus that
causes an exanthem called ‘fifth disease’ in young children. It
can cause flulike symptoms and joint pain in adults. Parvovirus
B19 infection during pregnancy can be associated with fetal mar-
row aplasia, hydrops and fetal demise. A study of women with
fetal loss before week 22 showed a small increase in the odds of
Immunoglobulin M positivity for parvovirus.107a A 1990 paper
estimated that abnormal outcomes might occur in 9% of pregnan-
cies infected with parvovirus B19,107b but other estimates were
from 1.7 to 4.2%.108,109 Maternal infection between 9 and 20
weeks’ gestation was associated with hydrops in 10 of 94 fetuses,
or 10.6% with a 95% CI of 5.2% to 18.7%.109  fetal alcohol syndrome, consist of facial dysmorphism (smooth
Toxoplasmosis. Toxoplasma gondii, the agent associated with
toxoplasmosis, is a parasite that completes its life cycle in the cat.
Toxoplasma bradyzoites can be found in muscle and give rise to
infection after the consumption of raw meat. Cats contract toxo-
plasmosis from eating raw meat after which oocysts are excreted in
their faeces. Sporulated oocysts can be present in water or in the
air from contamination with cat faeces, and oocysts in the soil may
be ingested by people who handle food without properly washing
their hands after gardening. Congenital toxoplasmosis has been
associated with deafness, microcephaly, cognitive impairment and
chorioretinitis in the offspring.
In a French study, nonimmune women were screened
monthly for toxoplasma antibody seroconversion.81Among
603 confirmed maternal infections, most of which were treated
with spiramycin and or pyrimethamine–sulfadiazine, fetal
transmission occurred in 29% (95% CI, 25%–33%). Fetal
transmission increased with increasing gestational age from a
low of 6% at 13 weeks to 72% at 36 weeks. Among congeni-
tally infected children, 27% had chorioretinal lesions, intra-
cranial calcification or hydrocephaly. Clinical signs were more
common when maternal infection occurred early than when
age and fetal infection increases with gestational age at mater-
nal infection and the relationship between fetal infection and
clinical symptoms in infants decreases with gestational age at
infection, the incidence of clinical signs in infants after mater-
nal infection reaches a peak of 10% when maternal infection
occurred at 24 to 30 weeks of gestation. 
Selected Recreational Exposures
Tobacco. Cigarette smoke consists of a mixture of gases, pri-
marily carbon monoxide, and particulate substances (tobacco
tar) composed of over 4000 different chemical constituents.110
tory and gastrointestinal tracts. Tobacco use has been associated
with spontaneous abortion, ectopic pregnancy, placental abrup-
tion, fetal growth restriction, preterm delivery, specific congenital
malformations, and sudden infant death syndrome (SIDS).
Women who smoke have lighter neonates, on average 200
g, with a clear dose–response relationship. This relationship is
thought to be due to placental changes in smokers that limit
uterine blood flow.111 Intrauterine growth restriction is 2.5 times
higher in mothers who smoke.112 Smoking cessation in the first
trimester decreases the risk for intrauterine growth restriction.111
Smoking in the first trimester has been associated with an
increased risk for cleft palate in individual studies and a meta-
analysis of 24 publications.113,114 The risk may be restricted to
individuals with specific gene variations (e.g. GSST1) that encode
A systematic review of 39 studies showed an adjusted odds
ratio of 2.08 (95% CI, 1.83–2.38)116 for cigarette smoking and
SIDS. A 2011 meta-analysis of 96 studies attributed 4% to 7% of
all stillbirths to maternal smoking in high-income countries. In
disadvantaged populations, maternal smoking could contribute to
20% of all stillbirths.117 
Ethanol. The association of alcoholism during pregnancy and
poor newborn condition has been described for centuries,118 but
systematic descriptions did not appear until 1968 in France119 and
1973 in the United States.120 The specific developmental effects
associated with alcohol abuse during pregnancy, sometimes called
philtrum, thin upper lip, small palpebral fissures), prenatal or
postnatal height or weight below the 10th percentile, head cir-
cumference below the 10th percentile, structural brain abnormali-
ties and cognitive deficits or developmental delay.121 A diagnosis
of fetal alcohol syndrome can be made in the presence of a history
of maternal alcohol use during pregnancy and absence of other
explanatory diagnoses because other disorders can be associated
with the features seen in fetal alcohol syndrome. Some of the other
features described in children considered to have ethanol-related
abnormalities include maxillary hypoplasia, atrial septal defect,
cleft lip, joint abnormalities and abnormalities of vertebrae or ribs.
Maternal risk factors for having a child with fetal alcohol syn-
drome include age older than 30 years, low socioeconomic status,
a previous child with fetal alcohol syndrome, undernutrition and
genetic predisposition.122

The term fetal alcohol spectrum disorder (FASD) is used to
denote the wider array of abnormalities associated with maternal
ethanol consumption during pregnancy. Each individual with
FASD experiences a unique combination of day-to-day challenges
that may include medical, behavioural, educational and social
problems. People with FASD may have difficulty in learning and
remembering, understanding and following directions, shifting
attention, controlling emotions and impulsivity, communicat-
ing and socialising, and performing daily life skills. FASD-related
brain damage causes people to make bad decisions, repeat the same
mistakes, trust the wrong people and have difficulty understand-
ing the consequences of their actions. The incidence of FASDs has
been estimated at 2.4% to 4.8%,123 and clinicians are encouraged
to screen for ethanol use in nonpregnant and pregnant patients
to decrease the considerable burden of ethanol-associated adverse
pregnancy outcome.
The adverse neonatal effects of ethanol have been associated
with chronic heavy alcohol use, defined as more than 5 drinks
per day, which can produce fetal alcohol syndrome in up to
40% of offspring,124 or binge drinking, defined as drinking 5
or more drinks at one time, which has less consistently been
associated with neurocognitive abnormalities.125 A safe level of
ethanol consumption during pregnancy has not been defined.
There are reports that miscarriage is increased in women who
drink 1 oz of absolute ethanol as little as twice a week.126 Neu-
robehavioural effects of ethanol at about 1 drink daily have been
reported but not consistently. Because of uncertainty about the
level of ethanol consumption that is without risk, the US Sur-
geon General in 2005 recommended that pregnant women not
drink any ethanol.127  such as the European Network of Teratogen Information services,
Cocaine. Cocaine is a local anaesthetic with limited medi-
cal value. It is a short-acting stimulant in the CNS and is used
recreationally. It has been associated with preterm delivery,
growth restriction, placental abruption, spontaneous rup-
ture of membranes and abnormal behavioural testing in the
CHAPTER 4 Teratology 37
offspring.128-130 A prospective study of 717 children born to
cocaine-using mothers found no increased risk for birth defects
but noted an increased risk for growth restriction and aberrant
neurologic symptoms in neonates.130
Cocaine effects on neurobehavioural development have been
inconsistent, affecting behavioural regulation, inhibitory con-
trol and attention, problem solving and abstract reasoning, and
language development.131–133 Interpretation of these studies is
difficult because of the inconsistency of the results and possible
residual confounding. 
Conclusion
The idea that external factors can affect the developing human
fetus was novel until the middle of the 20th century during the
rubella epidemic when an Australian physician noted an increase
in congenital cataracts. We now realise, that among other factors,
medication, infections, environmental agents and recreational
exposures may increase the risk for both structural birth defects
and other distinct forms of developmental toxicity such as sponta-
neous abortions and growth restriction when the exposure level is
sufficiently high and the timing is right.
At baseline, there is a 2% to 4% risk for structural anoma-
lies diagnosed at birth. Most structural anomalies do not have a
known cause, and with advances in the field of human genetics,
we are finding that many more have a genetic cause. Chemically
induced structural anomalies, including those caused by a medi-
cation exposure, occur in fewer than 1% of cases. For help with
patient counselling, it is advisable to use up-to-date resources
Organization of Teratology Information Specialists, Reprotox,
Teris, and Lactmed.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 21. Chen C, Jensen BK, Mistry G, et al. Negligi-
ble systemic absorption of topical isotretinoin
for misdiagnosed ectopic pregnancy. Int J
Gynaecol Obstet. 2007;99(3):253–255.
cream: implications for teratogenicity. J Clin 40. Svirsky R, Rozovski U, Vaknin Z, et al. The
1. Centers for Disease Control and Prevention
Pharmacol. 1997;37(4):279–284. safety of conception occurring shortly after
(CDC). Update on overall prevalence of major
22. Holzgreve W, Carey JC, Hall BD. Warfarin- methotrexate treatment of an ectopic preg-
birth defects—Atlanta, Georgia, 1978–2005.
induced fetal abnormalities. Lancet Lond Engl. nancy. Reprod Toxicol. 2009;27(1):85–87.
MMWR Morb Mortal Wkly Rep. 2008;57(1):
1976;2(7991):914–915. 41. Yip SK, Tse AO, Haines CJ, Chung TK.
1–5.
23. Hall JG, Pauli RM, Wilson KM. Maternal Misoprostol’s effect on uterine arterial blood
2. US Food and Drug Administration. Reviewer
and fetal sequelae of anticoagulation during flow and fetal heart rate in early pregnancy.
Guidance: Evaluating the Risks of Drug
pregnancy. Am J Med. 1980;68(1):122–140. Obstet Gynecol. 2000;95(2):232–235.
Exposure in Human Pregnancies. http://www.
24. Schaefer C, Hannemann D, Meister R, 42. Vargas FR, Schuler-Faccini L, Brunoni D,
fda.gov/downloads/Drugs/.../Guidances/
et  al. Vitamin K antagonists and pregnancy et  al. Prenatal exposure to misoprostol and
ucm071645.pdf.
outcome. A multi-centre prospective study. vascular disruption defects: a case-control
3. Webster WS. Teratogen update: congenital
Thromb Haemost. 2006;95(6):949–957. study. Am J Med Genet. 2000;95(4):302–
rubella. Teratology. 1998;58(1):13–23.
25. Malik HT, Sepehripour AH, Shipolini AR, 306.
4. Wilson JG. Mechanisms of teratogenesis. Am
McCormack DJ. Is there a suitable method 43. Gonzalez CH, Marques-Dias MJ, Kim CA,
J Anat. 1973;136(2):129–131.
of anticoagulation in pregnant patients with et  al. Congenital abnormalities in Brazilian
5. Parisot P, Mesbah K, Théveniau-Ruissy M,
mechanical prosthetic heart valves? Interact children associated with misoprostol misuse in
Kelly RG. Tbx1, subpulmonary myocardium
Cardiovasc Thorac Surg. 2012;15(3):484–488. first trimester of pregnancy. Lancet Lond Engl.
and conotruncal congenital heart defects. Birth
26. Meister R, Schaefer C. Statistical methods 1998;351(9116):1624–1627.
Defects Res A Clin Mol Teratol. 2011;91(6):
for estimating the probability of spontaneous 44. Song J. Use of misoprostol in obstet-
477–484.
abortion in observational studies–analyzing rics and gynecology. Obstet Gynecol Surv.
6. Smithells RW, Newman CG. Recogni-
pregnancies exposed to coumarin derivatives. 2000;55(8):503–510.
tion of thalidomide defects. J Med Genet.
Reprod Toxicol. 2008;26(1):31–35. 45. Goldberg AB, Greenberg MB, Darney PD.
1992;29(10):716–723.
27. McLintock C. Anticoagulant choices in Misoprostol and pregnancy. N Engl J Med.
7. Causation in teratology-related litigation. Birth
pregnant women with mechanical heart 2001;344(1):38–47.
Defects Res A Clin Mol Teratol. 2005;73(6):
valves: balancing maternal and fetal risks— 46. Gonzalez CH, Vargas FR, Perez AB, et al. Limb
421–423.
the difference the dose makes. Thromb Res. deficiency with or without Möbius sequence in
8. Otake M, Schull WJ. In utero exposure to
2013;131(suppl 1):S8–S10. seven Brazilian children associated with miso-
A-bomb radiation and mental retardation;
28. Basu S, Aggarwal P, Kakani N, Kumar A. prostol use in the first trimester of pregnancy.
a reassessment. Br J Radiol. 1984;57(677):
Low-dose maternal warfarin intake result- Am J Med Genet. 1993;47(1):59–64.
409–414.
ing in fetal warfarin syndrome: in search for 47. Castilla EE, Orioli IM. Teratogenicity of
9. Barish RJ. In-flight radiation exposure dur-
a safe anticoagulant regimen during preg- misoprostol: data from the Latin-American
ing pregnancy. Obstet Gynecol. 2004;103(6):
nancy. Birth Defects Res A Clin Mol Teratol. Collaborative Study of Congenital Mal-
1326–1330.
2016;106(2):142–147. formations (ECLAMC). Am J Med Genet.
10. Carey JC, Martinez L, Balken E, et al. Deter-
29. Iturbe-Alessio I, Fonseca MC, Mutchinik O, 1994;51(2):161–162.
mination of human teratogenicity by the astute
et al. Risks of anticoagulant therapy in preg- 48. Hofmeyr GJ, Milos D, Nikodem VC, de Jager
clinician method: review of illustrative agents
nant women with artificial heart valves. N M. Limb reduction anomaly after failed miso-
and a proposal of guidelines. Birth Defects Res
Engl J Med. 1986;315(22):1390–1393. prostol abortion. South Afr Med J Suid-Afr
A Clin Mol Teratol. 2009;85(1):63–68.
30. Thiersch JB, Philips FS. Effect of 4-amino- Tydskr Vir Geneeskd. 1998;88(5):566–567.
11. Hill AB. The environment and disease: asso-
pteroylglutamic acid (aminopterin) on early 49. Genest DR, Di Salvo D, Rosenblatt MJ,
ciation or causation? Proc R Soc Med. 1965;58:
pregnancy. Proc Soc Exp Biol. 1950;74(1):204– Holmes LB. Terminal transverse limb defects
295–300.
208. with tethering and omphalocele in a 17 week
12. Centers for Disease Control and Prevention.
31. Thiersch JB. Therapeutic abortions with a folic fetus following first trimester misoprostol
Surgeon General’s Report: Executive Summary.
acid antagonist, 4-aminopteroylglutamic acid exposure. Clin Dysmorphol. 1999;8(1):53–58.
https://www.cdc.gov/tobacco/data_statistics/
(4-amino P.G.A) administered by the oral route. 50. Pastuszak AL, Schüler L, Speck-Martins CE,
sgr/2004/pdfs/executivesummary.pdf.
Am J Obstet Gynecol. 1952;63(6):1298–1304. et  al. Use of misoprostol during pregnancy
13. Lacroix I, Damase-Michel C, Lapeyre-Mestre
32. Meltzer HJ. Congenital anomalies due and Möbius’ syndrome in infants. N Engl J
M, Montastruc JL. Prescription of drugs dur-
to attempted abortion with 4-aminop- Med. 1998;338(26):1881–1885.
ing pregnancy in France. Lancet Lond Engl.
teroglutamic acid. J Am Med Assoc. 51. Marques-Dias MJ, Gonzalez CH, Rosemberg
2000;356(9243):1735–1736.
1956;161(13):1253. S. Möbius sequence in children exposed in
14. Adam MP, Polifka JE, Friedman JM. Evolv-
33. Warkany J, Beaudry PH, Hornstein S. utero to misoprostol: neuropathological study
ing knowledge of the teratogenicity of medica-
Attempted abortion with aminopterin of three cases. Birth Defects Res A Clin Mol
tions in human pregnancy. Am J Med Genet C
(4-amino-pteroylglutamic acid); malfor- Teratol. 2003;67(12):1002–1007.
Semin Med Genet. 2011;157C(3):175–182.
mations of the child. AMA J Dis Child. 52. Orioli IM, Castilla EE. Epidemiological assess-
15. Mitchell AA, Gilboa SM, Werler MM, et al.
1959;97(3):274–281. ment of misoprostol teratogenicity. BJOG Int
Medication use during pregnancy, with par-
34. Shaw EB, Steinback HL. Aminopterin- J Obstet Gynaecol. 2000;107(4):519–523.
ticular focus on prescription drugs: 1976–
induced fetal malformation. Survival of infant 53. Miller MT, Ventura L, Strömland K. Tha-
2008. Am J Obstet Gynecol. 2011;205(1):51.
after attempted abortion. Am J Dis Child lidomide and misoprostol: ophthalmologic
e1–e8.
1960. 1968;115(4):477–482. manifestations and associations both expected
16. Celgene Risk Management. https://www.celg
35. Feldkamp M, Carey JC. Clinical teratology and unexpected. Birth Defects Res A Clin Mol
eneriskmanagement.com/REMSPortal/rems/
counseling and consultation case report: low Teratol. 2009;85(8):667–676.
portal/REMSPortal.portal
dose methotrexate exposure in the early weeks 54. da Silva Dal Pizzol T. Knop FP, Mengue SS.
17. Rizzo R, Lammer EJ, Parano E, et  al. Limb
of pregnancy. Teratology. 1993;47(6):533–539. Prenatal exposure to misoprostol and congeni-
reduction defects in humans associated with
36. Hyoun SC, Običan SG, Scialli AR. Teratogen tal anomalies: systematic review and meta-
prenatal isotretinoin exposure. Teratology.
update: methotrexate. Birth Defects Res A Clin analysis. Reprod Toxicol. 2006;22(4):666–671.
1991;44(6):599–604.
Mol Teratol. 2012;94(4):187–207. 55. Miura K, Sekine T, Iida A, et  al. Salt-los-
18. Lammer EJ, Chen DT, Hoar RM, et  al.
37. Adam MP, Manning MA, Beck AE, et  al. ing nephrogenic diabetes insipidus caused
Retinoic acid embryopathy. N Engl J Med.
Methotrexate/misoprostol embryopathy: report by fetal exposure to angiotensin receptor
1985;313(14):837–841.
of four cases resulting from failed medical abor- blocker. Pediatr Nephrol Berl Ger. 2009;24(6):
19. Dai WS, LaBraico JM, Stern RS. Epidemiol-
tion. Am J Med Genet A. 2003;123A(1):72–78. 1235–1238.
ogy of isotretinoin exposure during pregnancy.
38. Chapa JB, Hibbard JU, Weber EM, et al. Pre- 56. Diav-Citrin O, Shechtman S, Halberstadt Y,
J Am Acad Dermatol. 1992;26(4):599–606.
natal diagnosis of methotrexate embryopathy. et al. Pregnancy outcome after in utero expo-
20. Adams J, Lammer EJ. Neurobehavioral
Obstet Gynecol. 2003;101(5 Pt 2):1104–1107. sure to angiotensin converting enzyme inhibi-
teratology of isotretinoin. Reprod Toxicol.
39. Usta IM, Nassar AH, Yunis KA, Abu-Musa tors or angiotensin receptor blockers. Reprod
1993;7(2):175–177.
AA. Methotrexate embryopathy after therapy Toxicol. 2011;31(4):540–545.

37.e1
37.e2 References
57. Caton AR, Bell EM, Druschel CM, et  al.
Antihypertensive medication use during preg-
nancy and the risk of cardiovascular malfor-
mations. Hypertension. 2009;54(1):63–70.
58. Li D-K, Yang C, Andrade S, et al. Maternal
exposure to angiotensin converting enzyme
inhibitors in the first trimester and risk of mal-
formations in offspring: a retrospective cohort
study. BMJ. 2011;343:5931.
59. Lennestål R, Otterblad Olausson P, Källén
B. Maternal use of antihypertensive drugs
in early pregnancy and delivery outcome,
notably the presence of congenital heart
defects in the infants. Eur J Clin Pharmacol.
2009;65(6):615–625.
60. Hünseler C, Paneitz A, Friedrich D,
et  al. Angiotensin II receptor blocker
induced fetopathy: 7 cases. Klin Pädiatr.
2011;223(1):10–14.
61. Alwan S, Polifka JE, Friedman JM. Angioten-
sin II receptor antagonist treatment during
pregnancy. Birth Defects Res A Clin Mol Tera-
tol. 2005;73(2):123–130.
62. Weinstein MR, Goldfield M. Cardiovascular
malformations with lithium use during preg-
nancy. Am J Psychiatry. 1975;132(5):529–531.
63. Shepard TH, Brent RL, Friedman JM, et al.
Update on new developments in the study
of human teratogens. Teratology. 2002;65(4):
153–161.
64. Yacobi S, Ornoy A. Is lithium a real teratogen?
What can we conclude from the prospective
versus retrospective studies? A review. Isr J Psy-
chiatry Relat Sci. 2008;45(2):95–106.
65. Sifontis NM, Coscia LA, Constantinescu
S, et  al. Pregnancy outcomes in solid organ
transplant recipients with exposure to myco-
phenolate mofetil or sirolimus. Transplanta-
tion. 2006;82(12):1698–1702.
66. Coscia LA, Constantinescu S, Moritz MJ,
et al. Report from the National Transplanta-
tion Pregnancy Registry (NTPR): outcomes
of pregnancy after transplantation. Clin
Transpl. 2008:89–105.
67. Mohamed-Ahmed O, Nelson-Piercy C,
Bramham K, et  al. Pregnancy outcomes in
liver and cardiothoracic transplant recipi-
ents: a UK national cohort study. PloS One.
2014;9(2):e89151.
68. Hernández-Díaz S, Smith CR, Shen A, et al.
Comparative safety of antiepileptic drugs dur-
ing pregnancy. Neurology. 2012;78(21):1692–
1699.
69. Meador KJ, Baker GA, Browning N, et  al.
Fetal antiepileptic drug exposure and cogni-
tive outcomes at age 6 years (NEAD study):
a prospective observational study. Lancet Neu-
rol. 2013;12(3):244–252.
70. Christensen J, Grønborg TK, Sørensen MJ,
et al. Prenatal valproate exposure and risk of
autism spectrum disorders and childhood
autism. JAMA. 2013;309(16):1696–1703.
71. McClain RM, Becker BA. Teratogenic-
ity, fetal toxicity, and placental transfer of
lead nitrate in rats. Toxicol Appl Pharmacol.
1975;31(1):72–82.
72. Baltrop D. Transfer of lead to the human feo-
tus. Mineral metabolism in pediatrics. Oxford,
United Kingdom: Blackwood Scientific Pub-
lishing; 1969.
73. Fahim MS, Fahim Z, Hall DG. Effects of
subtoxic lead levels on pregnant women in the
state of Missouri. Res Commun Chem Pathol
Pharmacol. 1976;13(2):309–331.
74. Nogaki K. On action of lead on body of lead
refinery workers: particularly conception,
pregnancy and parturition in case of females
and on vitality of their newborn. Igaku Ken-
kyu. 1958;27:1314–1338.
75. Chen P-C, Pan I-J, Wang J-D. Parental expo-
sure to lead and small for gestational age
births. Am J Ind Med. 2006;49(6):417–422.
76. Jelliffe-Pawlowski LL, Miles SQ, Courtney JG,
et al. Effect of magnitude and timing of mater-
nal pregnancy blood lead (Pb) levels on birth
outcomes. J Perinatol. 2006;26(3):154–162.
77. Perkins M, Wright RO, Amarasiriwardena CJ,
et al. Very low maternal lead level in pregnancy
and birth outcomes in an eastern Massachu-
setts population. Ann Epidemiol. 2014;24(12):
915–919.
78. Bloom MS, Buck Louis GM, Sundaram R,
et  al. Birth outcomes and background expo-
sures to select elements, the Longitudinal
Investigation of Fertility and the Environment
(LIFE). Environ Res. 2015;138:118–129.
79. Taylor CM, Golding J, Emond AM. Adverse
effects of maternal lead levels on birth out-
comes in the ALSPAC study: a prospective
birth cohort study. BJOG Int J Obstet Gynae-
col. 2015;122(3):322–328.
80. Dietrich KN, Berger OG, Succop PA, et  al.
The developmental consequences of low to
moderate prenatal and postnatal lead expo-
sure: intellectual attainment in the Cincinnati
Lead Study Cohort following school entry.
Neurotoxicol Teratol. 1993;15(1):37–44.
81. Bellinger D, Leviton A, Waternaux C,
et  al. Longitudinal analyses of prena-
tal and postnatal lead exposure and early
cognitive development. N Engl J Med.
1987;316(17):1037–1043.
82. Dietrich KN, Krafft KM, Bornschein RL,
et  al. Low-level fetal lead exposure effect on
neurobehavioral development in early infancy.
Pediatrics. 1987;80(5):721–730.
83. Hertz-Picciotto I, Schramm M, Watt-Morse
M, et al. Patterns and determinants of blood
lead during pregnancy. Am J Epidemiol.
2000;152(9):829–837.
84. Centers for Disease Control and Prevention.
Guidelines for the Identification and Manage-
ment of lead Exposure in Pregnant and Lactat-
ing Women. http://www.cdc.gov/nceh/lead/p
ublications/leadandpregnancy2010.pdf.
85. Harada M. Minamata disease: methylmercury
poisoning in Japan caused by environmental
pollution. Crit Rev Toxicol. 1995;25(1):1–24.
86. Royal College of Obstetricians & Gynaecolo-
gists Healthy Eating and Vitamin Supplements
in Pregnancy. https://www.rcog.org.uk/global
assets/documents/patients/patient-informati
on-leaflets/pregnancy/pi-healthy-eating-and-
vitamin-supplements-in-pregnancy.pdf
87. American College of Obstetricians and Gyne-
cologists Nutrition During Pregnancy. http://
www.acog.org/Patients/FAQs/Nutrition-Dur-
ing-Pregnancy
88. Committee Opinion No. 656: Guidelines for
Diagnostic Imaging During Pregnancy and Lac-
tation. Obstet Gynecol. 2016;127(2):e75–e80.
89. Brent RL. Counseling patients exposed to
ionizing radiation during pregnancy. Rev
Panam Salud Pública Pan Am J Public Health.
2006;20(2–3):198–204.
90. Brent RL. Carcinogenic risks of prenatal ion-
izing radiation. Semin Fetal Neonatal Med.
2014;19(3):203–213.
91. Groen RS, Bae JY, Lim KJ. Fear of the
unknown: ionizing radiation exposure during
pregnancy. Am J Obstet Gynecol. 2012;206(6):
456–462.
92. F  AA Civil Aeromedical Institute. http://jag.ca
mi.jccbi.gov/cariprofile.asp.
93. Chen J, Mares V. Estimate of doses to the
fetus during commercial flights. Health Phys.
2008;95(4):407–412.
94. Kal HB, Struikmans H. Radiotherapy dur-
ing pregnancy: fact and fiction. Lancet Oncol.
2005;6(5):328–333.
95. Webster WS. Teratogen update: congenital
rubella. Teratology. 1998;58(1):13–23.
96. Rubella Wesselhoeft C. (German measles). N
Engl J Med. 1947;236(26):978–988.
97. Parkman PD. Making vaccination policy:
the experience with rubella. Clin Infect Dis
Off Publ Infect Dis Soc Am. 1999;28(suppl
2):S140–S146.
98. Laforet EG, Lynch CL. Multiple congenital
defects following maternal varicella; report
of a case. N Engl J Med. 1947;236(15):
534–537.
99. Paryani SG, Arvin AM. Intrauterine infec-
tion with varicella-zoster virus after maternal
varicella. N Engl J Med. 1986;314(24):1542–
1546.
100. Pastuszak AL, Levy M, Schick B, et al. Out-
come after maternal varicella infection in the
first 20 weeks of pregnancy. N Engl J Med.
1994;330(13):901–905.
101. Daniel Y, Gull I, Peyser MR, Lessing JB. Con-
genital cytomegalovirus infection. Eur J Obstet
Gynecol Reprod Biol. 1995;63(1):7–16.
102. Pass RF, Stagno S, Myers GJ, Alford CA. Out-
come of symptomatic congenital cytomegalovi-
rus infection: results of long-term longitudinal
follow-up. Pediatrics. 1980;66(5):758–762.
103. Brasil P, Pereira JP, Moreira ME, et al. Zika virus
infection in pregnant women in Rio de Janeiro.
N Engl J Med. 2016;375(24):2321–2334.
104. Cauchemez S, Besnard M, Bompard P,
et  al. Association between Zika virus and
microcephaly in French Polynesia, 2013–15:
a retrospective study. Lancet Lond Engl.
2016;387(10033):2125–2132.
105. Johansson MA, Mier-Y-Teran-Romero L,
et  al. Zika and the risk of microcephaly. N
Engl J Med. 2016;375(1):1–4.
106. Pacheco O, Beltrán M, Nelson CA, et  al.
Zika virus disease in Colombia—preliminary
report. N Engl J Med. 2016. [Epub ahead of
print].
107a. Lassen J, Jensen AKV, Bager P, et al. Parvo-
virus B19 infection in the first trimester of
pregnancy and risk of fetal loss: a population-
based case-control study. Am J Epidemiol.
2012;176(9):803–807.
107b. Prospetive study of human parvovirus (B19)
infection in pregnancy. Public Health Labora-
tory Service Working Party on Fifth Disease.
BMJ. 1990;300. http://doi.org/10.1136/bmj.
300.6733.1166. (Published 05 May 1990).
108. Gratacós E, Torres PJ, Vidal J, et  al. The inci-
dence of human parvovirus B19 infection
during pregnancy and its impact on perinatal
outcome. J Infect Dis. 1995;171(5):1360–1363.
109. Enders M, Klingel K, Weidner A, et al. Risk of
fetal hydrops and non-hydropic late intrauter-
ine fetal death after gestational parvovirus B19
infection. J Clin Virol. 2010;49(3):163–168.
110. Rogers JM. Tobacco and pregnancy: overview
of exposures and effects. Birth Defects Res C
Embryo Today Rev. 2008;84(1):1–15.
111. Rogers JM. Tobacco and pregnancy. Reprod
Toxicol. 2009;28(2):152–160.
112. Werler MM. Teratogen update: smoking and
reproductive outcomes. Teratology. 1997;55(6):
382–388.

113. Shi M, Wehby GL, Murray JC. Review on
genetic variants and maternal smoking in
the etiology of oral clefts and other birth
defects. Birth Defects Res C Embryo Today Rev.
2008;84(1):16–29.
114. Little J, Cardy A, Munger RG. Tobacco
smoking and oral clefts: a meta-analysis. Bull
World Health Organ. 2004;82(3):213–218.
115. Shi M, Christensen K, Weinberg CR, et al.
Orofacial cleft risk is increased with mater-
nal smoking and specific detoxification-gene
variants. Am J Hum Genet. 2007;80(1):
76–90.
116. Anderson HR, Cook DG. Passive smoking
and sudden infant death syndrome: review
of the epidemiological evidence. Thorax.
1997;52(11):1003–1009.
117. Flenady V, Koopmans L, Middleton P, et al.
Major risk factors for stillbirth in high-income
countries: a systematic review and meta-analy-
sis. Lancet Lond Engl. 2011;377(9774):1331–
1340.
118. Streissguth AP. Fetal alcohol syndrome: an
epidemiologic perspective. Am J Epidemiol.
1978;107(6):467–478.
119. Lemoine P, Harousseau H, Borteyru JP,
Menuet JC. Children of alcoholic parents—
observed anomalies: discussion of 127 cases.
Ther Drug Monit. 2003;25(2):132–136.
120. Jones KL, Smith DW, Ulleland CN, Streiss-
guth P. Pattern of malformation in offspring
of chronic alcoholic mothers. Lancet Lond
Engl. 1973;1(7815):1267–1271.
121.  Centers for Disease Control and Preven-
tion. Fetal Alcohol Syndrome: Guidelines for
Referral. http://www.cdc.gov/ncbddd/fasd/do
cuments/fas_guidelines_accessible.pdf.
122. Jones KL. The effects of alcohol on fetal devel-
opment. Birth Defects Res C Embryo Today Rev.
2011;93(1):3–11.
123. May PA, Baete A, Russo J, et  al. Prevalence
and characteristics of fetal alcohol spectrum
disorders. Pediatrics. 2014;134(5):855–866.
124. Jones KL, Smith DW, Streissguth AP, Myri-
anthopoulos NC. Outcome in offspring of
chronic alcoholic women. Lancet Lond Engl.
1974;1(7866):1076–1078.
125. Conover EA, Jones KL. Safety concerns
regarding binge drinking in pregnancy: a
review. Birth Defects Res A Clin Mol Teratol.
2012;94(8):570–575.
126. Windham GC, Fenster L, Swan SH. Moder-
ate maternal and paternal alcohol consump-
tion and the risk of spontaneous abortion.
Epidemiol Camb Mass. 1992;3(4):364–370.
127.  Centers for Disease Control and Preven-
tion. Advisory on Alcohol Use in Pregnancy:
A 2005 Message to Women from the U.S.
References 37.e3
Surgeon General. http://www.cdc.gov/ncbdd
d/fasd/documents/sg-advisory.pdf.
128. Gouin K, Murphy K, Shah PS. Knowledge
Synthesis group on Determinants of Low Birth
Weight and Preterm Births. Effects of cocaine
use during pregnancy on low birthweight and
preterm birth: systematic review and meta-
analyses. Am J Obstet Gynecol. 2011;204(4):
340.e1–e12.
129. Addis A, Moretti ME, Ahmed Syed F, et al.
Fetal effects of cocaine: an updated meta-anal-
ysis. Reprod Toxicol. 2001;15(4):341–369.
130. Bauer CR, Langer JC, Shankaran S, et  al.
Acute neonatal effects of cocaine exposure
during pregnancy. Arch Pediatr Adolesc Med.
2005;159(9):824–834.
131. Accornero VH, Anthony JC, Morrow CE,
et  al. Estimated effect of prenatal cocaine
exposure on examiner-rated behavior at age 7
years. Neurotoxicol Teratol. 2011;33(3):370–
378.
132. Carmody DP, Bennett DS, Lewis M. The
effects of prenatal cocaine exposure and gen-
der on inhibitory control and attention. Neu-
rotoxicol Teratol. 2011;33(1):61–68.
133. Richardson GA, Goldschmidt L, Larkby C,
Day NL. Effects of prenatal cocaine exposure
on adolescent development. Neurotoxicol Tera-
tol. 2015;49:41–48.
5
Early Pregnancy Failure
DAVOR JURKOVIC AND KUHAN RAJAH
KEY POINTS
• M iscarriage and ectopic pregnancy are the commonest early
pregnancy complications.
• The diagnosis of early pregnancy failure should be made on
transvaginal ultrasound scan given its high diagnostic sensi-
tivity and specificity.
• Sporadic chromosomal abnormalities are the overriding
cause of miscarriage.
• Early pregnancy failure should be managed in a dedicated
early pregnancy unit.
• Management of early pregnancy failure can be expectant,
medical or surgical depending on the clinical situation and
patient preference.
Miscarriage
Miscarriage is the most frequent complication in pregnancy. It has
been reported that 12% to 24% of women who have missed a
menstrual period and had a positive pregnancy test result experi-
ence the loss of a pregnancy.1 The rate of miscarriage reduces as
the gestational age increases. Three percent of women having a
routine first trimester ultrasound between 10 and 13 weeks are
diagnosed with a delayed miscarriage, and the incidence of a sec-
ond trimester miscarriage has been reported as between 1% to
4%.2,3 Approximately 25% to 50% of women experience at least
one miscarriage during their reproductive years.4 An estimated
125,000 miscarriages occur every year in the United Kingdom,
and these account for more than 50,000 admissions.5,6
The majority of first trimester miscarriages resolve spontaneously
without causing any maternal morbidity or requiring treatment.
However, because of their high incidence, miscarriages and the associ-
ated costs of investigation, hospital admission, treatment and follow-
up are a significant burden. Miscarriage has a negative impact on the
quality of life of women. It signifies a loss of a baby, even in early gesta-
tions, and is a stressful and sad time for the women and their partners.
The maternal mortality rates after miscarriage in the United King-
dom ranged from 0.05 to 0.22 per 100,000 pregnancies between
1985 and 2008. Haemorrhage and sepsis, which mainly occurred
after second trimester losses, were the most common causes of death.7  ticularly if disease control is poor before conception.17
Definition of Miscarriage
Miscarriage is the spontaneous loss of an intrauterine pregnancy
that occurs before 24 weeks’ gestation, the current limit of viability.8
38
Miscarriages have been described as early and late, with an early
miscarriage occurring up to 12 weeks’ gestation and a late miscar-
riage occurring after 12 weeks’ and before 24 weeks’ gestation.6 
Aetiology of Early Miscarriage
Chromosomal Abnormalities
The majority of first trimester miscarriages are caused by chromo-
somal abnormalities. Chromosomal abnormalities are detected in
up to 85% of pregnancy tissue analysed after a spontaneous miscar-
riage.9-11 Fetal malformations were seen in 85% of early miscarriages
in a study that assessed fetal morphology by embryoscopy before
surgical management.12 Roughly 70% of chromosomal abnor-
malities are accounted for by trisomies, and most trisomies involve
chromosome 16, 21 and 22. An estimated 20% are accounted for
by triploidies and 10% by monosomy X. The risk for miscarriage
increases dramatically with increasing maternal age. The risk for mis-
carriage is up to 15% in women up to the age of 34 years. However,
it increases to 25% at 35 to 39 years, 51% at 40 to 44 years and more
than 90% in women aged 45 years and older. The risk for trisomies
increases with maternal age, but the rate of nontrisomic and euploid
miscarriages does not vary significantly with maternal age.13,14 
Maternal Medical Condition
The risk for miscarriage is higher in women with thyroid dysfunc-
tion and thyroid autoimmunity. The presence of thyroperoxidase
antibodies was shown in a large meta-analysis to increase the risk
for miscarriage significantly (odds ratio (OR), 3.73; 95% confi-
dence interval (CI) 1.8–7.6).15 A large prospective study showed
that the risk for miscarriage nearly doubled in women with
a thyroid-stimulating level greater than 2.5 miU/L even in the
absence of thyroperoxidase antibodies.16 The Thyroid AntiBodies
and LEvoThyroxine (TABLET) trial is a large multicentre, dou-
ble-blind, placebo-controlled trial currently being carried out in
England and Scotland. The trial is looking at whether the chances
of delivery beyond 34 weeks’ are increased in women who have
thyroperoxidase antibodies but are euthyroid by taking 50 mcg of
Thyroxine daily (https://www.trials.bham.ac.uk/tablet). The rate
of miscarriage is also higher in women with diabetes mellitus, par-
 
Congenital Uterine Anomaly
A systematic review looking at the reproductive outcomes of
women with congenital uterine anomalies showed that the

presence of a uterine septum increases the risk for a first trimester
miscarriage (risk ratio, 2.89; 95% CI, 2.02–4.14).18 
Lifestyle Factors
There is no proven association between miscarriage and smoking,
and this was confirmed in a prospective study of 24,608 preg-
nancies.19 Obesity is linked with a greater risk for miscarriage. A
systematic review with a cohort of 28,538 women showed that
the miscarriage rate after spontaneous conception was higher in
women with a body mass index (BMI) of 28 kg/m2 or greater
than in women with a BMI less than 25 kg/m2 (13.6% vs 10.7%;
OR,1.31; 95% CI, 1.18–1.46).20 A national Danish birth cohort
study showed that alcohol increases the risk for miscarriage in
even small amounts and that the risk increased with increasing
alcohol consumption.21 
Clinical Symptoms and Findings of
Miscarriage
Approximately one in five women experience vaginal bleeding
and abdominal pain in the first trimester.6 The bleeding that
women experience in early pregnancy can vary from vaginal
spotting to severe haemorrhage causing shock. Vaginal bleed-
ing and the loss of pregnancy symptoms can suggest a mis-
carriage, but miscarriage is not diagnosed clinically. Almost
50% of pregnancies that are complicated by bleeding in the
early stages will continue to develop normally beyond the first
trimester.22
A miscarriage cannot be accurately diagnosed based on clinical
symptoms and findings on vaginal digital and speculum exami-
nation alone. A transvaginal ultrasound should be performed to
make an accurate diagnosis. A large Dutch study showed that a
diagnosis of miscarriage made on clinical symptoms and findings
was erroneous in more than 50% of cases.23 Another study showed
that 40% of women who were diagnosed as having a complete
miscarriage clinically were subsequently shown to have retained
products of conception.24
A speculum examination is necessary in women presenting
with heavy vaginal bleeding or who show signs of haemodynamic
instability because it may facilitate immediate removal of prod-
ucts of conception from the cervix. It can, however, be omitted in
women who present with lighter bleeding because it does not aid
in making an accurate diagnosis.  cavity, may also appear similar to a gestation sac. A pseudosac
Ultrasound Diagnosis of Miscarriage
Transvaginal ultrasonography is the accepted primary investigation
for suspected early pregnancy complications.25,26 The location and
viability of a pregnancy have to be determined when performing
an ultrasound scan in early pregnancy. The morphological appear-
ance of pregnancy is the only criteria taken into account for an
ultrasound diagnosis of a miscarriage. It is imperative to know the
features of a normal intrauterine pregnancy in order to accurately
diagnose early pregnancy complications.
Normal Intrauterine Pregnancy
A normal intrauterine pregnancy is located within the uterine cav-
ity, which starts at the level of the internal cervical os and extends
to the tubal ostia. The trophoblast should not extend beyond the
CHAPTER 5  Early Pregnancy Failure 39
• Fig. 5.1  A normal intrauterine pregnancy at 4 weeks’ gestation.
endometrial–myometrial junction.27,28 The transvaginal probe is
moved in the transverse plane from the internal os all the way to
the fundus to identify an intrauterine pregnancy. The location of
the gestation sac beneath the endometrial surface is seen in the
longitudinal view. The intrauterine location of the gestation scan
is finally confirmed by establishing a communication between the
cervical canal and uterine cavity.29 Endometrial thickness does not
aid in the diagnosis of a pregnancy’s location or viability.30 The
cervix, caesarean section scar if present, myometrium and inter-
stitial portions of the fallopian tubes are systematically assessed
by moving the transvaginal probe from side to side and up and
down.29
In a pregnancy that is developing normally, a gestation sac is
first visualised at 2 weeks and 3 days after conception, that is,
at 4 weeks and 3 days in a woman with regular 28-day cycles
(Fig. 5.1).31 The gestation sac typically appears as circular struc-
ture with a thick echogenic outer rim and a clear anechoic cen-
tre, which signifies the early chorionic activity. The sac is buried
into the decidualised endometrium, and its location is just below
the midline echo.29 Myometrial cysts which are associated with
adenomyosis may appear similar to an early gestation sac. Myo-
metrial cysts are located beyond the endometrial–­myometrial
junction, allowing differentiation from a gestation sac.32 A pseu-
dosac, which is an accumulation of blood within the uterine
appears avascular on Doppler examination and is encircled by a
single decidual layer. The shape of a pseudosac may also change
during the scan. These features differ from that of a normal ges-
tation sac, which has a stable shape, tends to demonstrate strong
peripheral blood flow and is surrounded by a double decidual
layer (Fig. 5.2).29
The mean gestation sac diameter is calculated by taking the
average of three perpendicular diameters measured from the inner
margin of the echogenic rim. The gestation sac grows roughly 1
mm per day in the early stages of pregnancy. The yolk sac can be
visualised within the gestation sac from 5 weeks’ gestation. The
technique of measuring the yolk sac is similar to that of the ges-
tation sac but with the measurements taken from the middle of
the yolk sac wall.29 The embryo is first visible at 5 weeks and 5
days’ gestation. The embryo at its early stages typically appears
as a linear echogenic structure next to the yolk sac. The vitelline
duct connects the embryo to the yolk sac. The crown (head) can
40 SE C T I O N 1     Early Fetal Development
A B
• Fig. 5.2  A, A pseudosac. B, A normal uterine pregnancy.
• Fig. 5.3  A normal intrauterine pregnancy with yolk sac, embryo and
amniotic sac at 7 weeks’ gestation.
be distinguished from the rump (trunk) from 7 weeks’ gestation
(Fig. 5.3). The crown-length measurement should be made in the
sagittal section of the embryo, ensuring the yolk sac is excluded in
the measurement.33
Embryonic cardiac activity can be visualised from 5 weeks and
5 days’ gestation, when the embryo measures 2 to 5 mm in length.
It is vital not to misinterpret background movement and mater-
nal pulsation as embryonic cardiac activity. The heart rate should
be measured using M-mode (Fig. 5.4). Pulsed Doppler examina-
tion should not be used in the first trimester because it produces
high-energy acoustic output.34 It has been reported that a heart
rate below 100 beats/min is suggestive of a pregnancy that is not
developing normally.35,36 The heart rate could, however, be below
100 beats/min in an early normal pregnancy and then rise rapidly
around 6 to 7 weeks’ gestation. The amniotic sac becomes visible
from 7 weeks’ gestation.29
The amniotic sac should be measured in a similar manner
to the yolk sac, with the measurement taken from the middle
of the amniotic sac wall. The spine and rhombencephalon can
be distinguished and the umbilical cord can be visualised from
7 weeks’ gestation. The forebrain, midbrain, hindbrain and
skull are evident at 8 weeks’ gestation. At the same time, the
limb buds start to grow, the amniotic sac expands, the vitelline
duct and umbilical cord lengthen and a midgut hernia becomes
apparent.29  ception or have conceived having had less than three menstrual
• Fig. 5.4  Embryonic heart rate measurement using M-mode.
Multiple Pregnancy
The first evidence of a multiple pregnancy is the presence of more
than one gestation sac around 5 weeks’ gestation. There is not
always a correlation between the number of embryos in a multiple
pregnancy with the number of gestation sacs and yolk sacs.31 The
entire gestation sac needs to be systematically examined when the
pregnancy progresses beyond 6 weeks’ gestation to ensure that all
embryos present are detected. The amnion is seen separate to the
embryo at 7 weeks’ gestation, and this is when amnionicity should
be assessed. The chorion and amnion are not fused at this stage, and
the chorionicity and amnionicity can be accurately established.29 
Early Embryonic Demise
Early embryonic demise describes the early stage in the course
of a miscarriage. An intact gestation sac is still visible within the
uterine cavity, and there is either no embryo present within the
gestation sac or there is no cardiac activity in a visualised embryo
(Fig. 5.5). The main challenge in to avoid confusing a normal
early pregnancy with a miscarriage. The risk for diagnostic error
is significant when making a diagnosis of early embryonic demise
because the diagnosis is based on negative findings. Women who
have irregular menstrual cycles, are unsure of the date of their
last menstrual period, have conceived while on hormonal contra-
 CHAPTER 5  Early Pregnancy Failure 41

• Fig. 5.5  A small embryo without cardiac activity and no increased vas- • Fig. 5.6  Highly vascular tissue within the uterine cavity on Doppler
cularity on Doppler examination and a large amniotic sac, confirming the examination, confirming the diagnosis of an incomplete miscarriage.
diagnosis of an early embryonic demise.

periods since their last pregnancy are at increased risk for being
given a wrong diagnosis. The risk for diagnostic error is also greater
in the presence of congenital uterine anomalies, uterine fibroids,
adhesions after abdominal surgery, altered uterine position after
pelvic surgery and a retroverted uterus.
A wide range of cut-off points for the size of the embryo
and gestation sac to be used when making a diagnosis of early
embryonic demise have been proposed over the years.37 The
2012 National Institute of Clinical Excellence (NICE) guide-
line on the diagnosis and initial management of ectopic preg-
nancy and miscarriage recommends that an early embryonic
demise should be suspected when the mean sac diameter is
25.0 mm or greater and there is no visible embryo and when
the crown–rump length is 7.0 mm or greater and there is no
visible cardiac activity. The guideline also recommends that a
second opinion on the viability of the pregnancy is sought or
a repeat scan is performed 7 to 14 days later before making a • Fig. 5.7  An irregular gestation sac.
confirmed diagnosis of a miscarriage. The reason for this is that
the chief cause of misdiagnosis is operator error, and this can inconclusive or nondiagnostic if the pregnancy has not been con-
occur with any cut-off value.6  firmed to be intrauterine previously. 

Incomplete Miscarriage Prediction of Miscarriage

Incomplete miscarriage describes the presence of products of con-
ception within the uterine cavity in the absence of an intact gesta-
tion sac. It can be sometimes difficult to differentiate trophoblastic
tissue from blood clots within the uterine cavity, which are often
present in women who are bleeding. Retained products of concep-
tion are typically seen as hyperechoic tissue which is well defined
and shows increased vascular blood flow on colour Doppler
examination (Fig. 5.6). Contrastingly, blood clots appear avascu-
lar on colour Doppler examination and are not well defined. It is
challenging to diagnose an incomplete miscarriage, and currently
there are no accepted criteria for making the diagnosis.29,38  gestation sac (Fig. 5.7); a gestation sac with a thin trophoblastic
Complete Miscarriage
Complete miscarriage is diagnosed when there is no evidence of
pregnancy tissue within the uterine cavity. The diagnosis can only
be made if there has been a previous ultrasound scan confirming
an intrauterine pregnancy. The pregnancy should be described as
Ultrasound Features
The presence of embryonic cardiac activity at the first ultrasound
scan does not always indicate that the pregnancy will develop nor-
mally. There are morphological features which suggest the preg-
nancy may not develop normally, and assessment of these features
allows appropriate counselling. These features are, however, not
diagnostic of a miscarriage, and further assessment of the preg-
nancy is usually required. The morphological features suggestive
of an increased risk for early pregnancy failure include an irregular
layer; the presence of an amniotic sac and the absence of embry-
onic cardiac activity; a disproportionately large gestation sac, yolk
sac or amniotic sac relative to the size of the embryo; a discrep-
ancy between the gestational age based on the crown–rump length
and the gestational age based on the last menstrual period; and an
embryonic heart rate below the fifth centile for gestational age or
less than 85 beats/min.39-42 
42 SE C T I O N 1     Early Fetal Development
Biochemical Markers
Biochemical markers play a role in cases in which the ultrasound
scan is nondiagnostic but are not routinely used to diagnose early
pregnancy failure. Human chorionic gonadotrophin (hCG) levels
in maternal serum double every 1.4 to 1.6 days from the point of
first detection to the 35th day of pregnancy and double every 2.0
to 2.7 days from the 35th to the 42nd day of pregnancy.43 Slower
hCG doubling times is associated with miscarriage, and declining
hCG levels is highly accurate in diagnosing a complete miscarriage
after an inconclusive scan.44-46 A serum progesterone level below
16 nmol/L is highly suggestive that the pregnancy is not viable,
but a diagnosis of miscarriage should not be made based on a
progesterone level alone.47,48  tional trophoblastic disease.6
Management of Miscarriage
Expectant Management
Expectant management has become an increasingly popu-
lar option over the past decade or so and is chosen by women
who desire a natural approach. The primary disadvantage with
expectant management is uncertainty over the timescale and
final outcome.49
Expectant management via the use of placebo has been shown
in randomised trials to be successful in 29% to 42% of women
with an early embryonic demise up to 12 weeks’ gestation and
55% to 86% of women with an incomplete miscarriage.50-54
Expectant management was shown to have a higher rate of
unplanned emergency intervention (35% vs 18%; relative risk
(RR), 2.28; CI, 1.93–2.7) and higher rate of blood transfusion
(1.6% vs 0.4%; RR 3.39; CI, 1.08–10.61) compared with active
management (medical or surgical) in a meta-analysis of published
studies. There was, however, no significant difference in infection
rates.55
The 2012 NICE guideline on the diagnosis and initial man-
agement of ectopic pregnancy and miscarriage recommends that
women be offered expectant management for 7 to 14 days as first-
line management. Waiting for longer than 2 weeks is not risky as
long as there are no signs of infection, and the chances of complete
resolution increase with length of follow-up.6  mal embryo.68 Antiphospholipid syndrome, which describes the
Medical Management
Medical management avoids the need for surgery and the associ-
ated risks in more than 70% of women with an early embryonic
demise up to 12 weeks’ gestation.50-54 The success rates of medical
management of an incomplete miscarriage are also high but do
not differ significantly comparison with expectant management.52
Approximately 20% to 30% of women choose to have medical
management.55,56
The prostaglandin analogue misoprostol is the most com-
monly used drug in medical management. It can be administered
in single or divided doses and via the oral, vaginal, sublingual or
rectal route but is only licensed for use orally.57,58 The use of mife-
pristone, an antiprogesterone drug, before the administration of
misoprostol does not increase the success rates significantly.59 The
main side effects with the use of misoprostol are nausea, fever,
diarrhoea and vomiting.51-53 Bleeding typically starts within a few
hours of misoprostol’s being administered. The bleeding can, how-
ever, continue for up to 3 weeks, and women should be reassessed
if they bleed for longer than 3 weeks.6 One percent of women
undergoing medical management will require emergency surgery
because of heavy bleeding.60 
Surgical Management
Surgical management can be performed by suction curettage
in an operating theatre under general anaesthesia or by manual
vacuum aspiration in the outpatient setting under local anaesthe-
sia. Women can be offered surgical management on an elective
basis unless they present with excessive vaginal bleeding, demon-
strate signs of haemodynamic instability or show signs of having
infected retained products of conception. Surgical management is
the management of choice in women suspected of having gesta-
A meta-analysis of clinical trials showed that women undergo-
ing medical management had a higher unplanned intervention
rate (21.3% vs 2.5%; RR, 8.13; CI, 6.26–10.55) compared with
women undergoing surgical management. Women also bled for
longer when they chose to have medical instead of surgical man-
agement (median, 11.0 vs 8.0 days).61 The rates of infection and
blood transfusion were not significantly different with surgical or
medical management.6
The complications with surgery include cervical laceration,
uterine perforation, excessive bleeding and intrauterine adhesions.
The rates of these complications are 2% to 8%.61-65 
Recurrent Miscarriage
Recurrent miscarriage, which is defined as three or more con-
secutive miscarriages, affects 1% of women of reproductive age.
The risk for a further miscarriage is approximately 40% after three
consecutive pregnancy losses.13 Women can experience recurrent
miscarriage despite having a previous live birth.66 Chromosomal
abnormalities are the overriding reason for early pregnancy loss. In
around 4% of couples with a history of recurrent miscarriage, at
least one partner will carry a chromosomal anomaly.67 The chro-
mosomal anomaly may be a balanced or Robertsonian translo-
cation, and the carrier is phenotypically normal. Approximately
50% to 70% of gametes will inherit an unbalanced chromosomal
complement during meiosis, resulting in a chromosomally abnor-
association between antiphospholipid antibodies (lupus anticoag-
ulant, anticardiolipin antibodies and anti-B2 glycoprotein-I anti-
bodies) and vascular thrombosis or adverse pregnancy outcomes,
is the most treatable cause of recurrent miscarriage. Adverse preg-
nancy outcomes include three or more consecutive miscarriages
before 10 weeks’ gestation, loss of one or more morphologically
normal fetus after 10 weeks’ gestation and one or more preterm
deliveries of a morphologically normal fetus before 34 weeks’ ges-
tation because of placental insufficiency.69 Antiphospholipid anti-
bodies are present in 15% of women with recurrent miscarriage
and only in 2% of women with a low-risk obstetric history. The
risk for miscarriage in a subsequent pregnancy is as high as 90%
without pharmacological treatment.70 A combination of aspirin
and unfractionated heparin has been shown to increase the chances
of a live birth to 70%.71 There is a fourfold higher prevalence of
congenital uterine anomalies in women with recurrent miscarriage
compared with women with a low-risk obstetric history according
to studies that used three-dimensional ultrasound to detect uterine
anomalies. The congenital uterine anomalies are also more severe
in the recurrent miscarriage group compared with the low-risk
group.72,73 A large meta-analysis showed an association between

• Fig. 5.8  Complete hydatidiform mole at 8 weeks’ gestation. The ultra-
sound image shows extensive cystic changes in the placental tissue.
factor V Leiden, activated protein C resistance, prothrombin gene
mutation and protein S deficiency and recurrent miscarriage.74  deaths from an ectopic pregnancy despite making up only 2.5%
Molar Pregnancy
Molar pregnancy is a diagnosis made histologically and can be
divided into complete and partial moles according to histologic
and genetic features. Roughly 80% to 95% of complete molar
pregnancies are detected on ultrasound. Thick and cystic tissue
within the uterine cavity without evidence of a gestation sac is
suggestive of a complete molar pregnancy (Fig. 5.8). An intact
gestation sac with cystic placental changes raises the suspicion of
a partial molar pregnancy. Only 20% to 30% of partial molar
pregnancies are detected on ultrasound. An accurate and early
diagnosis allows the appropriate management and follow-up,
and this is important because of the possibility of gestational
trophoblastic neoplasia (persistent gestational trophoblastic dis-
ease, invasive hydatidiform mole, choriocarcinoma and placen-
tal site trophoblastic tumour).75,76 The incidence in the United
Kingdom of gestational trophoblastic disease, which includes
molar pregnancy and gestational trophoblastic neoplasia, is 1 per
714 live births.77 Complete moles are androgenic and almost
always diploid. They arise mainly after reduplication without
cell cytokinesis after monospermic fertilisation or more rarely
from dispermic fertilisation of an anucleate oocyte. Partial moles
almost always occur after dispermic fertilisation of an ovum.78,79
The majority of molar pregnancies present with clinical signs and
ultrasound findings of early pregnancy failure. Suction curettage
ideally under ultrasound guidance is the recommended manage-
ment of a molar pregnancy. Products of conception must be sent
for histologic examination to make a diagnosis. All women with
a molar pregnancy need to be registered with a Gestational Tro-
phoblastic Disease Screening Centre for serial measurement of
serum or urine hCG. About 5% to 8% of women with gesta-
tional trophoblastic disease require chemotherapy for persistent
disease.79  an intrauterine contraceptive device in situ (OR, 10.6; 95%
Ectopic Pregnancy
An ectopic pregnancy is any pregnancy that implants outside
the uterine cavity. The prevalence of ectopic pregnancy in the
United Kingdom is 1.1% with nearly 12,000 ectopic pregnancies
CHAPTER 5  Early Pregnancy Failure 43
diagnosed each year.4 Women undergoing in  vitro fertilisation
have a higher ectopic pregnancy rate of up to 2%.80 The maternal
mortality rate is 0.2 per 1000 cases of ectopic pregnancies.6 Even
though the mortality rate is very low, expensive diagnostic tests
and treatment prove a significant burden.81
A total of 93% to 98% of ectopic pregnancies implant within
the fallopian tubes, making the fallopian tubes the most common
site for an ectopic pregnancy. As a result, the terms ectopic preg-
nancy and tubal pregnancy are often used interchangeably.82-85
A total of 5% to 7% of ectopic pregnancies implant outside
the uterine cavity but within the walls of the uterus. The ‘non-
tubal’ ectopic pregnancies include cervical, caesarean section
scar, intramural and interstitial pregnancies. Ectopic pregnan-
cies outside the tubes and uterus include ovarian and abdomi-
nal pregnancies. These ectopic pregnancies are more difficult to
diagnose than tubal pregnancies. This often leads to a delay in
diagnosis and late presentation after sudden rupture. The mor-
tality rate is consequently high with ‘nontubal’ ectopic pregnan-
cies. Interstitial ectopic pregnancies are particularly associated
with a high mortality rate and account for almost 20% of all
of all ectopic pregnancies.4
A heterotopic pregnancy is a concomitant intrauterine and
extrauterine pregnancy. It occurs in 0.3% to 0.8% spontaneous
pregnancies and 1% to 3% of pregnancies after assisted reproduc-
tive techniques.86
Risk Factors for Ectopic Pregnancy
A previous ectopic pregnancy, evidence of tubal pathology, previ-
ous tubal surgery and exposure to diethylstilboestrol are strongly
associated with ectopic pregnancy. Moderate risk factors for
ectopic pregnancy include a history of genital infections, includ-
ing Chlamydia and gonorrhoea, infertility and multiple sexual
partners.87
A large French case-control study identified women who are
smokers or ex-smokers and women who have been previously
been diagnosed with a sexually transmitted disease to have a
significantly increased risk for developing an ectopic pregnancy.
Other risk factors identified in this study are previous tubal sur-
gery, history of infertility and increased maternal age. However,
roughly 24% of women who were diagnosed with an ectopic
pregnancy in the study from France had no identifiable risk
factors.88
All forms of contraception reduce the risk for an ectopic
pregnancy because they reduce overall pregnancy rates. The
risk for developing an ectopic pregnancy when contraception
fails varies depending on the method of contraception used.
The risk for ectopic pregnancy is particularly high in women
who become pregnant after tubal ligation (OR, 9.3; 95% CI,
4.9–18.0).87 The 10-year cumulative risk for ectopic pregnancy
after tubal ligation was shown to be 7.3 per 1000 procedures in
a multicentre prospective cohort study.89 The risk for ectopic
pregnancy is also significantly increased in women who have
CI, 7.66–10.74).90 About 6% to 10% of women who become
pregnant while on the progesterone-only pill develop an ecto-
pic pregnancy.
The risk for ectopic pregnancy in women who use the combined
oral contraceptive pill, condoms or emergency hormonal contra-
ception is similar to women not using any contraception.91,92 
44 SE C T I O N 1     Early Fetal Development

A B
• Fig. 5.9  Tubal ectopic pregnancy. An empty uterine cavity (A) with a gestation sac within the left fal-
lopian tube (B) and evidence of blood in the pouch of Douglas and the uterovesical pouch.

Clinical Symptoms and Findings of Ectopic

Pregnancy
The clinical presentation of ectopic pregnancy is variable.
Women with an ectopic pregnancy have been described as pre-
senting with the triad of amenorrhoea, vaginal bleeding and pel-
vic or abdominal pain. This is, however, not always the case, and
one study showed that around 30% of women with an ectopic
pregnancy did not present with this triad.93 Vaginal bleeding in
the form of brownish vaginal discharge is typically the earliest
symptom, which often starts soon after the missed menstrual
period. The vaginal bleeding can occasionally be heavy, and this
can lead to the misdiagnosis of a miscarriage. A total of 10% to
20% of women with an ectopic pregnancy do not experience
vaginal bleeding.82
Abdominal pain is often a late symptom and usually occurs
because of tubal distension, bleeding through the fimbrial end
into the peritoneal cavity from tubal miscarriage or tubal rupture. • Fig. 5.10  A case of heterotopic pregnancy. A small gestation sac is seen
Abdominal pain from tubal rupture tends to be more intense, and implanted normally within the uterine cavity (left). A gestational sac con-
abdominal palpation may detect signs of peritonism. Shoulder tip taining a yolk sac is also seen lateral to the uterus.
pain, which characteristically reflects irritation of the diaphragm,
is a sign of major intraabdominal bleeding. Nearly 10% of women
with an ectopic pregnancy do not develop abdominal pain.82,94
Ultrasound Diagnosis of Ectopic Pregnancy
Severe intraabdominal bleeding can cause nausea, vomiting Criteria for the diagnosis of an ectopic pregnancy were first
and diarrhoea, which can erroneously suggest a gastrointestinal described in 1969.97 An ectopic pregnancy was initially sus-
(GI) disorder and delay the diagnosis of a ruptured ectopic pected when there was no conclusive evidence of an intrauterine
pregnancy. In the 2006 to 2008 Confidential Enquiry into pregnancy on scan. High-resolution transvaginal ultrasound has
Maternal and Child Health (CEMACH) report, four of six changed the diagnostic approach to the direct visualisation of the
women who died from an ectopic pregnancy since 1997 were ectopic pregnancy (Figs. 5.9 and 5.10).98 Transabdominal scan-
misdiagnosed initially as having a GI disorder. A key recom- ning is inferior to transvaginal scanning in the diagnosis of an ecto-
mendation from the last two CEMACH reports is that sudden pic pregnancy with early studies showing that the sensitivity with
GI symptoms should alert to the possible diagnosis of ectopic the transabdominal approach is 77% to 80% and the sensitivity
pregnancy.82 with the transvaginal approach is 88% to 90%.26,99 The sensitivity
The diagnostic benefit of vaginal examination including specu- with transvaginal scanning has increased further as the ultrasound
lum examination and bimanual palpation of pelvic organs is lim- machines and expertise of operators have improved.98,100 
ited. A vaginal examination has traditionally been performed as
part of the assessment when an early pregnancy complication is
suspected. The findings of cervical motion tenderness, adnexal
Tubal Ectopic Pregnancy
tenderness or an adnexal mass are nonspecific signs which do not Numerous observational studies have shown that an adnexal mass
aid in making a diagnosis. A total of 36% of women with an ecto- on transvaginal ultrasound is highly specific for a tubal preg-
pic pregnancy lack adnexal tenderness on vaginal examination. A nancy.100 A large meta-analysis showed that an adnexal mass other
transvaginal ultrasound should be the primary investigation car- than a simple cyst separate to the ovary is highly specific (98.9%)
ried out.92,94-96  and sensitive (84.4%) for the diagnosis of a tubal pregnancy.101

Uterine cavity
distended with blood
Gestation sac
with embryo
• Fig. 5.11  Caesarean section scar ectopic pregnancy. A gestation sac
with an embryo is seen implanted anteriorly into a deficient caesarean sec-
tion scar. The uterine cavity is distended with blood.
Similar results with 99% specificity and 88% sensitivity were
reported in a more recent systematic review.86 Tubal pregnancies
can be categorised based on their morphological appearance into
five categories, which are inhomogeneous swelling, empty gesta-
tion sac, gestation sac with yolk sac, gestation sac with an embryo
without cardiac activity and gestation sac containing a live embryo.
Complex adnexal pathology and large, tender, hyperstimulated
ovaries may make diagnosis more difficult. About 78% of tubal
pregnancies after spontaneous conception occur on the same side
of the corpus luteum. The adnexal mass thought to represent an
ectopic pregnancy needs to seen to move separately from the ovary
during palpation with the ultrasound probe.102,103 Echogenic fluid
is seen in the pelvis of 28% to 56% of women with an ectopic
pregnancy. This correlates with the finding of haematoperitoneum
at surgery but may occur as a result of bleeding through the fim-
brial end into the peritoneal cavity from tubal miscarriage or rup-
tured haemorrhagic ovarian cyst rather than tubal rupture.104,105
The presence of blood clots in the pouch of Douglas, blood in the
uterovesical pouch and blood in the upper abdomen signifies a
progressively greater amount of intraabdominal bleeding.  to comply with the follow-up for expectant or medical manage-
Nontubal Ectopic Pregnancy
Caesarean section scar pregnancies occur when the gestation sac
implants in a deficient caesarean section scar (Fig. 5.11). They
occur in approximately 1 in 1800 pregnancies. Caesarean section
scar pregnancies are typically located close to the internal os and
can progress into the second trimester or even rarely into a term
pregnancy, making them difficult to manage.
An interstitial pregnancy occurs when the gestation sac
implants within the interstitial portion of the fallopian tube. The
characteristic finding on ultrasound is the proximal aspect of the
interstitial portion of the fallopian tube communicating with the
medial aspect of the gestation sac and the lateral aspect of the
uterine cavity. Three-dimensional ultrasound scanning may aid in
making the diagnosis (Fig. 5.12). The gestation sac implants low
in the cervix in cervical pregnancies. It is vital to differentiate a
cervical pregnancy from a gestation sac that is passing through the
cervix in an intrauterine miscarriage. The sac might move with
gentle pressure from the probe, the internal cervical os may be
open and there will be a lack of peritrophoblastic blood flow in a
CHAPTER 5  Early Pregnancy Failure 45
Gestation sac
• Fig. 5.12  Interstitial ectopic pregnancy. Three-dimensional view of a ges-
tation sac implanted within the interstitial portion of the right fallopian tube.
miscarriage of an intrauterine pregnancy. Ovarian pregnancies are
characterised by the gestation sac surrounded by healthy ovarian
tissue with the two being inseparable on palpation.29 
Management of Tubal Ectopic Pregnancy
Surgical management.
The role of surgery in modern practice has evolved from being
the main diagnostic modality to being a main treatment option.
NICE recommends surgery as a first-line treatment option when
there is significant pain, an adnexal mass measuring 35 mm or
larger and a gestation sac containing a live embryo and when the
serum hCG level is 5000 iU/L or higher.6 Surgery is also indicated
when there is evidence of haemodynamic instability or significant
intraabdominal bleeding on ultrasound, the woman is unable
ment and it is a heterotopic pregnancy with a viable intrauterine
pregnancy. Laparoscopic surgery has replaced open surgery in the
treatment of ectopic pregnancy. However, laparotomy may be a
safer option than laparoscopy when there is a large intraabdominal
bleed and achieving immediate haemostasis is imperative.106
Salpingectomy, which is the partial or complete removal of the
fallopian tube, and salpingotomy, which is the removal of the preg-
nancy tissue through a linear incision on the fallopian tube and
conservation of the tube, are the two options available with either
laparoscopic or open surgical management. The Royal College of
Obstetricians and Gynaecologists (RCOG) recommends that a
salpingotomy is performed to conserve the tube if the contralateral
tube is diseased. Indications for salpingectomy include tubal rup-
ture, severe tubal damage, recurrence of ectopic pregnancy in the
same tube, lack of surgical expertise to perform a salpingotomy,
inability to achieve haemostasis after salpingotomy and no desire
for a future pregnancy.106 There is an up to 20% risk for residual
trophoblastic tissue after salpingotomy, which may necessitate
further treatment. A large randomised control trial showed that
cumulative ongoing pregnancy rate after spontaneous concep-
tion was not improved in women who underwent salpingotomy
46 SE C T I O N 1     Early Fetal Development
compared with women who underwent salpingectomy provided
the contralateral tube appeared normal at surgery.107  of a Shirodkar cervical cerclage is a very effective method of pre-
Medical management. Medical management with metho-
trexate (MTX) is viewed as an acceptable option when women
are largely asymptomatic, the ectopic pregnancy is small and
the serum hCG level is low. NICE recommends that medical
management can be offered to women when the pain is not sig-
nificant, the serum hCG level is less than 5000 iU/L but ideally
less than 1500 iU/L, the ectopic pregnancy is unruptured and
measures less than 35 mm, the gestation sac does not contain a
live embryo, there is no evidence of an intrauterine pregnancy
and the woman is able to comply with follow-up. Only 25%
to 30% of all women with ectopic pregnancies satisfy the crite-
ria for medical management, making its role limited in clinical
practice.108,109
Medical management with a single dose of systemic MTX was
shown to be significantly less effective than laparoscopic salpin-
gotomy (OR, 0.38; 95% CI, 0.20–0.71) in a meta-analysis.110
Medical management is more cost effective than surgery when the
serum hCG level is less than 1500 iU/L, and only a single dose of
MTX is required. The cost of medical management rises when the
hCG is higher than 1500 iU/L because women are more likely to
require multiple doses of MTX.110,111
The concerns surrounding medical management include the
need for several visits and repeated measurement of serum hCG
until the level falls below 20 iU/L, the need for repeated scans in
women who develop abdominal pain to exclude tubal rupture and
intraabdominal haemorrhage, the need to delay a further preg-
nancy for a minimum of 3 months and dose-related side effects,
including conjunctivitis, GI mucosal inflammation, deranged
liver function and bone marrow suppression.108,112  intrauterine from ectopic pregnancies.125 Changes is serum hCG
Expectant management. Expectant management has been
found to be effective in the management of ectopic pregnancy in
women who present with low hCG levels in a number of obser-
vational studies.113,114 A randomised control trial of women
with an ectopic pregnancy on ultrasound and a plateauing serum
hCG less than 1500 iU and an inconclusive scan and plateau-
ing serum hCG less than 2000 iU found that there was no dif-
ference in the success rates between medical management with
single dose MTX and expectant management (RR, 1.3; 96% CI,
0.9–1.8).115
The success rate of expectant management has been shown to
be around 70%.113,116 A retrospective study of women managed
expectantly showed that the median interval between maximum
hCG levels and prepregnancy levels was 18.0 days.117
Expectant management has advantages over medical manage-
ment, including not having to delay a further pregnancy for a
minimum of 3 months and avoiding the side effects of MTX, and
is becoming an increasingly popular choice. Patient selection is
key, however, and expectant management is most suitable when
the pain is not significant, the serum hCG level is less than 1500
iU/L, the ectopic pregnancy measures less than 30 mm, the gesta-
tion sac does not contain a live embryo, there is no evidence of
significant haematoperitoneum and the woman is able to comply
with follow-up.  a dedicated early pregnancy unit by health care professionals with
Management of Caesarean Section Scar
Pregnancy
Dilatation and curettage is the main surgical management of
caesarean section scar pregnancies. This can be associated with
uncontrolled bleeding from myometrial involvement.118 Insertion
venting excessive bleeding.
Systemic or transvaginal local injection of MTX is effective
in stopping the proliferation of trophoblastic tissue in caesarean
section scar pregnancies. It can, however, take several months
for the trophoblastic tissue to resolve, often causing intermittent
and even heavy vaginal bleeding. This limits the use of medical
management.
A caesarean hysterectomy is often required in caesarean sec-
tion scar pregnancies which evolve into placenta praevia accreta
or percreta.119 
Pregnancy of Unknown Location
The diagnosis of pregnancy of unknown location or an incon-
clusive scan is made when there is no evidence of an intrauter-
ine or extrauterine pregnancy on ultrasound in women with
a positive pregnancy test result. About 8% to 31% of women
with suspected early pregnancy complications will have an ini-
tial ultrasound scan that is inconclusive. Approximately 7% to
20% of women with an inconclusive scan will have an ectopic
pregnancy.120-123 The number of inconclusive scans depends on
a few factors, including the level of skill and experience of the
operator, the quality of the ultrasound machine and whether
a transvaginal or transabdominal scan is performed.124 Serum
hCG and progesterone levels are used to help in making a diag-
nosis when scan is inconclusive. A single hCG reading above
a certain value when a pregnancy is expected to be located on
ultrasound (discriminatory zone) does not aid in differentiating
levels do not help in determining the location of a pregnancy
because patterns of hCG secretion in ectopic pregnancy, normal
intrauterine pregnancy and failing intrauterine pregnancy can be
similar.126 A declining serum hCG level on consecutive measure-
ments or a single low initial serum progesterone level helps to
identify women in whom the pregnancy is spontaneously resolv-
ing irrespective of the location of the pregnancy. The risk for
these women requiring any form of medical intervention is low,
and they do not require further routine follow-up.82,126,127 
Conclusion
Miscarriage is the most frequent complication in pregnancy. It is
a stressful time for couples and a significant burden to the health
service. Miscarriages can be managed expectantly, medically and
surgically depending on the clinical situation and the wishes of the
woman. Ectopic pregnancy is another major early pregnancy com-
plication. Most ectopic pregnancies implant in the fallopian tubes,
and the majority are managed surgically. Nontubal ectopic pregnan-
cies are more difficult to diagnose, and this often leads to a delayed
diagnosis. Both miscarriage and ectopic pregnancy can potentially
cause significant morbidity and even mortality. Women with a sus-
pected early pregnancy problem should therefore be managed in
experience in diagnosing and managing miscarriage and ectopic
pregnancy. A transvaginal ultrasound examination should be carried
out as the primary investigation because of its high sensitivity and
specificity in diagnosing early pregnancy complications.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Galindo A, Burguillo AG, Azriel S, Fuente
Pde L. Outcome of fetuses in women with
36. Schats R, Jansen CA, Wladimiroff JW.
Embryonic heart activity: appearance and
pregestational diabetes mellitus. J Perinat development in early human pregnancy. Br J
1. Prendeville W. Epidemiology of miscarriage.
Med. 2006;34:323–331. Obstet Gynecol. 1990;97:989–994.
In: Grudzinskas JG, O’Brien PMS, eds. Prob-
18. Chan YY, Jayaprakasan K, Zamora J, et  al. 37. Jeve Y, Rana R, Bhide A, Thangaratinam S.
lems in Early Pregnancy: Advances in Diagno-
Reproductive outcomes in women with con- Accuracy of first-trimester ultrasound in the
sis and Management. London: RCOG Press;
genital uterine anomalies: a systematic review. diagnosis of early embryonic demise: a sys-
1997:1–15.
Ultrasound Obstet Gynecol. 2011;38:371–382. tematic review. Ultrasound Obstet Gynecol.
2. Pandya PP, Snijders RJ, Psara N, et  al. The
19. Wisborg K, Kesmodel U, Henriksen TB, et al. 2011;38:489–496.
prevalence of non-viable pregnancy at 10–13
A prospective study of maternal smoking and 38. Jurkovic D. Three-dimensional ultrasound in
weeks of gestation. Ultrasound Obstet Gynecol.
spontaneous abortion. Acta Obstet Gynecol gynecology: a critical evaluation. Ultrasound
1996;7:170–173.
Scand. 2003;82:936–941. Obstet Gynecol. 2002;19:109–117.
3. Regan L, Rai R. Epidemiology and the medi-
20. Boots C, Stephenson MD. Does obesity 39. Reljic M. The significance of crown-rump
cal causes of miscarriage. Baillieres Best Pract
increase the risk of miscarriage in spontaneous length measurement for predicting adverse
Res Clin Obstet Gynaecol. 2000;14:839–854.
conception: a systematic review. Semin Reprod pregnancy outcome of threatened abortion.
4. Stephenson M, Kutteh W. Evaluation and
Med. 2011;29:507–513. Ultrasound Obstet Gynecol. 2002;17:510–512.
management of recurrent early pregnancy
21. Andersen AM, Andersen PK, Olsen J, et  al. 40. Dickey R, Olar T, Taylor S, et al. Relationship
loss. Clin Obstet Gynecol. 2007;50:132–145.
Moderate alcohol intake during pregnancy of small gestational sac-crown-rump length
5. Health and Social Care Information Centre.
and the risk of fetal death. Int J Epidemiol. difference to abortion and abortus karyotypes.
Hospital episode statistics. NHS maternity sta-
2012;41:405–413. Obstet Gynecol. 1992;79:554–557.
tistics. 2011–12. https://catalogue.ic.nhs.uk/
22. Everett C. Incidence and outcome of bleed- 41. Nazari A, Check J, Epstein R, et al. Relation-
publications/hospital/maternity/nhs-mater-
ing before the 20th week of pregnancy: pro- ship of small-for-dates sac size to crown-rump
eng-2011-2012/nhs-mate-eng-2011-2012-
spective study from general practice. BMJ. length and spontaneous abortion in patients
rep.pdf.
1997;315:32–34. with a known date of ovulation. Obstet Gyne-
6. National Institute for Health and Care Excel-
23. Wieringa-de Waard M, Bonsel GJ, Ankum col. 1991;78:369–373.
lence. Ectopic Pregnancy and Miscarriage:
WM, et  al. Threatened miscarriage in gen- 42. Laboda L, Estroff J, Benacerraf B. First trimes-
Diagnosis and Initial Management in Early
eral practice: diagnostic value of history ter bradycardia. A sign of impending fetal loss.
Pregnancy of Ectopic Pregnancy and Miscar-
and physical examination. Br J Gen Pract. J Ultrasound Med. 1989;8:561–563.
riage. London: National Institute for Health
2002;52:825–829. 43. Pittaway DE, Reish RL, Wentz AC. Doubling
and Care Excellence; 2012.
24. Alcazar JI, Baldonado C, Laperte C. The times of human chorionic gonadotrophin
7. Saving Mothers’ Lives. The eighth report of the
reliability of transvaginal ultrasonography increase in early viable intrauterine pregnan-
confidential enquiries into maternal deaths in
to detect retained tissue after spontaneous cies. Am J Obstet Gynecol. 1985;152:299–302.
the united kingdom. BJOG. 2011;118(suppl
first-trimester abortion, clinically thought 44. Lower AM, Yovich JL. The value of serum
1):81–84.
to be complete. Ultrasound Obstet Gynecol. levels of oestradiol, progesterone and beta-
8. Costeloe KL, Hennessy EM, Haider S, et al.
1995;6:126–129. human chorionic gonadotrophin in the pre-
Short term outcomes after extreme preterm
25. Shillito J, Walker JJ. Early pregnancy assess- diction of early pregnancy loss. Hum Reprod.
birth in England: comparison of two birth
ment units. Br J Hosp Med. 1997;58:505–509. 1992;7:711–717.
cohorts in 1995 and 2006 (the EPICure stud-
26. Cacciatore B, Stenman UH, Ylõstalo P. Com- 45. Hahlin M, Thorburn J, Bryman I. The expect-
ies). BMJ. 2012;345:e7976.
parison of abdominal and vaginal sonography ant management of early pregnancies of
9. Ohno M, Maeda T, Matsunobu A. A cyto-
in suspected ectopic pregnancy. Obstet Gyne- uncertain site. Hum Reprod. 1995;1:1223–
genetic study of spontaneous abortions with
col. 1989;73:770–774. 1227.
direct analysis of chorionic villi. Obstet Gynae-
27. Jurkovic D, Mavrelos D. Catch me if you can: 46. Condous G, Kirk E, Van Calster B, et  al.
col. 1991;77:394–398.
ultrasound diagnosis of ectopic pregnancy. Failing pregnancies of unknown location: a
10. Skinner J, Luettich K, Ring M, et al. Analysis
Ultrasound Obstet Gynecol. 2007;30:1–7. prospective evaluation of human chorionic
of fetal DNA in the maternal venous blood for
28. Naftalin J, Jurkovic D. The endometrial-­ gonadotrophin ratio. BJOG. 2006;113:521–
abnormalities of chromosomes 13, 16, 18 and
myometrial junction: a fresh look at a busy 527.
21 in first-trimester spontaneous miscarriage.
crossing. Ultrasound Obstet Gynecol. 2009;34: 47. Verhaegen J, Gallos ID, van Mello NM, et al.
J Obstet Gynaecol. 2003;2:228–232.
1–11. Accuracy if single progesterone test to predict
11. Van den Berg MM, van Maarle MC, van Wely
29. Knez J, Day A, Jurkovic D. Ultrasound imag- early pregnancy outcome in women with pain
M, Goddjin M. Genetics of early miscarriage.
ing in the management of bleeding and pain or bleeding: meta-analysis of cohort studies.
Biochim Biophys Acta. 2012;1822:1951–1959.
in early pregnancy. Best Pract Res Clin Obstet BMJ. 2012;345:e6077.
12. Philipp T, Philipp K, Reiner A, et al. Embryo-
Gynaecol. 2014;28(5):621–636. 48. Cordina M, Schramm-Gajraj K, Ross J, et al.
scopic and cytogenetic analysis of 233 missed
30. Mehta T, Levine D, McArdle C. Lack of sen- Introduction of a single visit protocol in the
abortions: factors involved in the pathogenesis
sitivity if endometrial thickness in predicting management if selected patients with preg-
of development defects of early failed preg-
the presence of an ectopic pregnancy. J Ultra- nancy of unknown location: a prospective
nancies. Hum Reprod. 2003;18:1724–1732.
sound Med. 1999;18:117–122. study. BJOG. 2011;118:693–697.
13.  Nybo Andersen AM, Wohlfahrt J, Chris-
31. Day A, Jurkovic D. The role of ultrasound 49. Luise C, Jermy K, May C, et  al. Expectant
tens P, et  al. Maternal age and fetal loss:
in early pregnancy after assisted conception. management of incomplete, spontaneous
population based register linkage study. BMJ.
In: Jauniaux E, Rizk B, eds. Pregnancy After first trimester miscarriage: outcome accord-
2000;320:1708–1712.
Assisted Reproductive Technology. Cambridge: ing to initial ultrasound criteria and value of
14. Hassold T, Chiu D. Maternal age-specific
Cambridge University Press; 2012. follow-up visits. Ultrasound Obstet Gynecol.
rates of numerical chromosome abnormalities
32. Naftalin J, Hoo W, Pateman K, et  al. How 2002;19:580–582.
with special reference to trisomy. Hum Genet.
common is adenomyosis? A prospective 50. Blohm F, Friden BE, Milsom I, et al. A ran-
1985;70:11–17.
study of prevalence using transvaginal ultra- domised double blind trial comparing miso-
15.  van den Boogaard E, Vissenberg R, Land
sound in a gynaecology clinic. Hum Reprod. prostol or placebo in the management of early
JA, et  al. Significance of (sub)clinical thy-
2012;27:3432–3439. miscarriage. BJOG. 2005;8:1090–1095.
roid dysfunction and thyroid autoimmunity
33. Robinson H, Fleming J. A critical evaluation 51. Kovavisarach E, Sathapanachai U. Intravagi-
before conception and in early pregnancy:
of sonar crown-rump length measurement. Br nal 400 microgram misoprostol for pregnancy
a systematic review. Hum Reprod Update.
J Obstet Gynecol. 1975;82:70. termination in cases of blighted ovum: a ran-
2011;17:605–619.
34.  ISOUG bioeffects and safety committee. domised controlled trial. Aust N Z J Obstet
16. Negro R, Schwartz A, Gismondi R, et  al.
Safety statement, 2000 reconfirmed 2003. Gynaecol. 2002;2:161–163.
Increased pregnancy loss rate in thyroid
Ultrasound Obstet Gynecol. 2003;21:100. 52. Bagratee JS, Khullar V, Regan L, et al. A ran-
antibody negative women with TSH lev-
35. Doubilet P, Benson C. Embryonic heart rate domized controlled trial comparing medical
els between 2.5 and 5.0 in the first trimes-
in the early first trimester: what rate is normal? and expectant management of first trimester
ter of pregnancy. J Clin Endocrinol Metabol.
J Ultrasound Med. 1995;14:431–434. miscarriage. Hum Reprod. 2004;2:266–271.
2010;95:44–48.

46.e1
46.e2 References
53. Lister MS, Shaffer LE, Bell JG, et al. Random-
ized, double-blind, placebo-controlled trial
of vaginal misoprostol for management of
early pregnancy failure. Am J Obstet Gynecol.
2005;4:1338–1343.
54. Wood SL, Brain PH. Medical management of
missed abortion: a randomized clinical trial.
Obstet Gynecol. 2002;4:563–566.
55. El-Refaey H, Hinshaw K, Henshaw R,
et al. Medical management of missed abor-
tion and anembryonic pregnancy. BMJ.
1992;305:1399.
56. Shankar M, Economides DL, Sabin CA, et al.
Outpatient medical management of missed
miscarriage using misoprostol. J Obstet Gyn-
aecol. 2007;27:283–286.
57. Ngoc NT, Blum J, Westheimer E, et al. Medical
treatment of missed abortion using misoprostol.
Int J Gynaecol Obstet. 2004;87:138–142.
58. Tang OS, Ho PC. The use of misoprostol
for early pregnancy failure. Curr Opin Obstet
Gynecol. 2006;18:581–586.
59. Stockheim D, Machtinger R, Wiser A, et al. A
randomized prospective study of misoprostol
or mifepristone followed by misoprostol when
needed for the treatment of women with early
pregnancy failure. Fertil Steril. 2006;4:959–
960.
60. Gronlund A, Gronlund L, Clevin L, et  al.
Management of missed abortion: comparison
of medical treatment with either mifepristone
+ misoprostol or misoprostol alone with sur-
gical evacuation. Acta Obstet Gynecol Scand.
2002;8:1060–1065.
61. Trinder J, Brocklehurst P, Porter R, et  al.
Management of miscarriage: expectant, medi-
cal or surgical? Results of randomised con-
trolled trial [miscarriage treatment (MIST)
trial]. BMJ. 2006;7552:1235.
62. Chung TK, Lee DT, Cheung LP, et al. Spon-
taneous abortion: a randomized, controlled
trial comparing surgical evacuation with con-
servative management using misoprostol. Fer-
til Steril. 1999;6:1054–1059.
63. Egarter C, Lederhilger J, Kurz C, et  al.
Gemeprost for first trimester missed abortion.
Arch Gynecol Obstet. 1995;1:29–32.
64. Graziosi GCM, Mol BWJ, Reuwer PJH, et al.
Misoprostol versus curettage in women with
early pregnancy failure after initial expect-
ant management: a randomized trial. Hum
Reprod. 2004;8. 1894–189.
65. Muffley PE, Stitely ML, Gherman RB. Early
intrauterine pregnancy failure: a randomized
trial of medical versus surgical treatment. Am
J Obstet Gynecol. 2002;2:321–325.
66. Clifford K, Rai R, Regan L. Future pregnancy
outcome in unexplained recurrent first trimes-
ter miscarriage. Hum Reprod. 1997;12:387–
389.
67. Clifford K, Rai R, Watson H, Regan L. An
informative protocol for the investigation of
recurrent miscarriage: preliminary experi-
ence of 500 consecutive cases. Hum Reprod.
1994;9:1328–1332.
68. Stephenson MD, Sierra S. Reproductive
outcomes in recurrent pregnancy loss asso-
ciated with a parental carrier of a structural
chromosome rearrangement. Hum Reprod.
2006;21:1076–1082.
69. Royal College of Obstetricians and Gynaeco-
logists. The investigations and treatment of
couples with recurrent first-trimester and sec-
ond trimester miscarriage. Guideline No 17.
2011. https://www.rcog.org.uk/globalassets/
documents/guidelines/gtg_17.pdf.
70. Rai RS, Clifford K, Cohen H, Regan L.
High prospective fetal loss rate in untreated
pregnancies of women with recurrent miscar-
riage and antiphospholipid antibodies. Hum
Reprod. 1995;10:3301–3304.
71. Empson M, Lassere M, Craig JC, Scott JR.
Recurrent pregnancy loss with antiphospho-
lipid antibody: a systematic review of therapeu-
tic trials. Obstet Gynecol. 2002;99:135–144.
72. Salim R, Regan L, Woelfer B, et al. A compar-
ative study of the morphology of congenital
uterine anomalies in women with and without
a history of recurrent first trimester miscar-
riage. Hum Reprod. 2003;18:162–166.
73. Salim R, Woelfer B, Backos M, et al. Repro-
ducibility of three-dimensional ultrasound
diagnosis of congenital uterine anomalies.
Ultrasound Obstet Gynecol. 2003;21:578–582.
74. Rey E, Kahn SR, David M, Shrier I. Throm-
philic disorders and fetal loss: a meta-analysis.
Lancet. 2003;361:901–908.
75. Fowler DJ, Lindsay I, Seckl ML, et al. Routine
pre-evacuation ultrasound diagnosis of hyda-
tidiform mole: experience of more than 1000
cases from a regional referral centre. Ultra-
sound Obstet Gynecol. 2006;27:56–60.
76. Kirk E, Papageorghiou AT, Condous G, et al.
The accuracy of first trimester ultrasound in
the diagnosis of hydatidiform mole. Ultra-
sound Obstet Gynecol. 2007;29:70–75.
77. Tham BWL, Everard JE, Tidy JA, et al. Gesta-
tional trophoblastic disease in the Asian popu-
lation of Northern England and North Wales.
BJOG. 2003;110:555–559.
78. Devrientd K. Hydatidiform mole and trip-
loidy: the role of genomic imprinting in
placental development. Hum Reprod Update.
2005;11:137–142.
79. Royal College of Obstetricians and Gynaecolo-
gists. The management of gestational tropho-
blastic disease. Guideline No 38. 2010. https://
www.rcog.org.uk/globalassets/documents/guid
elines/gt38managementgestational0210.pdf.
80. Santos-Ribeiro S, Tournaye H, Polyzos NP.
Trends in ectopic pregnancy rates following
assisted reproductive technologies in the UK: a
12-year nationwide analysis including 160,000
pregnancies. Hum Reprod. 2016;31:393–402.
81. Wedderburn CJ, Warner P, Graham B, et al.
Economic evaluation of diagnosing and exclud-
ing ectopic pregnancy. Hum Reprod. 2010;25:
328–333.
82. Jurkovic D, Wilkinson H. Diagnosis and
management of ectopic pregnancy. BMJ.
2011;10:342.
83. Bouyer J, Coste J, Fernandez H, et  al. Sites
of ectopic pregnancy: a 10 year population-
based study of 1800 cases. Hum Reprod. 2002;
17:224–230.
84. Walker J. Ectopic pregnancy. Clin Obstet
Gynecol. 2007;50:89–99.
85. Varma R, Gupta J. Tubal ectopic pregnancy.
Clin Evid (Online). 2009;4:1–16.
86. Crochet JR, Bastian LA, Chireau MW. Does
this woman have an ectopic pregnancy? The
rational clinical examination systematic
review. JAMA. 2013;309:1722–1729.
87. Ankum WM, Mol BW, Van der Veen
F, Bossuyt PM. Risk factors for ectopic
pregnancy: a meta-analysis. Fertil Steril.
1996;65:1093–1099.
88. Bouyer J, Coste J, Shojaei T, et al. Risk fac-
tors for ectopic pregnancy: a comprehensive
analysis based on large case-control, popula-
tion-based study in France. Am J Epidemiol.
2003;157:185–194.
89. Peterson HB, Xia Z, Hughes JM, et  al. The
risk of ectopic pregnancy after tubal sterilisa-
tion. N Engl J Med. 1997;336:762–767.
90. Xiong X, Buekens P, Wollast E. IUD use
and the risk of ectopic pregnancy: a meta-
analysis of case-control studies. Contraception.
1995;52:23–34.
91. Furlong LA. Ectopic pregnancy risk when
contraception fails: a review. J Reprod Med.
2002;47:881–885.
92. McCann MF, Potter LS. Progestin-only oral
contraception: a comprehensive review. Con-
traception. 1994;50(6 suppl 1):S1–S195.
93. Weckstein LN, Boucher AR, Tucker H, et al.
Accurate diagnosis of early ectopic pregnancy.
Obstet Gynecol. 1985;65:393–397.
94. Dart RG, Kaplan B, Varaklis K. Predictive
value of history and physical examination in
patients with suspected ectopic pregnancy.
Ann Emerg Med. 1999;33:283–290.
95. Jurkovic D. Ectopic pregnancy. In: Edmonds
DK, ed. Dewhurst’s Textbook of Obstetrics
and Gynaecology. 8th ed. New Jersey: Wiley-­
Blackwell; 2012:245–254.
96. Mol BW, Hajenius P, Engelsbel S, et  al.
Should patients who are suspected of hav-
ing an ectopic pregnancy undergo a physi-
cal examination? Fertil Steril. 1997;71:
155–157.
97. Kobayashi M, Hellman LM, Fillisti LP.
Ultrasound: an aid in the diagnosis of ectopic
pregnancy. Am J Obstet Gynecol. 1969;103:
1131–1140.
98. Condous G, Okaro E, Khalid A, et  al. The
accuracy of transvaginal sonography for the
diagnosis of ectopic pregnancy prior to sur-
gery. Hum Reprod. 2005;10:1404–1409.
99. Valenzano M, Anserini P, Remorgida V,
et al. Transabdominal and transvaginal ultra-
sonographic diagnosis of ectopic pregnancy:
clinical implications. Gynecol Obstet Invest.
1991;31:8–11.
100. Kirk E, Papageorghiou AT, Condous G, et al.
The diagnostic effectiveness of an initial trans-
vaginal scan in detecting ectopic pregnancy.
Hum Reprod. 2007;22:2824–2828.
101. Brown DL, Doubilet PM. Transvaginal
sonography for diagnosing ectopic pregnancy.
J Ultrasound Med. 1994;13:259–266.
102. Timor-Tritsch I, Yeh M, Peisner D, et al. The
use of transvaginal ultrasonography in the
diagnosis of ectopic pregnancy. Am J Obstet
Gynecol. 1989;161:157–161.
103. Jurkovic D, Bourne T, Jauniaux E, et al. Trans-
vaginal color Doppler study of blood flow in
ectopic pregnancies. Fertil Steril. 1992;57:
68–73.
104. Fleischer AC, Pennell RG, McKee MS,
et  al. Ectopic pregnancy: features at trans-
vaginal sonography. Radiology. 1990;174:
375–378.
105. Nyberg DA, Hughes MP, Mack LA, et  al.
Extra-uterine findings of ectopic pregnancy
at transvaginal US: importance of echogenic
fluid. Radiology. 1991;178:823–826.
106. Royal College of Obstetricians and Gynaeco-
logists. The management of tubal pregnancy.
Guideline No 21. 2004. https://www.rcog.
org.uk/globalassets/documents/guidelines/
gtg21_230611.pdf.
107. Mol F, van Mello NM, Strandell A, et al. Sal-
pingotomy versus salpingectomy in women
with tubal pregnancy (ESEP Study): an open-
label, multicentre, randomised controlled
trial. Lancet. 2014;383:1483–1489.

108. Hajenius PJ, Engelsbel S, Mol BW, et al. Ran-
domised trial of systemic methotrexate versus
laparoscopic salpingostomy in tubal preg-
nancy. Lancet. 1997;350:774–779.
109. Sowter MC, Farquhar CM, Petrie KJ, Gudex
G. A randomised trial comparing single dose
methotrexate and laparoscopic surgery for the
treatment of unruptured tubal pregnancy.
BJOG. 2001;108:192–203.
110. Hajenius PJ, Mol F, Mol BWJ, et al. Interven-
tions for tubal ectopic pregnancy. Cochrane
Database Syst Rev. 2007;1:CD000324.
111. Sowter M, Farquhar C, Gudex G. An eco-
nomic evaluation of single dose methotrexate
and laparoscopic surgery for the treatment
of unruptured ectopic pregnancy. BJOG.
2001;108:204–212.
112. Barnhart K, Coutifaris C, Esposito M. The
pharmacology of methotrexate. Expert Opin
Pharmacother. 2001;2:409–417.
113. Mavrelos D, Nicks H, Jamil A, et al. Efficacy
and safety of a clinical protocol for expectant
management of selected women diagnosed
with a tubal ectopic pregnancy. Ultrasound
Obstet Gynecol. 2013;42:102–107.
114. Elson J, Tailor A, Banerjee S, et  al. Expect-
ant management of tubal ectopic pregnancy:
prediction of successful outcome using deci-
sion tree analysis. Ultrasound Obstet Gynecol.
2004;23:552–556.
115. Mello NM, Mol F, Verhoeve HR, et al. Meth-
otrexate or expectant management in women
with an ectopic pregnancy or pregnancy of
unknown location and lower serum hCG con-
centrations? A randomized comparison. Hum
Reprod. 2013;28:60–67.
116. Korhonen J, Stenman U, Ylostalo P. Low
dose methotrexate with expectant manage-
ment of ectopic pregnancy. Obstet Gynecol.
1996;88:775–778.
117. Mavrelos D, Memtsa M, Helmy S, et  al. β-
hCG resolution times during expectant man-
agement of tubal ectopic pregnancies. BMC
Women’s Health. 2015;15:43.
118. Jurkovic D, Hacket E, Campbell S. Diagno-
sis and treatment of early cervical pregnancy:
a review and report of two cases treated
conservatively. Ultrasound Obstet Gynecol.
1996;8:373–380.
119. Ben Nagi J, Ofili-Yebovi D, Marsh M,
Jurkovic D. First-trimester caesarean scar
pregnancy evolving into placenta previa/
accrete at term. J Ultrasound Med. 2005;24:
1569–1573.
120. Banerjee S, Aslam N, Woelfer B, et al. Expect-
ant management of early pregnancies of
unknown location: a prospective evaluation of
methods to predict spontaneous resolution of
pregnancy. BJOG. 2001;10:158–163.
References 46.e3
121. Condous G, Kirk E, Lu C, et al. Diagnostic
accuracy of varying discriminatory zones for
the prediction of ectopic pregnancy in women
with a pregnancy of unknown location. Ultra-
sound Obstet Gynecol. 2005;26:770–775.
122. Hahlin M, Thornburn J, Bryman I. The expect-
ant management of early pregnancies of uncer-
tain site. Hum Reprod. 1995;10:1223–1227.
123. Kirk E, Condous G, Van Calster B, et  al.
Rationalizing the follow-up of pregnancies
of unknown location. Hum Reprod. 2007;
22:1744–1750.
124. Condous G, Timmerman D, Goldstein S,
et al. Pregnancies of unknown location: con-
sensus statement. Ultrasound Obstet Gynecol.
2006;28:121–122.
125. Wang R, Reynolds TA, West HH, et al. Use
of a β-hCG discriminatory zone with a bed-
side pelvic ultrasonography. Ann Emerg Med.
2011;58(1):12–20.
126. Murray H, Baakdah H, Bardell T, Tulandi T.
Diagnosis and treatment of ectopic pregnancy.
CMAJ. 2005;173:905–912.
127. Cordina M, Schramm-Gajraj Ross J, et  al.
Introduction of a single visit protocol in the
management of selected patients with preg-
nancy of unknown location: a prospective
study. BJOG. 2011;118(6):693–697.
6
The Immunology of Implantation
ASHLEY MOFFETT, Y.W. LOKE AND ANDREW SHARKEY
KEY POINTS
• The extravillous pathway of trophoblast differentiation is es-
sential for the development of the fetoplacental blood supply.
• As they invade into the maternal decidua, extravillous tropho-
blast cells express a unique array of human leukocyte antigen
(HLA) class I molecules, HLA-G, HLA-E and HLA-C.
• The main population of maternal immune cells in the decidua
during placentation are uterine natural killer (uNK) cells.
• Interaction between polymorphic killer immunoglobulin-
like receptors (KIRs) on maternal uNK cells and their HLA-C
ligands on fetal trophoblast cells may regulate the depth and
extent of vascular modification by trophoblast.
• KIR–HLA-C interactions resulting in uNK inhibition are associ-
ated with reduced trophoblast invasion and increased risk for
the great obstetric syndromes (GOS): pre-eclampsia, stillbirth
and fetal growth restriction.
• Conversely, KIR–HLA-C interactions that activate uNK are
associated with increased birth weight and higher risk for
obstructed labour. Hence, the maternal immune system plays
a role in regulating human birth weight.
Introduction
The traditional way to study pregnancy immunology follows
the classical transplantation model, which views the fetus as an
allograft. A more recent approach focuses on the unique, local
uterine immune response to the implanting placenta. This
requires a detailed knowledge of implantation and placental struc-
ture because this impacts greatly on the type of immune response
produced by the mother. At the implantation site, cells from the
mother and the fetus intermingle during pregnancy. Unravelling
what happens here is crucial to our understanding of why some
human pregnancies are successful but others are not.  become incorporated into the vessel wall. At around 10 weeks of
Nidation
The invasive implantation undertaken by the human embryo
brings fetally derived trophoblast cells into direct contact with
maternal cells in the uterine mucosa. Initial contact is followed
by adhesion between the embryonic trophectoderm of the blas-
tocyst and the uterine surface epithelium.1 As the blastocyst pen-
etrates through the surface epithelium into the uterine mucosa,
this trophectoderm layer differentiates into an outer multinucle-
ated syncytiotrophoblast (primitive syncytium) and an inner layer
48
of primitive mononuclear cytotrophoblast. Lacunae soon appear
in the syncytium, and these rapidly enlarge by fusing with each
other. The uteroplacental circulation is potentially established
when this lacuna system erodes through the uterine capillaries.
The intervillous space of the definitive placenta is a derivation of
these lacunae.
The subsequent differentiation of trophoblast occurs along
two main pathways, villous and extravillous (Fig. 6.1). Villous
trophoblast is in contact with maternal blood in the intervillous
space, and its main functions are transport of nutrients and oxy-
gen to the fetus and secretion of hormones. In contrast, extravil-
lous trophoblast is involved in the establishment of the placental
blood supply and intermingles with maternal uterine tissues.2
At the tips of some chorionic villi, cytotrophoblast cells prolifer-
ate into cytotrophoblast columns that anchor these villi to the
underlying decidua. From these columns, individual trophoblast
cells break off to invade the decidua. These interstitial extravil-
lous trophoblast cells appear to move towards the decidual
spiral arteries, encircling these vessels, which then show endo-
thelial swelling and a characteristic ‘fibrinoid’ destruction of
the smooth muscle of the media. How trophoblast cells induce
these changes in the vessel wall is unknown. When migrating
trophoblast cells reach the decidual–myometrial junction, many
become multinucleated placental bed giant cells. These can be
regarded as the endpoint of the extravillous pathway of tropho-
blast differentiation.
Cytotrophoblast columns that lie over the openings of the
decidual spiral arteries form a plug of cells that are known as
endovascular trophoblast. Early in gestation, these plugs occlude
the lumen of the vessels (see Fig. 6.1A). This limits the influx of
blood in the first trimester so that there is only seepage of serum
into the intervillous space. This means that early in pregnancy,
the embryo in the first trimester exists in a low-oxygen environ-
ment.3 From these plugs, some endovascular trophoblasts move
down the inside of the artery, replacing the endothelium, and
gestation, the endovascular plugs disperse, and maternal blood
flow to the intervillous space is established.
Transformation of the spiral arteries by trophoblast is crucial to
successful implantation because these changes convert the arter-
ies from muscular vessels into flaccid sacs capable of transmitting
the increased blood flow required for the developing fetoplacental
unit. Failure of this arterial transformation will result in reduced
conductance and poor perfusion of the placenta, which will affect
the development of the villous tree. This in turn will lead to clini-
cal conditions such as miscarriage, stillbirth, fetal growth restric-
tion and pre-eclampsia (Fig. 6.2).4 
 CHAPTER 6  The Immunology of Implantation 49

Villous Villous stem cell

ST

Villous
CT cytotrophoblast (CT)
COL
TS

Extravillous Villous
cytotrophoblast syncytiotrophoblast
ET column (COL) (ST)
Epithelial
gland
A
F
Trophoblast shell
IT (TS)
uNK
IT
A F
Endovascular
Interstitial trophoblast
trophoblast
(IT)
(ET)

GC
T M Placental giant cell
(GC)
A B
• Fig. 6.1  Schematic representation of the implantation site. A, Placental villi (top) are shown with anchor-
ing cytotrophoblast cell columns and trophoblast invasion into the maternal decidua (bottom). Maternal
blood from decidual spiral arteries (A) fills the intervillous space in direct contact with syncytiotrophoblast
(ST). Distinct trophoblast populations are shown. Villous trophoblast comprises: cytotrophoblast (CT) syn-
cytiotrophoblast form the two layers covering placental villi and do not stain for human leukocyte antigen
(HLA)-G. Extravillous trophoblast includes cytotrophoblast cell columns (COL), interstitial trophoblast (IT),
endovascular trophoblast (ET) and placental bed giant cells (GCs); all stain strongly for HLA-G. Anchoring
cell columns coalesce to form a continuous trophoblast shell (TS). From this shell, interstitial trophoblasts
(ITs) invade through the decidual stroma to encircle and destroy the arterial media, which is replaced by
fibrinoid material (F). ETs move in retrograde fashion down spiral arteries, displacing endothelial cells. On
reaching the inner layer of the myometrium, trophoblast cells differentiate to multinuclear giant cells (GCs).
The inset shows a representation of cellular interactions within the decidua. ITs are seen between large
decidual stromal cells (S). Maternal leukocytes present are mainly uterine natural killer NK (uNK) cells with
a few macrophages (Ms) and occasional T cells (Ts).  B, Pathways of trophoblast differentiation and tro-
phoblast subtypes at the implantation site. (Panel A adapted from Moffett-King A. Natural killer cells and
pregnancy. Nat Rev Immunol 2(9):656–663, 2002.)

Decidualisation Current opinion favours the view that decidualisation facilitates

The uterine endometrial mucosa into which trophoblast invades
is transformed into decidua during pregnancy.5 Morphologi-
cally, the most obvious changes occur in the stromal cells, which
become rounded and glycogen rich. There is also infiltration
by large numbers of bone marrow–derived cells. These changes
begin during the luteal phase of the menstrual cycle (predecidual
change), but if pregnancy occurs, the decidualisation process
continues. This is unlike the situation in most other species in
which decidualisation only begins at implantation. Decidualisa-
tion is under the control of sex hormones oestrogen and proges-
terone. Both glandular and stromal cells of the endometrium
increasingly express oestrogen and progesterone receptors until
the time of ovulation, and expression then declines soon after
in the glands. Expression of progesterone receptors continues in
the stroma throughout the secretory phase and in early decidua.
Prolonged exposure to progesterone results in large, rounded
cells that secrete high levels of prolactin and insulin growth
factor binding protein-1. Other changes include secretion of
interleukin-15 (IL-15), metalloproteinases and chemokines
and the laying down of a pericellular rim of matrix proteins,
particularly fibronectin.
implantation by providing an appropriate substrate for tropho-
blast migration and a fertile soil for nourishment of the develop-
ing fetus throughout gestation. However, it is also possible that
decidua provides a restraining influence against overinvasion by
trophoblast.4,5 This accords with observations that, in situations in
which decidualisation is inadequate, such as in ectopic pregnan-
cies or implantation over a previous caesarean section scar, tro-
phoblast invasion is unrestrained, leading to conditions such as
placenta accreta. It is likely that decidua provides a balance, allow-
ing migration of trophoblast but only to a certain depth. Thus
mammalian reproduction may be considered as a parental tug-
of-war between the requirements of the fetus to derive as much
nourishment as possible from the mother and the defence of the
mother to reduce this nutritional burden for the sake of her own
health and for future pregnancies. 
Trophoblast Interaction with
Extracellular Matrix
Cell migration depends on the expression of adhesion molecules
which bind to extracellular matrix (ECM) proteins. For example,
50 SE C T I O N 2     The Placenta

Chorion
Myometrium Amnion
Amniotic cavity
Decidua parietalis
Cervical canal
Allantoic vessels in umbilical cord
Placenta vascularised by
allantoic vessels
Decidua basalis
Remnants of yolk sac
Radial artery Arcuate artery

A Uterine artery

Fetus
Placenta Placenta Placental villous
Villous tree has fewer
trophoblast cell Maternal blood in branches because of
intervillous space altered blood flow
characteristics
Spiral arterial Extravillous Decidua basalis
wall replaced by trophoblast
trophoblast cells cells (interstitial) Spiral artery remains
(endovascular) narrowed in this
Placental bed segment
Decidua basalis giant cells
Basal artery
Media Media
Endothelium Myometrium Endothelium

Radial artery Radial artery

B Arcuate artery C Arcuate artery

• Fig. 6.2  Disorders of human pregnancy resulting from abnormal placentation. A, The blood supply to
a human pregnant uterus. B, Normal pregnancy. Maternal blood flow to the intervillous space begins at
around 10 weeks’ gestation. The spiral arteries of the placental bed are converted to uteroplacental arter-
ies by the action of migratory extravillous trophoblast cells. Both the arterial media and the endothelium
are disrupted by trophoblast cells, converting the artery into a wide-calibre vessel that can deliver blood
to the intervillous space at low pressure. The small basal arteries are not involved and remain as nutritive
vessels to the inner myometrium and decidua basalis. C, Pre-eclampsia and fetal growth restriction. When
trophoblast cell invasion is inadequate, there is deficient transformation of the spiral arteries. The disturbed
pattern of blood flow leads to reduced growth of the branches of the placental villous tree, which results
in poor fetal growth.

the physiological migration of epithelial cells in wound healing
and the pathological invasion of cancer cells require cell–matrix
interactions. Trophoblast migration into decidua appears to use
similar mechanisms. There are four families of adhesion mol-
ecules, of which the most important for adhesion to the ECM are
the integrins. These are transmembrane glycoproteins consisting
of noncovalently associated α and β subunits. Different α and β
subunits exist and the way they combine determines the ligand
specificity of the integrin. For example, the heterodimers α1β1
and α6β4 are receptors for the ECM protein, laminin, while
α5β1, α4β1 and α4β7 bind fibronectin.
Using monoclonal antibodies specific for various subunits, the
pattern of expression of integrins by different trophoblast popula-
tions at the implantation site is now well documented. The α6β4
integrin is expressed on the villous cytotrophoblast layer and the
cytotrophoblast cells of the cell columns nearest the villous core.
This integrin disappears further out in the cell columns to be
replaced by the heterodimers α5β1 that continue to be expressed
by the interstitial trophoblast invading into decidua. Thus the
α6β4 laminin receptor is downregulated with a concomitant
upregulation of the α5β1 fibronectin receptor as trophoblast
invades the decidua. This observation is similar to that seen dur-
ing the healing of a skin wound where the sessile keratinocytes
that form the normal epidermis express α6β4, but keratinocytes
which migrate to close over the wound express α5β1. Binding of
trophoblast to fibronectin results in signalling through integrins
to the trophoblast cell with changes in gene expression that will
affect trophoblast function.6 In pre-eclampsia, trophoblast fails to
downregulate β4 as seen in normal pregnancy, indicating that dys-
regulation of these integrins could contribute to the inadequate
trophoblast invasion of decidua associated with this pathological
condition. 
Matrix Degradation by Trophoblast
Besides adhesion to ECM proteins, cellular migration also requires
degradation of the matrix. This involves the production of pro-
teolytic enzymes by the migrating cell.7 The two main groups of
enzymes are members of the plasminogen activator (PA) system
of serine proteases and the family of matrix metalloproteinases
(MMPs). The MMP family comprises three main classes based on
their substrate specificities: the collagenases, the gelatinases and
 CHAPTER 6  The Immunology of Implantation 51

the stromelysins. There is an intricate interaction between the PA
and MMP systems, and together they can break down the major
components of ECM. The activity of these proteases is subjected
to close control by specific inhibitors. There are two inhibitors for
PA (PAI), designated as PAI-1 and PAI-2, and two tissue inhibi-
tors for MMP (TIMP), designated as TIMP-1 and TIMP-2.
Trophoblast cells possess proteolytic activity which can be dem-
onstrated in vitro by their digestion of the surrounding matrix on
which the cells are seeded. Zymogram studies have shown that tro-
phoblast cells produce a wide array of proteases, this production
being greater in first-trimester trophoblast compared with tropho-
blast later in gestation, which therefore mirrors the invasive capacity
of early trophoblast. These observations have led to the conclusion
that PA, MMP, PAI and TIMP together provide an intricate net-
work that controls matrix degradation during trophoblast invasion.
Although it is clear that changes in integrin and protease expres-
sion play an important role in regulating trophoblast differentiation
and invasion, the factors that control these changes in normal and
pathological pregnancies are poorly understood.  the predominant cell type is natural killer (NK) cells, with rela-
Trophoblast Expression of Major
Histocompatibility Complex Antigens
Another group of molecules that alter expression as trophoblast
invasion occurs are the major histocompatibility complex (MHC)
class I and class II antigens. These serve as important recognition
molecules for immune cells. There is now good evidence that inter-
actions between MHC antigens and maternal immune cells may
regulate the extent of trophoblast invasion. In humans, these MHC
molecules are known as human leukocyte antigens (HLAs). HLA
class I antigens are expressed on nearly all nucleated cells and class
II antigens on specialised cells involved in antigen presentation such
as dendritic cells and activated macrophages. Both HLA class I and
class II antigens are highly polymorphic and incompatibility for these
antigens between donor and recipient is the basis of graft rejection.
None of the trophoblast cell populations express HLA class II
antigens, and villous trophoblast is also negative for HLA class
I. Maternal T cells cannot therefore directly recognise paternal
alloantigens presented by HLA class I on villous trophoblast.8
However, extravillous trophoblast cells that invade decidua and
interact with uterine tissues express an unusual array of HLA class
I antigens. Presently, there are six HLA class I loci that code for
an expressed protein: three classical loci (HLA-A, -B and -C) and
three nonclassical loci (HLA-E, -F and -G). Normal somatic cells
express HLA-A, -B -C, and the antigens expressed by extravil-
lous trophoblast are HLA-C, -E and -G.4 Of these only HLA-C
is highly polymorphic and varies significantly among pregnancies
(Table 6.1). By contrast, HLA-E and -G are essentially invari-
ant. In healthy individuals, the expression of HLA-G appears to
be restricted to extravillous trophoblast, and this suggests that it
might have a role to play in implantation. 
Leukocyte Populations in Decidua
Analysis of the leukocyte populations in decidua has shown that
tively few classical lymphocytes, T or B cells.9 The uterine NK
(uNK) cells have prominent cytoplasmic granules and the unusual
phenotype of CD56bright CD16-ve. This differentiates them from
classical NK cells in peripheral blood, which are CD56dim
CD16+. Unlike blood NK cells, uNK cells are weakly cytotoxic
against normal NK targets and do not kill trophoblast cells.10 The
number of these NK cells in the uterine mucosa varies through-
out the menstrual cycle. They are sparse during the proliferative
phase, increase significantly by the secretory phase and remain in
high numbers in decidua during early gestation. Current evidence
suggests their recruitment is hormonally controlled most probably
by IL-15 secretion by stromal cells in response to progesterone.
The numbers then decline as pregnancy progresses, and very few
cells remain by term. During the first trimester, these NK cells are
particularly abundant in the decidua basalis in close contact with
invading trophoblast cells. This temporal and spatial association
with the implanting placenta has led to the proposal that these
uNK cells might play an important role in the control of tropho-
blast migration and differentiation. 

TABLE HLA Class I Polymorphism: Expression on Somatic and Extravillous Trophoblast Cells and Corresponding
6.1 Major Receptors
HLA Protein Sequences Somatic Cells Trophoblast Receptors on
HLA-A 2396 +++ - T cells (TCR)
HLA-B 3131 +++ - T cells (TCR)
HLA-C 2089 + +++ NK cells
C1 epitope, KIR2DL2/3
C2 epitope, KIR2DL1 or
KIR2DS1
T cells (TCR; rare)
HLA-E 7 + + NK cells CD94/NKG2A/D
HLA-F 4 (+) - (Poorly defined)
KIR3DS1, LILRB1
HLA-G 16 - +++ NK and myeloid cells
LILRB1, LILRB2, KIR2DL4?

+, Low expression; +++, high expression; HLA, human leukocyte antigen; NK, natural killer; TCR, T cell receptor.
From Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE: The IPD and IPD-IMGT/HLA Database: allele variant databases. Nucleic Acids Research (2015) 43:D423–431.
  
52 SE C T I O N 2     The Placenta

Some
C1 C2
C1/C2

3DL3 2DL3 2DL1 2DL4 3DL1 2DS4 3DL2

KIR haplotype
A

3DL3 2DS2 2DL2 2DL1 2DL4 3DS1 2DL5 2DS5 2DS1 3DL2
KIR haplotype
B

C1 C2 C2

Framework Activating Inhibitory Inactivated in

genes receptors receptors >60% of individuals

• Fig. 6.3  Representative killer immunoglobulin-like receptor (KIR) A and B haplotypes of the KIR gene
family with known binding of human leukocyte antigen (HLA)-C epitopes (C1 or C2) depicted above their
cognate receptors. KIR2DS4 binds a few C1 and C2 allotypes, but more than 60% of individuals have a
truncated form of KIR2DS4. KIR2DS4 only recognises some HLA allotypes carrying that epitope. Frame-
work KIR genes that are present in all haplotypes are shown as black boxes. Activating KIRs are shown
as blue boxes and inhibitory KIR as red boxes.

Uterine Natural Killer Cell Recognition of Maternal KIR–Fetal HLA-C Combinations

Trophoblast
Uterine NK cells express an array of receptors, some of which are
known to bind to the HLA class I molecules expressed by extravil-
lous trophoblast.4 Unlike blood NK cells, all uNK cells express
high levels of the C-type lectin family member CD94/NKG2A,
which binds to HLA-E, resulting in inhibition of NK-cell cyto-
toxicity. Neither ligand or receptor shows significant polymor-
phism, and this is likely to be the signal that stops uNK cells
from killing trophoblast and the maternal cells in the decidua.
Uterine NK cells also express members of the killer immunoglob-
ulin-like receptors (KIRs) family of receptors. These are carried
together as a haplotype on chromosome 19 and have different
binding specificities.11 The KIR also differ in the length of their
cytoplasmic tail that either results in an inhibitory or an activat-
ing signal to the NK cell. Those that have a short tail (S) are
activating (KIR2DS), and those that are long (L) are inhibitory
(KIR2DL) receptors. In all populations, there are two main KIR
haplotypes, A and B; these differ in the presence of additional
activating receptors in the B haplotype. HLA-C, which is the only
polymorphic MHC class I molecule expressed by extravillous tro-
phoblast, is the dominant ligand for several KIR receptors. These
HLA-C allotypes all fall into two groups, C1 and C2, based on a
dimorphism at amino acid 80 of the α1 domain. KIR that bind
HLA-C distinguish between C1 and C2 as mutually exclusive
epitopes as shown in Fig. 6.3. The maternal–fetal immunologic
interaction that occurs at the site of implantation between uNK
and trophoblast therefore involves two gene systems, maternal
KIR and fetal HLA-C molecules. Because these are both poly-
morphic and both maternal and paternal HLA-C allotypes are
expressed on trophoblasts, the exact KIR–HLA-C interaction dif-
fers in each pregnancy. Some KIR–HLA-C combinations appear
to be more favourable to trophoblast invasion than others, thus
affecting reproductive outcome.  type and a fetus with an HLA-C allele bearing a C2 epitope. The
Influence Reproductive Success
Genetic studies of large pregnancy cohorts have now shown
that mothers with two KIR A haplotypes (KIR AA genotype)
are at increased risk for disorders of pregnancy, including pre-
eclampsia and other GOS if the fetus carries an HLA-C allele
with a C2 epitope inherited from the father.12 Conversely,
mothers with a KIR B haplotype (containing activating
KIR2DS1 that can also bind C2 epitopes) are at low risk,
but instead these mothers have an increased risk for deliver-
ing a large baby.13 When the fetus is C1/C1 homozygous,
the mother’s KIR genotype has no effect, so a C2 epitope is
the crucial fetal ligand (Fig. 6.4). Three major conditions of
pregnancy–recurrent miscarriage, FGR and pre-eclampsia–all
show the same association. Overall our results suggest that
receptor–ligand interactions leading to strong uNK inhibition
result in decreased trophoblast invasion and compromised
fetal development. Findings in mice support this. Binding of
the inhibitory receptor Ly49A on murine uNK cells to a single
extra MHC molecule on trophoblast results in decreased uterine
vascular remodelling and reduced fetal growth.14
A key question remains: how does the presence of the KIR B
haplotype reduce this risk? In Europeans, the protective genes on
the KIR B haplotype map to the region where the activating KIR for
C2 (KIR2DS1) is located. Protection from pre-eclampsia is likely
to be due to counterbalancing uNK activation when KIR2DS1
binds C2. Indeed, when KIR2DS1 on uNK cells binds to C2,
this increases secretion of cytokines that enhance trophoblast inva-
sion in vitro.15 If trophoblast invasion in vivo is correspondingly
enhanced, this could lead to improved placental perfusion and
better fetal growth. In support of this model, we find that high-
birth-weight pregnancies are associated with mothers who have
inherited the activating receptor KIR2DS1 on the KIR B haplo-
 CHAPTER 6  The Immunology of Implantation 53

Baby’s HLA-C effect is significant resulting in an estimated average increase in

Mother’s KIR birth weight of some 200 g.13 Conversely, KIR AA mothers who
haplotype have two copies of KIR2DL1 the inhibitory receptor for C2 and
C1 C1 C1 C2 C2 C2 have a fetus with a C2 epitope show reduced birth weight com-
KIR2DL1 pared with fetuses that lack C2. These effects on both large and
↓
A small babies are most significant when the fetal C2-bearing allele
A is paternally derived (Fig. 6.5).
↑
Pregnancies at both extremes of birth weight more likely to
↓ experience serious obstetric complications. Large babies are at
A risk for fetal obstruction, which can result in prolonged labour,
B fetal death from asphyxia and postpartum haemorrhage.16 On
↑ ↑
KIR2DS1 the other hand, when spiral artery modification is inadequate,
↓ ↓ poor placental perfusion can manifest as increased risk for pre-
B eclampsia, recurrent miscarriage or FGR, with increased mater-
B
↑ ↑ nal and fetal morbidity. Because humans have a large brain and
KIR2DS1 narrow pelvis relative to other primates, the obstetric dilemma is
• Fig. 6.4 Certain combinations of maternal killer immunoglobulin-like particularly acute, and there is strong selective pressure to main-
receptor (KIR) and fetal human leukocyte antigen (HLA)-C genotypes tain human birth weight between these two extremes. Although
increase susceptibility to pre-eclampsia, recurrent miscarriage or fetal many genes and environmental influences have an impact on
growth restriction. A cross (x) indicates the increased risk for a poor clinical fetal growth, the genetic studies suggest that KIR–HLA-C inter-
outcome. Maternal KIR A haplotype carries the inhibitory KIR2DL1 that actions play a role in maintaining an optimal birth weight in
can bind the C2 epitope carried by some fetal HLA-C alleles. KIR B haplo-
human populations.
types can also include the activating KIR2DS1 that binds C2.

0.4 Birth weight frequency

Special care transfer
frequency

0.3
Population frequency

0.2

0.1

0.0
1000 2000 3000 4000 5000 6000 Birth weight (g)
Low Normal High
Increased frequency of Increased frequency of
KIR AA+ paternal C2 KIR2DS1+ paternal C2

Poor spiral artery Normal Exceptional spiral artery

transformation transformation transformation?
• Fig. 6.5  The presence of maternal KIR2DS1 is associated with increased birth weight if fetus has inher-
ited a human leukocyte antigen (HLA)-C allele with a C2 epitope. Distribution of birth weights in Norwegian
MoBa cohort is shown together with percentage of babies transferred to special care baby unit. The cohort
was divided into high- (>90th percentile), normal- (6th–89th percentile) and low- (<5th percentile) birth-
weight babies. Whereas small babies show increased frequency of maternal killer immunoglobulin-like
receptor (KIR) AA and fetal C2, frequency of KIR2DS1 and fetal C2 is higher in large babies compared with
normal pregnancies. The schematic below indicates the extent of maternal spiral artery vascular conversion
corresponding to each condition. The enhanced vascular conversion depicted in mothers with KIR2DS1 and
a fetus with C2 is hypothesised. Evidence directly demonstrating improved blood supply to the placenta in
such pregnancies is not yet available. (Original data from Hiby SE, Apps R, Chazara O, et al. Maternal KIR in
combination with paternal HLA-C2 regulate human birth weight. J Immunol 192:5069–5073, 2014.)
54 SE C T I O N 2     The Placenta
The third HLA class I molecule expressed by trophoblast is
HLA-G, which binds with high affinity to leukocyte immuno-
globulin-like receptors (LILRs) expressed by myelomonocytic
cells. This interaction results in the induction of a ‘tolerogenic’
population of dendritic cells which, in a transplantation setting,
leads to tolerance. The idea that the placenta itself (via HLA-G)
is modifying the maternal immune reactivity locally in the uterus
to downregulate damaging alloreactive T-cell responses during
pregnancy is attractive. Thus HLA-G could act as a ‘placental’
signal to the decidual innate immune system through LILRB1 on
myelomonocytic cells to induce pregnancy-specific immune func-
tions in the uterus.4,17
Adults homozygous for HLA-G null alleles have been iden-
tified, showing that expression of membrane-bound HLA-G
by trophoblast is not essential for successful pregnancy.18 It
is likely that HLA-G provides only one of several redundant
mechanisms, including absence of HLA-A or HLA-B on tro-
phoblast, that favour T-cell tolerance in the decidua. Migration
of antigen presenting cells from decidua to local lymph nodes
is poor, and chemokines that recruit T cells are epigenetically
silenced. These effects work together with antigen-independent
mechanisms such as expression of indoleamine 2,3-dioxygen-
ase (IDO), galectins and secretion of immunosuppressive cyto-
kines such as transforming growth factor β, to create a local
tolerogenic immune environment.11 This favours the genera-
tion of regulatory T cells (Tregs) that could act to limit the
development of effector T cells at the maternal–fetal interface.
Although Treg depletion in mice leads to fetal loss, indicating
an important role, the mechanism and specificity of these cells
are unclear. Tregs are also generated in HLA-C–mismatched
human pregnancies to a greater degree than in HLA-C–
matched pregnancies, but there still is no convincing evidence
to date that demonstrates that T cell–mediated mechanisms
are responsible for human pregnancy failure.19 Further work is
required to understand the role played by T cells at the mater-
nofetal interface.  Self-assessment questions available at ExpertConsult.com.
Conclusion
Adequate trophoblast invasion and vascular remodelling is
required for proper placental and fetal growth. In addition,
epidemiological data have shown that growth retardation in
utero is associated with increased incidence of certain diseases
in adulthood. Thus any dysregulation of placentation has far-
reaching consequences. Recent genetic and functional studies
provide strong evidence that interactions of KIR on uNK cells
with HLA-C on trophoblast play an important role in regulating
the depth of trophoblast invasion. KIR–HLA-C combinations
that cause excessive uNK inhibition are associated with reduced
vascular remodelling and increased risk for GOS, including
pre-eclampsia, recurrent miscarriage, stillbirth and FGR. Con-
versely, combinations that enhance uNK activation may con-
tribute to increased birth weight. A direct effect of uNK cells
on spiral artery structure and function is also likely. The rela-
tive importance of interactions between the three components–
uNK cells, trophoblast and arteries–probably varies in different
species. It is also clear that maternal recognition of pregnancy
results in changes in decidual T cells, including Tregs, but there
is no compelling evidence that maternal T cells specific for fetal
HLA molecules are directly involved in pregnancy disorders.9,19
Whatever mechanisms are involved, the maternal immune system
must provide a balance between fetal intrusion into the mother’s
resources and the need to protect the mother from excessive fetal
demand. In studying this, the view of the uterus as a ‘privileged
site’ is no longer valid because all anatomical sites have unique
immune features, and this applies particularly to mucosal sur-
faces. The unusual features of the decidua result in a peaceful
physiological environment in which specific combinations of
maternal KIR and HLA-C contribute to successful reproduction
and act to maintain birth weight between two extremes.
Access the complete reference list online at ExpertConsult.com.
References of metzincin proteases in trophoblast biol-
ogy and placental development: a focus on
14. Kieckbusch J, Gaynor LM, Moffett A, et  al.
MHC-dependent inhibition of uterine NK
ADAM12. Placenta. 2015;36(suppl 1):S11– cells impedes fetal growth and decidual vascu-
1. Cha J, Sun X, Dey SK. Mechanisms of implan-
S19. lar remodelling. Nat Commun. 2014;5:3359.
tation: strategies for successful pregnancy. Nat
8. Nancy P, Erlebacher A. T cell behavior at 15. Xiong S, Sharkey AM, Kennedy PR, et  al.
Med. 2012;18:1754–1767.
the maternal-fetal interface. Int J Dev Biol. Maternal uterine NK cell-activating receptor
2. Pijnenborg R, Vercruysse L, Hanssens M. The
2014;58:189–198. KIR2DS1 enhances placentation. J Clin Invest.
uterine spiral arteries in human pregnancy:
9. Moffett A, Colucci F. Uterine NK cells: active 2013;123:4264–4272.
facts and controversies. Placenta. 2006;27:
regulators at the maternal-fetal interface. J Clin 16. Wittman AB, Wall LL. The evolutionary ori-
939–958.
Invest. 2014;124:1872–1879. gins of obstructed labor: bipedalism, encepha-
3. Burton GJ, Jauniaux E, Watson AL. Maternal
10. King A, Birkby C, Loke YW. Early human lization, and the human obstetric dilemma.
arterial connections to the placental intervil-
decidual cells exhibit NK activity against the Obstet Gynecol Surv. 2007;62:739–748.
lous space during the first trimester of human
K562 cell line but not against first trimester tro- 17. Li C, Houser BL, Nicotra ML, et al. HLA-G
pregnancy: the Boyd collection revisited. Am J
phoblast. Cell Immunol. 1989;118:337–344. homodimer-induced cytokine secretion
Obstet Gynecol. 1999;181:718–724.
11. Moffett A, Hiby SE. How does the maternal through HLA-G receptors on human decidual
4. Moffett-King A. Natural killer cells and preg-
immune system contribute to the development macrophages and natural killer cells. Proc Natl
nancy. Nat Rev Immunol. 2002;2:656–663.
of pre-eclampsia? Placenta. 2007;28(suppl Acad Sci U S A. 2009;106:5767–5772.
5. Gellersen B, Brosens IA, Brosens JJ. Decidu-
A):S51–S56. 18. Ober C, Aldrich C, Rosinsky B, et  al. HLA-
alization of the human endometrium: mecha-
12. Hiby SE, Walker JJ, O’shaughnessy KM, G1 protein expression is not essential for fetal
nisms, functions, and clinical perspectives.
et  al. Combinations of maternal KIR and survival. Placenta. 1998;19:127–132.
Semin Reprod Med. 2007;25:445–453.
fetal HLA-C genes influence the risk of pre- 19. Tilburgs T, Scherjon SA, van der Mast BJ,
6. Pollheimer J, Fock V, Knöfler M. Review:
eclampsia and reproductive success. J Exp Med. et  al. Fetal-maternal HLA-C mismatch is
the ADAM metalloproteinases—novel regu-
2004;200:957–9565. associated with decidual T cell activation and
lators of trophoblast invasion? Placenta.
13. Hiby SE, Apps R, Chazara O, et  al. Mater- induction of functional T regulatory cells. J
2014;35(suppl):S57–S63.
nal KIR in combination with paternal HLA- Reprod Immunol. 2009;82:148–157.
7. Aghababaei M, Beristain AG. The Elsevier Tro-
C2 regulate human birth weight. J Immunol.
phoblast Research Award Lecture: importance
2014;192:5069–5073.

54.e1
7
Development of the Placenta and Its
Circulation
CAROLINE E. DUNK, BERTHOLD HUPPERTZ AND JOHN C. KINGDOM
KEY POINTS
• T he development and structure of the human haemochorial
placenta
• The development of the uteroplacental and fetoplacental
circulations
Introduction
This chapter highlights areas in which developmental placental
biology directly impinges on clinical practice. Pregnancies com-
plicated by the pathologies of stillbirth, severe preeclampsia and
intrauterine growth restriction (IUGR), especially those that
deliver before 34 weeks, are associated with significant gross and
microscopic placental pathology (covered in Chapter 9). Prenatal
diagnosis of pregnancies at risk for these severe complications is
now possible by identifying markers of ‘placental insufficiency’
at the 20-week stage.1 Unfortunately, at the current time, new
biomarkers derived from maternal blood samples do not show
adequate power of prediction in the first or second trimester.2
According to the American College of Obstetricians and Gynecol-
ogists, ‘current predictive tests for preeclampsia may harm more
women than they benefit because of their low positive predictive
value (PPV)’ (https://www.acog.org/Womens-Health/Preeclamp-
sia-and-Hypertension-in-Pregnancy). This situation underscores
the need for a greater understanding of normal placental develop-
ment and thus a better appreciation of the developmental origins
of placental pathology. More detailed information can be found in
a review by Dunk and colleagues.3  trophoblast remains undelivered in the placental bed, normally
The Placenta at Delivery
A full-term human placenta is a disclike, circular organ com-
monly inspected in its ‘inverted’ state from the fetal, or chorionic
plate, surface. The chorionic plate is a fibrous disc into which the
umbilical arteries ramify as chorionic plate vessels, branching in a
dichotomous fashion, each penetrating the plate to enter the stem
(truncus) of a villous tree, and accompanied by a vein draining
oxygenated blood back towards the umbilical vein. The chorionic
plate is covered by the amniotic membrane, which is normally
glossy and can easily be peeled off. At the placental margin, the
chorionic plate thickens to form a ring and continues and fuses
with the basal plate to form the chorion laeve, the fetal mem-
branes. The gross structure of a full-term placenta is illustrated in
cross section in Fig. 7.1.
Macroscopic abnormalities, such as succenturiate lobes, vela-
mentous cord insertion or circumvallate margin (in which the ring
is undergrown by villous trees and the intervillous space and the
membranes insert medial to the edge of the chorionic plate) are
identified from this aspect and occur in approximately 10% of
cases at term.4 In isolation these abnormalities have weak associa-
tions with low birth weight or preeclampsia5 and may therefore be
ignored if other aspects of placentation are normal. Nevertheless,
experience in second trimester ultrasonographic examination of
the placenta is increasing,1 with rare but clinically relevant prob-
lems, such as vasa praevia, being identified by ultrasound resulting
in dramatic improvements in perinatal survival.6
The fetal surface of the placenta may also be inspected at deliv-
ery for evidence of intraamniotic infection, such as cloudy yellow
malodorous discolouration, or chronic staining by meconium,
when clinically relevant. Successful diagnosis of chorioamnion-
itis at delivery is facilitated by collection of fluid or pus (for gas
chromatography), a membrane roll (from rupture point to edge
of placenta) and a sample of umbilical cord into sterile contain-
ers before transport of the placenta to the pathology department.
Cloudy nodular discolourations of the amnion, termed amnion
nodosum, are indicative of prolonged rupture of the membranes
and oligohydramnios.7
The maternal surface of the placenta (referred to as the
basal plate) is an artificial surface in that delivery of the ‘pla-
centa’ requires the organ to be cleaved from the uterine wall
through the basal plate (see Fig. 7.1). In this sense, part of the
regressing over the ensuing weeks. The basal plate is a there-
fore heterogeneous mixture of trophoblastic and decidual cells,
embedded in large amounts of extracellular debris, fibrinoid and
blood clot. The basal surface is divided by a system of grooves
into 10 to 40 elevated areas referred to as the maternal lobes of
the placenta. These correspond approximately to the underlying
arrangement of villous trees with three or four trees per lobe in
the centre; small lobes at the periphery of the placenta are gener-
ally occupied only by a single villous tree. Only the latter cor-
respond to what has been described as a placentome8: a villous
tree together with its surrounding part of the intervillous space
and its corresponding uteroplacental vessels. Terms such as ‘fetal
placenta’ for the chorionic plate (including villous trees) and
55
56 SE C T I O N 2     The Placenta

P M CL A

UC

MZ

CP

IVS

BP
J

• Fig. 7.1  A nearly mature human placenta in situ. On the fetal side, the amnion (A) covers the chorionic
plate (CP), from which the umbilical cord (UC) continues towards the fetus. From the CP, the villous trees
extend into the intervillous space (IVS), which is filled with maternal blood. On the maternal side, the basal
plate (BP) demarcates the placenta from the placental bed, which defines that part of the uterine wall
directly underneath the placenta. Within the placental bed, trophoblast invasion takes place, and transfor-
mation of spiral arteries can be found. At the margin of the placenta, the CP and BP fuse to circumvent
the IVS and to generate the fetal membranes. CL, chorion laeve; J, junctional zone; M, myometrium; MZ,
marginal zone between placenta and fetal membranes, with obliterated intervillous space and ghost villi;
P, serosa; S, septum. (With permission from Kaufmann P, Scheffen I. Placental development. In Neonatal
and Fetal Medicine—Physiology and Pathophysiology, R Pollin, W Fox (eds.). Orlando: WB Saunders,
1992.)

‘maternal placental’ for the basal plate (including uteroplacen-
tal vessels invaded by trophoblast) are tempting from a clinical
perspective, as will be discussed in relation to Doppler studies.9
However, because it is impossible to physically separate the fetal
and maternal cellular constituents, this concept must be viewed
with caution.
Many academic departments take an active interest in placen-
tal genetics, structure and function as they relate to development,
pathology and the new science of perinatal programming. Sam-
pling of the placenta at delivery is thus an increasingly important
task. Because the organ is inherently heterogeneous, a strategy of
systematic random block sampling, used in studies that use stere-
ology methods,10 is preferred. When the focus is on vascular struc-
ture and villous development, the best approach is to immediately
clamp the cord root (to prevent fetoplacental vascular collapse)
and allow the organ to fix for several days in formaldehyde.11
However, because both mRNA’s and proteins are rapidly degraded
after delivery, especially in the metabolically active villous tropho-
blast, when studies at a protein, molecular, or ultrastructural level
are undertaken, a better approach is to open the placenta at ran-
dom sites from the basal plate to excise samples of villous tissue
that can be divided and rapidly frozen (for protein extraction) or
placed into RNA fixative.
Sampling of the placental bed is far more challenging than
that of the delivered placenta. Nevertheless, the introduction of
punch biopsy of the placental bed now permits several samples to
be taken, including samples from women after vaginal deliveries
and earlier gestation miscarriages.12 
Haemochorial Placental Blood Flow
The human placenta is termed haemochorial because it provides
direct contact between maternal blood and the chorionic (fetal)
villi in the intervillous space (see Fig. 7.1). Maternal blood leaves
the openings of the transformed spiral arteries and circulates
around the villi. Some villi anchor the villous trees to the basal
plate, but the bulk of the term placenta comprises trees of gas-
exchanging terminal villi floating in maternal blood. The classic
injection studies of spiral arteries by Wigglesworth indicated that
a majority of the 60 to 70 villous trees at term, branching from
the chorionic plate, are maternally perfused from their centres,
thus creating hollow-centred structures.13 This concept was sub-
stantiated by Schuhmann’s description of the 50 to 100 maternal
arterial inlets as being located near the centres of the villous trees.8
The 50 to 200 maternal venous outlets per placenta at term are
thought to be arranged around the periphery of the villous trees
 CHAPTER 7  Development of the Placenta and Its Circulation
such that each villous tree is perfused in a centrifugal manner.
The radioangiographic studies conducted in humans14 support
the placentome concept which can now be imaged using colour-
flow Doppler imaging to study intraplacental blood flow relation-
ships.15 The maternal arterial jet flows rapidly upwards through
the central cavity, dispersing in a centrifugal manner to perfuse
the surrounding well developed and more densely packed villi of
mature intermediate and terminal type (see later).16 Short-term
reduced intervillous perfusion of a placentome results in tempo-
rary hypoxia because fetal perfusion continues to remove oxygen
bound to fetal haemoglobin. In response, the stem villous arterioles
constrict to reduce the rate of removal of oxygen until intervillous
oxygen tension equilibrates again. In this way, the individual plac-
entomes self-regulate to maximise maternal–fetal exchange. Per-
sistent lack of intervillous perfusion can cause stem villous arterial
constriction or thrombosis.17 However, the central portions of the
placenta have the largest placentomes and the most-transformed
spiral arteries, so central vascular pathology is therefore rare in
normal pregnancy. By contrast, spiral artery thrombosis and vil-
lous infarction are often found at the margins of the placental disc
after a term delivery, with no apparent ill effects.
It is the uniquely invasive properties of the extravillous tro-
phoblast (EVT) that transform the uterine spiral arteries, which
result in the haemochorial arrangement that characterises human
placentation. The process of invasion is precisely controlled, and
it is thought that the disordered EVT proliferation and migration,
in combination with the placental vascular pathology, relates to
major clinical conditions such as preeclampsia and IUGR (inad-
equate invasion) or placenta percreta (excessive invasion) and is
discussed in detail in Chapter 9. Please see the online video files
associated with Chapter 9 for more information.  villi provide the cellular sources for this invasive process. The cell
Early Stages of Placental Development
Placental development begins with blastocyst attachment to the
uterine wall. At this stage, the first extraembryonic cell lineage dif-
ferentiates and is termed the trophoblast. Signals from the embryo
(inner cell mass), including fibroblast growth factor 4 (FGF4),
expand the population of trophoblast stem (TS) cells that are
capable of differentiating along both the extravillous and villous
pathways of development. Considerable knowledge of trophoblast
differentiation has been obtained using transgenic mice and mouse
TS cells and is summarised in Knott and Paul 2004.18 Blastocyst
symmetry is directed by the inner cell mass because only those
trophoblast cells overlying the inner cell mass make direct contact
with the uterine epithelium (Fig. 7.2A). Abnormal orientation of
these structures likely causes abnormalities in the site of umbilical
cord insertion into the placental disc and are more common in
pregnancies arising from in vitro fertilisation (IVF).19
The prelacunar stage of development (Fig. 7.2B) is character-
ised by the formation of an outer shell of syncytiotrophoblast that
is distinct from the later villous syncytiotrophoblast by being able
to penetrate the uterine epithelium and embed the conceptus in
the uterine stroma. The more proximal trophoblast cell popula-
tion is referred to as cytotrophoblast and is positioned between the
syncytiotrophoblast and embryoblast. The cytotrophoblast layer is
assumed to be a multipotent stem cell population capable of sub-
sequently producing each type of trophoblast, as in mice. Around
day 14 postconception, the conceptus is fully embedded within
uterine tissues, and the syncytiotrophoblast starts to develop fluid-
filled spaces termed lacunae (Fig. 7.2C). The lacunae gradually
coalesce to form one large intervillous space of the placenta. This
57
lacunar stage results in the formation of syncytiotrophoblast col-
umns, referred to as trabeculae, which reach from the embryonic
side of the placenta to the maternal decidual tissues.7 The devel-
opment of the lacunae and the establishment of the intervillous
space compartmentalise the growing placenta as follows:
• The site of attachment, comprising anchoring villi and the
basal plate
• The lacunar spaces, forming the intervillous space
• Branches that derive from the trabeculae develop into floating
villi
• The embryonic side develops into the chorionic plate
Further invasion of the maternal tissues by placental tropho-
blast cells is necessary so as to transform the spiral arteries of the
uteroplacental circulation as well as the uterine glands. The first
step is the streaming of cytotrophoblast cells down the centre
of the syncytiotrophoblast trabeculae, creating a new lineage of
extravillous cytotrophoblast that is in direct contact with mater-
nal tissues beyond the initial wave of syncytiotrophoblast invasion
(Fig. 7.2D–F). Cytotrophoblast at the tips of the trabeculae, their
maternal ends now referred to as anchoring villi, form trophoblast
cell columns. The proximal cells proliferate as the source of all
subsequent subtypes of invasive EVT cells. Initially, the invasion
of maternal decidual tissues by cytotrophoblast begins in the con-
nective tissue (interstitial EVT) followed by the walls of the spiral
arteries and uterine veins (endovascular trophoblast cells)20a,b or
the epithelium of uterine glands.21
Trophoblast invasion into maternal tissues is not restricted to
the process of implantation and early placentation but is a con-
tinuous process throughout pregnancy, serving a number of pur-
poses. The trophoblastic cell columns at the base of the anchoring
columns do not contain stroma because they were not excavated
by mesenchyme during formation of the villous trees. The cyto-
trophoblast cells composing these cell columns, together with tro-
phoblast cells in the placental bed, basal plate, chorionic plate and
membranes, are collectively described as EVT cells.20a,b
The trophoblastic cell columns resting on the basal plate
should be viewed as a rapidly proliferating zone from which
EVT cells migrate continuously into maternal tissues (Fig.
7.3). However, as soon as the EVT cells leave this proliferative
zone, they leave the cell division cycle and change to an inva-
sive phenotype. Their pattern of integrin expression, secretion
of proteolytic enzymes and production of extracellular matrix
(ECM) proteins is strikingly similar to that of malignant
tumour cells.20a,b Fortunately, they differ in one fundamental
aspect: they do not proliferate during invasion. This is one of
the reasons why the depth of invasion into maternal tissues
is limited. If deportation into the maternal circulation occurs,
metastatic growth is impossible because these cells have lost
their generative potency.
Invasion by EVT serves three very different purposes, namely
adhesion of the placenta to the uterine wall, adaptation of uterine
glands to enable histotrophic nutrition during the first trimester
of pregnancy and adaptation of uteroplacental arteries and veins,
enabling haemochorial nutrition to meet the fetal requirements
later in gestation.20b 
Phenotypes of Extravillous Trophoblast
Among the invasive trophoblast cells, a subset of cells secretes huge
amounts of ECM (composed of laminins, collagen IV, fibronectins,
vitronectin and heparan sulphate) known as matrix-type fibrinoid22
58 SE C T I O N 2     The Placenta

Blastocyst
Embryoblast
Cytotrophoblast Uterine epithelium
Syncytiotrophoblast

Uterine decidua

Endometrial capillaries
and glands
A

C
uE E Me

S
L

D
B C

Me Me
C

C
Me C Me C
I III
II
IVS IVS IVS
S
S
S
D
EVT D D
EVT EVT
D E F
• Fig. 7.2  Early placental development. A, The blastocyst is covered by a monolayer of trophoblast cells
that encircle a fluid-filled cavity, the blastocoel, and the developing embryoblast. A crucial developmental
prerequisite for implantation is a next differentiation step of those trophoblast cells in direct contact to the
uterine epithelium. Only syncytial fusion of these cells to develop a first syncytiotrophoblast enables the
blastocyst to penetrate the uterine epithelium and to further implant into the maternal uterine tissues.  B,
During the prelacunar stage, the syncytiotrophoblast (S) has penetrated the uterine epithelium (uE) and
has reached the decidua (D), continuing the direct contact to maternal cells. The layer of cytotrophoblast
cells (C) does not have direct contact to maternal cells but lies in second row between syncytiotropho-
blast and embryo (E).  C, During the lacunar stage, first fluid-filled spaces, lacunae (L) appear within the
mass of the syncytiotrophoblast, grow and flow together to finally generate one large fluid-filled space, the
intervillous space. Between embryo and cytotrophoblast, extraembryonic mesenchyme (Me) has spread
out.  D–F, Villous stages. The further development of the placenta is shown by the black rectangle in C.
The cytotrophoblast (C) begins to penetrate into the syncytiotrophoblast (S) and reaches the opposite side
of the placenta and thus reaches maternal tissues of the decidua (D). Cytotrophoblast cells that leave the
proper placenta are termed extravillous trophoblast (EVT). Within the placenta first sprouting of the syncy-
tiotrophoblast can be found (D), protruding towards the intervillous space (IVS). This is the development
of the first primary villi (I). A few days later, the extraembryonic mesenchyme (Me) starts to penetrate the
syncytiotrophoblast as well and displaces the cytotrophoblast from the core of the trabeculae and primary
villi. This leads to the development of the secondary villi (II) (E). Slightly later, first blood vessels develop
within the placental mesenchyme and lead to the formation of tertiary villi (III) (F).

into which they are embedded. The EVT cells adhere to the ECM
via the surface expression of molecules known as integrins. Like-
wise, the endometrial stromal cells adhere via similar mechanisms;
root-like projections of EVT, together with its associated matrix,
penetrate into the inner third of the myometrium, thereby ensuring
anchoring of the placenta. Adhesion of the placenta by the gluelike
ECM depends on the viability of the EVT cells expressing integrins;
in this sense, it is reversible at delivery. This anchoring process is
essential; otherwise, entry of maternal blood into the intervillous
space at high velocity would shear off the whole placenta.
 CHAPTER 7  Development of the Placenta and Its Circulation
Anchoring villus
∗ ran sulphate and vitronectin, and (iii) fibronectins and fibrillin
∗ ∗ ∗ ∗ ∗ ∗
Proliferating ∗
extravillous ∗ ∗
trophoblast
∗
Vascular
Invasive
trophoblast
extravillous
trophoblast
Decidua
• Fig. 7.3  Trophoblastic cell column. Schematic drawing of a trophoblastic
cell column connecting a villous stem (above) to the basal plate (below).
Proliferating stem cells (asterisk) of the extravillous trophoblast. The arrow
symbolises the invasive pathway. Fibrin-type fibrinoid (line shading). Extra-
cellular matrix (light point shading).
Along the invasive pathway through the decidual intersti-
tium, EVT cells show morphologically and functionally differ-
ent phenotypes. These different phenotypes display a varying
behaviour regarding contact to maternal cells, secretion of
matrix and invasiveness. At present, the molecular basis of
these different phenotypes is not known, although it has been
described in the related mouse placenta.23 Such knowledge
may in the future help understanding of the pathways that pre-
vent this physiologic process from occurring in certain specific
diseases, such as abruption (premature separation), preeclamp-
sia and IUGR.
In a normal intrauterine pregnancy, EVT cells that are invad-
ing the decidual interstitium can be subdivided into three mor-
phologically and functionally different subtypes.
Large Polygonal Cells
This subtype of large polygonal EVT cells corresponds to the
well described former X-cells.7 Compared with the subset of
small spindle-shaped cells, the relative number of this subtype
increases from 45% at weeks 9 to 12 to 69% at weeks 16 to 24
and makes up about 89% in weeks 31 to 39.24 Hence, this sub-
type is the prevailing phenotype of EVT at the time of delivery.
These cells are evenly distributed throughout the basal plate as
well as along the route of invasion reaching the inner third of
the myometrium. Morphologically, these cells are large, polygo-
nal, uninucleated EVT cells displaying big, irregularly shaped,
intensely staining nuclei. No other subtype of all trophoblast
cells displays a stronger immunoreactivity for cytokeratin 7 than
59
these large polygonal cells. At the same time, they are always
immunonegative for proliferation markers such as anti-Ki 67.
These cells secrete the typical ECM of EVT, the matrix-type
fibrinoid.25 This basement membrane-like ECM comprises
three different patches of matrix molecules: (i) collagen IV and
laminin, (ii) an amorphous ground substance containing hepa-
embedded in the same amorphous ground substance.25,26 The
large cells fix themselves within their self-secreted matrix by
expression and exposition of the respective integrins, such as
α5/β1, α1/β1 and α-v/β3/5 integrins, reviewed by Harris and
colleagues.27 These cells organise themselves in clusters, which
are usually void of maternal tissue components but filled with
matrix-type fibrinoid. These features do not support the classical
thinking that the large polygonal cells (‘X-cells’) are highly inva-
sive cells. Rather, it appears as if this subtype of interstitial EVT
may have a function in fixing and adhering the placenta to the
uterine wall by secreting matrix-type fibrinoid, which has been
termed the ‘trophoblast glue’.28 
Small Spindle-Shaped Extravillous
Trophoblast Cells
This subtype of the interstitial trophoblast is also negative for
proliferation markers and only moderately immunoreactive for
cytokeratin 7 and can be found from the transitional zone of
trophoblastic cell columns reaching the inner third of the myo-
metrium. Hence, this subtype shares the spatial distribution
pattern with the subtype of the large polygonal cells. Oppos-
ing the large polygonal subtype, this small subtype displays a
decrease in numbers towards term from 55% in the first trimes-
ter to 31% in the second trimester reaching only about 11% at
term.24 Structurally, this subtype is characterised by elongated,
partly filiform cell bodies mostly oriented radially to the uterine
wall containing small ovoid nuclei. This small subtype usually
forms loosely arranged arrays of cells, surrounded by only very
little matrix.
There are only a few descriptions of these small spindle-
shaped trophoblast cells so far,24 which may be because these
small, usually filiform cells easily escape the investigators’ atten-
tion, the reason being that they are rarely represented in one
section in full length. The small cells only secrete little amounts
of self-secreted matrix, which is mostly composed of cellular
and oncofetal isoforms of fibronectin.25 At the same time, these
cells only express ‘interstitial’ integrins such as α5/β1 and α-v-
integrins. The predominant expression of ‘interstitial’ integrins
in combination with the expression of oncofetal fibronectins is
an essential mechanism of trophoblast invasiveness.25 Interac-
tions between α5/β1 integrins and fibronectins are crucial for
trophoblast invasion.27
In the past decade, an integrin switch has been described dur-
ing the course of trophoblast invasion.27 In light of the data pre-
sented, this phenomenon could be explained as follows: The small
subtype of cells expresses only ‘interstitial’ integrins and prevails
in deeper zones of the invasive pathway because this phenotype is
really invasive. The large subtype additionally expresses ‘epithelial’
integrins. Therefore the integrin switch may relate to the termi-
nal differentiation of the motile spindle-shaped form to the non-
motile polygonal trophoblast cells. This process occurs over the
course of gestation and may account for the increasing percent-
age of polygonal trophoblast cells and corresponding decrease in
spindle-shaped trophoblast cells. 
60 SE C T I O N 2     The Placenta
Multinucleated Giant Cells
This subtype prevails in the depth of the placental bed at the border
between decidua and myometrium and does not show any prolif-
erative activity. A distinct difference to the earlier mentioned sub-
types is that these cells contain more than 1 and up to 10 irregularly
shaped nuclei of varying size, leading to a much larger volume with
a diameter ranging between 50 and 100 μm.24 These multinucle-
ated cells are either immunonegative for cytokeratin 7 or show few
spots of reactivity and thus may easily be missed during superficial
inspection of immunohistochemical sections of the placental bed.
Although a fusion event of such cells has never been observed, it is
generally accepted that fusion of interstitial EVT cells does occur.  trophoblast initially comes into contact with the superficial cap-
Regulation of Trophoblast Invasion
The invasive EVT cells finally reach the inner third of the myo-
metrium, where they can be found between the layers of smooth
muscle cells. Arrest at this stage is crucial so that pathological pla-
cental invasion does not occur. Some insight into the mechanisms
that permit further trophoblast invasion into the uterus has come
from studies in placenta accreta specimens.29
Hypotheses Favouring Extrinsic Factors:
• T rigger gradient: EVT cells need a trigger to be invasive. This
trigger is derived from the mesenchymal stroma of anchoring
villi. Cell invasion is therefore limited by a requirement for this
diffusible factor.
• Cellular interaction: Cells within the myometrium (smooth
muscle cells, specific immune cells) or endometrium may arrest
invasion.  • Reduction of the amount of free radicals to protect the embryo
Hypotheses Favouring Intrinsic Factors and
Programs:
• A poptosis: Programmed cell death occurs in EVT cells.30,31 The
rates of apoptosis differ significantly among studies because of
technical and sampling limitations. Therefore at present, the
role of apoptosis as a critical regulator of trophoblast invasion
is uncertain.
• Polyploidisation: The subtype of large polygonal trophoblast
cells is probably differentiated from the small spindle-shaped
cells by polyploidisation.32 This leads to the noninvasive phe-
notype that secretes ample matrix and anchors the placenta to
its implantation site.
• Syncytial fusion: It is generally accepted that the multinucle-
ated giant cells derive from syncytial fusion of their mono-
nucleated counterparts.7 The noninvasive multinucleated cells
accumulate close to the border between decidua and myome-
trium and may act as a trap for the trophoblast cells that try
to trespass into deeper layers. Cell–cell fusion likely occurs via
expression and interaction of the fusogenic proteins syncytin
with its receptor ASCT-2 and connexin 43.33 These terminally
differentiated multinucleated structures may participate in the
maternal recognition and adaptation to pregnancy.
The invasive nature of haemochorial placentation is pre-
cisely regulated under normal circumstances; when it occurs to
excess, EVT cells may invade deeply into (accreta) or through
(percreta) the uterine wall, such that physiological separation of
the placenta can no longer occur. Invasive placentation is more
common in a scarred uterus from multiple previous caesarean
sections34 and in pregnancies after successful endoscopic surgery
for Asherman syndrome (uterine adhesion from patchy loss of
endometrium).35
Conversely, a failure of this invasive anchoring process predis-
poses to premature placental separation (abruption), either during
the antepartum period or during labour. These important conditions
underscore the importance of understanding how EVT invasion and
production of an adhesive matrix is disturbed in these conditions. 
Placental Perfusion During Embryogenesis
During implantation, the expanding and invading early syncytio-
illary system of the decidua underneath the uterine epithelium.
Capillaries may leak maternal erythrocytes into the lacunar spaces
of the placenta, resulting in the presence of a few erythrocytes in
the early intervillous space. However, the predominant observa-
tion is that the stromal spiral arteries and arterioles are blocked by
EVT, and no arterial connections are made between the maternal
circulation and the primitive intervillous space.36 This apparent
lack of intervillous blood perfusion during the first trimester of
pregnancy has been shown by transcervical endoscopic observa-
tions37 and Doppler ultrasound38 of the intervillous space. Dur-
ing embryogenesis, the intervillous space is filled with a clear
fluid known as uterine milk comprising filtered maternal plasma
and uterine gland secretion products rich in lipids, nutrients and
growth factors crucial for placental and embryonic development.39
Occlusion of maternal arteries during implantation is designed
to facilitate embryogenesis in a low-oxygen environment below
20 mm Hg until week 10 of gestation40 and has the following
advantages41:
from teratogenesis during this critical phase of tissue and organ
development.
• Mammalian cells grow much faster under low oxygen com-
pared with higher oxygen tensions. Embryo development is
characterised by rapid cell division, and thus low oxygen is
ideal to generate an environment to achieve and maintain a
high level of cell divisions.
• Connection between placental and embryonic vessels is not
fully established before week 7 of gestation. Hence, there is no
need to perfuse the placenta with maternal blood to feed the
embryo before this time.
• Perfusion of maternal plasma without blood cells may protect
the early villous syncytiotrophoblast from direct contact with
circulating maternal immune cells. 
Transformation of the Uteroplacental
Arteries
Starting from the interstitial route of invasion, a subset of EVT
cells penetrates the walls of uterine veins and spiral arteries.20b
This subset of invasive EVT cells is termed the endovascular tro-
phoblast. The endovascular trophoblast cells intravasate from the
decidual interstitium into the lumen of the arteries, where they
may migrate inside the arterial lumen along the arterial wall and
later may even extravasate to focally reenter the walls of the spi-
ral arteries.7 The major role of the endovascular trophoblast is to
transform the distal spiral arteries into dilated segments that facili-
tate increased uteroplacental blood flow. The transformation of
spiral arteries is divided into three stages (Fig. 7.4).
 CHAPTER 7  Development of the Placenta and Its Circulation 61

1
5
2
8 6

7
B C

12

9 11

10

D E
• Fig. 7.4  Transformation of spiral arteries. A, An untransformed endometrial spiral artery. B, First trans-
formation of spiral arteries early during pregnancy in the absence of trophoblast invasion. Decidual natural
killer cells (1) and macrophages (2) accumulate around and penetrate the arterial wall. The muscle within
the arterial wall is reduced, and a first widening of the lumen can be seen. These events are preparative
steps towards trophoblast infiltration of the arterial walls.  C, Beginning during the third week of preg-
nancy, invasion of extravillous trophoblast cells starts. At the lower tip of anchoring villi (3), trophoblastic
cell columns form (4). These columns are the source of interstitial trophoblast cells (5) that migrate into
the connective tissues of the decidua (6). Some trophoblast cells reach the upper third of the myometrium
(7), and others take a side route and penetrate the spiral arteries (8).  D, The trophoblast cells that have
reached the spiral arteries are termed endovascular trophoblast (9), which can be found in the wall as
well as in the lumen of spiral arteries. Within the lumen, they build plugs (10) that during the first trimester
hinder maternal blood cells to flow into the intervillous space.  E, Only around the end of the first trimester
do the trophoblastic plugs disintegrate. Now maternal blood containing blood cells flow through the maxi-
mally enlarged uteroplacental arteries (11) and reach the intervillous space (12) of the placenta. The endo-
vascular trophoblast relines the transformed artery, and the immune cells move back into the decidua.

1. Maternal factors induce the first changes of uterine spiral arter-
ies (Fig. 7.4A) very early during pregnancy, still in the absence
of any invading trophoblast in their vicinity, the vessels begin
to dilate via a disorganisation of vascular smooth muscle cells
and altered endothelial cell morphology. Recently, a number
of studies have shown that the maternal immune cells of the
decidua are responsible for these changes. They accumulate
around the decidual arterioles and play an active role in the
early stages of transformation of these vessels through the
secretion of growth factors and matrix metalloproteinases to
degrade the ECM (Fig. 7.4B).42-44
2. At the same time, the EVT begins to separate from the anchor-
ing cell column and invade the decidua interstitially. There is
some evidence to suggest that the interstitial EVT can pen-
etrate the maternal vasculature and contribute to the degra-
dation of the vessel wall (Fig. 7.4C). Where anchoring cell
columns form in the vicinity of a decidual vessel, endovascu-
lar EVT cells accumulate in a plug in the proximal portion of
62 SE C T I O N 2     The Placenta

the decidual arteries and prevent early entry of maternal blood
(Fig. 7.4D).45-47
3. Endovascular trophoblast then migrates down the lumen of the
vessel, relining the wall as they go, resulting in remodelling of the
uteroplacental arteries (Fig. 7.4E). As they reline the vessel, they
begin to express endothelial markers such as platelet endothelial
cell adhesion molecule-1 (PECAM-1) and α-vβ3 integrin.48
This further dilation of the arteries results in lumen diameters
several times greater than the original. The reduced activity of
smooth muscle cells and the loss of elastic fibres are clear indica-
tions of the disruptive properties of the decidual leukocytes and
endovascular trophoblast in the vessel media.
The number of uteroplacental spiral arteries supplying the inter-
villous space reduces as pregnancy advances because of vascular
obliteration, such that by term, the intervillous space is perfused by
approximately 10 spiral arteries49 However, intervillous blood flow
increases considerably because of these physiological changes in the
uteroplacental arteries, including loss of autonomic innervation.50
The dilated and denervated uteroplacental arteries are released from
autonomic and local vasomotor influences such that intervillous per-
fusion is regulated by systemic arterial pressure. Differential regula-
tion of uteroplacental blood flow by the mother is no longer possible,
so hypotension, such as after the insertion of an epidural anaesthetic
during labour, may cause temporary fetal distress because of a fall in
uteroplacental perfusion, which is then corrected by intravenous crys-
talloid volume expansion and ephedrine. The uteroplacental veins,
approximately 50 to 200 in number, drain the intervillous space from
the margins of the fetal cotyledons, thus surrounding the basal parts of
intervillous space of the placenta (Fig. 7.4E). Recent novel work in
both primates and humans using microbubble contrast-enhanced
ultrasound has demonstrated the onset of blood flow into the
intervillous space at around 11 weeks of gestation.56
As detailed, there is a gradient in trophoblast development dur-
ing the first trimester of pregnancy with the most advanced stages
in the centre below the embryo and the less advanced stages in the
periphery underlying the uterine epithelium (Fig. 7.5).41 Simi-
larly, the plugging of the spiral arteries follows this gradient with
the deepest invasion and the highest number of EVT plugs found
in the centre of the placental bed. The low number of invading
cells in the peripheral part of the placenta together with the super-
ficial invasion of spiral arteries enables maternal blood cells to
override the outer trophoblastic plugs first, even well before 10
weeks of gestation. The inflow of maternal blood and blood cells
at this time results in enormous oxidative stress in these peripheral
parts of the placenta, leading to damage of villous trophoblast.
Intervillous
Villous space
Amnion
Chorion Uterine
trees epithelium
Amniotic
Decidua cavity
Embryo
Spiral artery

the villous trees. In contrast to the arteries, the veins are only invaded
by EVT but not transformed20b and obviously are not involved in the Umbilical
regulation of the intervillous blood flow.7 Placenta cord
The virtual absence of endovascular trophoblast invasion char-
acterises the placental bed in chromosomally normal late first
trimester miscarriages.12 Focal inadequate occlusion of invaded
endometrial stromal vessels by EVT results in a premature entry
of oxygenated maternal blood into the intervillous space, arrest-
ing trophoblast proliferation, villous angiogenesis and therefore
overall development of the chorioallantoic placenta. This is rec- Trophoblastic cell Interstitial extravillous Endovascular extravillous
ognised clinically by transvaginal ultrasound51 and by low levels columns trophoblast trophoblast
of trophoblast-derived pregnancy-associated placental protein A
A (PAPP-A) in maternal blood.52 By contrast, no abnormalities
of trophoblast invasion are found in early first trimester losses.53
Less severe reductions in trophoblast invasion characterise IUGR
and early onset preeclamptic pregnancies31,54 and are discussed
in detail in Chapter 9. Please see the online video files associated
with Chapter 9 for more information. 

Flow of Maternal Blood into the

Intervillous Space
During the course of the first trimester, EVT generated from
the anchoring cell columns penetrates the decidua and comes
into contact with the uterine spiral arteries to begin the process
of physiological transformation of these vessels. The EVT reor-
ganises the vessel walls and aggregates in the lumen of these ves-
sels, resulting in the plugging of the distal portions of the vessels
(Fig. 7.4D).55 Hence, there is no free communication of maternal
blood cells between these eroded vessels and the intervillous space
of the placenta until the end of the first trimester. Only then do
the intraarterial plugs of trophoblast become permeable and begin
to dislocate. This finally enables maternal blood cells to enter the
B
• Fig. 7.5  Onset of placental perfusion. Placental blood flow in the first
trimester. At this time, the spiral arterioles beneath the definitive placenta
are occluded by endovascular trophoblast (A). As a result, maternal blood
bypasses the intervillous space but can be seen to flow in the myometrium
using colour Doppler ultrasound (asterisk) (B). Note that the fetoplacental
circulation is established at this time and is detected by colour flow in the
umbilical cord (+).
 CHAPTER 7  Development of the Placenta and Its Circulation
Subsequently, degeneration of villi can be observed at these sites.
Thus in the peripheral parts of the placenta below the uterine epi-
thelium, villi regress, resulting in a secondary smoothing of the
chorion and hence the development of the chorion laeve, the fetal
membranes (reviewed by Burton and Jauniaux57). The reorganisa-
tion of villous tissues within the placenta at the end of the first
trimester results in the definitive disc-shaped placenta with the
central and remaining part of the villous tissues developing into
the chorion frondosum.
The temporal difference between the two onsets of blood flow
into the different parts of the placenta may play an essential role
in the generation of the decisive shape of the human placenta.
Before 10 weeks of gestation, villi regress with increasing oxygen
concentrations, and the same phenomenon between weeks 10 and
12 (increasing oxygen concentrations) provides a stimulus for tro-
phoblast differentiation. It has to be stressed, however, that also
at that time, oxidative stress within the placenta occurs, partly
resulting in damage of the syncytiotrophoblast.58 Premature entry
of maternal blood into the intervillous space can be detected by
colour Doppler ultrasound and is observed in nonviable pregnan-
cies destined for miscarriage.57 Although many miscarriages are
aneuploid, this phenomenon is seen in euploid miscarriages and
may therefore represent early pregnancy failure because of a lack
of adequate initial endovascular occlusion of stromal blood ves-
sels. Nonlethal variants of this may result in villous obliteration
and ‘chorion regression syndrome’ whereby severe preterm IUGR
placentas are at the less than 10th centile for weight and have
eccentric cord insertions. This pathology of pregnancy is discussed
in detail in Chapter 9.
The removal of the centrally located plugs within the spiral
arteries is achieved only after the flow of maternal blood in the
peripheral parts of the placenta has been established. With the
onset of maternal blood flow into the placenta, a direct contact
between maternal blood cells and the fetal syncytiotrophoblast is
established, resulting in a more than threefold increase in intra-
placental oxygen concentrations, from less than 20 mm Hg to
about 60 mm Hg.58 EVT-mediated transformation of the mater-
nal spiral arteries is largely complete by 16 to 18 weeks of gesta-
tion, but some modifications may continue to term. The EVT
penetrates and transforms the uterine spiral arteries as far as the
first third of the myometrium. Modelling of the vessel rheology
at term has shown that this widening of the vessel is associated
with a 5- to 10-fold dilation of the vessel mouth and a slowing
of the blood flow from a speed of 2 to 3m/s to 10 cm/s.59 This
low-speed, low-pressure uteroplacental circulation facilitates the
efficient exchange of oxygen and carbon dioxide and nutrients and
waste between the maternal and fetal circulations.
Failure of uterine spiral artery transformation detected by per-
sistent high-impedance uterine artery waveforms with preserva-
tion of the early diastolic notch on the placental side of the uterus
is associated with placental complications of pregnancy such as
IUGR, preeclampsia and abruption.60,61 It is suggested that these
pathologies may be caused by the profound consequences of this
failure on the blood flow into the intervillous space. Modelling
suggests that in a nontransformed vessel blood will enter the inter-
villous space in pulsatile jets at speed of 1 to 2 m/s, resulting in
damage to the placental villi and formation of echogenic cysts and
ischemia reperfusion of the intervillous space.59 A recent three-
dimensional power Doppler case control study has shown that
Doppler indices between 11 and 14 weeks of gestation reveal a
significantly lower placental VI, placental VFI, subplacental VI
and subplacental VFI in the first trimester in women who went
63
on to develop severe preeclampsia than control participants.62 The
noninvasive Doppler studies are supported by earlier histologic
studies demonstrating incomplete trophoblast invasion of spiral
arteries in these conditions.63,64 Please see the online video files
associated with Chapter 9 for more information. 
Development of Placental Villi
At about day 14 postconception, cytotrophoblast cells within the
syncytial trabeculae begin to proliferate and push the syncytial
layer to generate protrusions that bulge into the intervillous space.
These pure trophoblastic structures are termed primary villi, which
are composed of an outer syncytial layer and a core filled with
mononucleated cytotrophoblast cells (Fig. 7.2D). Shortly after,
the mesenchymal cells from the extraembryonic mesoderm follow
the cytotrophoblast cells and migrate into the syncytial trabeculae,
this time displacing the cytotrophoblast cells from the core of the
trabeculae. The mesenchymal cells follow the cytotrophoblast into
the primary villi, fill them with a first connective tissue and hence
generate secondary villi (Fig. 7.2E). At around day 20 postcon-
ception, first primitive blood vessels and haemangioblastic stem
cells develop within the mesenchyme, generating tertiary villi (Fig.
7.2F).65 The development of the placental vascular bed is inde-
pendent of that of the embryo, and both vascular systems connect
to establish a fully embryoplacental circulation at around day 35
postconception.7 During the first trimester, haematopoiesis can be
observed within newly formed placental capillaries; thereafter, the
fetal liver takes over.66 By 12 to 13 weeks, most villi have trans-
formed to tertiary vascularised villi through an intense period of
branching angiogenesis. This is driven in part by the intraplacental
low oxygen tension caused by effective occlusion of the decidual
stromal blood vessels over an area of the uterine wall that is des-
tined to become the definitive placenta (see Fig. 7.5). 
Architecture of the Villous Trees
Placental villi are composed of two compartments, a superficial
layer of villous trophoblast comprising cytotrophoblast covered by
continuous syncytiotrophoblast, and the villous core, comprising
stroma and fetally derived blood vessels.7 Syncytiotrophoblast is
generated by syncytial fusion of a subset of villous cytotrophoblast
(Langhans cells). Cytotrophoblast numbers constantly increase
throughout the second and third trimesters as does the volume
of syncytiotrophoblast, to cover the exponentially growing spe-
cialised villous cores. Consequently, the cytotrophoblast popu-
lation becomes dispersed, and the syncytiotrophoblast becomes
thinner.67 Mitosis is confined to the cytotrophoblast layer, and
these cells are analysed for the preliminary karyotype by chorionic
villous sampling. Determination of fetal karyotype by culture of
villous explants depends on the slower multiplication of villous
stromal cells; because these are derived from embryonic (allantoic)
tissues, they more accurately reflect the karyotype in fetal tissues.68
From a functional perspective, fusion of newly formed cyto-
trophoblast into the outer syncytium results in the transfer of
fresh organelles, enzyme systems and messenger RNA transcripts
into the syncytium as well as the cytotrophoblast nuclei and is
essential to sustain the intense metabolic activity required for
maternofetal transfer processes and secretory and endocrine
functions. As a result of this continuous fusion, syncytial nuclei
are of different ages and display a variety of morphologies and
more condensed chromatin, suggesting that they are not tran-
scriptionally active. However, recent research has shown that a
64 SE C T I O N 2     The Placenta
significant proportion of the syncytial nuclei are themselves tran-
scriptionally active, expressing both RNA Pol I and II, and that
their numbers increase in proportion to the increasing tropho-
blast volume.69 Aged syncytial nuclei cluster together, known
as ‘syncytial knots’, and protrude into the intervillous space and
may break away; the resulting syncytial globules are deported
in the maternal blood, most becoming lodged in the pulmo-
nary capillary bed.7 True syncytial knots must be distinguished
from syncytial sprouts and false knots caused by sectioning arte-
facts; true knots do not contain transcriptionally active nuclei
and do contain effete damaged nuclei that stain positively for
8-oxo-deoxyguanosine70 (Fig. 7.6). As gestation advances, this
process of rejuvenation appears to slow down because the ratio
of cytotrophoblast to syncytial nuclei within individual terminal
villi decreases as syncytial knots become more common. However,
stereological analysis of the total trophoblast number shows that
the ratio between cytotrophoblast nuclei and syncytiotropho-
blast nuclei remains mostly constant throughout gestation with
a value of 9 at 13 to 16 weeks and again a value of 9 at 37 to 41
weeks of gestation.67
Because syncytial knots are engulfed by lung macrophages,71
they are found in uterine venous, but not arterial, blood of
pregnant women.72 Syncytial shedding and villous trophoblast
37 wk
vs vs
A B IVS
39 wk
IVS IVS
vs vs
C D
19 wk
vs
IVS
• Fig. 7.6  Identification of true syncytial knots and sprouts. False knots,
as identified by serial sectioning, contain a heterogeneous population of
8OHdG-positive and 8OHdG-negative nuclei (A and B). True knots con-
tained a high proportion of condensed nuclei staining intensely for 8OHdG,
which also display a condensed morphology (C and D). Sprouts contain
mostly 8OHdG-negative nuclei (E). IVS, Intervillous space; VS, villous
stroma. Scale bar = 20 μm. (With permission from Fogarty NM, Fergu-
son-Smith AC, Burton GJ. Syncytial knots (Tenney-Parker changes) in the
human placenta: evidence of loss of transcriptional activity and oxidative
damage. Am J Pathol 183(1):144–152, 2013.)
morphology are abnormal in severe forms of IUGR and pre-
eclampsia and are discussed in Chapter 9.
The fetal vessels within the stem villi comprise muscularised
arteries and veins. These lead into the elongated capillaries of the
mature intermediate and terminal villi, the latter providing a sur-
face for gas exchange thought to exceed 10 m2.7 The endothelium
of the fetal capillaries acts as a passive filter, limiting macromo-
lecular transfer across the vessel wall to molecules below 20 000
Da, depending on molecular charge.73 The contractile cells that
surround the walls of the arteries and arterioles of the stem villi
are of great interest clinically because reduced fetoplacental perfu-
sion, detected by Doppler ultrasound examination of the umbili-
cal arteries in vivo, is associated with poor fetal growth, fetal death
and perinatal loss.74 Because these vessels have no autonomic
innervation, blood flow must be regulated by local and systemic
vasomotor factors, together with the anatomical arrangements
and fetal cardiac output.75,76
Connective tissue cells within the stroma are heterogeneous in
nature, producing various connective tissue fibres, which increase
the mechanical stability of the villous core. In addition, the villous
core contains macrophages (Hofbauer cells) that are capable of
producing a variety of growth factors regulating growth and dif-
ferentiation of all villous components.77 
Villous Development
The importance of appreciating villous development is under-
scored by the significant alterations of the fetoplacental blood
vessels and villi in the various forms of IUGR resulting from pla-
cental insufficiency. For a detailed review of placental villous and
vascular development, see Kaufmann et  al.78 Five types of villi
have been distinguished on the basis of their calibre, stromal char-
acteristics and vessel structure (Fig. 7.7).
1. Stem villi represent the first 5 to 30 generations of unequal
dichotomous branching and serve to give mechanical support
Stem villi
Terminal villi
Immature intermediate villi Mesenchymal villi Mature intermediate villi
• Fig. 7.7  Villous types. Simplified representation of a distal part of a mature
placental villous tree, together with typical cross-sections of the various vil-
lous types. For details, see text. (With permission from Kaufmann P. Basic
morphology of the fetal and maternal circuits in the human placenta. Con-
trib Gynecol Obstet 13:5–17, 1985.)
 CHAPTER 7  Development of the Placenta and Its Circulation 65

to the villous trees. They range from about 100 μm to several
millimetres in diameter and are characterised by a compact
fibrous stroma containing centrally located arteries or larger
arterioles and veins or venules.
2. Mature intermediate villi (MIV), ranging from 80 to 120 μm
in diameter, arise from the ends of the last generation of stem
villi. These are often gently curving, and terminal villi arise at
intervals from the convex aspects of their surfaces. Internally,
they consist of a loose stromal core, and embedded within this
are narrow arterioles, characterised by a single layer of contrac-
tile cells leading into long capillaries.
3. Terminal villi are the final branches of the villous tree and,
from a physiological viewpoint, are the most important com-
ponent. They are short stubby protuberances, up to 200 μm in
length with a diameter of 50–100 μm, arising from the surface
of the MIV. Their characterising feature is the high degree of
capillarisation; more than 50% of terminal villous volume is
represented by capillaries (Fig. 7.8).
The thickness of the syncytiotrophoblast is not uniform over
the terminal villous surface; rather, there are areas where the
trophoblast is extremely attenuated, devoid of syncytial nuclei
(Fig. 7.9), known as the ‘vasculosyncytial membrane’ (VSM).
Underlying such areas are dilated segments of fetal capillar-
ies referred to as ‘sinusoids’, where the diffusional distance
between maternal and fetal plasma is reduced to as little as 0.5
to 2.0 μm. As gestational age advances towards term, the pro-
portion of villous surface area occupied by VSM increases. At
other points on the villous surface, the syncytiotrophoblast is
relatively thick containing clusters of syncytial nuclei. These are
the most important sites of metabolic and endocrine activity.
4. Immature intermediate villi (IIV) represent peripheral con-
tinuations of stem villi that are in the process of development.
Thus, whilst common in immature placentas characteristically
lacking terminal villi, their distribution in the mature organ
is generally limited to the central regions of the lobules sur-
rounding the central cavities. These villi are recognised by
the characteristically loose reticular meshwork in the stroma,
in which numerous macrophages (Hofbauer cells) are found.
Embedded amongst the stromal cells are arterioles and venules,
confirming that these villi are the forerunners of stem villi. It is
important that their presence in normal term pregnancy is rec-
ognised and that they are not incorrectly interpreted as oede-
matous villi.
5. Mesenchymal villi. This is again a transient population, seen
predominantly in the earliest stages of pregnancy when these
villi are the precursors of IIV. In a more mature placenta, they
are inconspicuous, usually situated at the surfaces of IIV, where
they represent the points of villous sprouting and development.
The development of the villous tree starts by the formation of
syncytial or trophoblastic sprouts; in early gestation, these are seen
arising in an apparently random pattern from the surfaces of mes-
enchymal and IIV. These primary syncytiotrophoblastic sprouts are
invaded centrally by cytotrophoblast, followed by a second central
invasion by mesenchyme, to form secondary villi; the latter dif-
ferentiates into stroma and capillaries, resulting in tertiary (mesen-
chymal) villi. Most villi at the end of the first trimester are of this
type. As development proceeds into the second trimester, mesen-
chymal villi transform into IIV. New syncytial sprouts continue
to form from the IIV until they themselves are transformed into
terminally-differentiated stem villi. Thus growth of the placental
villous trees is controlled by the activity of the IIV. This concept is
important to appreciate because the clinical literature79-81 errone-
ously refers to primary, secondary and tertiary stem villi; in truth,
the number of generations of stem villi is variable, ranging from
5 to 30 generations, because of the local activity of the IIV. This
mechanism of placental villous growth is illustrated in Fig. 7.10. By
the onset of the third trimester, the immature villous types (mesen-
chymal and IIV) have transformed into their mature counterparts
(MIV and stem villi, respectively). The MIV are by definition the
structure that makes the terminal villi, where gas exchange and
nutrient transfer takes place, primarily across the ‘vasculosyncytial
membrane’–the functional structure of the placenta.7

E D C B
• Fig. 7.8  Fetal vascularisation of terminal villi. Cast of vessels from a group of terminal villi (A). Corre-
sponding semi-thin sections of the transition to a mature intermediate villus (B), the basis of the branching
terminal villi (C), a single terminal villus near its tip (D) and a flat section of the terminal villous tip (E). These
pictures demonstrate the structural variability of terminal villi; they all have in common that fetal capillaries
and the highly dilated sinusoids amount to more than 50% of the stromal volume as long as postpartal
collapse can be avoided by early fixation. Magnification ×300. (With permission from Kaufmann P, Luck-
hardt M, Leiser R. Three-dimensional representation of the fetal vessel system in the human placenta.
Trophoblast Res 3:113–137, 1988.)
66 SE C T I O N 2     The Placenta

VSM

VSM
S

C VSM

H
VSM
CT

SI
VSM

VSM
• Fig. 7.9  Ultrastructure of the terminal villi. Electron micrograph of a terminal villus from a fully mature pla-
centa showing capillaries (Cs) and sinusoids (SIs). The sparse connective tissue is composed of macro-
phages (Hs) and fibroblasts (Fs). Magnification ×1400. CT, Cytotrophoblastic cell; S, syncytiotrophoblast;
VSM, vasculosyncytial membrane. (Modified with permission from Becker V, Schiebler TH, Kubli F. Die
Plazenta des Menschen. Stuttgart, Germany: Thieme Verlag, 1981.)

Formation of terminal villi is thought to be the result of stim-

1st and 2nd trimesters
ulated capillary growth (Fig. 7.11). Within MIV, arterioles give
way to long, slender capillaries. Under normal conditions, elonga-
Trophoblastic tion of these capillaries is increased during the third trimester and
sprouts
Villous exceeds that of the containing villi. As a result, capillary coils are
sprouts Stem formed, which protrude from the surface, raising an attenuated
villi
Immature covering of trophoblast before them. In this way, terminal villi are
intermediate formed, and the same capillary may run through several terminal
villi
Mesenchymal villi in series before communicating with a venule. The degree of
villi
capillarisation and formation of terminal villi is regulated by non-
branching angiogenesis and therefore indirectly by local oxygen
partial pressure and is summarised by Kingdom and Kaufman.82
3rd trimester The switch in differentiation pathways at the start of the third
Trophoblastic
trimester is of key importance in placental development. If this
sprouts takes place too early or is arrested, terminal villi will not form in
Villous normal amounts, and overgrowth as seen in chronic fetal anaemia,
sprouts Stem
Immature villi
will result in an excessively thick placenta, excessive trophoblast
intermediate shedding and the mirror-syndrome form of preeclampsia.7 These
villi changes are discussed in Chapter 9. 
Mesenchymal
villi

Mature
The Placental Barrier
intermediate
villi Maternal and fetal blood is separated by the following layers (see
Fig. 7.9):
1. Syncytiotrophoblast. This continuous layer of villous tropho-
blast does not possess any lateral cell borders and thus repre-
Terminal sents a single layer containing millions of nuclei and covering
villi
all villi of a single placenta. The syncytiotrophoblast represents
the outermost layer of the placental villi and is the fetal layer in
direct contact with maternal blood and blood cells.
• Fig. 7. 10  Routes of villous development during early and late pregnancy.
White arrows indicate transformation of one villous type into another. Black
2. Cytotrophoblast. The initially complete layer of mononucle-
arrows indicate new production of villi or sprouts along the surface of other ated cytotrophoblast cells (first trimester) becomes discontinu-
villi. (Modified with permission from Castellucci M, Scheper M, Scheffen I, ous later during pregnancy (second and third trimesters).
et al. The development of the human placental villous tree. Anat Embryol 3. Basement membrane. The epithelial-like villous trophoblast
(Berl) 181(2):117–128, 1990.) rests on a basement membrane that is typically composed of
 CHAPTER 7  Development of the Placenta and Its Circulation
1 Integrity of the Placental Barrier
2 median value for gestation) with no associated fetal malformation
3
4
• Fig. 7.11  Simplified diagram of the terminal villous development in relation
to capillary growth. Varying degrees of imbalance between villous and capil-
lary growth result in different types of terminal villous development, such as
terminal villi deficiency (1), normal mature placenta (2), hypermaturity (3), and
hypoxic hypervascularisation (e.g., preeclampsia or maternal anaemia) (4),
the conditions 1 and 3 are found, for example, in intrauterine growth restric-
tion combined with absent end-diastolic flow velocity (AEDFV). (Modified
and extended from Kaufmann, P., Bruns, U., Leiser, R., Luckhardt, M., and
Winterhager, E. (1985) The fetal vascularisation of term human placental villi.
II. Intermediate and terminal villi. Anat. Embryol. 173, 203-214.)
laminins and collagen IV. This trophoblastic basement mem-
brane may fuse with the basement membrane of the endo-
thelium of the placental capillaries and sinusoids in the last
trimester because of thinning of the stroma.
4. Stromal connective tissue. The basement membranes of
trophoblast and capillaries are separated by connective tissue
derived from the extraembryonic mesoderm.
5. Fetal endothelium. The cytoplasm of the endothelium
becomes thinner in the third trimester because of capillary loop
sinusoid formation at the apex of terminal villi.
These changes gradually increase the conductance of the pla-
centa to oxygen and permit exponential growth of the fetus in the
third trimester. The maternofetal diffusion distance, which is 50
to 100 μm in the first trimester, is eventually reduced to 4 to 5 μm
and even lower at term by the following mechanisms.7
• The thickness of the villous trophoblast is reduced from 20 μm
in early pregnancy to about 3.5 μm at term. The vasculosyncy-
tial membrane reduces this to 0.5 to 2.0 μm.
• At sites of the vasculosyncytial membrane, cytotrophoblasts
disappear and are found in niches where they do not disturb
the diffusion and transport between the maternal and fetal
blood system.
• The mean villous diameter decreases as the villi differentiate.
In addition, the blood vessels inside the villi become demus-
cularised and reside directly under the trophoblast basement
membrane.  illaries, so that impedance or flow is regulated by autonomically
67
α-Fetoprotein (AFP) is produced by the fetal liver. Its concentra-
tion in the fetal blood is 50,000 times higher than in the maternal
blood because the trophoblastic barrier is generally not permeable
to proteins.83 The introduction of midtrimester maternal serum
AFP screening programs for the detection of fetal spina bifida led
to the observation that elevated levels of AFP (>2 multiples of the
had an increased risk for adverse outcomes such as preeclamp-
sia, IUGR and antepartum fetal death.84 The implication is that
increased placental permeability may lead to impaired pregnancy
outcome. Abnormal Doppler values and abnormal placental shape
identify those at greatest risk for perinatal death and preterm deliv-
ery from placental damage. Villous repair after loss of trophoblast
involves deposition of fibrin, and in vitro studies using horseradish
peroxidase confirm these as sites of increased permeability and so
are responsible for the increased release of AFP into the maternal
circulation.85 In a normal term placenta, fibrin deposits that may
be responsible for the passage of macromolecules cover approxi-
mately 7% of the villous surface.
Another paratrophoblastic transfer route for smaller molecules
is provided by the transtrophoblastic channels, approximately 20
nm in diameter and seen only by electron microscopy. These exist
to allow transfer of water-soluble, lipophobic molecules with an
effective molecular diameter smaller than 1.5 nm and may be
important for the regulation of fluid balance. Under certain cir-
cumstances such as fetal hydrops, increased fetal venous pressure
or reduced fetal oncotic pressure, these channels dilate such that
not only water but also fetal proteins may pass into the maternal
circulation.83 
Physiology of Fetoplacental Blood Flow
Fetal size and thus oxygen and nutritional demands rapidly out-
strip growth of the placenta such that by term, 1 g of placenta
supports 6 g of fetus. To meet these demands, the peripheral vil-
lous placenta differentiates such that by term, the proportion of
descending aortic blood flow entering the umbilical arteries is
increased to 40%, and the diffusive capacity is increased 10-fold.
These alterations are almost wholly dependent on the exponen-
tial elaboration of terminal villi in the second half of pregnancy.
As in the uteroplacental circulation, which becomes ‘denervated’
by trophoblast, the villous tree remains free of fetal autonomic
influences and is dependent upon fetal cardiac output because it is
devoid of nerves. The fetoplacental circulation competes with the
lower fetal body for aortic blood. The umbilical arteries receive
this large proportion of descending aortic blood flow because of
low impedance.7
Doppler studies of the umbilical arteries indicate a progressive
fall in fetoplacental vascular impedance, reflected by increasing
end-diastolic flow velocity. During the first trimester, end-diastolic
velocities are absent, becoming consistently present by 14 weeks of
gestation. Thereafter the steady rise in end-diastolic velocity par-
allels differentiation of the villous tree into its mature form. The
dramatic changes in peripheral capillarisation of villi throughout
pregnancy (see Fig. 7.11) contribute to changes in umbilical artery
blood flow in addition to the local vasomotor regulatory process
in muscularised stem villous arterioles.75
Systemic vascular beds have a relatively short distance between
arterioles and venules and these are bridged by many parallel cap-
68 SE C T I O N 2     The Placenta
innervated precapillary sphincters; these structures regulate blood
flow across a wide range, for example, in muscle that may exercise or
rest. By contrast, fetoplacental blood flow must be constant and ever
increasing; the capillary bed of the peripheral villi is much longer
than in muscle (2000–4000 μm), less richly branched, and is focally
dilated into sinusoids within terminal villi (see Figs. 7.8 and 7.11).7  stillbirth and premature death. In the near future, more wide-
Conclusions
This chapter has aimed to discuss the clinical relevance of normal
human placental development for obstetrics. Furthermore, a wide
range of important clinical problems, such as adult cardiac disease,
have their origins in placental maldevelopment and pathology.
Increasing interest in placental research and important contribu-
tions by clinicians, especially collaboration to obtain Doppler and
real-time ultrasound information of the placenta just before deliv-
ery, has led to important advances in our understanding of the
pathological basis of placental insufficiency syndromes that cause
spread acceptance amongst maternal-fetal medicine clinicians of
the value of making a prenatal diagnosis of placental pathology
may lead to advances in the therapeutic options for at-risk women.
Access the complete reference list online at ExpertConsult.com
Self-assessment questions available at ExpertConsult.com
References singleton pregnancies obtained by in vitro fer-
tilization and embryo transfer. Obstet Gynecol.
34. Silver RM, Landon MB, Rouse DJ, et  al.
Maternal morbidity associated with multiple
1990;76(1):61–64. repeat caesarean deliveries. Obstet Gynecol.
1. Toal M, Chan C, Fallah S, et  al. Usefulness
20a. Kaufmann P, Castellucci M. Extravillous tro- 2006;107(6):1226–1232.
of a placental profile in high-risk pregnancies.
phoblast in the human placenta. Trophoblast 35. Fernandez H, Al-Najjar F, Chauveaud-
Am J Obstet Gynecol. 2007;196(4):363. e1–e7.
Res. 1997;10:21–65; Lambling A, et al. Fertility after treatment of
2. Zhong Y, Zhu F, Ding Y. Serum screening in
20b. Moser G, Windsperger KPollheimer J, de Asherman’s syndrome stage 3 and 4. J Minim
first trimester to predict pre-eclampsia, small
Sousa Lopes SC, Huppertz B. Human tro- Invasive Gynecol. 2006;13(5):398–402.
for gestational age and preterm delivery: sys-
phoblast invasion: new and unexpected 36. Burton GJ, Jauniaux E, Watson AL. Maternal
tematic review and meta-analysis. BMC Preg-
routes and functions. Histochem Cell Biol. arterial connections to the placental intervil-
nancy Childbirth. 2015;15:191.
2018;150(4):361–370. lous space during the first trimester of human
3. Dunk C, Huppertz B, Kingdom J. Fetal medi-
21. Moser G, Gauster M, Orendi K, et al. Endo- pregnancy: the Boyd collection revisited. Am J
cine. In: Rodeck CH, Whittle MJ, eds. Devel-
glandular trophoblast, an alternative route of Obstet Gynecol. 1999;181(3):718–724.
opment of the Placenta and Its Circulation. 2nd
trophoblast invasion? analysis with novel con- 37. Schapps JP, Hustin J. In  vivo aspect of the
ed. St. Louis: Elsevier; 2007.
frontation co-culture models. Hum Reprod. maternal-trophoblastic border during the
4. Torpin R. The Human Placenta. Springfield,
2010;25(5):1127–1136. first trimester of gestation. Trophoblast Res.
IL: Charles C Thomas; 1969.
22. Frank HG, Malekzadeh F, Kertschanska S, 1988;3:39–48.
5. Liu CC, Pretorius DH, Scioscia AL, et  al.
et al. Immunohistochemistry of two different 38. Jauniaux E, Jurkovic D, Campbell S, et  al.
Sonographic prenatal diagnosis of marginal
types of placental fibrinoid. Acta Anat (Basel). Investigation of placental circulations by color
placental cord insertion: clinical importance.
1994;150(1):55–68. Doppler ultrasonography. Am J Obstet Gyne-
J Ultrasound Med. 2002;21(6):627–632.
23. Simmons DG, Fortier AL, Cross JC. Diverse col. 1991;164(2): 486–468.
6. Silver RM. Abnormal placentation: placenta
subtypes and developmental origins of tropho- 39. Jauniaux E, Gulbis B, Burton GJ. The human
previa, vasa previa, and placenta accreta.
blast giant cells in the mouse placenta. Dev first trimester gestational sac limits rather than
Obstet Gynecol. 2015;126(3):654–668.
Biol. 2007;304(2):567–578. facilitates oxygen transfer to the foetus—a
7. Benirschke K, Kaufmann P. Pathology of the
24. Kemp B, Kertschanska S, Kadyrov M, et  al. review. Placenta. 2003;24(suppl A):S86–S93.
Human Placenta. 4th ed. New York: Springer-
Invasive depth of extravillous trophoblast cor- 40. Burton G.J., Jaunaiux E.. Maternal vascu-
Verlag; 2006.
relates with cellular phenotype: a comparison larisation of the human placenta: does the
8. Plazenton Schuhmann. Begriff, entstehung,
of intra- and extrauterine implantation sites. embryo develop in a hypoxic environment?
funktionelle anatomie. In: Becker V, Schie-
Histochem Cell Biol. 2002;117(5):401–414. Gynecol Obstet Fertil. 2001;29(78):503–508.
bler TH, Kubli F, eds. Die Plazenta des Men-
25. Huppertz B, Kertschanska S, Frank HG, 41. Huppertz B, Gauster M, Orendi K, et  al.
schen. Stuttgart, Germany: Thieme Verlag;
et al. Extracellular matrix components of the Oxygen as modulator of trophoblast invasion.
1981:199–207.
placental extravillous trophoblast: immunocy- J Anat. 2009;215(1):14–20.
9. Zimmermann P, Eirio V, Koskinen J, et  al.
tochemistry and ultrastructural distribution. 42. Hazan AD, Smith SD, Jones RL, et al. Vascular-
Doppler assessment of the uterine and utero-
Histochem Cell Biol. 1996;106(3):291–301. leukocyte interactions: mechanisms of human
placental circulation in the second trimester in
26. Frank HG, Huppertz B, Kertschanska S, decidual spiral artery remodeling in vitro. Am J
pregnancies at high risk for pre-eclampsia and/
et  al. Anti-adhesive glycosylation of fibro- Pathol. 2010;177(2):1017–1030.
or intrauterine growth retardation: compari-
nectin-like molecules in human placental 43. Lima PD, Zhang J, Dunk C, et  al. Leuko-
son and correlation between different Dop-
matrix-type fibrinoid. Histochem Cell Biol. cyte driven-decidual angiogenesis in early
pler parameters. Ultrasound Obstet Gynecol.
1995;104(4):317–329. pregnancy. Cell Mol Immunol. 2014;11(6):
1997;9(5):330–338.
27. Harris LK, Jones CJ, Aplin JD. Adhe- 522–537.
10. Mayhew TM. Stereology and the pla-
sion molecules in human trophoblast—a 44. Smith SD, Choudhury RH, Matos P, et  al.
centa: where’s the point? a review. Placenta.
review. II. extravillous trophoblast. Placenta. Changes in vascular extracellular matrix
2006;27(suppl A):S17–S25.
2009;30(4):299–304. composition during decidual spiral arteriole
11. Egbor M, Ansari T, Morris N, et al. Morpho-
28. Feinberg RF, Kliman HJ, Lockwood CJ. remodeling in early human pregnancy. Histol
metric placental villous and vascular abnor-
Is oncofetal fibronectin a trophoblast glue Histopathol. 2016;31(5):557–571.
malities in early- and late-onset pre-eclampsia
for human implantation? Am J Pathol. 45. Smith SD, Dunk CE, Aplin JD, et al. Evidence
with and without fetal growth restriction.
1991;138(3):537–543. for immune cell involvement in decidual spi-
BJOG. 2006;113(5):580–589.
29.  Tseng JJ, Chou MM. Differential expression ral arteriole remodeling in early human preg-
12. Ball E, Bulmer JN, Ayis S, et al. Late sporadic
of growth-, angiogenesis- and invasion-related nancy. Am J Pathol. 2009;174(5):1959–1971.
miscarriage is associated with abnormalities in
factors in the development of placenta accreta. 46. Smith SD, Choudhury RH, Matos P, et  al.
spiral artery transformation and trophoblast
Taiwan J Obstet Gynecol. 2006;45(2):100–106. Changes in vascular extracellular matrix
invasion. J Pathol. 2006;208(4):535–542.
30. Kadyrov M, Kingdom JC, Huppertz B. composition during decidual spiral arteriole
13. Wigglesworth JS. Vascular organization of the
Divergent trophoblast invasion and apop- remodeling in early human pregnancy. Histol
human placenta. Nature. 1967;216(5120):
tosis in placental bed spiral arteries from Histopathol. 2016;31(5):557–571.
1120–1121.
pregnancies complicated by maternal ane- 47. Kaufmann P, Black S, Huppertz B. Endovas-
14. Borell U, Fernstrom I, Westman A. Eine arte-
mia and early-onset preeclampsia/intrauter- cular trophoblast invasion: implications for the
riographische studie des plazentarkreilaufs.
ine growth restriction. Am J Obstet Gynecol. pathogenesis of intrauterine growth retardation
Geburtshilfe Frauenheilhd. 1958;18:1–9.
2006;194(2):557–563. and preeclampsia. Biol Reprod. 2003;69(1):
15. Konje JC, Huppertz B, Bell SC, et  al.
31. Kadyrov M, Schmitz C, Black S, et  al. Pre- 1–7.
3-dimensional colour power angiography for
eclampsia and maternal anaemia display 48. Zhou Y, Fisher SJ, Janatpour M, et al. Human
staging human placental development. Lancet.
reduced apoptosis and opposite invasive phe- cytotrophoblasts adopt a vascular pheno-
2003;362(9391):1199–1201.
notypes of extravillous trophoblast. Placenta. type as they differentiate. A strategy for suc-
16. Moll W. Physiologie der maternen plazentaren
2003;24(5):540–548. cessful endovascular invasion? J Clin Invest.
durchblutung. In: Becker V, Schiebler TH,
32. Zybina TG, Frank HG, Biesterfeld S, 1997;99(9):2139–2151.
Kubli F, eds. Die Plazenta des Menschen. Stutt-
Kaufmann P. Genome multiplication of 49. Boyd JD, Hamilton WJ. The Human Placenta.
gart, Germany: Thieme Verlag; 1981:172–194.
extravillous trophoblast cells in human pla- Cambridge, United Kingdom: Heffer; 1970.
17. McDermott M, Gillan JE. Chronic reduction
centa in the course of differentiation and inva- 50. Brosens I, Robertson WB, Dixon HG. The
in fetal blood flow is associated with placental
sion into endometrium and myometrium. II. physiological response of the vessels of the
infarction. Placenta. 1995;16(2):165–170.
Mechanisms of polyploidization. Tsitologiia. placental bed to normal pregnancy. J Pathol
18. Knott JG, Paul S. Transcriptional regula-
2004;46(7):640–648. Bacteriol. 1967;93(2):569–579.
tors of the trophoblast lineage in mammals
33. Dunk CE, Gellhaus A, Drewlo S, et  al. 51. Jauniaux E, Greenwold N, Hempstock J, et al.
with hemochorial placentation. Reproduction.
The molecular role of connexin 43 in Comparison of ultrasonographic and Dop-
2014;148(6):R121–R136.
human trophoblast cell fusion. Biol Reprod. pler mapping of the intervillous circulation in
19. Jauniaux E, Englert Y, Vanesse M, et  al.
2012;86(4):115. normal and abnormal early pregnancies. Fertil
Pathologic features of placentas from
Steril. 2003;79(1):100–106.

68.e1
68.e2 References
52. Tong S, Marjono B, Mulvey S, et al. Low levels
of pregnancy-associated plasma protein-A in
asymptomatic women destined for miscarriage.
Fertil Steril. 2004;82(5):1468–1470.
53. Ball E, Robson SC, Ayis S, et al. Early embry-
onic demise: no evidence of abnormal spiral
artery transformation or trophoblast invasion.
J Pathol. 2006;208(4):528–534.
54. Brosens I. The uteroplacental vessels at term—
the distribution and extent of physiological
changes. Trophoblast Res. 1988;3:61–67.
55. Burton GJ, Hempstock J, Jauniaux E. Oxygen,
early embryonic metabolism and free radical-
mediated embryopathies. Reprod Biomed
Online. 2003;6(1):84–96.
56. Roberts VH, Lo JO, Salati JA, et  al. Quan-
titative assessment of placental perfusion by
contrast-enhanced ultrasound in macaques
and human subjects. Am J Obstet Gynecol.
2016;214(3):369. e1–e8.
57. Burton GJ, Jauniaux E. Placental oxidative
stress: from miscarriage to preeclampsia. J Soc
Gynecol Investig. 2004;11(6):342–352.
58. Jauniaux E, Watson A, Burton G. Evaluation
of respiratory gases and acid-base gradients in
human fetal fluids and uteroplacental tissue
between 7 and 16 weeks’ gestation. Am J Obstet
Gynecol. 2001;184(5):998–1003.
59. Burton GJ, Woods AW, Jauniaux E, et  al.
Rheological and physiological consequences
of conversion of the maternal spiral arteries for
uteroplacental blood flow during human preg-
nancy. Placenta. 2009;30(6):473–482.
60. Bewley S, Cooper D, Campbell S. Doppler
investigation of uteroplacental blood flow
resistance in the second trimester: a screen-
ing study for pre-eclampsia and intrauterine
growth retardation. Br J Obstet Gynaecol.
1991;98(9):871–879.
61. Viero S, Chaddha V, Alkazaleh F, et al. Prog-
nostic value of placental ultrasound in preg-
nancies complicated by absent end-diastolic
flow velocity in the umbilical arteries. Placenta.
2004;25(8-9):735–741.
62. Demers S, Girard M, Roberge S, et  al. First-
trimester placental and myometrial blood per-
fusion measured by three-dimensional power
Doppler in preeclampsia. Am J Perinatol.
2015;32(10):920–926.
63. Brosens IA, Robertson WB, Dixon HG. The
role of the spiral arteries in the pathogenesis of
pre-eclampsia. J Pathol. 1970;101(4): Pvi.
64. Khong TY, De Wolf F, Robertson WB,
Brosens I. Inadequate maternal vascular
response to placentation in pregnancies com-
plicated by pre-eclampsia and by small-for-
gestational age infants. Br J Obstet Gynaecol.
1986;93(10):1049–1059.
65. Demir R, Kaufmann P, Castellucci M,
et  al. Fetal vasculogenesis and angiogenesis
in human placental villi. Acta Anat (Basel).
1989;136(3):190–203.
66. Aplin JD, Whittaker H, Jana Lim YT, et  al.
Hemangioblastic foci in human first trimester
placenta: distribution and gestational profile.
Placenta. 2015;36(10):1069–1077.
67. Mayhew TM. A stereological perspective on
placental morphology in normal and compli-
cated pregnancies. J Anat. 2009;215(1):77–90.
68. Wilkins-Haug L, Roberts DJ, Morton CC.
Confined placental mosaicism and intrauter-
ine growth retardation: a case-control analysis
of placentas at delivery. Am J Obstet Gynecol.
1995;172(1 Pt 1):44–50.
69. Fogarty NM, Mayhew TM, Ferguson-Smith
AC, Burton GJ. A quantitative analysis of tran-
scriptionally active syncytiotrophoblast nuclei
across human gestation. J Anat. 2011;219(5):
601–610.
70. Fogarty NM, Ferguson-Smith AC, Burton GJ.
Syncytial knots (Tenney-Parker changes) in
the human placenta: evidence of loss of tran-
scriptional activity and oxidative damage. Am J
Pathol. 2013;183(1):144–152.
71. Lee W, Ginsburg KA, Cotton DB, Kaufman
RH. Squamous and trophoblastic cells in the
maternal pulmonary circulation identified by
invasive hemodynamic monitoring during
the peripartum period. Am J Obstet Gynecol.
1986;155(5):999–1001.
72. Johansen M, Redman CW, Wilkins T, Sargent
IL. Trophoblast deportation in human preg-
nancy—its relevance for pre-eclampsia. Pla-
centa. 1999;20(7):531–539.
73. Sibley CP, Bauman KF, Firth JA. Molecular
charge as a determinant of macromolecule
permeability across the fetal capillary endothe-
lium of the guinea-pig placenta. Cell Tissue Res.
1983;229(2):365–377.
74. Alfirevic Z, Neilson JP. Doppler ultrasonog-
raphy in high-risk pregnancies: systematic
review with meta-analysis. Am J Obstet Gynecol.
1995;172(5):1379–1387.
75. Kingdom JC, Burrell SJ, Kaufmann P. Pathol-
ogy and clinical implications of abnormal
umbilical artery Doppler waveforms. Ultra-
sound Obstet Gynecol. 1997;9(4):271–286.
76. Su EJ. Role of the fetoplacental endothelium in
fetal growth restriction with abnormal umbili-
cal artery Doppler velocimetry. Am J Obstet
Gynecol. 2015;213(suppl 4):S123–S130.
77. Castellucci M, Muhlhauser J, Zaccheo D. The
Hofbauer cell: the macrophage of the human
placenta. In: Andreani D, Bompiani GD, Di
Mario U, et al., eds. Immunobiology of Normal
and Diabetic Pregnancy. New York: John Wiley;
1990:135–144.
78. Kaufmann P, Mayhew TM, Charnock-Jones
DS. Aspects of human fetoplacental vasculo-
genesis and angiogenesis. II. Changes during
normal pregnancy. Placenta. 2004;25(2-3):
114–126.
79. Bracero LA, Beneck D, Kirshenbaum N, et al.
Doppler velocimetry and placental disease. Am
J Obstet Gynecol. 1989;161(2):388–393.
80. Giles WB, Trudinger BJ, Baird PJ. Fetal umbil-
ical artery flow velocity waveforms and pla-
cental resistance: pathological correlation. Br J
Obstet Gynaecol. 1985;92(1):31–38.
81. McCowan LM, Mullen BM, Ritchie K.
Umbilical artery flow velocity waveforms and
the placental vascular bed. Am J Obstet Gynecol.
1987;157(4 Pt 1):900–902.
82. Kingdom JC, Kaufmann P. Oxygen and pla-
cental villous development: origins of fetal
hypoxia. Placenta. 1997;18(8):613–621; dis-
cussion 623–626.
83. Kaufmann P, Schroder H, Leichtweiss HP.
Fluid shift across the placenta: II. Fetomaternal
transfer of horseradish peroxidase in the guinea
pig. Placenta. 1982;3(4):339–348.
84. Alkazaleh F, Chaddha V, Viero S, et  al. Sec-
ond-trimester prediction of severe placental
complications in women with combined eleva-
tions in alpha-fetoprotein and human cho-
rionic gonadotrophin. Am J Obstet Gynecol.
2006;194(3):821–827.
85. Brownbill P, Edwards D, Jones C, et al. Mecha-
nisms of alphafetoprotein transfer in the perfused
human placental cotyledon from uncompli-
cated pregnancy. J Clin Invest. 1995;96(5):
2220–2226.
86. Kaufmann P, Scheffen I. Placental develop-
ment. In: Pollin R, Fox W, eds. Neonatal and
Fetal Medicine—Physiology and Pathophysiol-
ogy. Orlando: WB Saunders; 1992.
87. Kaufmann P. Basic morphology of the fetal
and maternal circuits in the human placenta.
Contrib Gynecol Obstet. 1985;13:5–17.
88. Kaufmann P, Luckhardt M, Leiser R. Three-
dimensional representation of the fetal vessel
system in the human placenta. Trophoblast Res.
1988;3:113–137.
89. Becker V, Schiebler TH, Kubli F. Die Plazenta
Des Menschen. Stuttgart, Germany: Thieme
Verlag; 1981.
90. Castellucci M, Scheper M, Scheffen I, et al. The
development of the human placental villous tree.
Anat Embryol (Berl). 1990;181(2):117–128.
8
Placental Function in Maternofetal
Exchange
COLIN SIBLEY AND MARK DILWOR TH
KEY POINTS
• M aternofetal exchange across the placenta provides the
solutes and water needed for fetal development and growth
and enables the waste products of fetal metabolism to be
transferred to the maternal circulation.
• The placental exchange barrier consists of the syncytio-
trophoblast epithelial cell layer, basement membrane and
connective tissue, and the fetal capillary endothelium. All
contribute to the barrier, but the syncytiotrophoblast is prob-
ably the most important locus of regulation of maternofetal
exchange.
• Driving forces for maternofetal exchange are, depending
on the molecule in question, electrochemical gradients or
hydrostatic gradients (or both) between maternal and fetal
circulations.
• The placenta is highly permeable to small lipophilic mol-
ecules such as oxygen. These therefore rapidly cross the
exchange barrier with the rate of transfer being mainly
dependent on uterine and umbilical blood flow.
• The placenta has a low permeability to larger hydrophilic
molecules. These therefore only diffuse slowly across the
placenta with the rate of transfer being more dependent on
the properties of the placental barrier rather than blood flow.
• Transfer of a hydrophilic molecule is likely to require selective
transporter proteins in the plasma membranes of the syncy-
tiotrophoblast (channels or carriers) or, for larger molecules,
vesicles enabling endocytosis at one membrane and exocyto-
sis at the other.
• Placental dysfunction includes abnormalities in maternofetal
exchange and can lead to pathologies, including fetal growth
restriction (FGR). Such dysfunction may be stratified into
vascular defects with abnormal blood flow or nonvascular
defects with abnormalities of the syncytiotrophoblast. Devel-
opment of new treatments for FGR will need to target these
different phenotypes of placental dysfunction.
Introduction
This chapter summarises current understanding of the mechanisms
of maternofetal exchange across the placenta. A full description
of these mechanisms would combine details of the physiological
processes involved with information about the identities, proper-
ties and genetic control of the relevant molecular species. How-
ever, there remains much to be learnt before such a comprehensive
review is possible. Therefore we describe the key principles
required for a full understanding of the maternofetal exchange
of any solute and then provide examples of how these apply to
selected substances. Finally, we consider the clinical relevance
of maternofetal exchange in relation to fetal growth restriction.
Additional detailed coverage of aspects of placental transfer which
are beyond the scope of this chapter may be found elsewhere.1-5
A variety of species have been used to study placental transport,
but considerable care must be taken in extrapolating from these to
the human because of the great diversity of placental morphology
and function.4 Animal studies do provide an important founda-
tion for understanding placental function,6 but here we focus on
work on human placenta. A variety of in vitro and in vivo tech-
niques have been used to study the human placenta, and these are
considered in detail elsewhere.4,5,7,8 Complete characterisation of
a transport mechanism should broadly include four sets of infor-
mation: (i) the amount of substance transferred per unit time and
per unit surface area, the flux, in both maternofetal and fetoma-
ternal directions, the difference between the two giving the mag-
nitude and direction of the net flux; (ii) the magnitude and factors
controlling the driving force for the transfer of a substance (e.g.,
plasma concentrations along the length of the exchange surface
and blood flow); (iii) the route of transfer, for example, whether
across the plasma membranes and through the cytosol (transcel-
lular route) or via extracellular water-filled channels (paracellular
route); and (iv) the role and contribution of the placenta’s own
metabolic processes. Historically, measurement of flux was the
focus of attention, but more recently, the focus has shifted to the
cellular and molecular aspects of transport with the use of in vitro
and molecular techniques. However, there is an ongoing require-
ment for the physiological relevance of mechanistic components
deduced from in  vitro techniques to be reconciled with overall
flux and accretion. It must also be borne in mind that transport
by components of the placenta does not always lead to transfer
across the placenta because some of the transport will satisfy the
metabolic needs of the placenta itself. 
The Placental Exchange Barrier
The human placenta is of the haemochorial type so that blood
delivered into the intervillous space via the spiral arteries (Fig. 8.1)
69
70 SE C T I O N 2     The Placenta

directly bathes the syncytiotrophoblast lining of the villi (i.e., there
is no endothelium separating blood from this epithelium). The
syncytiotrophoblast is also unusual in that it a true multinucleated
syncytium with no lateral intercellular spaces akin to those found
in other epithelia (but see later discussion on paracellular routes)
and is usually considered to be the main barrier to exchange. It has
two plasma membranes: the microvillous, maternal-facing plasma
membrane (MVM) and the fetal-facing basal plasma membrane
(BM). Underlying the syncytiotrophoblast, there is an extracel-
lular matrix (ECM) of connective tissue and finally the capillary
endothelium bathed in fetal blood. Although ECM will not present
a major barrier to most solutes, it is likely that, as in other epithelia,
it will create a slow-moving pool of fluid (an ‘unstrirred layer’) that
will affect the nature of electrochemical gradients across the syncy-
tiotrophoblast. The fetal capillary endothelium has lateral intercel-
lular spaces through which small molecules can diffuse. However,
the diffusion of large proteins, known to cross the placenta, such
as immunoglobulin G and alpha-fetoprotein, is restricted through
these spaces so that the endothelium is a significant barrier to such
molecules.9 

Types of Exchange Mechanisms

The different types of mechanisms of exchange across the placenta
VT are summarised in Fig 8.2.
MVM The placental exchange barrier limits solute transfer to varying
degrees. Lipophilic substances (e.g., oxygen) that dissolve readily
BM in the plasma membrane will rapidly diffuse across the barrier.6
On the other hand, for hydrophilic substances (e.g., sodium ions,
amino acids), the placenta is a significant barrier to transfer. How-
ever, this barrier is not absolute because there are multiple path-
ways available for hydrophilic solutes to pass across the placenta,
including pores, channels, carriers (including cotransporters and
exchangers), pumps and vesicles. These can be matched to mecha-
nisms such as filtration (pores), diffusion (pores and channels),
UC facilitated diffusion and secondary active transport (carriers), pri-
FC mary active transport (pumps) and endocytosis and exocytosis
(vesicles).
N
Pores allow for the movement of solute and solvent through a
paracellular, extracellular water-filled pathway such that the trans-
ferred substances do not have to cross any plasma membranes to
SA
traverse a layer of tissue. Pores may allow transfer by diffusion of
IVS
solute alone or, by bulk flow, of solute and solvent together. The
extent of diffusion through pores can be altered by changes in elec-
trochemical gradients and, in the case of bulk flow, by changes in
• Fig. 8.1  The placental barrier. This primarily consists of the syncytio-
plasma hydrostatic and osmotic pressures.10 There is clear physi-
trophoblast and the fetal capillary (FC) endothelium. Of these structures,
it is primarily the two polarised plasma membranes, the microvillous
ological evidence, both in vivo and in vitro, that transfer through
(MVM) and the basal plasma membrane (BM) of the syncytiotrophoblast, pores does occur in human placentas11-13 as well as in the placen-
that restrict the transfer of molecules like glucose and amino acids. IVS, tas of several other species.14 Because the syncytiotrophoblast is a
intervillous space; N, nucleus of syncytiotrophoblast; SA, spiral artery; true syncytium as described earlier, the morphological correlates
UC; umbilical cord; VT, villous tree. of the pores are unclear. However, there is evidence that naturally

Amino
O2 Mannitol Glucose acid Na+ K+

ATP Ca+
MVM

Na+
Ca+

Na+ Ca+
H+
ATP
ATP BM
IgG

Na+ K+ Cl−, K+
a b c d e f g h i
• Fig. 8.2  Schematic of the major transfer mechanisms across the microvillous membrane (MVM) and
basal membrane (BM) of the syncytiotrophoblast with examples of the solutes transferred. (a) Diffusion of
relatively lipophilic substances; (b) paracellular route for hydrophilic substances; (c) facilitated diffusion; (d)
cotransport; (e) exchange; (f and h) active transport; (g) ion channels and (i) endocytosis-exocytosis. ATP,
Adenosine triphosphate; IgG, immunoglobulin G. (Reproduced and adapted from Desforges M, Sibley CP.
Placental nutrient supply and fetal growth. Int J Dev Biol. 2010;54(2-3):377–390.)

occurring areas of syncytial denudation, found in all normal pla-
centas, could provide a large pore with contributions from other
extracellular fluid-containing routes.15 This paracellular route of
transfer is quantitatively of major importance for the transfer
of small hydrophilic solutes such as calcium ions and chloride
ions.16,17 Of course, the transcellular route through the syncytio-
trophoblast, using channels and carriers, is likely to be qualita-
tively of greater importance, allowing fine tuning of net flux.
Channels are integral membrane proteins through which
ions may diffuse down electrochemical gradients either into or
out of cells. Although passive, the diffusion of solute through
channels is selective, gated and saturable and may be function-
ally asymmetric. These properties allow cells to modify the extent
of inward and outward solute movements caused by diffusion
in response to homeostatic signals mediated by intracellular,
autocrine, paracrine and endocrine agents and by effects at the
genome. This probably allows a range of normal processes and a
broad repertoire of reactions to abnormal processes.
Carriers are integral membrane proteins that selectively com-
bine with a solute and can carry it from one side of the membrane
to the other. The combining site is only exposed to one side of
the membrane at a time. Channels and carriers show different
behaviours. In general terms, if a solute is added to the far side
(the trans side) of the membrane, then the combining site will
be able to return to the near side (the cis side) more quickly than
it would otherwise. The combining site will be on the near side
more often and will remove solute more frequently. A carrier will
thus carry more solute if it is ‘transstimulated’, but a channel will
not respond in this way. Some carriers can transport more than
one solute at a time. A cotransporter carries two solutes in the
same direction; an exchanger swaps solutes. This allows cells to
coordinate the movement of disparate solutes.
Pumps carry solutes against concentration gradients. This is
called ‘active transport’ because energy (as adenosine triphosphate
(ATP)) is used up in the process. A good example is the active
extrusion of sodium by cells in exchange for potassium on the
Na+/K+-ATPase (sodium pump). This is primary active transport.
Carriers can harness the gradients generated by pumps by linking
solute movements to sodium movements. This is secondary active
transport, which allows cells to move solutes against concentration
gradients and thus to control their surroundings more subtly than
if diffusion gradients were the only forces present.
Vesicles are formed on one side of an epithelium such as the
syncytiotrophoblast by invagination of the plasma membrane
and, on the opposite side of the cell, fuse with the plasma mem-
brane and open onto the extracellular space. Solute and water may
be taken up into the forming vesicle by simple entrapment (‘fluid-
phase’ endocytosis), or solute may be taken up specifically by
binding to receptors on the surface of the area of membrane about
to vesiculate. Vesicles may move around the cytoplasm randomly
by Brownian motion or may be directed by the cytoskeleton.  the supply of some substances is vastly in excess of fetal accretion,24
Factors Affecting Maternofetal Exchange
Most placental exchange is driven by diffusion or modifica-
tions of this process. Factors affecting diffusion will thus affect
the magnitude of net flux. The rate of diffusion is determined by
the concentration gradient across the barrier and the permeabil-
ity of the barrier and its components. Different substances have
different concentration gradients.3 Permeability varies among
solutes according to their size, shape and lipophilicity (perme-
ability to hydrophobic molecules being much greater than that
CHAPTER 8  Placental Function in Maternofetal Exchange 71
of hydrophilic ones as mentioned earlier). The permeability of the
placenta to hydrophilic solutes increases towards term in various
animals,18 although this has not been studied in humans.
A potential difference across the exchange barrier will affect the
transfer of charged solutes. Potential differences between mother
and fetus have been measured in some species,19 but it is unclear
whether these are generated by the placenta. In humans, a poten-
tial difference has been measured in vitro both across the MVM
and across the entire exchange barrier of isolated mature interme-
diate villi derived from term placentas (magnitude ∼4 mV; fetal
side, negative.)20 In vivo, a small maternofetal potential difference
was measured in women in the third trimester21 of similar mag-
nitude to that found in vitro. However, at term, there is reported
to be no significant maternofetal potential difference.22 This ques-
tion of the magnitude and polarity of potential difference across
the placental exchange barrier is difficult to address experimentally
in humans but is of fundamental importance in understanding
the driving forces for ions and other charged solutes.
The pattern and magnitude of blood flow affects exchange.23
A hydrophobic substance (e.g., oxygen) crosses the membrane so
quickly that it has effectively gone from the maternal side of the
placenta as soon as it arrives; the rate-limiting steps of transfer will
be the rate at which it arrives and the rate at which it is taken away.
The transfer of such substances is said to be ‘flow limited’; if pla-
cental blood flow is deranged, oxygen delivery, for example, will
be impaired. Furthermore, the pattern of blood flow will affect the
efficiency of exchange. If the blood flows are in opposite directions
(countercurrent flows), the exchange will be more efficient than if
they are in the same direction (concurrent flows). The human pla-
centa is thought to have an intermediate arrangement in efficiency
called ‘multivillous pool flow’.23 A hydrophilic substance, on the
other hand (e.g., an amino acid) will have much lower perme-
ability across the placenta, transfer is slow and its concentration
in the maternal circulation hardly changes across the exchange
barrier. The transfer of such substances is therefore relatively unaf-
fected by blood flow, and their transfer is said to be ‘membrane’
or ‘diffusion’ limited. It is important to understand the distinction
between flow- and membrane-limited diffusion when considering
the phenotypes of placental dysfunction as related to FGR (see
final section of this chapter).
The placenta is a metabolically active organ, which significantly
affects the traffic of solutes such as oxygen, amino acids and carbo-
hydrates. Control of placental metabolism, as well as of transport,
by hormonal, genetic or intrinsic means, by the mother or fetus, is
likely to be of considerable importance.
Finally, it should be noted that although the placenta shares
many characteristics with other tissues (e.g., sodium pumps and
intracellular signalling apparatus), we can identify features which
are less prominent in other organs, and these need to be mentioned
as a background to any discussion of function or pathology. First,
but other fluxes are more obviously related to fetal requirements.
Second, transfer represents diverse phenomena; some substances
(e.g., glucose) are transferred by one or two mechanisms only, and
alterations in these mechanisms are relatively easy to detect. Other
substances (e.g., sodium) are transferred by many specific mecha-
nisms, none of which is dominant, and alterations in these systems
are more difficult to detect. Water flux, which is greater than for
any other molecule,25 appears to be particularly complex. There
is evidence in rats26,27 that net water flux may be the balance of
osmotic flow in the maternofetal direction following active trans-
port of ions and bulk flow in the fetomaternal direction down
72 SE C T I O N 2     The Placenta
a hydrostatic pressure gradient. Alteration in water transfer may
thus reflect a wide range of changes and is therefore not simple to
understand and will not be easy to manipulate.  Fetal plasma (umbilical vein) Pco2 at term is between 38 and 45
Specific Examples of Transferred Substances
Respiratory Gas Exchange
Gas exchange at the placenta is determined by the following
factors:
(i) the concentration gradient of the gas across the placenta;
(ii) the gas-carrying capacities of fetal and maternal blood;
(iii) the rates of fetal and maternal placental blood flow;
(iv) the relative spatial orientation of the two blood flows; and
(v) the permeability properties and surface area of the mem-
branes involved.
Membrane permeability and total surface area change over ges-
tation but cannot respond to short-term perturbations, and the
relative orientation of blood flows is also fixed. Blood flow in the
intervillous space is thought not to occur before 10 to 12 weeks
of gestation.28 After intervillous blood flow is established, transfer
processes seem to fit a multivillous pool flow model best.23 Blood
flows and concentration gradients can change, in the short term,
in normal physiological states and, in the long term, in abnormal
states such as preeclampsia and FGR.
Oxygen supply. Throughout pregnancy, fetal blood has a
higher haemoglobin (Hb) concentration than maternal blood
and a higher affinity for oxygen (O2) because of a lower affin-
ity for 2,3-diphosphoglycerate.29 Whereas fetal blood at term has
an O2-carrying capacity of 25 mL/dL maternal blood can carry
only 15 mL/dL. There are many ways of expressing O2 exchange,
but the crucial physiological variable is fetal O2 uptake because it
determines the capacity for oxidative metabolism. In sheep, for
example, homeostatic mechanisms keep this variable remarkably
constant in the face of wide variations in other variables (see earlier
discussion). It is not until fetal O2 uptake drops below a criti-
cal level (0.6 mmol O2/min/kg) that metabolic acidosis ensues.
A small drop in umbilical artery O2 content results in a large
increase in the amount of O2 transferred from mother to fetus.
This is due to the relative characteristics of HbA and HbF O2 dis-
sociation curves for the mother and fetus, respectively. The result
of this relationship is that the single most important determinant
of O2 transfer from maternal to fetal blood is the O2 content of
blood perfusing the placenta from the umbilical arteries. This
mechanism keeps fetal O2 uptake relatively constant during short-
term changes in umbilical and uterine blood flow via compen-
satory changes in fractional O2 extraction.30 Although responses
to short-term hypoxia are seen in clinical situations such as cord
compression, maternal exercise, fetal activity or uterine contrac-
tions, no effect on long-term outcome has been demonstrated.
However, long-term hypoxia is important to fetal well-being as
demonstrated by studies on women living at high altitudes.31  porter in the BM, but not MVM, is reduced in pregnancies with
Carbon dioxide removal. Most carbon dioxide (CO2) in
the blood is hydrated to hydrogen ions (H+) and bicarbonate
(HCO3− ) ions; this conversion is catalysed by carbonic anhy-
drase (CA) inside red blood cells. Some CO2 is associated with
deoxygenated Hb, as carbamino-Hb, and only a small amount
is in solution. In the guinea pig, transfer of HCO3− relies on the
presence of CA on both MVM and BM,32 suggesting that HCO3−
must be converted back to CO2 before transfer across the pla-
centa. Carbon dioxide is lipid soluble, so it is readily transferred
across the placenta by diffusion. In the same study, a small amount
of transfer of the HCO3− ion, in exchange for lactate or chloride
ions, was seen when CA activity was inhibited with acetazolamide.
mm Hg,33 but maternal arterial Pco2 is between 26 and 34 mm
Hg34 (lower than prepregnancy values because of hyperventila-
tion). This concentration gradient therefore drives CO2 transfer.
However, because diffusion of the small, lipophilic CO2 molecule
is rapid, relative fetal and maternal placental blood flows are the
critical determinants of its rate of transfer. Consequently, reduced
uteroplacental or umbilical blood flow leads to respiratory acido-
sis in the fetus; this is rapidly corrected if normal blood flow is
reestablished. 
Acid–Base Balance
The role of the placenta in fetal acid–base balance is poorly under-
stood. Mechanisms for regulating acid–base status are a dynamic
element of fetal metabolism. For example, as normal gestation
proceeds, the pH of blood in the umbilical vessels falls.35 These
changes do not occur in isolation. Po2 also declines during normal
gestation, and fetal Hb rises.35 Acid equivalents produced by the
fetus during metabolism cannot be eliminated by CO2 transfer
across the placenta and require transport of protons from fetal to
maternal circulations or of bicarbonate in the reverse direction.
Although there is good evidence of a Cl−/HCO3− exchanger in the
synctriotrophoblast,17,36,37 its role in HCO3− transport has not
been elucidated. Better studied is the sodium-proton exchanger
(NHE) of which several isoforms have been identified in the
human placenta.38,39 NHE is highly active in the MVM, where it
exchanges Na+, moving down its concentration gradient into the
syncytiotrophoblast, for H+ which is therefore eliminated into the
maternal circulation. NHE expression and activity on the MVM
is lower in FGR pregnancies than those with normally grown
babies,40 and this could contribute to the acidosis that some FGR
babies develop.
Lactate is traditionally viewed as a byproduct of anaerobic res-
piration. However, in a fetus, lactate may be an important source
of energy for the fetus and the placenta even when oxygen and
glucose supplies are adequate.41 Umbilical venous lactate concen-
trations are higher than the umbilical arterial levels, and both are
higher than the levels in the maternal circulation.41 This suggests
that lactate is secreted into both circulations by the placenta.42
Small-for-gestational age (SGA) fetuses have lower umbilical arte-
rial and venous Po2 and pH values with higher Pco2 and lactate
values than in normally grown fetuses.35,42 This elevated lactate
concentration might suggest a reduced oxidative capacity in SGA
fetuses; the lower Po2 and raised Pco2 are likely to be indicative of
reduced placental blood flow. There is good evidence that lactate
is taken up by both MVM and BM of the syncytiotrophoblast
via isoforms 1 and 4 of the lactate/H+ co-transporter (also known
as the monocarboxylate transporter).43 The activity of this trans-
FGR,44 and this may contribute to the Lacticacidaemia associated
with this condition. 
Ions
Because the permeability of the human placenta to small hydro-
philic substances is so high, it seems likely that the major compo-
nent of both maternofetal and fetomaternal fluxes of ions are by
diffusion through a paracellular route. As already noted, under-
standing the exact contribution of such diffusion to ion exchange
 CHAPTER 8  Placental Function in Maternofetal Exchange 73

is hampered by the lack of a clear understanding of the mater-
nofetal potential difference Furthermore, the presence of specific
ion transporters in the MVM and BM of the syncytiotrophoblast
suggests that there is at least a small transcellular component of
exchange which, being regulatable, might be qualitatively most
important.
In rats, there is good evidence that sodium is actively trans-
ported to the fetus.45 Analysis of the classical data of Flexner and
colleagues, obtained by in  vivo measurements of Na transfer,24
suggests that although the bulk of sodium transfer to the fetus
might be by passive means, the human placenta also does not act
solely as a simple filter of sodium. Several routes for transport of
sodium have been demonstrated in human placental preparations,
and these include sodium channels in the MVM,46 the NHE as
described earlier, Na+/amino acid cotransport (see later section on
amino acids) and the Na+/K+-ATPase.47 However, all of these may
contribute to placental homeostasis as well as to fetal growth. Cer-
tainly Na+/K+-ATPase in the syncytiotrophoblast has a key role in
cellular homeostasis, as in all cells. Interestingly, the activity of this
transporter on the MVM, like that of NHE, is reduced in FGR,47
and this could impair the functioning of all Na+-coupled solute
transporters in this condition.
Chloride transfer across the placenta has been poorly explored.
There is evidence that the bulk of maternofetal chloride transfer is
by passive diffusion but that transcellular routes do contribute a
quantitatively small fraction.17 Several such routes have been identi-
fied, including channels48 and the Cl−/HCO3− exchanger described
earlier, but the relative contribution of each of these to maternofetal
exchange rather than syncytiotrophoblast homeostasis is unknown.
Placental transfer of the divalent cations calcium, magnesium
and phosphorus involves common features. First, transport from
a mother to her fetus is against a concentration gradient, the con-
centration of each of these solutes being higher in fetal plasma
than maternal49 this suggests that active transport mechanisms
underlie the placental transfer of all three ions. Second, the net
placental transport of each of the divalent ions increases over the
last third of gestation, coincident with the mineralisation of the
fetal skeleton.49 This coordinated gestational increase in placental
transfer, despite different transport mechanisms being involved
for the individual ions, is remarkable. Unfortunately, the means
by which this coordination is brought about is not yet known.
Although phosphorous and magnesium transfer are poorly under-
stood, the mechanism of calcium transfer across the human pla-
centa has been reasonably well described.
Fetal plasma concentrations of total and ionised calcium are
higher than maternal, and there is strong evidence that there is
active, transcellular transport of this cation across the placenta.4
This transcellular transport of calcium most likely involves three
steps (Fig. 8.3). The first of these is being transfer from the mater-
nal blood to the trophoblastic cytosol across the MVM of the
syncytiotrophoblast. The electrochemical gradient for calcium
movement across this plasma membrane is favourable: the median
potential difference across the MVM, when measured in  vitro,
was –22 mV (trophoblast negative),20 and the intracellular ‘free’
(ionised) calcium concentration is likely to be of the order of
10–7 M,50 four orders of magnitude lower than the extracellular
‘free’ calcium concentration of 10–3 M in plasma. This favour-
able inwardly directed electrochemical gradient makes channels

Ca2+ [Ca2+] = 1.2 mmol/L

Ca2+
Maternal
TRPV6

side

Microvillous
membrane
Fetomaternal

Maternofetal

Maternofetal

Calbindin-D9K or D28K
[Ca2+] =

ATP Ca2+ ADP + Pi 0.1 µmol/L

PMCA Basal membrane

Fetal side

Ca2+

Paracellular Transcellular [Ca2+] = 1.4 mmol/L

• Fig. 8.3  Schematic showing the paracellular and transcellular routes of Ca2+ transfer across the syn-
cytiotrophoblast. Paracellularly, there are maternofetal and fetomaternal fluxes, though the fetomaternal
flux is lower because of the prevailing electrochemical gradient. Transcellularly, Ca2+ enters the syncy-
tiotrophoblast across the microvillous plasma membrane (MVM) down an electrochemical gradient from
maternal blood (∼1.2 mmol/L) into the trophoblastic cytosol (0.1 μmol/L). This diffusion is likely to involve
channels such as the transient potential vanilloid type 6 (TRPV6). When inside the cytosol of the tro-
phoblast, Ca2+ binds to a calcium binding protein, with calbindin-D9K (and calbindin-D28K) thought to be
important for intracellular buffering of Ca2+. At the basal membrane (BM), Ca2+ is effluxed across the BM
against an electrochemical gradient and into fetal blood through the actions of the plasma membrane
calcium ATPase (PMCA). This process maintains a relatively hypercalcaemic (∼1.4 mmol/L) environment
in fetal versus maternal blood. ADP, Adenosine diphosphate; ATP, adenosine triphosphate.
74 SE C T I O N 2     The Placenta
the likely route of calcium diffusion into the syncytiotropho-
blast across the MVM, and evidence from mice shows that the
Ca2+ selective channel, transient receptor potential, vanilloid 6
(TRPV6) plays such a role in this species.51 Furthermore, this
channel is expressed in human syncytiotrophoblasts.52 Although
it seems that TRPV6 is an excellent candidate for the Ca2+ entry
route, it should be noted that other Ca2+ selective channels are
expressed in human syncytiotrophoblasts, suggesting multiple
routes of entry.52
The second step in the transcellular transfer of calcium across
the placenta involves translocation across the trophoblast cytosol,
without generating marked fluctuations in intracellular calcium
concentration. This is likely to be achieved mainly by the pres-
ence of various calcium-binding proteins. In particular, the role
of a 9-kDa calcium-binding protein (calbindin-D9k) has been the
focus of several studies. In rat placentas, the mRNA expression of
calbindin-D9k increases markedly over the last third of gestation,
and this induction is temporally associated with the gestational
increase in the unidirectional maternofetal clearance of calcium
across this tissue.53 This suggests that the protein is stoichio-
metrically involved in transplacental calcium transport. However,
calbindin-D9k knockout mice do not have defective placental cal-
cium transport,54 perhaps because of compensation by other pro-
teins. This protein is present in human trophoblast cells55 as are
other calcium-binding proteins such as calbindin-D28k,56 which
may also be involved in transcellular calcium transfer across the
syncytiotrophoblast.
The third step in the transcellular transfer of calcium across the
placenta involves efflux of calcium from the trophoblast cytosol
across the BM and hence across the fetal capillary endothelium
into fetal plasma. Because this step involves transport against a
chemical gradient, an active mechanism is implicated. Indeed,
the transfer of calcium across the BM in the human placenta is
now known to occur via the plasma membrane calcium ATPase
(PMCA or Ca2+-ATPase).57,58
As already noted, there is a marked increase in calcium flux
across the placenta to the fetus towards the end of gestation, pre-
sumably to enable the rapid skeletal mineralisation that happens
at this time. In rats, this increase in calcium transport appears to
be mediated by a sharp increase in calbindin-D9k expression,53 but
in human placentas, calcium pump activity on the BM increases
significantly from 32 to 37 weeks.59 The latter might be regulated
by parathyroid hormone-related peptide (PTHrP): the midmol-
ecule fragment 38 to 94 of this hormone does acutely stimulate
the activity of human placental calcium pump activity in vitro.60  centration gradient. This can be achieved by accumulative trans-
Glucose
Glucose is the primary metabolic substrate for energy in both
fetuses and placentas. In human placentas, uptake of radiolabelled
d-glucose or methyl d-glucose, a nonmetabolisable analogue,
across the MVM and BM of the syncytiotrophoblast, was found
to be rapid, stereospecific, Na+-independent and inhibitable by
cytochalasin B, phloretin and phlorizin.61,62 These are character-
istics of the GLUT family of Na+-independent sugar transporters
which undoubtedly play the major role in transplacental glucose
transfer.
At least four different GLUT isoforms are expressed in the
syncytiotrophoblast of a first trimester placenta (GLUT1, 3, 4
and 12).63,64 GLUT1 is the predominant isoform in the syncy-
tiotrophoblast at term.63,65 GLUT3 is also expressed at term in
the MVM but at much lower levels than in the first trimester,66
suggesting it is of greater importance in early pregnancy. Both first
trimester and term human placentas show greater GLUT1 expres-
sion on the MVM compared with the BM.65,67 This asymmetry in
GLUT1 distribution together with the much greater surface area
of the MVM may well result in syncytial glucose concentrations
approaching maternal, providing a maximal gradient for transfer
to the fetus.
The driving force for facilitated glucose transfer across the pla-
centa is the maternofetal concentration gradient with fetal whole
blood or plasma concentrations being lower than maternal.68
From the Km values for the glucose transporters in the MVM and
BM of the human syncytiotrophoblast, 31 mM61 and 23 mM,62
respectively, it can be concluded that not only is transport likely
to be linearly related to maternal concentration in the physiologi-
cal range but also that transplacental glucose transport will not be
saturated under physiological conditions. A consequence of this is
that maternal hyperglycaemia in diabetes with suboptimal meta-
bolic control will result in increased placental glucose transfer with
potentially increased fetal insulin secretion and fetal overgrowth.
GLUT4 and GLUT12 are sensitive to regulation by insulin,
and there is evidence that glucose uptake is stimulated by insulin
in the first trimester.63 However, GLUT1 and GLUT 3 are not
insulin sensitive, and to date, there is no good evidence for hor-
monal control of glucose transfer across late gestation placentas.69 
Amino Acids
Amino acids are required for fetal protein synthesis, as energy
substrates and for other functions such as cell volume regulation.
The concentration of almost all amino acids is higher in umbili-
cal venous blood than in maternal uterine arterial blood from the
midtrimester onwards, implying active transport from the mother
to the fetus across the syncytiotrophoblast.70 In support of this,
a wide range of amino acid transporters have now been charac-
terised in the MVM and BM of the syncytiotrophoblast5; each
transporter mediates the uptake of several different amino acids,
and each specific amino acid can be transported by several dif-
ferent transporters. Concentrations within placental tissue itself
are higher than in either maternal or fetal serum,71 so the inter-
esting question is how net flux from mother to the fetus occurs.
There appear to be three different classes of transporter present
in the syncytiotrophoblast which enable net flux – accumulative,
exchanger and facilitative72 (Fig. 8.4). The active step is at the
MVM, where amino acids have to transported against their con-
porters, which in many cases use the inwardly directed sodium
gradient created by Na+/K+ATPase in an energy-dependent pro-
cess. An example of such a secondary active process is the system
A amino acid transporter which co-transports alanine, serine or
glycine with sodium into the syncytiotrophoblast. Alternatively,
other transport systems such as system y+ for cationic amino acids
can use the electrical gradient across the MVM (inside negative)
to drive amino acid accumulation into the syncytiotrophoblast.
Exchangers can swap one amino acid for another across the plasma
membrane; for example, the steep outwardly directed gradient for
glycine across the MVM (arising from accumulative transport)
can enable accumulation of leucine by exchange on the system L
transporter. These exchange systems therefore cannot change the
total quantity of amino acid in the syncytiotrophoblast, but they
can change the composition.
For net flux to occur, amino acids have to leave the syncytiotro-
phoblast across the BM. Because of the high syncytiotrophoblast
 CHAPTER 8  Placental Function in Maternofetal Exchange 75

Microvillous Basal Fetal capillary

membrane membrane endothelium
Syncytiotrophoblast
Maternal blood Fetal blood
arterial Paracellular routes arterial

Accumulative Accumulative
Flow and mixing transporters transporters

Exchange Exchange

Flow
transporters transporters

Facilitative
transporters
Arrows indicate
movement of
amino acids
Venous Venous
• Fig. 8.4  Schematic of amino acid transfer routes across the syncytiotrophoblast. Amino acid transfer
is reliant upon a range of transport proteins/systems with different specificities and modes of action. The
complex interplay between these transporters is summarised above. (Replicated with permission from
Lewis RM, Brooks S, Crocker IP, et al. Review: modelling placental amino acid transfer—from transporters
to placental function. Placenta 2013;34(suppl):S46–S51.)

cytosol amino acid concentration, this is down a concentration
gradient, and recent evidence suggests that this uses both exchang-
ers and facilitated diffusion through specific efflux pathways.72,73
Interestingly accumulative transporters such as system A are also
expressed on the BM5; presumably, they have a role here in main-
taining the amino acid gradients required for the exchangers to
operate.
Although the fundamental steps involved in amino acid trans-
fer as described earlier now seem fairly clear, the overall mecha-
nism of net flux of the 20 amino acids required by a growing fetus
is complex. As noted, although the transporters are selective, they
have overlapping affinities for different amino acids, which there-
fore compete; there has to be integration between accumulative,
exchange and facilitative transporters as they each alter the quan-
tity and composition of amino acid in the syncytiotrophoblast;
finally, amino acids are used and metabolised by the syncytiotro-
phoblast and other placental cell types, again modulating transfer
to the fetus. In silico modelling of placental amino acid transfer
is now being used to resolve this complexity.74,75 This is particu-
larly important because abnormal placental amino acid transfer
appears to be an important component in the aetiology of FGR,
as described further later.
A number of hormones, growth factors and cytokines affect
placental amino acid transfer. For example, insulin, insulinlike
growth factor 1 (IGF-1), leptin, interleukin-6 and tumour necro-
sis factor alpha all upregulate system A transporter activity; cor-
ticosteroids appear to downregulate activity of this transporter,
and adiponectin inhibits insulin-stimulated amino acid transfer
(recently reviewed by Dimasuay and colleagues3). Such circulat-
ing signals and other factors affecting nutrient transfer, including
blood flow, oxygen and maternal nutrition, appear to be sensed
by the mammalian (or mechanistic) target of rapamycin (mTOR)
pathway.3 It is likely that mTOR has a key role in modulating
placental amino acid transfer both by modulating the activity of
amino acid transporters and by affecting the trafficking of these
transporters to the syncytiotrophoblast plasma membrane. In the
placental nutrient-sensing model, it is proposed3 that the syn-
cytiotrophoblast integrates signals from the maternal and fetal
compartment that modulate placental function and match fetal
growth to maternal nutrient availability. 
Immunoglobulin G
In women, passive immunity is conferred on the fetus by the selec-
tive transfer of immunoglobulin G (IgG) by the placenta. Selective
IgG transfer has been considered as involving an endocytosis–exo-
cytosis model with three steps: (i) IgG destined for transfer to the
fetus is taken up from maternal plasma by endocytosis via specific
Fc receptors in coated pits (specialised areas of plasma membrane
which have a fuzzy electron-dense outline, under the electron
microscope, because of a protein called ‘clathrin’) on the microvil-
lous plasma membrane of the syncytiotrophoblast; (ii) Fc receptor-­
bound IgG within coated vesicles, formed by invagination of the
coated pits, is protected from digestion by lysosomes within the
syncytiotrophoblast; and (iii) vesicles containing undigested IgG
fuse with the basal plasma membrane of the syncytiotrophoblast,
exocytose and release the IgG into the interstitial space.
Studies in which IgG in the syncytiotrophoblast has been local-
ised under the electron microscope, as well as biochemical experi-
ments, mainly support the first part of the model described,76-80
although not every study has found IgG in coated pits and vesi-
cles.81 Evidence suggests that this uptake involves the human neo-
natal Fcϒ receptor (hFcRn).82 Unlike other Fcϒ receptors, hFcRn
has a much higher affinity for IgG at pH 6.0 than it does at neu-
tral pH and is therefore unable to bind IgG at the extracellular
face of the MVM. It is therefore most likely that IgG in relatively
high concentrations in maternal plasma is taken up by fluid-phase
endocytosis and then binds to hFcRn in the acidic environment
76 SE C T I O N 2     The Placenta
of endosomes inside the syncytiotrophoblast. It is in this form that
IgG is transferred across the syncytiotrophoblast (step ii). Experi-
mental evidence for exocytosis in step (iii) of the model is equivo-
cal; direct evidence for exocytosis is weak.78-81
When IgG reaches the interstitial space on the fetal side of the
syncytiotrophoblast, it still has to traverse the basement mem-
brane and fetal capillary endothelium. Although the former does
not appear to present a significant barrier,78,81 the latter does9; the
available data suggest that transcytosis in vesicles is a more likely
route of transfer across the endothelium than is diffusion through
the lateral intercellular spaces between endothelial cells.78-80  membrane amino acid transporters and other nutrient transport-
Clinical Considerations: Maternofetal
Exchange and Fetal Growth Restriction
More than 95% of the solutes and water that constitute a term
baby is attained by net flux across the placenta over the course of
gestation (the remainder largely via yolk sac in early pregnancy and
a transamniotic route). Therefore, by definition, net flux across
the placenta over gestation in a pregnancy with a small baby will
have been lower compared with that in a pregnancy with a larger
baby. A key question is whether, in a pregnancy affected by FGR
(or in the opposite condition of fetal overgrowth), the altered net
flux across the placenta is a cause or consequence of the abnormal
fetal growth. There is in fact now considerable evidence to sup-
port a causal role of dysfunctional maternofetal exchange across
the placenta in FGR; indeed, evidence of placental dysfunction is
becoming the gold standard in separating cases of small but nor-
mal babies from those with FGR.83 However, it is also becoming
increasingly clear that abnormal placental transfer in FGR may
be stratified into at least two separate pathologies relating to the
concepts of blood flow limited and membrane limited transfer as
described earlier in the chapter.
Placental dysfunction in preeclampsia and FGR primar-
ily arises from inadequate trophoblast invasion and incomplete
maternal uterine spiral artery remodelling, resulting in impaired
development and function of the uteroplacental vasculature and
abnormal formation and renewal of the syncytiotrophoblast.84,85
Abnormal spiral artery transformation leads to ischaemia/reper-
fusion injury in the placenta, inducing oxidative and nitrative
stress.86 The free radicals generated may also impair syncytiotro-
phoblast development and function. This aetiology could lead to a
number of different placental abnormalities resulting in different
disease phenotypes of FGR. These can be broadly categorised as (i)
vascular FGR (caused by abnormal uterine and myometrial artery
function, with consequent inadequate blood flow to the placenta)
and (ii) nonvascular FGR (caused by defective syncytiotropho-
blast function).
Raised vascular resistance in FGR is detected clinically by
abnormal Doppler ultrasound flow-velocity waveforms in the
uterine and umbilical arteries.84 Abnormal vascular anatomy87
and impaired myometrial and chorionic plate vascular tone reg-
ulation88-91 probably underlie this raised vascular resistance. As
described previously, reduced blood flow through the placenta
particularly alters the diffusion gradient for transfer of small
lipophilic solutes such as O2 and CO2 across the placenta and so
directly restricts fetal growth.
However, FGR is found in the absence of abnormal uter-
ine or umbilical artery Doppler waveforms,83,85 suggesting that
reduced blood flow is not the direct cause of reduced maternofetal
exchange in such pregnancies. Furthermore, some features of
FGR cannot be ascribed to abnormal blood flow (e.g., umbilical
vein plasma amino acid concentrations in FGR are significantly
lower than those in normally grown babies)70; such relatively
large hydrophilic molecules would have too low a permeability
across the placenta for their transfer to be significantly affected
by flow. It therefore seems that there is a set of FGR pregnan-
cies in which defective syncytiotrophoblast function is the major
cause of reduced maternofetal transfer. This is supported by data
showing that the activity of several syncytiotrophoblast plasma
ers are decreased in FGR (reviewed by Hayward5). It is difficult to
show a direct cause-and-effect relationship between decreased pla-
cental transporter activity and reduced fetal growth. However, in
rats fed a low-protein diet, decreased placental amino acid trans-
porter activity precedes FGR,92 and in mice, genetically altering
the activity of the placental system A amino acid transporter is
followed by FGR.93
Changes in transporter activity may directly arise from free
radical damage to the syncytiotrophoblast in early pregnancy after
abnormal transformation of spiral arteries. Alternatively, placental
transporter activity is regulated by growth factors and hormones
such as IGF-1 and leptin, and it is clear that there are changes in
maternal plasma concentrations of these and of placental receptor
number and activity in FGR which would be compatible with
(e.g., reduced system A amino acid transporter activity3). A fur-
ther causative mechanism could be via nutrient sensing proteins in
the syncytiotrophoblast such as mTOR. In vitro experiments with
human placental fragments or trophoblast cells show that inhibi-
tion of mTOR markedly reduced amino acid transporter activ-
ity,94,95 and it has also been shown that placental mTOR signalling
activity is downregulated in FGR.95
Although the activity of some transporters is reduced in pla-
centas from FGR pregnancies, that of others is unchanged,5 and
indeed one, the syncytiotrophoblast BM Ca2+ATPase, is actually
increased.96 This suggests that the change in syncytiotrophoblast
transporter activity in FGR is discrete rather than being a result
of generalised tissue damage. There is evidence from both human
and animal studies that placental transporter activity adapts in
relation to fetal growth, putatively to return an abnormal growth
pattern back to normal (i.e., there is increased activity per mil-
ligram of placental membrane protein when growth is reduced
compared with normal and vice versa.97,98 It could be that non-
vascular FGR related to syncytiotrophoblast dysfunction is at least
in part because of a failure of such adaptation.
There are currently no treatments for FGR other than early
delivery. However, there are clinical trials in progress of two thera-
pies designed to improve uteroplacental blood flow – sildenafil
citrate99 and vascular endothelial growth factor delivered by gene
therapy.100 The outcomes of these trials will be complicated by the
presence or absence of patients with nonvascular syncytiotropho-
blast dysfunction as well as those with uterine and myometrial vas-
cular dysfunction. Future work needs to be directed at techniques
for stratifying pregnancies with FGR into these two groups, as
well as perhaps other subgroups of placental dysfunction. Placen-
tal hormone biomarkers such as placental growth factor (PIGF)
might be useful for this, but specific tools for determining pla-
cental exchange function directly are needed. Magnetic resonance
spectroscopy could be one such tool,101 as could measurements
of placental oxygen handling using magnetic resonance imag-
ing.102 After stratification, targeting of drugs to either vasculature

or syncytiotrophoblast would likely make therapies more effective
and reduce side effects; there are now exciting data to suggest that
this might be achieved by packaging drugs in nanoparticles coated
with tissue specific homing peptides.103  refined animal models. This will also be important in characteris-
Conclusions
Although understanding of placental function in maternofetal
exchange has markedly increased over the past 50 years, there
is still much to learn. Knowledge of the individual components
and factors that determine transfer of any particular solute is
now good but improved understanding of how these contribute
to net flux of that solute across the placenta is needed. Because of
CHAPTER 8  Placental Function in Maternofetal Exchange 77
the complexity involved and the difficulty in studying the whole
system in pregnant women, this will require new methods, which
will undoubtedly include in silico modelling alongside more
ing and stratifying placental dysfunction in relation to FGR and
fetal overgrowth and enabling targeted treatments. Regulation
of maternofetal exchange is undoubtedly important, and more
work is also needed in this area, although the placental nutrient-
sensing model provides a good foundation for further studies.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 19. McNaughton TG, Power GG. The maternal-
fetal electrical potential difference: new find-
37. Powell TL, Lundquist C, Doughty IM, et  al.
Mechanisms of chloride transport across the syn-
ings and a perspective. Placenta. 1991;12(3): cytiotrophoblast basal membrane in the human
1. Desforges M, Sibley CP. Placental nutri-
185–197. placenta. Placenta. 1998;19(4):315–321.
ent supply and fetal growth. Int J Dev Biol.
20. Greenwood SL, Boyd RD, Sibley CP. Trans- 38. Sibley CP, Glazier JD, Greenwood SL,
2010;54(2-3):377–390.
trophoblast and microvillus membrane poten- et  al. Regulation of placental transfer: the
2. Brett KE, Ferraro ZM, Yockell-Lelievre J, et al.
tial difference in mature intermediate human Na(+)/H(+) exchanger—a review. Placenta.
Maternal-fetal nutrient transport in pregnancy
placental villi. Am J Physiol. 1991;265(2 Pt 2002;23(suppl A):S39–S46.
pathologies: the role of the placenta. Int J Mol
1):C460–C466. 39. Speake PF, Mynett KJ, Glazier JD, et al. Activ-
Sci. 2014;15(9):16153–16185.
21. Stulc J, Svihovec J, Drabkova J, et al. Electri- ity and expression of Na+/H+ exchanger iso-
3. Dimasuay KG, Boeuf P, Powell TL, Jansson
cal potential difference across the mid-term forms in the syncytiotrophoblast of the human
T. Placental responses to changes in the mater-
human placenta. Acta Obstet Gynecol Scand. placenta. Pflugers Arch. 2005;450(2):123–130.
nal environment determine fetal growth. Fron-
1978;57(2):125–126. 40. Johansson M, Glazier JD, Sibley CP, et  al.
tiers Physiol. 2016;7:12.
22. Mellor DJ, Cockburn F, Lees MM, Blagden A. Activity and protein expression of the Na+/
4. Atkinson DE, Boyd RDH, Sibley CP. Placen-
Distribution of ions and electrical potential H+ exchanger is reduced in syncytiotropho-
tal transfer. In: Neill JD, ed. Knobil and Neil’s
differences between mother and fetus in the blast microvillous plasma membranes isolated
physiology of reproduction. 2nd ed. San Diego:
human at term. J Obstet Gynaecol Br Com- from preterm intrauterine growth restric-
Elsevier; 2006:2787–2846.
monw. 1969;76(11):993–998. tion pregnancies. J Clin Endocrinol Metab.
5. Hayward CE, Jones RL, Sibley CP. Mecha-
23. Schroder HJ. Comparative aspects of placen- 2002;87(12):5686–5694.
nisms of transfer across the human placenta.
tal exchange functions. Eur J Obstet Gynecol 41. Burd LI, Jones Jr MD, Simmons MA,
In: Polin RA, Abman SH, Rowitch DH, et al.,
Reprod Biol. 1995;63(1):81–90. et  al. Placental production and foetal utili-
eds. Fetal and neonatal physiology. 5th ed. Phil-
24. Flexner LB, Cowie DB, et al. The permeability sation of lactate and pyruvate. Nature.
adelphia: Elsevier; 2017:121–133.
of the human placenta to sodium in normal 1975;254(5502):710–711.
6. Dilworth MR, Sibley CP. Review: transport
and abnormal pregnancies and the supply of 42. Nicolaides KH, Economides DL, Soothill PW.
across the placenta of mice and women. Pla-
sodium to the human fetus as determined Blood gases, pH, and lactate in appropriate- and
centa. 2013;34(suppl):S34–S39.
with radioactive sodium. Am J Obstet Gynecol. small-for-gestational-age fetuses. Am J Obstet
7. Greenwood SL, Sibley CP. In  vitro methods
1948;55(3):469–480. Gynecol. 1989;161(4):996–1001.
for studying human placental amino acid
25. Barcroft J. Researches on prenatal life. Part 1. 43. Settle P, Mynett K, Speake P, et al. Polarized
transport placental villous fragments. Methods
Oxford, United Kingdom: Blackwell Scientific lactate transporter activity and expression in
Mol Med. 2006;122:253–264.
Publications; 1947. the syncytiotrophoblast of the term human
8. Glazier JD, Sibley CP. In  vitro methods for
26. Stulc J, Stulcova B. Asymmetrical transfer of placenta. Placenta. 2004;25(6):496–504.
studying human placental amino acid trans-
inert hydrophilic solutes across rat. Placenta 44. Settle P, Sibley CP, Doughty IM, et  al. Pla-
port: placental plasma membrane vesicles.
Am J Physiol. 1993;265(3 Pt 2):R670–R675. cental lactate transporter activity and expres-
Methods Mol Med. 2006;122:241–252.
27. Stulc J, Stulcova B. Effect of NaCl load sion in intrauterine growth restriction. J Soc
9. Firth JA, Leach L. Not trophoblast alone: a
administered to the fetus on the bidirectional Gynecol Investig. 2006;13(5):357–363.
review of the contribution of the fetal micro-
movement of 51Cr-EDTA across rat pla- 45. Stulc J, Stulcova B, Sibley CP. Evidence for
vasculature to transplacental exchange. Pla-
centa. Am J Physiol. 1996;270(5 Pt 2):R984– active maternal-fetal transport of Na+ across
centa. 1996;17(2-3):89–96.
R989. the placenta of the anaesthetized rat. J Physiol.
10. Faber JJ, Anderson DF. Model study of pla-
28. Jauniaux E, Jurkovic D, Campbell S. Current 1993;470:637–649.
cental water transfer and causes of fetal water
topic: in  vivo investigation of the placental 46. Marino GI, Kotsias BA. Expression of the epi-
disease in sheep. Am J Physiol. 1990;258(5 Pt
circulations by Doppler echography. Placenta. thelial sodium channel sensitive to amiloride
2):R1257–R1270.
1995;16(4):323–331. (ENaC) in normal and preeclamptic human
11. Willis DM, O’Grady JP, Faber JJ, Thorn-
29. Bauer C, Ludwig I, Ludwig M. Different placenta. Placenta. 2013;34(2):197–200.
burg KL. Diffusion permeability of cyano-
effects of 2.3 diphosphoglycerate and adenos- 47. Johansson M, Karlsson L, Wennergren M,
cobalamin in human placenta. Am J Physiol.
ine triphosphate on oxygen affinity of adult et al. Activity and protein expression of Na+/
1986;250(3 Pt 2):R459–R464.
and foetal human haemoglobin. Life Sci Pt 1 K+ ATPase are reduced in microvillous syn-
12. Thornburg KL, Burry KJ, Adams AK, et  al.
Physiol. 1968;7(23P1):1339–1343. cytiotrophoblast plasma membranes isolated
Permeability of placenta to inulin. Am J Obstet
30. Edelstone DI, Peticca BB, Goldblum LJ. from pregnancies complicated by intrauterine
Gynecol. 1988;158(5):1165–1169.
Effects of maternal oxygen administration on growth restriction. J Clin Endocrinol Metab.
13. Bain MD, Copas DK, Taylor A, et al. Perme-
fetal oxygenation during reductions in umbili- 2003;88(6):2831–2837.
ability of the human placenta in vivo to four
cal blood-flow in fetal lambs. Am J Obstet 48. Brown PD, Greenwood SL, Robinson J,
non-metabolized hydrophilic molecules. J
Gynecol. 1985;152(3):351–358. Boyd RD. Chloride channels of high conduc-
Physiol. 1990;431:505–513.
31. Zamudio S. High-altitude hypoxia and pre- tance in the microvillous membrane of term
14. Stulc J. Extracellular transport pathways
eclampsia. Front Biosci. 2007;1(12):2967–2977. human placenta. Placenta. 1993;14(1):103–115.
in the haemochorial placenta. Placenta.
32. Hatano H, Leichtweiss HP, Schroder H. 49. Husain SM, Mughal MZ. Mineral transport
1989;10(1):113–119.
Uptake of bicarbonate Co2 in the isolated across the placenta. Arch Dis Child. 1992;67(7
15. Brownbill P, Edwards D, Jones C, et  al.
guinea-pig placenta. Placenta. 1989;10(2): Spec No):874–878.
Mechanisms of alphafetoprotein transfer
213–221. 50. Juhlin C, Lundgren S, Johansson H, et  al.
in the perfused human placental cotyledon
33. Economides DL. Acid-base balance in the 500-Kilodalton calcium sensor regulat-
from uncomplicated pregnancy. J Clin Invest.
fetus. In: Hillier SG, Kitchener HC, Neilson ing cytoplasmic Ca2+ in cytotrophoblast
1995;96(5):2220–2226.
JP, eds. Scientific essentials of reproductive medi- cells of human placenta. J Biol Chem.
16. Stulc J, Stulcova B, Smid M, Sach I. Par-
cine. London: W.B. Saunders; 1996:376–385. 1990;265(14):8275–8279.
allel mechanisms of Ca++ transfer across
34. Robson SC. Maternal respiratory and cardio- 51. Suzuki Y, Kovacs CS, Takanaga H, et al. Cal-
the perfused human placental cotyledon.
vascular changes during pregnancy. In: Hillier cium channel TRPV6 is involved in murine
Am J Obstet Gynecol. 1994;170(1 Pt 1):
SG, Kitchener HC, Neilson JP, eds. Scientific maternal-fetal calcium transport. J Bone Miner
162–167.
Essentials of Reproductive Medicine. London: Res. 2008;23(8):1249–1256.
17. Doughty IM, Glazier JD, Greenwood SL,
W.B. Saunders; 1996:443–454. 52. Yang H, Kim TH, An BS, et  al. Differential
et  al. Mechanisms of maternofetal chloride
35. Soothill PW, Nicolaides KH, Rodeck CH, expression of calcium transport channels in
transfer across the human placenta per-
Campbell S. Effect of gestational age on placenta primary cells and tissues derived from
fused in  vitro. Am J Physiol. 1996;271(6 Pt
fetal and intervillous blood gas and acid- preeclamptic placenta. Mol Cell Endocrinol.
2):R1701–R1706.
base values in human pregnancy. Fetal Ther. 2013;367(1-2):21–30.
18. Sibley CP, Coan PM, Ferguson-Smith AC,
1986;1(4):168–175. 53. Glazier JD, Atkinson DE, Thornburg KL,
et al. Placental-specific insulin-like growth fac-
36. Shennan DB, Davis B, Boyd CA. Chloride et  al. Gestational changes in Ca2+ transport
tor 2 (Igf2) regulates the diffusional exchange
transport in human placental microvillus across rat placenta and mRNA for calbi-
characteristics of the mouse placenta. Proc Natl
membrane vesicles. I. Evidence for anion ndin9K and Ca(2+)-ATPase. Am J Physiol.
Acad Sci U S A 25. 2004;101(21):8204–8208.
exchange. Pflugers Arch. 1986;406(1):60–64. 1992;263(4 Pt 2):R930–R935.

77.e1
77.e2 References
54. Lee GS, Lee KY, Choi KC, et al. Phenotype
of a calbindin-D9k gene knockout is compen-
sated for by the induction of other calcium
transporter genes in a mouse model. J Bone
Miner Res. 2007;22(12):1968–1978.
55. Belkacemi L, Gariepy G, Mounier C, et  al.
Calbindin-D9k (CaBP9k) localization and
levels of expression in trophoblast cells
from human term placenta. Cell Tissue Res.
2004;315(1):107–117.
56. Belkacemi L, Gariepy G, Mounier C, et  al.
Expression of calbindin-D28k (CaBP28k) in
trophoblasts from human term placenta. Biol
Reprod 68(6):1943–1950.
57. Fisher GJ, Kelley LK, Smith CH. ATP-depen-
dent calcium transport across basal plasma
membranes of human placental trophoblast.
Am J Physiol. 1987;252(1 Pt 1):C38–C46.
58. Borke JL, Caride A, Verma AK, et al. Calcium
pump epitopes in placental trophoblast basal
plasma membranes. Am J Physiol. 1989;257(2
Pt 1):c341–c346.
59. Strid H, Powell TL. ATP-dependent Ca2+
transport is up-regulated during third trimes-
ter in human syncytiotrophoblast basal mem-
branes. Pediatr Res. 2000;48(1):58–63.
60. Strid H, Care A, Jansson T, Powell T. Para-
thyroid hormone-related peptide (38–94)
amide stimulates ATP-dependent calcium
transport in the basal plasma membrane of
the human syncytiotrophoblast. J Endocrinol.
2002;175(2):517–524.
61. Johnson LW, Smith CH. Monosaccha-
ride transport across microvillous mem-
brane of human placenta. Am J Physiol.
1980;238(5):C160–C168.
62. Johnson LW, Smith CH. Glucose transport
across the basal plasma membrane of human
placental syncytiotrophoblast. Biochimica et
Biophysica Acta 26. 1985;815(1):44–50.
63. Ericsson A, Hamark B, Powell TL, Jansson T.
Glucose transporter isoform 4 is expressed
in the syncytiotrophoblast of first trimester
human placenta. Hum Reprod. 2005;20(2):
521–530.
64. Gude NM, Stevenson JL, Rogers S, et  al.
GLUT12 expression in human placenta in
first trimester and term. Placenta. 2003;24(5):
566–570.
65. Jansson T, Wennergren M, Illsley NP. Glu-
cose transporter protein expression in human
placenta throughout gestation and in intra-
uterine growth retardation. J Clin Endocrinol
Metab. 1993;77(6):1554–1562.
66. Brown K, Heller DS, Zamudio S, Illsley
NP. Glucose transporter 3 (GLUT3) protein
expression in human placenta across gestation.
Placenta. 2011;32(12):1041–1049.
67. Hahn T, Hartmann M, Blaschitz A, et  al.
Localisation of the high affinity facilitative
glucose transporter protein GLUT 1 in the
placenta of human, marmoset monkey (Calli-
thrix jacchus) and rat at different developmen-
tal stages. Cell Tissue Res. 1995;280(1):49–57.
68. Ashmead GG, Kalhan SC, Lazebnik N, Nua-
mah IF. Maternal-fetal substrate relationships
in the third trimester in human pregnancy.
Gynecol Obstet Investig. 1993;35(1):18–22.
69. Ericsson A, Hamark B, Jansson N, et al. Hor-
monal regulation of glucose and system A
amino acid transport in first trimester placen-
tal villous fragments. Am J Physiol Regul Integr
Comp Physiol. 2005;288(3):R656–R662.
70. Cetin I, Marconi AM, Corbetta C, et al. Fetal
amino acids in normal pregnancies and in preg-
nancies complicated by intrauterine growth
retardation. Early Hum Dev. 1992;29(1-3):
183–186.
71. Sooranna SR, Burston D, Ramsay B, Steer PJ.
Free amino acid concentrations in human first
and third trimester placental villi. Placenta.
1994;15(7):747–751.
72. Lewis RM, Brooks S, Crocker IP, et al. Review:
Modelling placental amino acid transfer—
from transporters to placental function. Pla-
centa. 2013;34(suppl):S46–S51.
73. Cleal JK, Glazier JD, Ntani G, et  al. Facili-
tated transporters mediate net efflux of amino
acids to the fetus across the basal membrane
of the placental syncytiotrophoblast. J Physiol.
2011;589(Pt 4):987–997.
74. Widdows KL, Panitchob N, Crocker IP,
et  al. Integration of computational modeling
with membrane transport studies reveals new
insights into amino acid exchange transport
mechanisms. FASEB J. 2015;29(6):2583–
2594.
75. Panitchob N, Widdows KL, Crocker IP,
et  al. Computational modelling of amino
acid exchange and facilitated transport in
placental membrane vesicles. J Theoret Biol.
2015;21(365):352–364.
76. Pearse BM. Coated vesicles from human pla-
centa carry ferritin, transferrin, and immu-
noglobulin G. Proc Natl Acad Sci U S A.
1982;79(2):451–455.
77. Ockleford CD, Clint JM. The uptake of IgG
by human placental chorionic villi: a cor-
related autoradiographic and wide aperture
counting study. Placenta. 1980;1(2):91–111.
78. King BF. Absorption of peroxidase-conjugated
immunoglobulin G by human placenta: an
in vitro study. Placenta. 1982;3(4):395–406.
79. Leach L, Eaton BM, Firth JA, Contractor SF.
Immunogold localisation of endogenous
immunoglobulin-G in ultrathin frozen sec-
tions of the human placenta. Cell Tissue Res.
1989;257(3):603–607.
80. Leach L, Eaton BM, Firth JA, Contractor
SF. Immunocytochemical and labelled tracer
approaches to uptake and intracellular routing
of immunoglobulin-G (IgG) in the human
placenta. Histochem J. 1991;23(10):444–449.
81. Lin CT. Immunoelectron microscopy localiza-
tion of immunoglobulin G in human placenta.
J Histochem Cytochem. 1980;28(4):339–346.
82. Szlauer R, Ellinger I, Haider S, et al. Functional
expression of the human neonatal Fc-receptor,
hFcRn, in isolated cultured human syncytio-
trophoblasts. Placenta. 2009;30(6):507–515.
83. Benton S, McCowan L, Grynspan D, et  al.
Placental growth factor as a marker for placen-
tal intrauterine growth restriction. Reprod Sci.
2016;23. 306A–A.
84. Brosens I, Pijnenborg R,Vercruysse L, Romero R.
The “great obstetrical syndromes” are associ-
ated with disorders of deep placentation. Am J
Obstet Gynecol. 2011;204(3):193–201.
85. Sibley CP, Turner MA, Cetin I, et al. Placen-
tal phenotypes of intrauterine growth. Pediatr
Res. 2005;58(5):827–832.
86. Burton GJ, Jauniaux E. Oxidative stress. Best
Pract Res Clin Obstet Gynaecol. 2011;25(3):
287–299.
87. Junaid TO, Brownbill P, Chalmers N, et  al.
Fetoplacental vascular alterations associ-
ated with fetal growth restriction. Placenta.
2014;35(10):808–815.
88. Sweeney M, Wareing M, Mills TA, et  al.
Characterisation of tone oscillations in pla-
cental and myometrial arteries from nor-
mal pregnancies and those complicated by
pre-eclampsia and growth restriction. Pla-
centa. 2008;29(4):356–365.
89. Wareing M, Greenwood SL, Fyfe GK, Baker
PN. Reactivity of human placental chorionic
plate vessels from pregnancies complicated by
intrauterine growth restriction (IUGR). Biol
Reprod. 2006;75(4):518–523.
90. Mills TA, Greenwood SL, Johnstone ED,
et  al. Mechanical and receptor-mediated
responses of placental chorionic plate arteries
are altered in fetal growth restriction. Reprod
Sci. 2012;19(S3):314A–A.
91. Jones S, Bischof H, Lang I, et al. Dysregulated
flow-mediated vasodilatation in the human
placenta in fetal growth restriction. J Physiol.
2015;593(14):3077–3092.
92. Jansson N, Pettersson J, Haafiz A, et al. Down-
regulation of placental transport of amino
acids precedes the development of intrauter-
ine growth restriction in rats fed a low protein
diet. J Physiol. 2006;576(Pt 3):935–946.
93. Constancia M, Hemberger M, Hughes J, et al.
Placental-specific IGF-II is a major modu-
lator of placental and fetal growth. Nature.
2002;417(6892):945–948.
94. Roos S, Kanai Y, Prasad PD, et al. Regulation
of placental amino acid transporter activity by
mammalian target of rapamycin. Am J Physiol
Cell Physiol. 2009;296(1):C142–C150.
95. Roos S, Jansson N, Palmberg I, et  al. Mam-
malian target of rapamycin in the human
placenta regulates leucine transport and is
down-regulated in restricted fetal growth. J
Physiol. 2007;582(Pt 1):449–459.
96. Strid H, Bucht E, Jansson T, et al. ATP depen-
dent Ca2+ transport across basal membrane of
human syncytiotrophoblast in pregnancies
complicated by intrauterine growth restriction
or diabetes. Placenta. 2003;24(5):445–452.
97. Godfrey KM, Matthews N, Glazier J, et  al.
Neutral amino acid uptake by the microvil-
lous plasma membrane of the human pla-
centa is inversely related to fetal size at birth
in normal pregnancy. J Clin Endocrinol Metab.
1998;83(9):3320–3326.
98. Coan PM, Angiolini E, Sandovici I, et  al.
Adaptations in placental nutrient trans-
fer capacity to meet fetal growth demands
depend on placental size in mice. J Physiol.
2008;586(18):4567–4576.
99. Ganzevoort W, Alfirevic Z, von Dadelszen P,
et  al. STRIDER: Sildenafil Therapy In Dis-
mal prognosis Early-onset intrauterine growth
Restriction—a protocol for a systematic review
with individual participant data and aggregate
data meta-analysis and trial sequential analy-
sis. Syst Rev. 2014;3:23.
100. Sheppard M, Spencer RN, Ashcroft R, et  al.
Ethics and social acceptability of a proposed
clinical trial using maternal gene therapy to
treat severe early-onset fetal growth restric-
tion. Ultrasound Obstet Gynecol. 2016;47(4):
484–491.
101. Denison FC, Semple SI, Stock SJ, et al. Novel
use of proton magnetic resonance spectroscopy
(1HMRS) to non-invasively assess placental
metabolism. PloS One. 2012;7(8):e42926.
102. Huen I, Morris DM, Wright C, et  al. R1
and R2 * changes in the human placenta in
response to maternal oxygen challenge. Magn
Reson Med. 2013;70(5):1427–1433.
103. King A, Ndifon C, Lui S, et  al. Tumor-
homing peptides as tools for targeted deliv-
ery of payloads to the placenta. Sci Adv.
2016;2(5):e1600349.
9
Placental Pathology and Implications for
Fetal Medicine
NEIL J. SEBIRE AND JOHN C. KINGDOM
KEY POINTS
• Pathological examination of the placenta may provide use-
ful information regarding the underlying mechanisms of a
range of pregnancy complications that may guide future
management and improve understanding of disease patho-
physiology.
• Placentas should be submitted for examination by special-
ist pathologists in all complicated pregnancies according to
national and local guidelines.
• Interpretation of the clinical significance of many placental
histologic changes remains difficult, and novel approaches
are required for future development in addition to traditional
histologic evaluation.
• Paraffin-embedded tissue blocks are stable for many years
at room temperature and thus may be transferred to tertiary
pathology centres if required for reassessment, using ad-
ditional histologic or DNA methods, and may also be used for
medicolegal assessment of disease causation.
• Placental evaluation should be encouraged in all cases of
intrauterine death, regardless of whether formal postmortem
fetal examination is requested.
Introduction
It has long been recognised that a wide range of disorders of
pregnancy are related to changes affecting the placenta, and
our understanding of the underlying mechanisms of many
obstetric diseases, such as fetal growth restriction (FGR)
and preeclampsia, is largely derived from pathological stud-
ies of the delivered placenta. With this in mind, the potential
benefits of a specialised placental pathology service include
improved evaluation of pathophysiological processes in spe-
cific cases, which may affect subsequent management and
recurrence risk, and as a source of material for subsequent
research. This chapter provides an overview of the role of pla-
cental pathological assessment in modern fetal medicine, with
examples in relation to antenatal diagnosis, and suggests how
this area may develop in the near future. Extensive literature is
available regarding details of specific placental pathologies in
specialist texts.1-3  for disease).8
78
Placental Pathological Assessment
The yield of significant abnormal findings from placental pathology
examination is related to the underlying clinical circumstances, and
there are therefore several recommendations published regarding indi-
cations for formal placental pathological evaluation.4,5 These largely
include all preterm deliveries and otherwise complicated pregnancies,
either associated with maternal or fetal diseases, acute compromise to
fetal health or admission to the neonatal intensive care unit (NICU).
This policy results in examination of around 10% of placentas from
unselected low-risk pregnancies, a proportion that will obviously
be much greater in tertiary referral fetal medicine centres. In addi-
tion, protocols exist describing the suggested examination approach,
including macroscopic assessment, tissue sampling and subsequent
histologic evaluation to form an overall diagnostic opinion.6,7
In the majority of cases examined as part of clinical practice,
sampling of the umbilical cord, membranes and placental paren-
chyma (normal and abnormal), including any lesions, takes place
after a period of fixation, with subsequent processing and histologic
evaluation of haematoxylin and eosin–stained slides from each tis-
sue sample. Large placental tissue diagnostic archives are therefore
available but are limited by being composed primarily of formalin-
fixed paraffin-embedded (FFPE) blocks and slides rather than fro-
zen material that is typically obtained from fresh tissue. With the
introduction of novel methods of future investigation, it is likely
that routine storage of fresh placenta samples taken immediately at
delivery may be required for analysis (typically for protein, metab-
olite or RNA studies), with obvious resource implications.
Placental histologic sections are evaluated and the findings inter-
preted in the context of details in the clinical history such as gestational
age, pregnancy complications, birth details and the initial gross pla-
cental findings (Fig. 9.1). In this regard, placental pathology reporting
is in many ways more challenging than other areas such as tumour
pathology because there are few placental histologic changes that are
pathognomonic of a specific disease; rather, interpretation is based
upon constellations of features in relation to the clinical features that are
statistically associated with particular clinical presentations. Placental
features are therefore helpful for determining the broad mechanisms
of underlying pathological processes leading to overt clinical manifes-
tations so as to improve our understanding of the pathogenesis of a
variety of complications of pregnancy (e.g., early- and late-onset pre-
eclampsia, in which maternal factors can considerably affect the risk
 CHAPTER 9  Placental Pathology and Implications for Fetal Medicine 79

A B

C D
• Fig. 9.1  Photomicrographs of placental histology demonstrating extensive villitis of unknown aetiology.
(A and B; haematoxylin and eosin (H&E) original magnifications ×20 and ×100, respectively) and congeni-
tal toxoplasmosis. C and D; H&E original magnifications ×40 and ×200, respectively.)

However, because histologic evaluation involves subjective assess-

ment, a relationship exists between pathologist expertise or experi-
ence and placental reporting utility; 40% of placental cases reported
by nonspecialist pathologists were erroneous compared with subspe-
cialty assessment, including omissions and false-positive findings.9
It is therefore recommended that multidisciplinary placental teams
are established in specialist centres, with close interaction of the
obstetric and perinatal pathology reporting staff, and regular discus-
sion of findings in relation to both antenatal ultrasound findings
and clinical outcomes. It is hoped that recent efforts regarding con-
sensus statements for placental reporting will reduce interpatholo-
gist variability and allow improved studies of interpretation.10

Prenatal Assessment of Specific Placental

Pathologies
The ability to identify pregnancies with, or at risk for, a range
of placental pathologies has advanced considerably over the past
30 years, with the application of both real-time and colour Dop-
pler ultrasound to evaluate both the placenta and its maternal and
fetal circulations. The definitive placenta is formed by the end of
the first trimester, such that many aspects of gross anatomy, such
as shape, size, cord insertion and implantation site, can be deter-
mined from the first trimester, using either simple two-dimen-
sional methods11 or three-dimensional volume assessment.12
During the second trimester, the placenta is larger and thus easier
to assess using abdominal ultrasound imaging including Doppler
assessment of the uteroplacental circulation13 (Figs. 9.2 to 9.4).
Because maternal vascular malperfusion (MVM) is the most
common type of placental pathology associated with early-onset
preeclampsia and FGR, screening programs to identify women at
CI
• Fig. 9.2  Normal anterior placenta at 19 weeks’ gestation demonstrating
a central placental cord insertion and normal sonographic appearances.
most risk have focused on incorporating uterine artery Doppler
studies into screening algorithms that include clinical risk scores
and biomarkers, such as serum placenta growth factor (PlGF).14
This combined approach has the potential to provide improved
precision in screening for both preventable stillbirth caused by
placental disease15 and FGR,16 although to date, no interventions
have delivered improved perinatal outcome.
Many large-scale research screening programs such as those
referenced lack placental pathology findings, which is under-
standable because of the associated cost per case. However, the
80 SE C T I O N 2     The Placenta

A B

C D
• Fig. 9.3  Normal placental appearances on routine 20-week sonographic assessment, including various
methods of placental measurements.

A B

C D
• Fig. 9.4  Normal (A) and abnormal (B) uterine artery Doppler waveforms. Abnormal flow is associated
with fetal growth restriction with placental hyperinflation (C) caused by maternal vascular malperfusion and
placental infarction (D).

inherent variability in underlying placental pathophysiology
associated with stillbirth and FGR17 has the potential to con-
found the accuracy of screening. Uterine artery Doppler may
predict the placental features associated with MVM; however,
in a study of severe early-onset FGR pregnancies with abnormal
umbilical artery Doppler, 10% had normal uterine artery Dop-
pler studies; these had a greater risk for placental diseases unre-
lated to MVM but with significant recurrence rates (e.g., chronic
histiocytic intervillositis (CHI) and massive perivillous fibrin
deposition).18
 CHAPTER 9  Placental Pathology and Implications for Fetal Medicine
• Fig. 9.5 Abnormally appearing ‘hyperinflated’ placenta in association
with low first trimester serum placental associated protein A (PAPP-A) and
early-onset fetal growth restriction (see Video 9.1).
8 cm
• Fig. 9.6 Massive placentomegaly with nonimmune fetal hydrops at
32 weeks’ gestation caused by fetomaternal haemorrhage. The mother
showed features of ‘mirror syndrome’.
It has further been suggested that many placentas from early-
onset FGR or preeclampsia exhibit abnormalities of size, shape,
cord insertion or parenchyma, which may be detectable antena-
tally. For example, ‘chorion regression’ may be associated with a
‘jelly-like’ hyperinflated placenta,19 and several specific placental
pathologies have sonographically identifiable features. However,
the role of ‘placental sonography’ beyond individual case assess-
ment remains uncertain. Illustrative examples of the role of pla-
cental sonography in identifying pathologies of the placenta,
umbilical cord and membranes are provided (Figs. 9.5 and 9.6
and Video 9.1). 
Classification of Placental Lesions
One of the historical difficulties of interpreting literature relating
to placental pathology has been inconsistent use of terminologies
by clinicians, scientists and pathologists and use of multiple labels
for the same entity. To address this issue, there have been recent
81
 Classification of Placental Pathologies
• BOX 9.1 
Placental vascular processes
• Maternal stromal-vascular lesions
• Malperfusion (including distal villous hypoplasia, accelerated villous
maturation and infarct)
• Loss of integrity (including abruptio placenta and marginal
abruption)
• Fetal stromal-vascular lesions
• Developmental (including delayed villous maturation and dysmorphic
villi)
• Malperfusion (including global and segmental lesions)
• Loss of integrity (including fetal haemorrhage and fetomaternal
haemorrhage) 
Placental inflammatory-immune processes
• Infectious inflammatory lesions
• Acute (including maternal and fetal inflammatory responses)
• Chronic (including villitis and intervillositis)
• Immune or idiopathic inflammatory lesions
• Including villitis of unknown aetiology (chronic villitis, chronic
chorioamnionitis, lymphoplasmacytic deciduitis, eosinophil T-cell fetal
vasculitis) and chronic histiocytic intervillositis 
Other placental processes
• Massive perivillous fibrin(oid) deposition (maternal floor infarction)
• Morbidly adherent placentas (accreta)
• Meconium-associated changes
Adapted from Redline RW. Classification of placental lesions. Am J Obstet Gynecol 213
(4 suppl):S21-S28, 2015.
collaborative approaches to unify the use of terminology. The cur-
rently recommended classification system is being used in this
chapter (Box 9.1).10 
Interpretation of Lesions
Some placental lesions demonstrate characteristic and unique
histologic features, allowing definitive diagnosis regardless of
clinical circumstances or other factors. However, such enti-
ties represent only a minority of histologic changes identi-
fied in the placenta, with the majority of lesions also being
encountered in clinically uncomplicated normal pregnancies,
although being more or less frequent in association with spe-
cific pregnancy complications. This overlap results in consis-
tent data describing risks or odds ratios for the strength of
association among specific histologic features and specific
obstetric disorders on a population basis, but accurate inter-
pretation of the clinical significance of specific findings in an
individual case is fraught with difficulties. The details pro-
vided summarise the available data but should be interpreted
with these above in mind. 
Categories of Placental Pathologies
In this section, entities which are relatively common or important
are described, focusing particularly on their relationship to ante-
natal detection and management of common clinical conditions.
Extensive literature is available providing details of the full spec-
trum of pathologies.1-3 The categories broadly map to Box 9.1 but
for ease of discussion are described in terms of their mechanisms
and clinical significance.
82 SE C T I O N 2     The Placenta
Placental portion anterior
Succenturiate lobe posterior
• Fig. 9.7  Antenatal sonogram of a case of vasa previa with a posterior
succenturiate lobe (LT), with colour Doppler demonstrating fetal vessels
running outside of the placenta connecting the placental masses (inset)
(see Video 9.2).
Abnormalities of Placental Development
The details of normal placental development are described in
Chapter 7. There are a range of macroscopically identifiable dis-
orders which are believed to be a consequence of gross abnormal-
ities of the process of initial implantation or subsequent growth
of the placental disk, resulting in abnormalities in placental
shape, architecture or umbilical cord insertion site. These lesions
are not usually associated with specific histologic abnormalities
(although it has been suggested that abnormal placental regres-
sion may also be related to villous changes) but may be associ-
ated with increased risk for certain pregnancy complications.
Eccentric or velamentous insertion and vasa praevia. The
umbilical cord normally inserts into the central portion of the
placental disk chorionic plate. Minor degrees of peripheral
insertion are usually of no clinical significance, but because of
rarefaction of the process of dichotomous branching of the sur-
face chorionic plate arteries, the opposite side of the placenta
from a marginal cord may be hypovascularized, thus reducing
placental efficiency. At the extremes, umbilical cord–derived
vessels may leave the margins of the placenta, found in around
1% of pregnancies, termed velamentous insertion; a variant of
this is when vessels from a marginal cord insertion traverse
within the membranes to accessory, or succenturiate, lobes,
so as to connect them in a functional sense, to the fetus. The
term vasa previa describes this arrangement when the vessels
run closer to, or over, the internal os of the cervix. The fetus
is then at risk for hypovolemic shock during vaginal delivery
because these vessels may be damaged as labour advances, espe-
cially at the time of membrane rupture. Antenatal screening
using ultrasound (for variants of placental and cord anatomy)
and diagnosis (using transvaginal colour Doppler ultrasound)
are lifesaving for the fetus because elective caesarean section
increases fetal survival to more than 95% (Figs. 9.7 and 9.8
and Video 9.2).  trally perfused by a spiral artery branch. These functional units
Bilobata, succenturiata and other shape abnormalities.
Although the normal human placenta is discoid, there are numer-
ous variations in shape, most of which are not associated with
significant or consistent clinical complications. These include
• Fig. 9.8  Delivered placenta from a case of vasa previa with numerous
large chorionic vessels running within the fetal membranes.
placenta membranacea, in which placental villous tissue persists
extensively around the gestational sac; placenta fenestrate, in
which there is focal deficiency of parenchyma; placenta bilobata,
in which two distinct disks are present usually with central cord
insertion between the two; and placenta succenturiata, in which
one of more accessory lobes is present joined to the main disk by
intramembranous vessels. These deviations from a spherical pla-
centa and central cord insertion have been suggested to reduce
efficiency in some studies20 but do not threaten fetal survival
unless additional pathologies are present. However, because of the
abnormal anatomy, either placental parenchymal tissue or chori-
onic vessels may be present over the cervical os, with associated
risks of trauma and haemorrhage. 
Circummarginate or circumvallate placenta. As part of nor-
mal placental development, the edge of the placental parenchy-
mal disk (basal plate) corresponds to the edge of the chorionic
plate and hence the smooth junction of the amniotic cavity with
the placenta. If this process is defective the edge of the chori-
onic plate may no longer be sited over the placental parenchy-
mal edge, resulting in either a smooth or ridged, abnormally
sited junction (circummarginate and circumvallate placenta-
tion, respectively). To some degree, this affects around 1% to
5% of placentas with little functional significance but has been
associated with increased rates of antepartum haemorrhage and
preterm delivery. The normal marginal sinus, where intervillous
blood reenters the uteroplacental veins, can be imaged by ultra-
sound and may on occasion be prominent. This is of no conse-
quence unless sited close to the internal os but can be mistaken
for marginal abruption. 
Abnormalities of Placental Perfusion
To function normally, maternal blood must flow, at the appro-
priate rate and pressure, into and through the intervillous space
(uteroplacental circulation) surrounding the chorionic villi;
this blood supply is arranged as functional units, each cen-
may be termed placentomes, and up to 50 exist in a normal-
term placenta. Effective transplacental diffusion also requires
adequate perfusion from the fetus (fetoplacental circulation).
It will be apparent therefore that these processes may be
 CHAPTER 9  Placental Pathology and Implications for Fetal Medicine
defective at any level, resulting in chronic fetal hypoxia and
impaired fetal growth, and it is therefore logical to discuss
these according to the anatomical area predominantly affected.
Abnormalities of Uteroplacental Flow
Fetal growth restriction and preeclampsia. Pathological studies
of products of conception, delivered placentas and placental
bed biopsies have demonstrated that abnormalities of normal
establishment of the uteroplacental circulation are associated
with, and likely the underlying pathophysiological process
responsible for, a range of pregnancy complications ranging
from early pregnancy failure (miscarriage), preeclampsia and
FGR. It is now generally accepted that in the first trimester
decidual vessels become occluded by extravillous endovascular
trophoblast to protect the early conceptus from pressure
and oxygen-related damage, with failure of such ‘plugging’
one of the causes of miscarriage, for example, in association
with antiphospholipid antibodies. After initial endovascular
invasion, during the second trimester, the trophoblast masses
recanalize, and both endovascular and interstitial extravillous
trophoblast of the implantation site combine to convert the
distal muscular spiral artery branches into poorly muscularised,
low-resistance, high-flow uteroplacental vessels supplying
the intervillous space. Failure of this phase of development is
associated with abnormally reactive uteroplacental vessels with
increased flow resistance and reduced and abnormally pulsatile
intervillous flow.21 These changes have secondary effects
on chorionic villus structure and function, the combination
of which results in FGR, preeclampsia or both. Although in
most cases, the cause of the defective implantation remains
unknown, in a minority, there may be underlying conditions,
such as maternal connective tissue diseases, which are associated
with identical features.
Pathological evaluation of the delivered placenta in such cases
may demonstrate a range of histologic features, which are now
recognised as ‘typical’ changes of FGR or preeclampsia described
collectively as MVM. These changes include reduced placental
size and surface area, presence of decidual vasculopathy (fibri-
noid necrosis or macrophages and inflammatory cells within the
vessel wall (atherosis)), villous infarction, fetoplacental vasocon-
striction, reduced villous branching and hypovascularity, acceler-
ated maturation and a range of functional alterations. Although
many such changes are subjective and may be identified to some
degree in clinically uncomplicated pregnancies at term, the con-
stellation of all features, especially in iatrogenic preterm deliver-
ies, is highly suggestive of underlying MVM. The alterations in
maternoplacental flow or placental shape and size are detectable
antenatally based upon uterine artery Doppler and placental mor-
phology assessment, and the secondary changes in fetoplacental
flow, especially maldevelopment of the gas exchanging peripheral
villi, result in changes detectable using umbilical artery Doppler
sonography (Fig. 9.9).  For example, extensive entrapment of a long, hypercoiled umbil-
Abruption and retroplacental haemorrhage. The placenta is
normally firmly adherent to the uterus at the basal plate until the
third stage of labour. If abnormally premature separation occurs,
either centrally or at the margin, the consequence is retroplacen-
tal haemorrhage, which most often tracks along the uteropla-
cental junction, resulting in vaginal bleeding, but is occasionally
‘concealed’, being retained retroplacentally. In addition, because
separation has occurred, no functional uteroplacental circulation
remains in these areas, with associated complete loss of functional
83
• Fig. 9.9 Multifocal basal placental infarction presenting at 37 weeks’
gestation with the features of late-onset fetal growth restriction.
capacity of the supplied villous areas, which may result in isch-
aemic necrosis (infarction) of the overlying placenta. It should
be noted that although in some cases, unequivocal abnormal ret-
roplacental haemorrhage with secondary overlying changes may
be identified in the delivered placenta, in other cases, especially
with marginal separation and vaginal bleeding, the delivered
placenta may not demonstrate characteristic changes of abrup-
tion even in the presence of a typical clinical history. Ultrasound
may occasionally diagnose chronic abruption,22 although often
abruption is such an acute event in labour and delivery that clin-
ical management and delivery override the utility of ultrasound
imaging (Fig. 9.10). 
Abnormalities of Fetoplacental Flow
It has been demonstrated that after primary abnormalities of
uteroplacental perfusion, secondary changes in fetoplacental
perfusion develop, such as with typical FGR caused by MVM.
However, in addition, morphological changes may also occur
indicating reduced fetoplacental flow in the absence of any
maternal abnormalities. Such changes include either the direct
documentation of chorionic vascular thrombosis or the down-
stream villous effects of proximal fetovascular occlusion, namely
clusters of chorionic villi with normal intervillous space showing
intravascular karyorrhexis, syncytial knot formation and stromal
sclerosis, according to chronicity. Such changes are within the
spectrum of fetal vascular occlusion (FVO) or fetal thrombotic
vasculopathy (FTV). When focal, they are usually of no clini-
cal significance, even though they may be reported more com-
monly in certain scenarios, such as maternal diabetes mellitus,
but occasionally may be associated with underlying fetal visceral
thrombosis or placental functional consequences if extensive.
ical cord in association with FVO lesions may suggest causality
in stillbirth. 
Primary Abnormalities of Villous Development
In some circumstances of FGR or fetal distress, there are diffuse
changes affecting fetal chorionic villi which are not associated
with typical features of MVM, and it has been suggested that such
cases are caused by primary abnormalities of fetal development.
84 SE C T I O N 2     The Placenta

A PL B

Maternal surface

C Fetal surface
D
• Fig. 9.10  Sonographic identification of a consolidated asymptomatic central concealed abruption in a
clinic setting at 30 weeks’ gestation (A and B), which progressed after 2 inpatient days, precipitating cae-
sarean delivery after steroid administration for fetal lung maturity and a favourable outcome. C, Histopa-
thology. Contrast with bedside ultrasound findings in acute abruption in a labour and delivery setting (D).

Most cases of distal villous hypoplasia and villous hypermaturity
are now believed to be changes secondary to alterations in utero-
placental flow, although it has been suggested that some could
represent primary maldevelopment.
In contrast, a generalised disorder of villous development, distal
villous immaturity (DVI), is now well recognised, being identified
as a generalised increase in villous stroma with immature appear-
ing villi containing centrally located small capillaries with paucity
of normal vasculosyncytial membranes.23 The consequence of this
histologic finding is that the diffusion distance between maternal
and fetal erythrocytes is greatly increased, impairing conductance
of carbon dioxide and oxygen. DVI may therefore contribute to
some instances of antenatal stillbirth, especially with larger fetuses
in the context of diabetes.  finally the fetal circulation, represents an infective process, most
Abnormalities Primarily Affecting the
Intervillous Space
In addition to the maternal and fetal circulations, abnormalities
affecting the normal structure or function of the intervillous space
are rare but may occur and have distinctive histologic features.
CHI is a condition characterised by the presence of large numbers
of maternal histiocytes within the intervillous space, often asso-
ciated with fibrin deposition, in the absence of known infective
cause. The aetiology is unknown but is presumed autoimmune,
particularly in view of the findings that presentation may be
throughout pregnancy, from first trimester loss through to term,
with a greater than 50% recurrence risk.
Massive perivillous fibrin deposition (MPVFD) is charac-
terised by the majority of the intervillous space being involved
by perivillous fibrin, into which trophoblast proliferates, which
separates chorionic villi and prevents normal intervillous blood
flow. Again, the exact mechanism remains uncertain, but there is
a significant recurrence risk (20%). Both of these conditions are
only reliably diagnosed on histologic examination and have no
typical ultrasonographic appearances.24 
Inflammatory Lesions
Inflammatory lesions may be infectious or noninfectious, presumed
immune mediated.
Ascending genital tract infection. Inflammation affecting the
fetal membranes overlying the cervical os, with subsequent spread
to involve the membranes more diffusely, the amniotic cavity and
often with normal vaginal or cervical commensal organisms, the
condition representing a loss of normal balance between defence
mechanisms and colonisation. An initial localised maternal
inflammatory response of the fetal membranes occurs (chorioam-
nionitis), which may result in stimulation of the onset of labour,
or the infectious process may progress if delivery does not ensue,
until the fetus mounts a systemic inflammatory response (funisi-
tis). Ascending genital tract infection is therefore a major cause of
midtrimester pregnancy loss and severe preterm delivery and even
at term may act synergistically with other insults, such as hypoxia-
ischaemia, to cause neurological damage. Although some cases
may be associated with a systemic maternal response, with fever,
there is poor correlation between clinical and histologic features. 
Villitis or intervillositis caused by haematogenous infection.
Because maternal blood supplies the intervillous space, maternal
systemic diseases may involve the placenta, leading to either col-
lections of inflammatory cells or fibrin within the intervillous
 CHAPTER 9  Placental Pathology and Implications for Fetal Medicine
• Fig. 9.11  Sonographic appearance of a placental chorioangioma includ-
ing colour Doppler identification of a large functional shunt within a 6-cm
placental chorioangioma. The fetus demonstrated signs of high-output
cardiac failure (Video 9.3).
space (e.g., malaria), or inflammation of the villi (villitis; e.g.,
cytomegalovirus). When there is villitis from an infective cause,
which may be viral, bacterial or protozoal, the placenta usually
demonstrates patchy but diffuse involvement, with florid focal vil-
litis, which may even be associated with villous necrosis or granu-
loma formation. The pattern of tissue involvement may suggest
a particular organism as the aetiology, but confirmation should
always be based on additional ancillary investigations. In some
cases of viral infection, characteristic viral cellular inclusions may
be present, making the specific diagnosis more definitive.  (Fig. 9.11 and Video 9.3).
Villitis of unknown aetiology. Villitis indicates infiltration of
chorionic villi by inflammatory cells. As noted earlier, in some
cases, the pattern of inflammation may be characteristic and the
aetiology determined to be an infectious agent. However, in the
majority of cases in which villitis is identified, there is patchy infil-
tration of groups of chorionic villi by mononuclear inflammatory
cells, mainly lymphocytes, with some macrophages, but no other
specific findings and no infectious agent is identified. Such cases
are classified as villitis of unknown aetiology (VUE). VUE may be
present in clinically normal deliveries at term but is reported more
frequently in association with complications such as FGR and pre-
eclampsia. It is now established that the majority of infiltrating
cells in VUE are of maternal origin, and it has therefore been sug-
gested that this may represent a maternofetal immune-mediated
process, similar in concept to graft rejection25 (see Fig. 9.1).  PHM). It should be noted, however, that in the first trimester, the
Tumours and Tumourlike Lesions
There are few mass lesions affecting the placenta, but there are
several entities which may be detected on antenatal sonographic
examination, which have clear histologic correlates and effects on
clinical management.
Chorioangioma. By far the commonest ‘tumour’ of the pla-
centa is chorioangioma, which represents a benign proliferation
of villous blood vessels surrounded by expanded villous stromal
tissue and trophoblast. These lesions are often highly vascular
on imaging and when large can develop functional shunts of
85
• Fig. 9.12 Sonographic appearances of a placenta at 21 weeks’ ges-
tation with severe preeclampsia, growth restriction, abnormal umbilical
artery Doppler and bilateral cystic ovarian enlargement. Note the increased
thickness and multiple small cysts. Amniocentesis revealed triploidy (par-
tial hydatidiform mole), and the pregnancy was terminated by induction
of labour.
the fetoplacental circulation. Such lesions are most often situ-
ated beneath the chorionic plate, may be single or multiple, and
can vary in size from millimetres to more than 10 cm in diam-
eter. Small lesions appear to have no direct clinical significance,
but larger or more extensive lesions may be associated with
fetal cardiac failure, polyhydramnios or nonimmune hydrops.26
Chorioangiomas may infarct in utero, resulting in spontane-
ous resolution of high-output cardiac failure. Fetal interven-
tional techniques can also be used to occlude the aberrant
arteriovenous malformation and restore normal fetal physiology
 
Hydatidiform mole and intraplacental choriocarcinoma.
Hydatidiform moles (HMs) represent genetically abnormal con-
ceptions with relative overexpression of the paternal genome,
leading to villous hydropic change and abnormal trophoblast
hyperplasia. Depending on their pathological and genetic fea-
tures, HM may be complete (CHM; diploid) or partial (PHM;
triploid), with most cases presenting with vaginal bleeding or early
pregnancy failure. However, in some cases, such as mosaic HM
and HM with a normal co-twin, the pregnancy may continue into
the third trimester with coexistence of sonographically normal
placental tissue and other areas demonstrating marked hydropic
change. The main clinical significance of diagnosing HM is the
increased subsequent risk for persistent gestational trophoblastic
disease requiring chemotherapy (15% for CHM and 0.5% for
majority of HMs sonographically appear as early pregnancy fail-
ures and may not demonstrate significant sonographically detect-
able hydropic change27 (Fig. 9.12).
Very rarely, a focus of intraplacental choriocarcinoma may
develop within an otherwise unremarkable third trimester pla-
centa, which may lead to metastatic disease of the mother, fetus
or both. Such focal lesions are not detectable sonographically
and even on macroscopic examination of the delivered placenta
are indistinguishable from intervillous thrombi, infarct or other
lesion until the correct diagnosis is made after histologic evalua-
tion of the lesion. 
86 SE C T I O N 2     The Placenta
Placental mesenchymal dysplasia. Placental mesenchymal
dysplasia (PMD) is described here because it is increasingly rec-
ognised as a specific entity with distinctive pathological features
and because it may sonographically be confused with a placenta
affected by hydatidiform molar change. Typically, such cases dem-
onstrate a sonographically homogeneously enlarged placenta with
diffusely scattered hydropic cystic change in association with an
apparently structurally normal fetus. The placenta may appear
larger than the fetus. Histologically, such placentas demonstrate
characteristic hydropic change of stem villi, without trophoblast
hyperplasia, often in association with marked dilation of chorionic
plate vessels. It appears that PMD may represents androgenetic or
biparental mosaicism and in a minority of cases may be associ-
ated with underlying disorders such as fetal Beckwith-Wiedemann
syndrome.28 
Future Approaches
To date, placental pathology data has been almost exclusively based
on findings of subjective morphological studies describing the fre-
quency of various histologic lesions in specific groups. Although
this approach has led to important observations related to both
clinical care and underlying mechanisms of disease, further devel-
opments are likely to require additional approaches which provide
objective data to minimise the effects of nonblinding, unconscious
bias and may identify mechanistic rather than structural altera-
tions. Recent technological developments in -omic approaches,
such as genomics, proteomics, metabolomics and microbiomics,
will have profound effects on the evaluation of tissue samples in
disease, and such techniques are now being applied to the pla-
centa, and their findings are beginning to challenge our existing
paradigms of disease mechanisms and pathophysiology.29
However, with the introduction of such new capabilities, the
importance of a range of factors related to sample acquisition is
increasing because such factors may affect the interpretation of
findings. Examples are the precise geographical localisation of
sampling, in relation to the periphery, basal and chorionic plates,
and cord insertion; the timing of sampling in relation to deliv-
ery; the mode of delivery; the method of protein extraction; and
the temperature of storage and length of storage time. All these,
and likely many yet unrecognised, factors require modifications of
existing placental examination protocols, but such a multidimen-
sional approach will lead to exciting new discoveries in relation to
a wide range of placental related obstetric complications. 
Conclusion
Many pregnancy complications are caused by a variety of pla-
cental pathologies, some of which are detectable antenatally
through ultrasound examination. Histopathological examination
of the delivered placenta may allow both confirmation of spe-
cific diagnoses and identification and mechanisms of underlying
disease patterns and pathophysiology. Some pathological condi-
tions appear to be poorly detectable antenatally and, at present,
are only recognised on microscopic placental examination. It is
highly likely that in addition to these abnormalities described, a
range of placental functional disorders may also lead to pregnancy
complications, and in this context, the development of novel
additional investigations may allow more accurate detection of
these disorders and potentially their early antenatal detection and
prevention.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References
1. Fox H, Sebire NJ. Pathology of the Placenta.
St. Louis: Elsevier Saunders; 2007.
2. Benirschke K, Burton G. Pathology of the
Human Placenta. Berlin: Springer; 2012.
3. Kraus FT, Redline RW, Gersell DJ, Nelson
DM, Dicke JM. Placental Pathology (Atlas of
Nontumor Pathology), 1st ed. Washington:
American Registry of Pathology; 2005.
4. Hargitai B, Marton T, Cox PM. Best practice
no 178. Examination of the human placenta.
J Clin Pathol. 2004;57:785–792.
5. Langston C, Kaplan C, Macpherson T, et al.
14. Ghosh SK, Raheja S, Tuli A, et  al. Serum
Practice guideline for examination of the
placenta: developed by the Placental Pathol-
ogy Practice Guideline Development Task
Force of the College of American Patholo-
15. Bhide A, Thilaganathan B, Singh T, et  al.
gists. Arch Pathol Lab Med. 1997;121:449–
476.
6. Cox P, Evans C. Tissue Pathway for Histopath-
ological Examination of the Placenta. London;
2011.
7. Burton GJ, Sebire NJ, Myatt L, et al. Opti-
mising sample collection for placental
research. Placenta. 2014;35:9–22.
8. Raymond D, Peterson E. A critical review
of early-onset and late-onset preeclampsia.
Obstet Gynecol Surv. 2011;66:497–506.
9. Khong TY, Gordijn SJ. Quality of placen-
tal pathology reports. Pediatr Dev Pathol.
2003;6:54–58.
10. Redline RW. Classification of placental
lesions. Am J Obstet Gynecol. 2015;213(suppl
4):S21–S28.
11. Milligan N, Rowden M, Wright E, et  al.
Two-dimensional sonographic assessment of
maximum placental length and thickness in
the second trimester: a reproducibility study.
J Matern Fetal Neonatal Med. 2015;28:1653–
1659.
12. Schwartz N, Coletta J, Pessel C, et  al. Novel
3-dimensional placental measurements in early
pregnancy as predictors of adverse pregnancy
outcomes. J Ultrasound Med. 2010;29:1203–
1212.
13. Bahlmann F, Fittschen M, Reinhard I, et al.
Reference values for blood flow velocity in the
uterine artery in normal pregnancies from 18
weeks to 42 weeks of gestation calculated by
automatic Doppler waveform analysis. Ultra-
schall Med. 2012;33:258–264.
PLGF as a potential biomarker for predict-
ing the onset of preeclampsia. Arch Gynecol
Obstet. 2012;285:417–422.
Role of second-trimester uterine artery Dop-
pler in assessing stillbirth risk. Obstet Gynecol.
2012;119:256–261.
16. Crovetto F, Triunfo S, Crispi F, et al. First trimes-
ter screening with specific algorithms for early
and late onset fetal growth restriction. Ultrasound
Obstet Gynecol. 2016;48(3):340–348.
17. Walker MG, Fitzgerald B, Keating S, et  al.
Sex-specific basis of severe placental dysfunc-
tion leading to extreme preterm delivery. Pla-
centa. 2012;33:568–571.
18. Deter RL, Levytska K, Melamed N, et  al.
Classifying neonatal growth outcomes: use of
birth weight, placental evaluation and indi-
vidualized growth assessment. J Matern Fetal
Neonatal Med. 2016;29:3939–3949.
19. Porat S, Fitzgerald B, Wright E, et  al. Pla-
cental hyperinflation and the risk of adverse
perinatal outcome. Ultrasound Obstet Gynecol.
2013;42:315–321.
20. Salafia CM, Yampolsky M, Misra DP, et  al.
Placental surface shape, function, and effects
of maternal and fetal vascular pathology. Pla-
centa. 2010;31:958–962.
21. Burton GJ, Woods AW, Jauniaux E, King-
dom JCP. Rheological and physiological con-
sequences of conversion of the maternal spiral
arteries for uteroplacental blood flow during
human pregnancy. Placenta. 2009;30:473–482.
22. Walker M, Whittle W, Keating S, Kingdom J.
Sonographic diagnosis of chronic abruption. J
Obstet Gynaecol Can. 2010;32:1056–1058.
23. Fitzgerald B, Kingdom J, Keating S. Distal
villous hypoplasia. Diagnostic Histopathol.
2012;18:195–200.
24. Sebire NJ, Sepulveda W. Correlation of pla-
cental pathology with prenatal ultrasound
findings. J Clin Pathol. 2008;61:1276–1284.
25. Tamblyn JA, Lissauer DM, Powell R, et al. The
immunological basis of villitis of unknown eti-
ology [review]. Placenta. 2013;34:846–855.
26. Zanardini C, Papageorghiou A, Bhide A,
Thilaganathan B. Giant placental chorioan-
gioma: natural history and pregnancy out-
come. Ultrasound Obstet Gynecol. 2010;35:
332–336.
27. Fowler DJ, Lindsay I, Seckl MJ, Sebire NJ.
Routine pre-evacuation ultrasound diagno-
sis of hydatidiform mole: experience of more
than 1000 cases from a regional referral center.
Ultrasound Obstet Gynecol. 2006;27:56–60.
28. Kaiser-Rogers KA, McFadden DE, Livasy
CA, et al. Androgenetic/biparental mosaicism
causes placental mesenchymal dysplasia. J
Med Genet. 2006;43:187–192.
29. Law KP, Han T-L, Tong C, Baker PN. Mass
spectrometry-based proteomics for pre-eclamp-
sia and preterm birth. Int J Mol Sci. 2015;16:
10952–10985.
86.e1
10
Development of the Heart and
Cardiovascular System in Relation to
Cardiac Abnormalities
MICHAEL ASHWORTH
KEY POINTS
• T he heart is largely derived from mesoderm.
• A single heart tube forms with venous and arterial connec-
tions. It elongates by addition of cells at either end from the
surrounding mesenchyme.
• The tube folds to the right and lies in the pericardial cavity.
• The atria and ventricles form by ballooning from the tube;
septation of the atria and ventricles is largely achieved by this
method.
• The endocardial cushions at the atrioventricular (AV) junction
and outflow tract complete septation of the AV junction and
outflow tracts and give rise to the AV and arterial valves.
• The epicardium gives rise to the mesenchyme of the heart, the
veins and most of wall of the coronary arteries.
• The epicardial coronary arteries grow into the aortic root.
• The conduction tissue is derived from primitive myocardium
of the original heart tube.
• A complex series of venous precursors contribute to the ab-
dominal veins.
• Aortic arches 3, 4 and 6 give rises to the vessels of the head,
neck and thorax.
Introduction
The heart is an organ largely derived from mesoderm, with a
contribution from ectodermally derived neural crest cells. Its
development is critically dependent upon a cascade of events
tightly regulated in time and space.1 The first recognisable
sign of heart development in the embryo is the formation of
the heart tube within the lateral splanchnic mesoderm late in
the third week. Invisible to the classical morphologist are a
series of preceding genetic steps on which the morphological
changes are critically dependent. These include the specifica-
tion of precursor cells in the bilaminar embryonic disc, their
migration to the heart fields and the specification of cell types
within the developing heart. By the end of the seventh week,
the heart, although tiny, is essentially fully formed. It does not
develop in isolation but is intimately related to surrounding
88
structures such as the pharynx, and heart development is both
influenced by and influences these surrounding structures.
Unsurprisingly, defects in the heart are associated with defects
in these associated structures. From very early in its develop-
ment, the heart is a beating structure and contains a flowing
liquid, so mechanical forces are also important in shaping its
development.2
Heart development is contributed to by multiple genes,
many of which have multiple functions in development, with
considerable overlap.3 If a defect develops, it is likely to be
modified by growth and subsequent alterations in haemody-
namics. Because heart development is a sequential and complex
process, no single gene defect leads to a single specific heart
defect. There has been a proliferation of information on heart
development in recent decades, with genetic experiments in
mouse, chick and zebrafish embryos having determined details
of cell lineage. Although much remains unknown, the genetic
mechanisms regulating heart development are beginning to be
understood. 
Brief Overview of Heart Development
The bulk of the work on early cardiac development comes from
studies in fish, chick and mouse embryos.4-6
Within the cardiac crescent (the group of mesodermal cells
lying in the splanchnic mesoderm anterior to the buccopharyn-
geal membrane and that show patterns of gene expression com-
mitting them to cardiogenesis), paired endothelial-lined tubes
develop with their long axis in the long axis of the embryonic disc
(Fig. 10.1).
With folding of the embryo, these paired tubes fuse medi-
ally over part of their length to form the primitive single heart
tube. The surrounding mesoderm of the cardiac crescent dif-
ferentiates to provide an investing sleeve of myocardium, both
layers separated by extracellular matrix termed cardiac jelly.
The inflow is caudal, a continuation of the primitive veins,
and the outflow cranial and connected to the paired dorsal
aortae.
The pericardial cavity develops initially on the dorsal sur-
face of the developing heart as part of the intraembryonic coe-
lom. With folding of the embryo and formation of the single
 CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities
• Fig. 10.1  Schematic representation of a cross-section of the trilami-
nar embryo before embryonic folding. The coelomic cavity is developing
within the mesoderm, localising the paired heart tubes to the splanchnic
mesoderm on the ventral aspect. As the embryo folds, the lateral edges
are brought into apposition, the endoderm fuses to form the gut, and the
paired heart tubes fuse in the midline ventral to the gut. The coelom forms
the pericardial cavity. The ectoderm enfolds the other structures, and the
amniotic cavity extends completely to surround the embryo.
heart tube, the pericardial cavity comes to lie ventral to the
heart tube, which is connected dorsally to the mesenchyme by
the dorsal mesocardium. The septum transversum grows into
the intraembryonic coelom partially to divide the pericardial
cavity from the peritoneal cavity. The pericardial cavity is fur-
ther divided by ingrowth of tissue from the lateral body walls,
the pleuropericardial folds, that fuse with each other and the
foregut mesenchyme to completely enclose the pericardial cav-
ity and create two pleural cavities still joined to the peritoneal
cavity. The dorsal mesenchyme breaks down to leave the heart
connected to the body wall only at the arterial and venous
poles. The space thus created is the transverse pericardial sinus.
The primitive myocardium of this primary heart tube shows
regular contractions by the third week (embryonic age). This heart
tube elongates by addition of mesodermal cells at its two poles
(and from the dorsal mesocardium until that structure involutes)
and undergoes rightward looping (Fig. 10.2). The primitive heart
tube is the scaffold to which the atrial and ventricular chambers
are added by ballooning from its myocardium. Septa are formed,
dividing the right and left sides of the atria, ventricles and great
arteries.
Heart formation is completed with the development of valves,
the conduction system and the formation of the coronary arter-
ies by the ingrowth of extracardiac tissues derived from the neu-
ral crest and from the proepicardium situated in the septum
transversum.  utes to the formation of right ventricular and outflow tract
The Heart Fields
Heart progenitor cells are located in two small patches, one on
either side of the midline in the epiblast of the bilaminar embry-
onic disc, close to the cranial part of the primitive streak. The
earliest known committed cardiac precursors express the T-box
transcription factor Eomesodermin\Tbr2, which activates another
transcription factor MESP1.7 These progenitor cells migrate
together through the primitive streak and form two plates of lateral
mesoderm cells positioned anteriorly. Specification of cardiogenic
89
• Fig. 10.2  Looped heart. A dissection of an embryo of approximately 35
days. The pericardial sac has been opened to expose the heart. The heart
is looped to the right. The bulboventricular sulcus is seen as a notch on
the inferior surface. The inflow is located behind the ventricular mass and
the outflow is superior.
mesoderm has already started during this cellular migration. The
progenitor cells migrate such that the medial-lateral arrangement
of these cells will become the cranial-caudal axis of a linear heart
tube.
With the formation of the embryonic coelom, they occupy the
splanchnic mesoderm and fuse in the midline cranially to form
the cardiac crescent. The cells of this cardiac mesoderm express
the cardiac specific transcription regulator genes NKX2.5 and
GATA-4.8
The cells of the splanchnic mesoderm, one on each side of
the body, interact with adjacent tissues (Fig. 10.3). The splanch-
nic mesoderm is positioned adjacent to the foregut endoderm
(see Fig. 10.1), which is thought to provide inductive signals
to begin myocardial differentiation.9 Endoderm also has a
mechanical role in assembly of the heart tube. By its active con-
traction, it pulls the bilateral fields of cardiogenic mesoderm
towards the midline, permitting them to fuse and form a single
heart tube.
The primitive, single heart tube initially functions not so
much to support the embryonic circulation as to provide a scaf-
fold into which the cells from the second heart field migrate to
effect chamber formation. The second heart field cells are first
located medial to the cardiac crescent and subsequently lie in
mesoderm underlying the pharynx before they are added to the
heart.10 The cranial part of the second heart field, the anterior
heart field, which is identified by FGF10 expression, contrib-
myocardium. Cells in the posterior second heart field express
Isl1, but not anterior heart field markers, contribute to atrial
myocardium.
The nomenclature of the heart fields may be confusing10
because not all authors use exactly the same terms, and the heart
fields are dynamic, changing in position and with time.
The first and second heart fields develop sequentially, and
the anterior and posterior heart fields describe anatomical
positions.
It appears that the overall cellular environment is critical for
this process because cells taken from a different location or at a
90 SE C T I O N 3     Fetal Physiology and Pathology

+ –
BMP β-catenin/WNT
Pharynx\foregut Midline
FGF
+
–

WNT inhibitors

Activation of transcription factors

NKX2.5
TBX5
GATA4
MEF2C
SMARCD3 (BAF60C)

MESP1

Eomes

Second heart field

ISL1
TBX1
FGF8
FGF10

• Fig. 10.3  Activation of cardiac transcription factors. BMP, Bone morphogenetic protein; FGF, fibroblast
growth factor.

different developmental time point can contribute to the heart
when placed in the appropriate location.  diaphragm at the caudal aspect, and ventral to the foregut, as in
The Heart Tube
Within the cardiac crescent, the primitive heart tube forms. The
horseshoe-shaped cardiac crescent forms a tube with two cau-
dolateral inlets, or venous pole, and one craniomedial outlet,
or arterial pole. The process starts with the formation of endo-
thelial-lined channels in the mesenchyme that form a plexus
that eventually coalesces to form paired endothelial tubes. Fold-
ing of the embryo then brings these paired tubes together in
the midline where they fuse to form a single endothelial-lined
tube with a surrounding cuff of mesenchyme, separated from
it by extracellular matrix termed cardiac jelly. The endocardium
develops simultaneously with the myocardium and is a special-
ised endothelial cell type derived from the splanchnic mesoderm
that, via an unknown mechanism, differs from the myocardial
precursors.11
The linear heart tube subsequently grows by addition of cells
from a proliferating pool of precursors located external to it in the
heart fields and not by division of myocytes in situ. Being added
to the heart, their fate is not fixed, and their identity depends on
their eventual location.
The cardiac precursors are therefore initially situated cranial
and lateral to the future mouth but caudal to the mesoderm
that forms the transverse septum and contributes to the forma-
tion of the diaphragm. During the process of folding, the cardiac
precursors end up between the mouth on the cranial aspect, the
the adult situation.12
By folding of the embryo, the lateral parts of the cardiac
mesoderm are brought together, forming the ventral part of
the heart tube. The inner curvature of the cardiac crescent
forms the dorsal side of the tube and is contiguous with the
dorsal mesocardium, the attachment of the heart to the body
wall.
The peripheral part of the cardiac crescent will eventually face
the transverse septum and form the venous pole of the heart, and
the central part of the crescent, which forms the outflow tract,
is contiguous with the pharyngeal mesenchyme. This intimate
association of the cardiac and facial region during development
explains the high incidence of combined cardiac and facial mal-
formations in syndromic conditions.
Retinoic acid appears critical in specifying these posterior pre-
cursor cells to become the inflow, or venous parts of the heart, the
sinus venosus and atria, 
Contraction
The myocardium shows regular contractions by the third week,
meaning that the structural requirements for contraction, namely
contractile proteins, sarcoplasmic reticulum and gap junctions, are
already present in the myocardial cells of the primitive heart tube.
It would appear that the initial contractions, at least in the chick
embryo, are not essential for tissue oxygen and nutrient supply
 CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities 91

but likely have an important function providing mechanical stim-

uli for further development of the heart.2 

Looping of the Heart Tube

The myocardium of the primitive heart tube demonstrates TBX2
and TBX3 expression.13 Traditionally, five consecutive segments
of the tube are recognised: venous, atrial, left ventricular, right
ventricular and arterial. Between each of the segments is a transi-
tion zone – the sinoatrial ring, the atrioventricular (AV) canal,
the primary heart fold and the ventricular outflow tract. It is
important to recognise that the cardiac chambers develop as out-
growths from these segments and that the original segments form
the connections of the cardiac chambers rather than the chambers
themselves.
Looping of the straight heart tube follows activation of a • Fig. 10.4  Atrial and ventricular formation. The heart on approximately
gene cascade that determines right–left symmetry. Left–right day 37. The two atrial appendages can be seen on either side of the arte-
differentiation has already started at the time of gastrulation rial trunk that is positioned predominantly over the right ventricle. Septation
with the formation of the Hensen node. Nodal cilia beat in of the trunk is evident by the separate column of blood on the right side.
one direction and cause a gradient of molecules across the The ventricles can be seen ballooning out from the heart tube.
bilaminar embryo. These genes are already expressed in the car-
diac mesoderm before formation of the heart tube and include
NODAL, LEFTY and PITX2, of which PITX2 is the effector
gene. Furthermore, the direction of looping is not random but
is controlled by genes not yet understood and is not under the
control of PITX2.
The tube loops to the right (see Fig. 10.2), which causes the
transition zones to be brought into close proximity on the inner
curvature of the loop. This is absolutely essential for establishing
the correct connections of the chambers. Actin polymerisation-
driven myocardial cell shape changes have been found to contrib-
ute to the bending of the heart tube, but the torsional component
of looping is largely caused by forces from its encapsulating mem-
brane, the splanchnopleure.14
The looped heart tube has unidirectional blood flow, which
was originally thought to be peristaltic but is now regarded as
functioning as a Liebau pump (unidirectional and pulsatile flow
resulting when an elastic tube containing fluid is periodically
squeezed15). At this stage, the AV cushions function as primitive • Fig. 10.5  A 13-week fetus with left atrial isomerism. There were bilateral
valves to permit unidirectional flow of blood in the looped heart superior caval veins, complete atrioventricular septal defect and discor-
tube.16  dant ventriculoarterial connections with anterior aorta. The heart and lungs
are viewed from the front. Two morphologically left atrial appendages are
present enclosing the anteriorly situated aorta.
Development of the Chambers and Outflows
Both atria and ventricles develop by ballooning growth from
the heart tube (Fig. 10.4), the atria growing from the dorsal shows specific gene expression (ANF, CX40, CX43) but not
aspect and the ventricles from the ventral aspect. Atrial segment TBX2 and TBX3.17 The early markers of chamber formation
growth is bilateral and in parallel; hence, it is possible to develop remain restricted to the original trabeculated myocardium,
isomerism (Fig. 10.5). Ventricular segment growth is unilateral with NOTCH, ERBB, and Ephrin playing roles in trabecula-
and in sequence; therefore development of isomerism is not tion. The compact ventricular layer does not express ANF and
possible. CX40, but NOTCH, bone morphogenetic protein (BMP), and
The initially formed atrial myocardium only gives rise to the fibroblast growth factor play roles in compact myocardium
trabeculated, atrial appendages in the formed heart. All smooth- development.18
walled myocardium found in the full-grown heart is added later Myocardial ‘compaction’ is a slightly misleading term because it
during development from cells of the posterior heart field. His- is not a process by which the previously trabeculated myocardium
tologically, chamber formation becomes evident when the exten- becomes compact but rather one in which the myocardium of the
sive extracellular matrix between endocardium and myocardium, epicardial side of the ventricular wall proliferates to form the compact
known as cardiac jelly, disappears and trabeculations become evi- layer; when the compact outer layer starts to form, proliferation in
dent in both atria and ventricles.12 the ventricular trabeculations ceases.12 This process may be related
Numerous genes are involved in this process, the primi- to mechanical strain because there is a gradient of strain across the
tive myocardium of the primary heart tube expressing TBX2 ventricular wall, greatest on the inner surface and least on the outer
and TBX3, and the myocardium that forms the chambers surface. The curvature of the heart therefore depends on cell shape
92 SE C T I O N 3     Fetal Physiology and Pathology
changes at the cellular level caused by a complex interplay on hae-
modynamic shear stress, contractive wall strain and electrical activity.
It remains uncertain precisely how the left and right ventri-
cles achieve their different morphologies, although there are dif-
ferences in gene expression between the left and right ventricles,
with the cardiac transcription factor TBX5 expressed in a gradient
tapering off toward the right ventricle.19
Distal to the ventricle is the bulbus cordis. It is divided into
three components, the proximal part forming the trabeculated
part of the right ventricle, the midpart (conus cordis) forming the
outflow tract of both ventricles and the distal third the truncus
arteriosus. The bulbus cordis is demarcated externally from the
ventricle by the bulboventricular sulcus, and internally this is the
site of the interventricular foramen. 
Septation
Septation of the ventricles occurs between 5 and 7.5 weeks of
gestation. Initially, because the atrial segment is connected to
the left ventricular segment, there is no direct connection with
the right ventricle, but blood can flow from the atria to the
right ventricle during diastole via the interventricular foramen
(Fig. 10.6). Similarly, the outlet is connected initially solely to
the right ventricle, but blood flows from the left ventricle to the
outlet during systole via the interventricular foramen. The ven-
tricular septum grows from the apex of the heart loop between
the left and right ventricular segments, and its growth is largely
appositional caused by ballooning of both ventricles. The inter-
ventricular foramen lies above the interventricular septum.12  endocardial cushions are coloured blue. The dorsal and ventral atrioven-
Atrial Septation
The atria incorporate the draining veins and form a pair of
valves around the sinus venosus. Fusion of the anterior part
of these valves creates the septum spurium, which contributes
to closure of the atria. The primary atrial septum develops
to the right of this and grows downwards towards the fused
AV cushions (Fig. 10.7) with the gap between its free edge of
the primary septum and the AV cushions called the ostium
primum. There is a mesenchymal cap on the edge of the pri-
mary septum that is continuous ventrally with the ventral AV
cushion and dorsally with the dorsal mesenchymal protrusion
and the dorsal AV cushion.20 Before obliteration of the ostium
primum by fusion of the septum primum and the AV cush-
ions, an opening forms by fenestration in the primary septum
– the ostium secundum. The septum secundum then develops
on the right side of the septum primum, beginning at about
day 41, by infolding of the muscular wall of the atrium. The
septum secundum is thicker and more muscular than the sep-
tum primum. It grows downwards, and its anterior part fuses
with the endocardial cushions, but a defect remains – the oval
fossa.21 The ostium primum is actually beneath the free edge
of the septum primum and is obliterated when that edge fuses
with the endocardial cushions at about day 38. The sinus veno-
sus is incorporated into the right atrium, the coronary sinus
representing its left horn. The right valve of the sinus venosus
disappears in its upper part, but the lower part becomes the
valve of the inferior caval vein (Eustachian valve) and the valve
of the coronary sinus (thebesian valve) (Fig. 10.8). Occasion-
ally, a complete remnant of the right valve persists, termed a
Chiari network (Fig. 10.9). This may fill out like a sail and
extend through the right AV valve. The left valve of the sinus
• Fig. 10.6  The heart during septation. The parts of the primary heart tube
are coloured yellow. They form a tight curve on the inner aspect of the
heart. The sinus venous (guarded by its valves) enters the posterior aspect
the right atrium. The two atria have ballooned laterally, and the two ven-
tricles ventrally, from this tube. The primary foramen lies between the inner
curvature of the primary heart tube and the developing interventricular
septum. Through it, because of differential streaming of the blood, blood
flows from the right atrium to the right ventricle in diastole and from the
left ventricle to the outflow tract in systole (black arrows). The developing
tricular (AV) cushions divide the inflow from the atria. The primary atrial
septum is growing downwards, and its mesenchymal cap will fuse with the
AV cushions and the dorsal mesenchymal protrusion to seal the right from
the left AV junction. The spiral endocardial cushions in the outflow tract
have already fused distally and the zone of closure is moving proximally.
Their fusion with the atrioventricular cushions will seal the outflow tracts.
(Adapted from Sylva M, van den Hoff MJB, Moorman AFM. Development
of the human heart. Am J Med Genet 164A(6):1347–1371, 2014.)
venosus is incorporated into the developing septum secundum
but occasionally remnants of it persist as thin threads attached
to the right side of the septum.
The pulmonary vein enters the left part of the atrium and is
incorporated into it. The exact site of development of the pulmo-
nary vein remains controversial. The smooth wall of the body of the
left atrium results from incorporation of the pulmonary veins into
the atrium. Failure of connection of the pulmonary vein results in
total anomalous pulmonary venous connection (Fig. 10.10).
The atrial appendages hence represent the original parts of
the primitive atrium. The smooth-walled part of the right atrium
develops from incorporation of the sinus venosus and is termed
the sinus venarum. 
The Interventricular Septum
The greater part of the interventricular septum results from a
process of apposition because of ballooning of the ventricular
chambers, beginning at about day 26. The crest of the inter-
ventricular septum is at the site of the primary foramen and
retains the genetic expression pattern of the primitive heart tube
(expressing TBX3 and NOTCH1), fusing with the dorsal AV
cushion.22 
 A B

C D
• Fig. 10.7  Development of the interatrial septum. A, Diagrammatic view of the heart showing the right
and left atria and the left ventricle with posterior atrioventricular (AV) cushion. The primary septum (septum
primum) grows between the right and left atria. Its lower border grows towards the AV cushion, the gap
between them forming the ostium primum (B).  Fusion of the lower border of the primary septum and the
AV cushion eliminates the ostium primum. By the time they fuse, however, fenestrations have appeared in
the septum primum to permit right-to-left passage of blood in the atrium (C). These fenestrations coalesce
as the ostium secundum. A second septum, the septum secundum, grows to the right of the septum
primum and covers the ostium secundum, the septum primum forming the flap valve of the oval fossa
(D). The left valve of the sinus venosus fuses with the septum secundum. The upper part of the right valve
regresses, but the lower part forms the eustachian and thebesian valves.

• Fig. 10.8  Interatrial septum viewed from the right side. The structures
of the right side of the septum are well seen: oval fossa, coronary sinus,
eustachian valve and thebesian valve. In addition, there is a secundum
atrial septal defect where the flap valve of the oval fossa does not com-
pletely cover the orifice.
• Fig. 10.9  Chiari network. Delivery at 31 weeks’ gestation. Fetal hydrops.
Pulmonary stenosis and dysplastic tricuspid valve. The right atrium and
right ventricle are exposed to show a diaphanous, baglike membrane cov-
ering the right side of the interatrial septum, a remnant of the right valve of
the sinus venosus.
94 SE C T I O N 3     Fetal Physiology and Pathology

LUPV

LLPV RUPV

RLPV

• Fig. 10.11  Atrioventricular (AV) cushions. Left side of the AV junction at

about day 37. The ventricle is still highly trabeculated with commencement
of formation of an epicardial compact layer. The AV canal between the
• Fig. 10.10  Total anomalous pulmonary venous connection. A case of
atrial and ventricular chambers shows dorsal and ventral myxoid masses
right atrial isomerism in a fetus of 22 weeks. There were bilateral superior
(cushions) with a gap between them. These will eventually fuse to obliter-
caval veins, atrioventricular septal defect, transposition, pulmonary atre-
ate the central gap and create separate AV orifices to the right and to the
sia with major aortopulmonary collateral arteries, absent arterial duct and
left. The cushions also play a role in completing septation of the atria, the
asplenia. The pulmonary veins come to a cruciate confluence behind the
ventricles and the outflow tract. Separate cushions are also evident in the
heart, from which a vein ascends to insert into the left superior caval vein.
arterial trunk where they are just fusing to divide it.
LLPV, Left lower pulmonary vein; LUPV, left upper pulmonary veins; RLPV,
right lower pulmonary veins; RUPV, right upper pulmonary vein.

The Atrioventricular Junction

The AV valves develop form the two endocardial cushions.
During looping, the cardiac jelly is eliminated from much
of the heart tube but persists at the site of the endocardial
cushions at the AV junction as well as in the outflow tract.
The cushions are originally acellular, but beginning on day
26, the epithelial-to-mesenchymal transition produces cells
that populate the cushions. In the first stage of this process,
a subset of endocardial cells lining the AV junction and the
outflow tract transform into a mesenchymal phenotype medi-
ated by BMP through transforming growth factor β (TGFβ)
and Notch produced by the myocardium of the AV junction
and invade the cardiac jelly.23 This invasive, proliferating mes-
enchyme progressively remodels the matrix, and the resultant
cellular masses, now called cushions, continue to grow and • Fig. 10.12  Ventricular septal defect. A heart with tetralogy of Fallot, cut in
extend into the lumen (Fig. 10.11). In the AV canal, these a simulated echocardiographic long axis view. There is a ventricular septal
cushions form superiorly and inferiorly and fuse in the mid- defect with overriding of the aorta. The crest of the interventricular septum
line to create right and left AV orifices, differential growth forms the lower part of the defect.
of the right AV canal having brought the right atrium into
contact with the right ventricle. Fusion of the cushions with Even before septation, laminar blood flow ensures separation
the developing interventricular muscular septum and the of the right and left streams of blood. The primary foramen is that
atrial primary septum completes septation of the atrial and part of the primitive heart tube from which the ventricles balloon.
ventricular chambers. This complex process may be defective It is sometimes referred to as the interventricular foramen but is
and result in ventricular septal defects (perimembranous in not actually between the two ventricles but rather above them
this region) (Fig. 10.12). at the inner curvature of the heart (see Fig. 10.6). Blood flows
Lateral cushions also develop, attracting cells from the through this foramen from the right atrium to the right ventricle
epicardium. The dorsal mesenchymal protrusion, also called during diastole and from the left ventricle to the aorta during sys-
the vestibular spine, is contiguous with the dorsal AV cush- tole. This foramen becomes septated by the membranous septum
ion and the mesenchymal cap of the primary atrial septum in at about day 45. The AV canal develops ventral and dorsal cush-
the atria and is derived from extracardiac cells. Maldevelop- ions that separate the canal into right and left parts.
ment of the dorsal mesenchymal protrusion causes AV septal The outflow tract is separated by two ridges called the septal
defect (Figs. 10.13 and 10.14), and absence of the dorsal mes- and parietal outflow tract ridges (Figs. 10.16 and 10.17). In an
enchymal protrusion is seen in fetuses with Down syndrome adult heart, the membranous part of the ventricular septum is
(Fig. 10.15).24 the remnant of the fused AV and outflow tract cushions. It has
 CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities
• Fig. 10.13  Complete atrioventricular (AV) septal defect. Heart cut in a
simulated four chamber echocardiographic view. There is a common AV
junction with a common valve. The large defect lies between the lower
border of the interatrial septum and the upper border of the interventricular
septum.
• Fig. 10.14  Atrioventricular (AV) septal defect in left atrial isomerism. There
are bilateral left atrial appendages. There is also situ inversus. There is a
complete AV septal defect. There were also bilateral superior caval veins
and secundum atrial septal defect and aortic isthmus hypoplasia. The
shape of the heart (allowing for situs inversus) shows a striking similarity to
a heart at about 35 days (see Fig. 9.10.)
been proposed25 that the morphology of transposition of the
great arteries is a defect of laterality akin to the heterotaxy syn-
dromes (Fig. 10.18).  to the posterior aortic and anterior pulmonary leaflets. The cushion
Valves
The septal leaflet of the tricuspid valve and the aortic leaflet of
the mitral valve arise from fusion of the dorsal and ventral AV
cushions on the right and left sides, respectively.26 The septal
leaflet detaches from the myocardium by cellular apoptosis. The
mural leaflets of the two AV valves are formed from the lateral
AV cushions, and the ventricular space grows behind these mural
leaflets.27
95
• Fig. 10.15  Membranous interventricular septum, trisomy 21 in a male
infant aged 9 months who died from respiratory viral infection. There is
a secundum atrial septal defect, and histologically, the lungs showed
changes of pulmonary arterial hypertension. The right atrium and ventricle
have been opened to expose the septal structures. The membranous sep-
tum (black dots) is large, and there is discontinuity between the septal and
anterior leaflets of the tricuspid valve.
• Fig. 10.16  Outflow tract in an embryo at 37 days. The ventricular myo-
cardium is trabeculated. The outflow is connected largely to the develop-
ing right ventricle. Endocardial cushions are present in the outflow tract
that are growing toward one another but have not yet fused.
The semilunar valves form from the parietal and septal outflow
tract cushions and two intercalated ridges. The parietal outflow tract
cushion gives rise to the right aortic and pulmonary valve leaflets.
The septal outflow tract cushion gives rise to the left aortic and pul-
monary leaflets, and the right and left intercalated ridges give rise
at the distal margin undergoes apoptosis to achieve a cusp. The AV
and semilunar valves then mature by remodelling of their matrix. 
Outflow Tract
The outflow tract connects the developing ventricles to the aortic
sac that is connected to the symmetrical pharyngeal arch arteries.
Septation starts at day 32 and occurs distally to proximally (see
Figs. 10.16 and 10.17) with the cushions arranged in a spiral fash-
ion. The outflow tract myocardium becomes incorporated into the
96 SE C T I O N 3     Fetal Physiology and Pathology
• Fig. 10.17  Septation of the truncus arteriosus in an embryo at approxi-
mately day 37. The truncus is cut in cross section lying above the ventricu-
lar mass as it travels backwards. Four endocardial cushions are present,
two major and two lesser ones. The developing interventricular septum
can be seen in addition to the interventricular foramen lying superior to it.
• Fig. 10.18  Transposition of the great arteries. The heart is cut in a simu-
lated echocardiographic long-axis view. The aorta arises anteriorly from
the right ventricle and the pulmonary trunk posteriorly from the left ven-
tricle. The normal spiral relation of the great arteries to each other is abol-
ished. Instead they ascend in parallel. The pathogenesis of the defect is
regarded by some on morphological and epidemiological grounds as akin
to the heterotaxy syndromes in which there is disturbance of the normal
left–right patterning.
• Fig. 10.19  Truncus arteriosus. A common arterial trunk arises from the
heart from which the aorta and pulmonary arteries arise. Of necessity,
there is a ventricular septal defect. The defect arises from failure of fusion
of the outflow tract cushions.
developing right ventricle. The outflow tract is connected via the
aortic sac to arch arteries 3, 4 and 6. A protrusion of pharyngeal
mesoderm, the aortopulmonary septum, grows into the aortic sac
and connects distally to the spiralling outflow tract ridges, which
separates the sixth and fourth pharyngeal arch arteries.
The outflow tracts therefore have a complex origin, partly
from the primary heart tube and partly from ingrowth of cells
from two distinct sources, including the cardiac neural crest and
the second heart field. The complex interaction of all these tis-
sues gives rise to the ventricular outflow tracts, the arterial valves
and the intrapericardial parts of the aorta and pulmonary trunk
(Fig. 10.19).28 
Coronary Circulation
The coronary arteries and veins develop by both vasculogenesis
(the formation of vessels in situ) and angiogenesis (the formation
of new vessels by sprouting from existing vessels) from cells that
grow over the myocardium from the proepicardium (Fig. 10.20).
Both the endothelium and the medial smooth muscle of the coro-
nary arteries derive from this source (Fig. 10.21). These vessels
link up and grow to join with the aorta (Fig. 10.22).29
The pericardium develops as a sac around the developing heart
tube. Initially, the tube is connected to the posterior mediastinum
by the dorsal mesocardium, but this breaks down, permitting the
folding of the heart tube on which subsequent development is so
critically dependent. 
Conduction Tissue
The conduction tissue develops from the myocardium of the
primitive heart tube, being found in the transitional zones.30
Biomechanical factors play a critical role in induction and pat-
terning of the cardiac conduction system.2 The formation of
an insulating fibrous and fatty tissue plane between atrial and
ventricular myocardium occurs only after completion of septa-
tion, beginning at 7 weeks and largely complete by 12 weeks of
development. 
 CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities
• Fig. 10.20  Derivation of the epicardium. A schematic representation of
the developing heart showing the location of the proepicardial organ in the
pericardial cavity adjacent to the septum transversum. The cells migrate
over the surface of the heart and undergo epithelial-to-mesenchymal
transformation to form the epicardium, the stroma of the myocardium and
the coronary arteries. (Adapted from Pérez-Pomares JM, de la Pompa
JL, Franco D, et al. Congenital coronary artery anomalies: a bridge from
embryology to anatomy and pathophysiology—a position statement of the
development, anatomy, and pathology ESC Working Group. Cardiovasc
Res 109(2):204–216, 2016.)
Pulmonary Veins
The pulmonary vein develops and connects to the heart after the
formation of the initial heart tube and start of chamber forma-
tion. Mesenchyme dorsal to the heart differentiates into a vas-
cular plexus surrounding the embryonic foregut. At around day
30, the cranial component of this plexus connects as a solitary
pulmonary vein to the heart through the dorsal mesocardium
in the midline and cranial to the AV node. Failure of the vein
to connect with the atrium will result in anomalous pulmo-
nary venous connection (see Fig. 10.10). Although a midline
structure, eventually, the pulmonary vein will drain into the left
atrium because the primary atrial septum develops at the right
side. Following its connection to the developing left atrium, the
pulmonary vein and its bifurcations acquire a sleeve of myo-
cardium, which differentiate de novo. The transcription factor
PITX2c plays a crucial role in the differentiation of the pul-
monary vein myocardium.31 The muscularised pulmonary vein
is gradually incorporated into the left atrium, up to its second
division, resulting in four pulmonary orifices in the left atrium.
The pulmonary myocardial sleeves may extend upstream of the
pulmonary orifices.  to the cardinal veins. With growth of the liver, they form anasto-
The Venous System
There are three pairs of major veins in the embryo in the fifth
week.
97
• Fig. 10.21  Origin of the coronary circulation, showing the triple origin of
the coronary circulation. The coronary veins develop by budding from the
endothelium of the sinus venosus (orange). These small veins ramify in the
mesenchyme of the epicardium. The epicardial cells, transformed to mes-
enchyme (purple), form the tunica media and adventitia of the coronary
arteries. The endothelium of the coronary arteries derives largely from the
endocardium that extends through the trabeculations of the developing
myocardium of the ventricles and atria as sinsuoids (green). (Adapted from
Pérez-Pomares JM, de la Pompa JL, Franco D, et al. Congenital coronary
artery anomalies: a bridge from embryology to anatomy and pathophysi-
ology—a position statement of the development, anatomy, and pathology
ESC Working Group. Cardiovasc Res 109(2):204–216, 2016.))
Vitelline Veins (Omphalomesenteric Veins)
These paired veins carry blood from the yolk sac to the sinus
venosus. Before joining the sinus medially, they break into a
plexus around the duodenum and cross the septum transversum.
Hepatocytes growing into the septum induce the hepatic sinu-
soids. The proximal right vitelline vein persists as the hepatic part
of the inferior vena cava, its distal part becomes the superior mes-
enteric vein and the anastomotic network around the duodenum
becomes the portal vein. The proximal part of the left vitelline
vein disappears 
Umbilical Veins
Paired veins join the sinus lateral to the vitelline veins and medial
moses with the hepatic sinusoids. The proximal parts of both right
and left veins disappear, and the distal left umbilical vein persists
and drains to the hepatic sinusoids. A direct anastomosis develops
between the left umbilical vein and the proximal right vitelline
vein – the venous duct (ductus venosus). 
98 SE C T I O N 3     Fetal Physiology and Pathology

• Fig. 10.22  Origin of the coronary artery from the aorta. Section of the
developing aortic root of a mouse embryo showing the connection of the
coronary artery with the aortic lumen. The aortic valve still comprises cush-
ions that have not undergone remodelling.

Cardinal Veins
The paired common cardinal veins join the sinus most later-
ally and are formed by the confluence of anterior cardinal veins
(draining the head) and posterior cardinal veins. From the fifth to
the seventh week, further veins form: the subcardinal veins drain-
ing mostly the kidneys, the supracradinal veins draining the body
wall by the intercostal veins following regression of the posterior
cardinal veins, and the sacrocardinal veins draining the lower
limbs. Anastomoses develop between the right and left sides.
The brachiocephalic vein is formed of the anastomosis
between the two anterior cardinal veins. The superior caval vein
is formed from the proximal right anterior cardinal vein and the • Fig. 10.23  Embryonic origin of Inferior caval vein. The inferior caval vein is
right common cardinal vein. The anastomosis between the two viewed from behind. The aorta is to its left. The iliac veins and inferior vena
subcardinal veins becomes the left renal vein and the left subcar- cava bifurcation derive from the post cardinal veins (orange). The segment
dinal vein and then regresses with its distal part, becoming the between the renal veins and the bifurcation derives from the supra cardinal
vein (yellow). The segment between the liver and the renal veins derives
left gonadal vein. The right subcardinal vein becomes the renal
from the subcardinal vein (green). The short segment between the liver
segment of the inferior caval vein, and it connects proximally and the right atrium derives from the right vitelline vein (red).
with the hepatic inferior caval vein derived from the right vitel-
line vein (Fig. 10.23).
The anastomosis between the sacrocardinal veins becomes the The Arterial System
left common iliac vein. The right supracardinal vein becomes the
supracardinal segment of the inferior caval vein. The azygos vein Folding of the embryo during the fourth week (days 22–24)
derives from the right supracardinal vein and part of the poste- causes the paired dorsal aortae attached to the cranial end of
rior cardinal vein. On the left side, the supracardinal vein between the heart to form a pair of dorsoventral loops – the first aor-
the fourth and seventh intercostal veins becomes the haemiazygos tic arches. The paired dorsal aortae fuse below the level of the
vein that drains to the azygos vein (Fig. 10.24). fourth thoracic segment and become connected with the umbili-
Initially, the veins draining to the heart are embedded in the cal arteries. Between days 26 and 29, four additional pairs of
mesenchyme at the venous pole of the heart with expansion of aortic arches develop in succession from cranial to caudal within
the pericardial cavity excavating the connecting veins from this the mesenchyme of the pharyngeal arches, connecting the aortic
mesenchyme. By this process, the common cardinal veins, which sac at the superior end of the truncus arteriosus to the dorsal
are the confluence of the left and right superior and inferior car- aortae – arches 2, 3, 4 and 6 (Fig. 10.25). No fifth aortic arch
dinal veins, become incorporated within the pericardial cavity. ever develops. The first two pairs of arch arteries regress while
They then acquire a sleeve of myocardium, and the confluence of the later arch arteries are forming. It is therefore the aortic arch
the systemic veins is then called the sinus venosus, or left and right arteries 3, 4 and 6 that give rise to the arteries of the head, neck
sinus horns, which both eventually connect to the right atrium.  and thorax (Table 10.1).32
CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities 99

A B
• Fig. 10.24  Azygos continuation of the inferior caval vein. Photograph (A) and drawing (B) of the main
features. Termination of pregnancy at 20 weeks’ gestation for left atrial isomerism. The thoracoabdominal
organs are viewed from behind. The aorta and caval vein ascend together through the diaphragm, the
vein as the azygos vein that enters the superior caval vein. In addition to left atrial isomerism, there were
complete atrioventricular septal defect, bilateral superior caval veins and situs inversus with polysplenia.
The hepatic veins drained independently to the right atrium.

B
• Fig. 10.25  A, Schematic representation of the aortic arches viewed from the left side. The head is to the
left and the lower body to the right. The arch arteries are symmetrically paired and develop form cranial to
caudal. No fifth arch ever develops. Not all the arches are present at the same time. The first and second
have regressed by the time the sixth arch develops.  B, Schematic representation of the aortic arches
following remodelling showing the persistence of some and involution of others. As in A, the specimen is
viewed from the left side with the head to the left of the field. The third arch derivatives are symmetrical,
but the derivatives of the fourth and sixth arches are asymmetrical.
100 SE C T I O N 3     Fetal Physiology and Pathology

TABLE Arterial Derivatives of the Embryonic Aortic

10.1 Arches
Embryonic
Arch Arterial Derivative Notes
I Maxillary artery
II Hyoid artery
Stapedial artery
III Common carotid artery Dorsal aorta gives rise to
First part of the internal remainder of internal
carotid artery carotid
IV Proximal right subclavian LSCA derives from the • Fig. 10.27  Retro-oesophageal right subclavian artery. The right subcla-
artery seventh intersegmen- vian artery derives from the seventh intersegmental artery. Normally, it con-
Aortic arch between the tal artery nects to the ventral part of the fourth arch artery. When it connects instead
LCCA and LSCA to the right dorsal aorta, because it is posteriorly situated, it courses
behind the gut and airway to reach its usual course.
VI Branch pulmonary arter-
ies and arterial duct

LCCA, left common carotid artery; LSCA, left subclavian artery.

  

• Fig. 10.28  Retro-oesophageal right subclavian artery. The normal right

• Fig. 10.26  Derivatives of the aortic arch arteries. The truncus arteriosus
gives rise to the most proximal segments of the aorta and pulmonary trunk
(orange). The aortic sac gives rise to the ascending aorta and brachioce-
phalic artery (green). The third aortic arches give rise to the internal carotid
arteries. The fourth aortic arch gives rise on the right side to the first part of
the subclavian artery and on the left to the segment of aortic arch between
the left common carotid and left subclavian arteries (purple). The distal
right subclavian artery and all the left subclavian artery derive from the sev-
enth intersegmental arteries (yellow). The pulmonary arteries and arterial
duct derive from the sixth aortic arch arteries (blue). The remainder of the
descending aorta (uncoloured) derives from the left dorsal aorta.
The dorsal aorta develops three sets of branches:
1. A series of ventral branches that supply the gut derivatives
derived from a network of vitelline arteries
2. Lateral branches that supply retroperitoneal structures such as
adrenals, kidneys and gonads
3. Dorsolateral intersegmental branches that penetrate between
the somite derivatives
The third arch on both right and left sides becomes the common
carotid and first part of the internal carotid artery (see Fig. 10.25).
The segments of dorsal aorta connecting arches 3 and 4 regress,
subclavian artery develops from the connection of the seventh interseg-
mental artery with the right fourth arch artery. In retro-oesophageal right
subclavian artery, this does not happen. Instead the seventh right interseg-
mental artery takes origin from the right dorsal aorta. In this photograph,
the thoracic contents are viewed from behind, and the right subclavian
artery is seen to take origin from the descending aorta on the left side and
to run behind the oesophagus to reach its normal location.
leaving the third aortic arch to supply blood to the head. The
fourth and sixth arches undergo asymmetrical remodelling to sup-
ply the upper extremities, dorsal aorta and lungs. The aortic sac
gives rise to the proximal aortic arch and brachiocephalic artery.
(Fig. 10.26). The left fourth arch becomes the aortic arch between
the left common carotid and left subclavian arteries and the most
cranial part of the descending aorta. A retro-oesophageal right sub-
clavian artery arises when the seventh intersegmental artery that
normally connects with the ventral part of the right fourth arch
connects instead to the persistent right dorsal aorta (Fig. 10.27).
A retro-oesophageal right subclavian artery is a common finding
in trisomy 21 (Fig. 10.28).
The right and left subclavian arteries derive from the seventh
intersegmental arteries. The sixth arch arteries provide the pulmo-
nary arteries, and the left arch provides the arterial duct.
Persistence of remnants of the aortic arches can cause vascular
rings that enclose the oesophagus and trachea (Figs. 10.29 and
10.30). There are multiple patterns described. Abnormal involu-
tion can also lead to interruption of the aortic arch (Fig. 10.31). 
 CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities 101

• Fig. 10.29  Vascular ring. The ascending aorta gives rise to left and right common carotid arteries and the right subclavian artery. It then arches over
the hilum of the right lung, and the descending aorta lies on the right side. The pulmonary trunk gives rise to the right and left pulmonary arteries, with a
left-sided arterial duct connecting the left subclavian artery to the left pulmonary artery. The left subclavian artery arises from the descending aorta via a so-
called retro-oesophageal diverticulum (diverticulum of Kommerell). There is thus a complete vascular ring which surrounds the trachea and oesophagus;
viewed posteriorly, the vessels display a Y-configuration formed by the aortic arch on the right and the left subclavian artery or diverticulum of Kommerell
on the left. The descending thoracic aorta more distally is situated in the midline, posterior to the oesophagus. No right-sided arterial duct was identified,
and there was no evidence of coarctation.

A B
• Fig. 10.30  Vascular ring in a male fetus aged 20 weeks. A, The thoracic contents are viewed from behind. There is a complete arterial ring around the
oesophagus and trachea. There is a right aortic arch with right-sided aorta but a left arterial duct and an isolated left subclavian artery. B, Isolated heart
with the trachea and oesophagus removed. The ring is formed by the right aortic arch and the left-sided arterial duct. The left subclavian artery arises
from the duct.
102 SE C T I O N 3     Fetal Physiology and Pathology

• Fig. 10.31  Interrupted aortic arch. The great vessels arise normally from the heart. The aorta gives rise to brachiocephalic and left common carotid arter-
ies, but the aortic isthmus is absent (asterisk), and the descending aorta and left subclavian artery are supplied from the pulmonary trunk via the arterial
duct. The patient was a neonate who collapsed suddenly and died. At postmortem, a ventricular septal defect and aortic stenosis were also seen.

Conclusion tube. A complex series of arteries and veins develops sequentially

The human heart develops over a 4-week period from a mass of
mesenchymal cells in the ventral embryo. An endothelial-lined
tube enveloped by muscle forms first and begins rhythmic con-
traction. The tube loops to the right, and from its walls, the atrial
and ventricular chambers balloon out. Mesenchymal protrusions
into the lumen effect separation of the right and left sides in addi-
tion to forming the AV and arterial valves. The conduction sys-
tem develops from the primitive myocardium of the original heart
and remodels to form the definitive arterial and venous system.
Defects in the process can cause defects of laterality (isomerism
and transposition) or failure of septation (atrial, AV and ventricu-
lar septal defects). Secondary haemodynamic changes can greatly
modify and exacerbate the original defect.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References Genet Am J Med Genet A. 2014;164A(6):
1347–1371.
23. MacGrogan D, Luna-Zurita L, de la Pompa JL.
Notch signaling in cardiac valve development
13. Miquerol L, Kelly RG. Organogenesis of the and disease. Birth Defects Res A Clin Mol Tera-
1. Bruneau BG. Signaling and transcriptional net-
vertebrate heart. Wiley Interdiscip Rev Dev Biol. tol. 2011;91:449–459.
works in heart development and regeneration.
2013;2:17–29. 24. Briggs LE, Kakarla J, Wessels A. The pathogen-
Cold Spring Harb Perspect Biol. 2013;5:1–18.
14. Taber LA, Voronov DA, Ramasubramanian A. esis of atrial and atrioventricular septal defects
2. Lindsey SE, Butcher JT, Yalcin HC. Mechani-
The role of mechanical forces in the torsional with special emphasis on the role of the dor-
cal regulation of cardiac development. Front
component of cardiac looping. Ann N Y Acad sal mesenchymal protrusion. Differentiation.
Physiol. 2014;5:318.
Sci. 2010;1188:103–110. 2012;84:117–130.
3. Su W, Zhu P, Wang R, et al. Congenital heart
15. Manner J, Wessel A, Yelbuz TM. How does the 25. Unolt M, Putotto C, Silvestri LM, et al. Trans-
diseases and their association with the variant
tubular embryonic heart work? Looking for the position of great arteries: new insights into the
distribution features on susceptibility genes.
physical mechanism generating unidirectional pathogenesis. Front Pediatr. 2013;1:11.
Clin Genet. 2017;91:349–354.
blood flow in the valveless embryonic heart 26. Hinton RB, Yutzey KE. Heart valve structure
4. Gut P, Reischauer S, Stainier DYR, Arnaout R.
tube. Dev Dyn. 2010;239:1035–1046. and function in development and disease.
Little fish, big data: zebrafish as a model for car-
16. Butcher JT, McQuinn TC, Sedmera D, et  al. Annu Rev Physiol. 2011;73:29–46.
diovascular and metabolic disease. Physiol Rev.
Transitions in early embryonic atrioventricular 27. Combs MD, Yutzey KE. Heart valve develop-
2017;97:889–938.
valvular function correspond with changes in ment: regulatory networks in development and
5. Kulesa PM, McKinney MC, McLennan R.
cushion biomechanics that are predictable by tis- disease. Circ Res. 2009;105:408–421.
Developmental imaging: the avian embryo
sue composition. Circ Res. 2007;100:1503–1511. 28. Anderson RH, Mori S, Spicer DE, et al. Devel-
hatches to the challenge. Birth Defects Res C
17. Moorman AFM, Christoffels VM. Cardiac opment and morphology of the ventricular
Embryo Today. 2013;99:121–133.
chamber formation: development, genes and outflow tracts. World J Pediatr Congenit Heart
6. Krishnan A, Samtani R, Dhanantwari P,
evolution. Physiol Rev. 2003;83:1223–1267. Surg. 2016;7:561–577.
et  al. A detailed comparison of mouse and
18. Samsa LA, Yang B, Liu J. Embryonic cardiac 29. Pérez-Pomares JM, de la Pompa JL, Franco D,
human cardiac development. Pediatr Res.
chamber maturation: trabeculation, conduc- et  al. Congenital coronary artery anomalies:
2014;76:500–507.
tion, and cardiomyocyte proliferation. Am J Med a bridge from embryology to anatomy and
7. Rana MS, Christoffels VM, Moorman AFM. A
Genet C Semin Med Genet. 2013;163C:157–168. pathophysiology—a position statement of the
molecular and genetic outline of cardiac mor-
19. Boogerd CJ, Evans SM. TBX5 and NuRD development, anatomy, and pathology ESC
phogenesis. Acta Physiol. 2013;207:588–615.
divide the heart. Dev Cell. 2016;36:242–244. working group. Cardiovasc Res. 2016;109(2):
8. Chung IM, Rajakumar G. Genetics of con-
20. Briggs LE, Kakarla J, Wessels A. The pathogen- 204–216.
genital heart defects: the NKX2–5 gene, a key
esis of atrial and atrioventricular septal defects 30. van Weerd JH, Christoffels VM. The forma-
player. Genes (Basel). 2016;7: pii, E6.
with special emphasis on the role of the dor- tion and function of the cardiac conduction
9. Lough J, Sugi Y. Endoderm and heart develop-
sal mesenchymal protrusion. Differentiation. system. Development. 2016;143:197–210.
ment. Dev Dyn. 2000;217:327–342.
2012;84:117–130. 31. Mommersteeg MT, Brown NA, Prall OW,
10. Abu-Issa R. Heart fields: spatial polarity and
21. Jensen B, Spicer DE, Sheppard MN, Anderson RH. et  al. Pitx2c and Nkx2-5 are required for the
temporal dynamics. Anat Rec (Hoboken).
Development of the atrial septum in relation to formation and identity of the pulmonary myo-
2014;297:175–182.
postnatal anatomy and interatrial communica- cardium. Circ Res. 2007;101:902–909.
11. Haack T, Abdelilah-Seyfried S. The force
tions. Heart. 2017;103:456–462. 32. Yashiro K, Shiratori H, Yashiro K, et al. Hae-
within: endocardial development, mechano-
22. Anderson RH, Brown NA, Mohun TJ. Insights modynamics determined by a genetic pro-
transduction and signalling during cardiac mor-
regarding the normal and abnormal formation gramme govern asymmetric development of
phogenesis. Development. 2016;143:373–386.
of the atrial and ventricular septal structures. the aortic arch. Nature. 2007;450:285–288.
12. Sylva M, van den Hoff MJB, Moorman AFM.
Clin Anat. 2016;29:290–304.
Development of the human heart. Am J Med

102.e1
11
Lung Growth and Maturation
RICHARD HARDING, ANNIE R.A. MCDOUGALL AND STUART B. HOOPER
KEY POINTS
• S urvival at birth depends upon the lung attaining an ad-
equate size and degree of structural maturity during fetal life.
This chapter deals with mechanisms underlying normal and
impaired lung growth and lung maturation before birth.
• The airways of the fetal lung contain a liquid that is actively
secreted by the epithelium; this ‘lung liquid’ causes the lung
to develop in an expanded state, which is necessary for nor-
mal lung growth and maturation.
• The degree of lung expansion in a fetus is determined by the
lung’s physical environment, including intrathoracic space,
fetal breathing movements (FBMs) and amniotic fluid volume.
Mechanical stress in lung tissue stimulates gene networks,
leading to tissue growth and differentiation. The long-term
absence of the physiological stretch stimulus leads to lung
hypoplasia.
• Clearance of lung liquid begins with the onset of labour
caused by (i) imposed fetal postural changes that cause loss
of lung liquid via the nose and mouth and (ii) active reabsorp-
tion across the lung epithelium. Luminal liquid remaining
after birth is cleared because of transpulmonary pressure
gradients generated by inspiration.
• Pulmonary blood flow (PBF) is generally low during fetal life
but can increase with FBMs. At birth, pulmonary vascular
resistance decreases markedly, thereby permitting increased
blood flow through the lungs, which is necessary for ad-
equate gas exchange.
• Lung aeration at birth underpins the cardiovascular transition
at birth, including the marked increase in PBF. With the loss
of umbilical venous return at birth, the increase in pulmonary
venous return takes over the critical role of supplying preload
for the left ventricle.
• Maturation of the lung in preparation for birth involves extra-
cellular matrix remodelling, alveolar epithelial cell differentia-
tion and surfactant production. These changes are driven by
mechanical stress in lung tissue and corticosteroid signalling.
Introduction
In fetal life, the lungs play no role in gas exchange, but at birth,
they must immediately take over from the placenta the critical
role of gas exchange. This transition is normally uneventful, which
is remarkable given that before birth, the lungs are liquid filled
with a low blood flow. For the lung to function as an organ of gas
exchange at birth, it must cease the secretion of fetal lung liquid,
and the airways must be cleared of luminal liquid; the lungs must
produce adequate amounts of surfactant; and pulmonary vascular
resistance (PVR) must be reduced, allowing them to receive the
entire output of the right ventricle. During normal fetal develop-
ment, the lung becomes progressively prepared for these dramatic
changes in physiology at birth. However, if lung growth or matu-
ration in utero is impaired or if an infant is born before term, the
newborn infant may develop respiratory distress syndrome (RDS).
This chapter focuses on the processes controlling prenatal lung
growth and maturation and highlights the physiological changes
that underpin the transition to newborn life. Some of the more
common respiratory complications in neonates and their fetal ori-
gins are discussed together with strategies for their treatment. 
Stages of Lung Development
Pulmonary morphologists recognise five or six major stages in
human lung development1 (Table 11.1).
Embryonic Stage (4–7 Weeks)
The lung first appears as an outgrowth of the primitive foregut
(i.e., endodermal tissue) at 22 to 26 days postconception. This bud
divides to form the left and right bronchi, which then undergo
dichotomous branching to form the major units of the bronchial
tree. During early embryonic development, epithelial cells that are
endodermal in origin form the developing ‘airways’ and grow into
the surrounding tissue, which is derived from splanchnic meso-
derm. This mesodermal tissue gives rise to the mesenchymal cells
that ultimately form the nonepithelial structures of the lung, includ-
ing blood and lymph vessels, airway cartilage and smooth muscle,
fibrous tissue and other components of the lung parenchyma. 
Pseudoglandular Stage (5–17 Weeks)
During the pseudoglandular stage, the lung resembles a typical
exocrine gland. The major bronchi and associated functional
units of the lung (i.e., acini) progressively form, accompanied by
branches of the pulmonary arterial tree. As a result, each major
‘airway’ is accompanied by a branch of the pulmonary artery. The
formation of each acinus (respiratory unit) results from repeated
branching of the distal extremities of blind-ending tubes or ‘air-
ways’ composed of epithelial cells (Fig. 11.1). This process of
branching (branching morphogenesis) is induced by airway epi-
thelial cells interacting with adjacent mesenchymal cells, which are
supplied by a loose network of capillaries (see Fig. 11.1). Airway
epithelial cells gradually differentiate (in a centrifugal direction)
103
104 SE C T I O N 3     Fetal Physiology and Pathology

TABLE
11.1 Stages of Lung Development in the Human
Stage Gestational Age Major Events
Embryonic 4–7 wk Appearance of ventral bud in
foregut. Epithelial tube branches
and grows into surrounding
mesenchyme. Vascular connec­
tions formed.
Pseudo­ 5–17 wk Development of bronchial tree, par­
glandular alleled by formation of vascular
tree. Lung periphery contains
parenchymal precursors.
Canalicular 16–26 wk Addition of further generations
of airways and vascular tree.
Differentiation of type I and type
II epithelial cells. Formation of
thin air–blood barrier. Start of
surfactant production.
Saccular 25–40 wk Formation of additional airway
stage (term) generations. Dilation of prospec­
tive gas-exchanging airspaces.
Maturation of surfactant system.
Alveolar 36 wk–18 mo Start of alveolar formation by
stage outgrowth of secondary septa.
Microvascular Birth–3 yr Change from double- to single-
maturation capillary network. Reduction in
interstitial tissue mass; fusion of
capillaries; preferential growth
of single-layered capillary
network areas.
Reproduced in modified form, with permission, from Burri P. In: Hanson MA, Spencer JAD,
Rodeck CH, Walters DV, eds. Fetus and Neonate. Physiology and Clinical Implications.
Cambridge, United Kingdom: Cambridge University Press; 1994:3–19.
  
into specific cell types: ciliated cells (by 11–13 weeks), goblet cells
and mucous glands.  terminal sacs.
lung tissue volume. During the terminal sac stage, the develop-
ment of secondary septa begins; these outgrowths from primary
septa will eventually subdivide the terminal sac into multiple alve-
oli (see Fig. 11.1). The primitive primary septa, which separate
adjacent saccules, are thicker than secondary septa and contain a
double capillary network rather than the single capillary layer of
the mature alveolus. Elastic fibres are formed by myofibroblasts
within secondary septa and are deposited at their tips, thereby
contributing to the inherent elastic (recoil) properties of the lung.
The epithelial cells become differentiated into type I and type II
epithelial cells. As a result of these structural changes, the separa-
tion between luminal ‘air’ and capillary blood becomes smaller,
thereby enhancing the ability of the lung to exchange respiratory
gases after birth. 
Alveolar Stage (36 Weeks of Gestation to 1–2
Years)
During the alveolar stage, terminal sacs become subdivided by the
outgrowth of secondary septa from the primary septa to form alve-
oli. Initially, these alveoli resemble shallow cups, but they deepen
because of elongation of the secondary septa. The alveolar walls
and the epithelial cells lining them become thinner, leading to
the formation of definitive alveoli. The mean alveolar diameter
increases greatly, from about 30 μm at 30 weeks to about 150 μm
at 40 weeks. The final stage of alveolar maturation involves the
restructuring of the capillary network, such that the more primi-
tive double capillary network lining the terminal sacs and alveoli
is transformed into a single layer of capillaries1 (see Fig. 11.1); this
marks the existence of definitive alveoli.
By the time of term birth, the human lung contains 20 to
50 million alveoli. An adult human lung contains approximately
300 million alveoli, indicating that most are formed postnatally.
The alveolar stage of lung development is thought to continue
for at least 1 to 2 years after birth, although some alveoli may
continue to be formed later in life. In species born at an ear-
lier stage of development (e.g., rats and mice), the alveolar stage
begins after birth; therefore, at birth, gas exchange occurs across
 

Canalicular Stage (16–26 Weeks) Pulmonary Circulation

During the canalicular stage, the airways widen and lengthen,
and mesenchymal tissue surrounding the distal airways becomes
attenuated (see Fig. 11.1). This process (canalisation) results in a
substantial increase in the ratio of lumen volume to tissue volume.
During the canalicular stage, the functional units of the lung are
formed, consisting of terminal bronchioles ending with expan-
sions that subsequently form terminal sacs (primitive alveoli).
A network of blood capillaries develops around the terminal air
sacs, increasing the proximity of blood capillaries to the epithe-
lial surface; this marks the beginning of the air–blood interface
that is required for effective gas exchange (see Fig. 11.1). Thus the
late canalicular stage is the earliest at which the lungs can support
independent life.  the bronchiolar and pulmonary veins. The pulmonary arteries
Terminal Sac Stage (28–40 Weeks)
The terminal sac, or saccular, stage of lung development sees a
progressive enlargement of the distal ‘air spaces’. This enlargement
results from further attenuation of perisaccular mesenchymal tis-
sue and leads to a further increase in luminal volume relative to
Structural Development
The structural development of the pulmonary vasculature has
recently been reviewed in detail.2,3 The lung develops with two
anatomically and functionally distinct vascular systems: the
pulmonary system, which supplies the gas-exchanging tissue
(alveoli), and the bronchial system, which perfuses the non–gas-
exchanging tissue of the lung. The arteries of the bronchial circu-
lation give rise to capillaries which supply the walls of the bronchi
and bronchioles but do not extend to the most peripheral gas-
exchanging parts of the bronchial tree; bronchial venous blood
returns via the pulmonary veins because of anastomoses between
develop a muscular wall except near the lung periphery, where the
arteries are only partially muscularised. Pulmonary veins show a
branching pattern similar to that of arteries but do not follow the
arteries and airways; rather, they tend to run at right angles in the
mesenchyme.
Arterioles are virtually absent in the adult pulmonary circula-
tion; therefore, pulmonary blood flow (PBF) is determined largely
 CHAPTER 11  Lung Growth and Maturation 105

A B

c
c

C D
• Fig. 11.1 Diagram showing development of lung parenchyma and its microvasculature. A, Pseudo-
glandular stage, during which epithelial tubes lined by columnar epithelial cells invade the mesenchyme,
which contains a loose network of blood capillaries (C). The remaining panels show further development
of structures enclosed by the frame in A. B, Canalicular stage, showing differentiation of ‘airspace’ epi-
thelium and expansion of airspaces resulting in attenuation of mesenchyme; capillaries are rearranged
around the epithelial tubes so that walls between ‘airspaces’ contain a double layer of capillaries. A thin
‘air’–blood interface develops, and types I and II epithelial cells become apparent. C, Terminal sac and
alveolar stages, showing development of secondary septa from primary septa; septa are primitive in that
they contain a double capillary network and a central layer of connective tissue. D, Mature lung, showing
thin interalveolar walls containing a single capillary layer. (Reproduced with permission from Burri PH. Fetal
and postnatal development of the lung. Ann Rev Physiol. 1984;46:617–628.)
106 SE C T I O N 3     Fetal Physiology and Pathology

Section of RBC

Blood–gas
Capillary barrier
endothelial cell
Type I alveolar Fused basement
epithelial cell membrane

Alveolus

Nucleus of type I
Capillary alveolar epithelial cell

Alveolus
1 m

Alveolus
• Fig. 11.2  An electron micrograph of an alveolus and an adjacent capillary, demonstrating the very thin
barrier that separates the airspace from the capillary lumen (air–blood barrier). Note that this barrier con-
sists of the attenuated cytoplasm of an alveolar epithelial cell and a capillary endothelial cell, which are
separated by their respective basement membranes that have fused (see inset). In this micrograph, the
attenuated cytoplasmic extension of the type I cell indicated extends around the entire alveolus. RBC,
Red blood cell.

by the resistance of the alveolar capillaries. In fetuses and new-
borns, however, the smooth muscle surrounding the small pulmo-
nary arteries is thicker than in an adult lung, relative to diameter,
and extends farther down the vascular tree. This likely contributes
to the high vascular resistance of the fetal lung and may be a con-
sequence of the high fetal pulmonary artery pressure (relative to
postnatally). In the first few weeks after birth, the arterial smooth
muscle thins, leading to a reduction in arterial wall thickness,
likely because of a reduction in pulmonary arterial pressure fol-
lowing the functional separation of the pulmonary and systemic
circulations.
The creation of an efficient gas exchange surface within
the lung depends upon the development of a dense capillary
bed in close proximity to the epithelium of the terminal sacs
or alveoli. Early in development, capillaries form a loose net-
work adjacent to the immature airspaces, but the two struc-
tures are usually separated by other cells and extracellular
matrix (ECM) components. During the canalicular stage, the
number of capillaries increases greatly, and they come into
close contact with the epithelium of the primitive air sacs.
By the terminal sac stage, the capillaries form a dense net-
work around these sacs and, with further attenuation of tissue
between adjacent sacs, the basement membrane underlying
the capillary endothelial cells fuses with the basement mem-
brane underlying the alveolar epithelial cells (Fig. 11.2). The
sites of basement membrane fusion are initially focal, but they
expand as the lung matures, providing a very thin (∼0.2 μm)
barrier for gas exchange.  Po2 similar to in fetuses greatly increases PVR.7 In fetal sheep,
Functional Development of the Pulmonary
Circulation
This topic has recently been reviewed in depth.3 At midgestation,
only 3% to 4% of total cardiac output perfuses the lung, and by
late gestation, this has risen to only 8% to 10%.3 In a fetus, most
(∼88%) of the right ventricular output is diverted away from the
lungs to the descending aorta via the ductus arteriosus. Mean
pulmonary arterial pressure in a near-term fetus is about 55 mm
Hg, which is about 5 mm Hg higher than mean aortic pressure,
thereby maintaining flow from the pulmonary to the systemic cir-
culation through the ductus arteriosus. Although PVR declines
progressively during fetal life due to a large increase in total cross-
sectional area of the pulmonary vascular bed, it remains much
higher (up to eightfold) just before birth than immediately after
birth.3
In fetuses, PVR is influenced by a range of factors, includ-
ing the physical and oxygen environments of pulmonary vessels
and the presence of vasoactive agents. Because this topic has been
extensively reviewed,3,4 only a brief outline will be provided here.
In fetal sheep, PVR is reduced by vigorous fetal breathing move-
ments (FBMs)5 and is closely related to lung liquid volume.6
Increasing the volume, and hence pressure, of liquid within the
terminal air sacs likely compresses the small pulmonary vessels
(mainly capillaries), thereby increasing PVR.6 The low Po2 of
blood perfusing the fetal lungs also contributes to a high PVR.
In postnatal lambs, perfusion of the lungs with blood having a
 CHAPTER 11  Lung Growth and Maturation 107

A B

• Fig. 11.3  A, Simultaneous phase-contrast x-ray images and angiograms of a newborn rabbit after uni-
lateral ventilation of the right lung, showing that global increases in pulmonary blood flow are independent
of lung aeration. The flow of iodine (contrast reagent; black) through vessels is equal in both the aerated
right lung and nonaerated left lung. B, Phase-contrast x-ray image of a spontaneously breathing new-
born rabbit in which the air–liquid boundary is visible. Complete aeration of the lungs has been achieved
(white speckle), down to the most distal gas-exchange regions (inset); single alveoli can be seen when
one airway is in projection. The role of inspiration in clearing lung liquid has been demonstrated using this
technique, showing that lung liquid can be completely cleared from the airways during the first three to five
breaths caused by the transpulmonary hydrostatic pressures generated during inspiration.

lowering and raising the Po2 of arterial blood (i.e., hypoxia and
hyperoxia) causes increases and decreases in PVR, respectively.3
The mechanism by which oxygen tension influences PVR is
unknown but may involve the release of prostacyclin (PGI2) and
endothelium-derived nitric oxide (NO), both of which affect vas-
cular smooth muscle.  (see Fig. 11.2), the creation at birth of an air–liquid interface in
Changes at Birth
Lung aeration is the primary trigger for the decrease in PVR at
birth, resulting in an 8- to 10-fold increase in PBF.6 Although the
increase in PBF is enhanced by increased oxygenation, it is not
dependent on an increase in oxygenation8,9 and occurs even if the
lungs are ventilated with 100% nitrogen.7 Furthermore, as partial
lung aeration leads to a global increase in PBF, increasing equally
in both aerated and nonaerated regions (Fig. 11.3), the increase
in PBF is not spatially related to aerated lung regions.10 This can
lead to a large ventilation/perfusion mismatch in the lung if it only
partially aerates at birth, which as argued later, is likely to be more
beneficial than harmful for the transitioning infant.
The mechanisms underlying the large fall in PVR at birth
are complex and probably involve alterations in the physical
environment, the oxygen environment and the balance between
vasodilator and vasoconstrictor substances. Because the alveolar
epithelium and capillary endothelium are mechanically coupled
the alveoli and the partial recoil of the lungs (see later) likely alter
the geometry of small pulmonary vessels, causing them to dilate.
Rhythmic expansion of the lungs at birth, increased oxygenation
and release of vasodilators such as PGI2, bradykinin (a potent
vasodilator in the fetus) and NO, are thought to mediate pulmo-
nary vasodilation after birth. Indeed, inhibition of NO synthe-
sis before birth attenuates the birth-induced increase in PBF and
results in pulmonary hypertension in the newborn.3
None of the above-mentioned mechanisms can readily explain
the global increase in PBF induced by partial lung aeration
because the increase occurs equally in aerated and nonaerated lung
regions and can be induced by ventilation with 100% N2 (see
Fig. 11.3).7,10 Although the mechanism is unknown, it has been
suggested that the clearance of airway liquid into the surrounding
108 SE C T I O N 3     Fetal Physiology and Pathology

perialveolar tissue could activate J-receptors which signal via vagal TABLE Composition of Fetal Lung Liquid in Comparison
afferents to effect global pulmonary vasodilation.10 11.2 to Fetal Arterial Blood and Amniotic Fluid
The increase in PBF at birth plays a critical role in the cardio-
vascular transition at birth. Before birth, because PBF is low, pre­ Parameter Arterial Blood Lung Liquid Amniotic Fluid
load for the left ventricle is predominantly derived from umbilical Na+ (mmol/L) 150 ± 0.7 150 ± 1.3 113 ± 6.5
venous return, which flows via the ductus venosus, inferior vena
cava and foramen ovale to enter the left atrium.11 Thus if the K+ (mmol/L) 4.8 ± 0.2 6.3 ± 0.7 7.6 ± 0.8
umbilical cord is clamped before the lungs have aerated and PBF Cl– (mmol/L) 107 ± 1.0 157.1 ± 4.1 87 ± 5
has increased, the left ventricle will be deprived of preload until
the lungs aerate and PBF increases.12,13 This can cause a sustained Ca2+ (mmol/L) 3.3 ± 0.1 0.8 ± 0.1 1.6 ± 0.1
decrease in cardiac output (by up to 50%) after birth and places HCO3− (mmol/L) 24 ± 1.2 2.8 ± 0.3 19 ± 3
the infant at high risk for systemic hypotension and hypoxic-isch-
emic brain injury. This is because an increase and redistribution Urea 291 ± 2 7.9 ± 2.7 10.5 ± 2.4
of cardiac output are the primary mechanisms that protect the Osmolality (mOsm) 291 ± 2 294 ± 2 265 ± 2
brain from oxygen deficiency during hypoxic-asphyxic episodes.14
Protein 4090 ± 260 27 ± 2 100 ± 10
On the other hand, aerating the lung and increasing PBF before
(mg/100 mL)
the umbilical cord is clamped allows the supply of preload blood
to the left ventricle to immediately switch from umbilical venous pH 7.34 ± 0.04 6.27 ± 0.5 7.02 ± 0.09
to pulmonary venous return without any decrease in supply.12 As
Values are derived from Adamson TM, Boyd RD, Platt HS, Strang LB. Composition of alveolar
a result, the characteristic decrease in cardiac output (often evi- liquid in the foetal lamb. J Physiol. 1969;204:159–168.
denced as a decrease in heart rate) is prevented when the onset of
ventilation precedes umbilical cord clamping.12 Furthermore, in
  
view of the vital role that a high PBF plays after birth in provid-
ing preload for the left ventricle and maintaining cardiac output,
it is logical for the increase in PBF to be independent of, and not a similar difference exists in humans. That is, the change in lung
linked (at least initially), to the degree of lung aeration.  luminal volume associated with individual FBM in late gesta-
tion is normally less than 1% of resting (baseline) luminal vol-
Fetal Breathing Movements ume. Thus FBM resemble postnatal breathing with an obstructed
upper airway; this presumably gives rise to the paradoxical nature
Episodes of breathing-like movements occur intermittently of FBM during which the chest wall retracts during inspiratory
throughout much of gestation in healthy mammalian fetuses.15 efforts and the abdominal wall moves out.21,22
These movements, termed ‘fetal breathing movements’, share key Fetal breathing movements are readily detected by ultrasound
features with postnatal breathing and are thought to be an early and, as a component of the fetal biophysical profile,23 have been
expression of coordinated, rhythmical activity in the brainstem used to assess fetal health. The incidence of FBMs is reduced in
regions responsible for the control of breathing. FBMs involve fetuses that are subjected to intrauterine stresses such as hypox-
rhythmic ‘inspiratory’ activation of the diaphragm and dilator mus- emia, hypoglycaemia, intrauterine infection, increased levels of
cles of the larynx.16 Expiratory muscles (e.g., intercostal muscles, prostaglandins and labour.15,24 FBMs may be absent or impaired
upper airway constrictors) are not significantly active during peri- in fetuses with congenital abnormalities of the nervous system or
ods of unstimulated FBM. Similar to postnatal breathing, FBMs are skeletal muscle. Maternal drug use can also abolish or attenuate
stimulated by increased arterial PaCO2 and by decreased pH in the FBM; for example, alcohol consumption, tobacco smoking and
blood and cerebrospinal fluid, probably via stimulation of central common sedatives and narcotics can inhibit FBM.
chemoreceptors.17 FBMs are also influenced by fetal behavioural Fetal breathing movements are now known to be critical for
states; they principally occur in association with a state resembling normal lung development; indeed, the prolonged absence of FBM
rapid eye movement (REM) sleep and are absent during the state results in hypoplastic lungs, in which the lungs are small and
resembling quiet (slow-wave or non-REM sleep.15 Whether the structurally immature.25,26 If severe, lung hypoplasia can result in
fetus is ever in a state of wakefulness is controversial; however, short respiratory insufficiency or death in the newborn. FBMs play a
periods of a fetal state of heightened activity resembling wakeful- critical role in maintaining the fetal lungs in an expanded state by
ness (low-voltage electrocortical activity, eye movements, fetal swal- opposing the inherent tendency of the fetal lung to recoil. Lung
lowing, head and neck movements) are usually accompanied by expansion during fetal life is known to be necessary for the normal
FBM. The suppression of FBM during fetal quiet (non-REM) sleep growth and structural maturation of the lungs; the role of FBMs
is of considerable interest because this suppression must cease after in maintaining lung expansion is discussed in more detail later. 
birth when breathing becomes continuous, regardless of sleep–
wake states. It is currently thought that breathing becomes continu-
ous after birth as a result of increased CO2 production and removal Fetal Lung Liquid
from putative inhibitory agents released by the placenta. Control of Fetal Lung Liquid Secretion
Fetal breathing movements differ from postnatal breathing in
that the ‘tidal volume’ is very small, and each ‘breath’ is essentially The future air spaces of the fetal lung contain a liquid that is
isovolumic; the low tidal volume of a fetus can be attributed to secreted by the pulmonary epithelium. This unique liquid (fetal
the high viscosity (and inertia) of water relative to air, and the lung liquid) is not inhaled amniotic fluid because it accumu-
high resistance of moving liquid through the respiratory tract.18 lates in the lungs when the trachea is obstructed,16 and it has
In ovine fetuses, the ‘tidal volume’ is normally less than 1 mL19 an ionic composition very different to that of amniotic fluid27
compared with 40 to 50 mL (8–10 mL/kg) in newborn lambs20; (Table 11.2). Measurements of unidirectional ion fluxes in fetal

sheep lungs indicate that lung liquid secretion results from the
net movement of chloride and sodium ions across the pulmonary
epithelium into the ‘airway’ lumen.28 This generates a transepi-
thelial osmotic gradient that promotes the movement of water
into the lung lumen. It is thought that Na+,K+-ATPase, located
on the basolateral surface of pulmonary epithelial cells, provides
the electrochemical gradient for Na+ to enter the cell coupled to
Cl–. The Cl– then exits the cell across its apical membrane and
enters the lung lumen down the transmembrane electrochemi-
cal gradient. The net movement of Cl– into the lung lumen pro-
vides an electrical gradient for Na+ to enter the lumen as well;
together these ionic movements create an osmotic gradient for
the movement of water from the cell into the lung lumen.28 After
being secreted, fetal lung liquid leaves the lung via the trachea and
enters the pharynx, from where it is either swallowed or enters the
amniotic sac.29,30
Although there are no data on lung liquid secretion for human
fetuses, fetal sheep secrete lung liquid at 3 to 4 mL/kg/h during
the last third of gestation before the onset of labour.31 The rate of
secretion is controlled by endocrine, metabolic and physical fac-
tors. Both adrenaline, acting via β-adrenergic receptors,32,33 and
arginine vasopressin34 are potent inhibitors of lung liquid secre-
tion in  vivo, possibly via activation of adenylate cyclase leading
to an increase in intracellular cyclic adenosine monophosphate
(cAMP) concentrations.35 Other hormones known to affect fetal
lung liquid secretion include cortisol and prolactin, both of which
increases lung liquid secretion in vivo.36-38
Because lung liquid secretion is an active process, it is inhib-
ited by hypoxaemia (acute or chronic),39,40 an effect which is
likely to be mediated by a reduction in oxygen availability or
associated changes in tissue pH rather than an increase in circu-
lating adrenaline and vasopressin.33,41,42 Physical factors such as
the pulmonary luminal liquid pressure also influence the secre-
tion of fetal lung liquid. A reduction in fetal lung liquid volume,
and hence a reduction in luminal pressure, increases lung liq-
uid secretion rates.43-45 In contrast, sustained increases in fetal
lung expansion, which increase luminal pressure, lead to either a
reduction46 or cessation of lung liquid secretion.5,47 The driving
force for the net movement of water across the lung epithelium
must be governed by the sum of all forces affecting liquid move-
ment. Under normal conditions, a small hydrostatic pressure
exists across the lungs because pressure within the lung lumen is
1 to 2 mm Hg greater than amniotic sac pressure.16 This small
pressure gradient likely opposes the osmotic pressure resulting
from the movement of chloride ions. The osmotic pressure pro-
moting lung liquid secretion must exceed the opposing hydro-
static pressure for liquid to cross the epithelium into the lung
lumen. Thus reductions or increases in the intraluminal pres-
sure, resulting from alterations in lung liquid volume, would be
expected to increase or reduce net lung liquid production rates
by altering the magnitude of the opposing hydrostatic pressure
(i.e., without directly affecting the ionic mechanism of lung liq-
uid secretion).  sodes is opposed by simultaneous rhythmic contractions of the
Control of Fetal Lung Liquid Volume
For most of gestation, the fetal lung develops in an expanded state,
and the volume of liquid within the future airways increases mark-
edly over the last half of gestation.48 Over the last third of ovine
gestation, lung liquid volume typically increases from 25 to 30
mL/kg to 45 to 50 mL/kg near term (145–150 days) (Fig. 11.4).
In contrast, the corresponding luminal volume of the air-filled
CHAPTER 11  Lung Growth and Maturation 109
19
50
38
Resting lung volume (mL/kg)
47 46 36
40 36
9
14
30
13 14
14
20
10
0 120 130 140 10 20 30 40
Gestational age Postnatal age
(days) (days)
Birth
• Fig. 11.4  The volume of fetal lung liquid, measured by dye dilution, dur-
ing the last 40 days of gestation in chronically catheterised fetal sheep in
utero. Measurements of functional residual capacity in postnatal lambs,
made using a He-dilution technique, have been included for comparison.20
Numbers represent the number of measurements made at each gesta-
tional or postnatal age.
lung (functional residual capacity (FRC)) is 15 to 20 mL/kg
immediately after birth in newborn rabbits49 and 20 to 25 mL/kg
in newborn lambs at 1 to 2 days of age.20 Thus, during late gesta-
tion, the fetal lung is hyperexpanded relative to the postnatal air-
filled lung (see Fig. 11.4). The reduction in lung luminal volume
(i.e., FRC) at birth is likely due to the formation at birth of an
air–liquid interface, and hence surface tension, which constitutes
the major part of the postnatal lung’s elastic recoil. Although the
presence of surfactant greatly reduces surface tension, it does not
eliminate it and, as a consequence, the lung tends to pull away
from the chest wall after birth. This increased recoil after birth
results in the formation of a subatmospheric pressure in both the
intrapleural and the perialveolar interstitial tissue space which is
not present in a fetus.13,16
During fetal life, lung liquid volume is largely maintained by
factors that control transpulmonary pressure and the efflux of liq-
uid from the lungs rather than by alterations in secretion rate.16
The rate of efflux of lung liquid is controlled by transpulmonary
pressure and the resistance of the upper airway, particularly the
muscles of the larynx.18,50 During ‘nonbreathing’ periods in the
fetus (i.e., during fetal ‘apnea’), tonic activity in the laryngeal con-
strictor muscles restricts lung liquid efflux and thereby opposes the
lung’s elastic recoil (Fig. 11.5). During periods of FBM, the glottis
dilates because of phasic activity of the laryngeal dilator muscles,
which reduces the resistance to lung liquid efflux. Consequently,
the combined effect of a lowered resistance to lung liquid efflux
and the lung’s elastic recoil increases liquid loss during periods
of FBM.30 However, the efflux of lung liquid during FBM epi-
diaphragm muscle,43,44 which limit the loss of lung liquid dur-
ing FBM episodes31 (see Fig. 11.5). Thus the high degree of lung
expansion in the fetus is apparently caused by (i) the low level
of elastic recoil (owing to the absence of an air–liquid interface),
(ii) the high resistance to lung liquid efflux offered by the larynx
during non-FBM periods and (iii) contractions of the diaphragm
muscle during FBM episodes which oppose lung liquid efflux
associated with rhythmic dilation of the glottis and reduced upper
airway resistance. 
110 SE C T I O N 3     Fetal Physiology and Pathology

Glottis adducted Dilated glottis

High resistance Low resistance
to liquid efflux to liquid efflux

Lung
Lung
liquid Intraluminal
Intraluminal liquid
pressure
distending
–0 mm Hg
pressure
1–2 mm Hg

A B
• Fig. 11.5  Control of fetal lung liquid volume during (A) periods of apnea and (B) periods of fetal breathing
movements (FBMs). During apnea, the glottis is actively adducted, which restricts the efflux of lung liquid
and promotes its accumulation within the future airways, thereby maintaining an intraluminal distending
pressure of 1 to 2 mm Hg above ambient pressure (i.e., amniotic sac pressure). During periods of FBM,
the glottis phasically dilates, which greatly reduces the resistance to lung liquid efflux. As a result, liquid
leaves the lungs at a higher rate, causing a reduction in lung liquid volume and the distending pressure (at
end-expiration) reduces to ambient pressure.

Clearance of Lung Liquid at Birth

Fetal lung liquid must be cleared from the airways at birth so that
effective pulmonary gas exchange can be established. This process
begins before birth and continues for some time after birth. Stud-
ies in fetal sheep show that lung liquid clearance begins with the
onset of labour48 and that in healthy fetuses with normal amniotic
fluid volumes, much of the liquid is cleared during labour and
after birth. It was previously considered that increased circulat-
ing concentrations of adrenaline and arginine vasopressin (AVP)
in fetal plasma37,38 are primarily responsible for clearing airway
liquid by activating amiloride-blockable sodium channels on the
luminal surface of pulmonary epithelial cells.51 Activation of these
channels increases Na+ and Na+-linked Cl– flux away from the
lung lumen into the interstitium, thereby reversing the osmotic
gradient across the epithelium and leading to lung liquid reab-
sorption.51 However, this mechanism is unlikely to be the only
mechanism, and recent evidence suggests that it has minimal
impact on airway liquid clearance at birth.52 Indeed, any factor
that increases transpulmonary pressure can contribute to the loss
of liquid during labour. For instance, in unstressed fetal sheep at
term, large amounts of lung liquid (∼50%) are lost during the
early stages of labour before fetal plasma adrenaline and AVP
concentrations increase.48 This reduction in lung liquid volume
is probably caused by compression of the lungs resulting from
postural changes imposed on the fetus when the uterine muscle
shortens during labour; it has been established that fetal trunk
flexion causes substantial loss of lung liquid.53
Phase contrast x-ray imaging studies in rabbits have demon-
strated that after birth, any liquid remaining in the airways is
cleared because of transpulmonary hydrostatic pressures gener-
ated during inspiration.52,54 Using this imaging technique, the
air–­liquid boundary is clearly visible and was observed to move
through the airways towards the distal gas exchange regions only
during inspiration (see Fig. 11.3). During expiration, the air–liq-
uid interface tended to move proximally, resulting in a gradual
decline in FRC between breaths.52,54 However, the subsequent
inspiration rapidly recleared this liquid, resulting in very rapid
lung aeration.54 Indeed, rabbit pups at term were observed to com-
pletely clear their airways of liquid within 3 to 5 breaths (∼20 sec),
resulting in the rapid formation of an FRC of about 15 mL/kg.
Although the liquid was found to clear very rapidly from the air-
ways, clearance from the surrounding perialveolar tissue is much
slower (∼4 hours).55 Interestingly, the coexistence of airway liquid
residing in the lung tissue and the presence of a gas volume fill-
ing the airways immediately after birth results in expansion of the
chest wall.52 This finding underlines the importance of having a
compliant chest wall at birth. The movement of airway liquid into
perialveolar lung tissue after birth is, in itself, known to increase
interstitial tissue pressure,55 which must increase the potential for
liquid to reenter the airways at FRC. This likely explains the grad-
ual reduction in FRC between breaths in spontaneously breathing
newborn rabbits.52,54 However, if the chest wall was less compli-
ant, interstitial tissue pressures resulting from airway liquid clear-
ance would increase to a higher level, which would increase the
pressure gradient for liquid to reflood the airways at FRC. 
Lung Growth
Regulation of Fetal Lung Growth
The degree of lung expansion in the fetus, and hence the degree of
lung tissue stretch, plays a critical role in the growth and matura-
tion of the fetal lung.56 Thus most clinical conditions that lead to
fetal lung hypoplasia do so by causing a prolonged reduction in

lung expansion.16,31 These include disorders that prevent the fetal
lung from expanding (e.g., diaphragmatic hernias, pleural fluid
accumulation), cause compression of the fetal chest (e.g., oligo-
hydramnios) and impair fetal skeletal muscle activity or bone (rib
cage) development.
The critical importance of fetal lung expansion in regulating
lung development was first demonstrated by experiments showing
profound changes in lung growth and maturation after prolonged,
experimentally induced alterations in lung liquid volume. Pro-
longed drainage of fetal lung liquid, which chronically reduces the
degree of lung expansion, causes a cessation of fetal lung growth57
and severe lung hypoplasia.45,58 In contrast, chronically increas-
ing the degree of fetal lung expansion (by obstructing the fetal
trachea) markedly increases fetal lung growth and enhances alveo-
larisation.57-59 The lung growth response to tracheal obstruction is
rapid and can cause an almost doubling in total lung cell number
within 7 days.57 The growth response, in terms of cell proliferation
rates, follows a specific time-course, with maximum rates being
detected at 1 to 2 days, which return to control levels by 10 days.47
Thus the processes leading to an acceleration in fetal lung growth
after an increase in lung expansion are only active within a rela-
tively narrow window of time.  lung expansion. That is, the lung hypoplasia most probably results
Mechanisms Relating Lung Stretch to Lung
Growth
The cellular and molecular processes mediating changes in fetal
lung growth in response to altered lung expansion are localised
and apparently restricted to expanded or deflated lung regions.59
This response is not unique to the lung as mechanical forces are
well known to affect DNA synthesis as well as the phenotypic
expression, via the activation or repression of genes, of many
different types of cells.60 However, the mechanisms by which
mechanical forces are translated into cellular responses (mecha-
notransduction) are poorly understood. It is likely that the dis-
tortion of lung tissue associated with a change in lung expansion
is transmitted via the ECM and causes changes in cell shape or
tension within the cytoskeleton. This stimulus may be translated
into a cellular response because of direct activation of stretch-sen-
sitive ion channels or to the direct activation of second messen-
ger systems (e.g., tyrosine kinases and phospholipases) associated
with the proteins that interlink ECM receptors (e.g., integrins)
with the cytoskeleton.61 It is also possible that distortion of a cell
may directly activate or inactivate genes or DNA synthesis via
changes in tension or orientation of the cytoskeleton or nucleo-
skeleton.62,63 This may regulate the access of transcription factors
to DNA via altering chromatin structure and acetylation of gene
promoters.64
It is possible that alterations in gene expression for a variety
of growth factors contribute to the pulmonary growth response
induced by changes in lung expansion, possibly by integrating
and propagating the response. Indeed, alterations in fetal lung
expansion induce corresponding changes in insulin-like growth
factor-II (IGF-II) gene expression in lung tissue.43,57 IGF-II has
potent mitogenic and differentiating activities and is thought to
play an important role in fetal growth. Similarly, intermittent
stretch of pulmonary epithelial cells in culture is a potent stimu-
lus for pulmonary DNA synthesis65 and increases platelet-derived
growth factor (PDGF) gene expression.65 Because the effect of
phasic stretch on DNA synthesis in cultured pulmonary epithe-
lial cells can be blocked by antisense oligonucleotides for PDGF,
PDGF must play a crucial role in this response.66 However, an
CHAPTER 11  Lung Growth and Maturation 111
in vivo study has failed to demonstrate an increase in PDGF and
IGF-II expression when cell proliferation rates are elevated in
response to an increase in fetal lung expansion.67 Furthermore,
a differential gene expression analysis, designed to identify genes
activated and repressed by increases in fetal lung expansion, failed
to identify a number of other growth factors thought to medi-
ate expansion-induced fetal lung growth.68 However, numer-
ous other genes were identified which are likely to be activated
directly by the mechanical stimulus and play a vital role in this
process.68,69 
Fetal Lung Hypoplasia
Lung hypoplasia at birth is a graded phenomenon and if severe
can increase the risk for neonatal morbidity and mortality.70 Fetal
lung hypoplasia has multiple causes, including congenital dia-
phragmatic hernia (CDH), oligohydramnios, fetal hydrothorax
and congenital cystic adenomatoid malformations (CCAM), as
well as fetal muscular and skeletal abnormalities.70 It is likely that
fetal lung hypoplasia associated with these prenatal conditions
has a common mechanism, namely a prolonged reduction in fetal
from the absence of a growth stimulus (i.e., tissue stretch) rather
than from an active inhibition of tissue growth.
The hypoplastic fetal lung is not simply small but is also
structurally immature. For example, hypoplastic lungs contain
reduced numbers of airways and alveoli and71,72 have a reduced
proportion of airspace, reduced elastin development, narrower
airways and altered vascular development.73 The maturation of
the respiratory epithelium is impaired, as evidenced by the per-
sistence of cuboidal cells, particularly in the peripheral parts of
the acinus.74 This structural immaturity is also associated with a
reduced size and effectiveness of the gas-exchange surface, result-
ing in impaired gas exchange that contributes to the neonatal
respiratory compromise. Another important factor that limits
gas exchange is increased PVR in hypoplastic lungs; this is likely
a result of altered structural and functional development of the
pulmonary vascular bed.73 The severity and range of pathological
changes in the lungs will depend on the duration and degree of
reduced lung expansion,75 and it is likely that some of these will
persist into postnatal life owing to the limited capacity for lung
regeneration after birth.
Because fetal lung growth is so sensitive to alterations in
lung expansion, tracheal obstruction has been used therapeuti-
cally to reverse lung growth deficits in human fetuses with severe
CDH.76,77 In utero, a balloon is deployed into the fetal trachea
via ultrasound-guided endoscopy. Trials so far have indicated that
fetal endoscopic tracheal occlusion significantly decreases mortal-
ity rates in babies born with CDH77-79 via reversal of the severe
lung hypoplasia normally seen in these babies. However, the
available evidence from animal studies suggests that a number of
unwanted side effects need to be considered. In particular, tracheal
obstruction leads to altered proportions of epithelial cell types in
the terminal airways, resulting in fewer type II cells58,80,81 and
hence reduced surfactant protein A, B, and C gene expression.82
For this reason, the balloon placed in the trachea is deflated before
delivery, which has been shown to restore type II cell numbers
and surfactant protein expression.81 However, more information
is needed regarding cardiorespiratory function in newborns with
CDH, whether or not they have been exposed to tracheal obstruc-
tion in utero, because current recommendations are based upon
expert opinion.83 
112 SE C T I O N 3     Fetal Physiology and Pathology
Lung Maturation
Structural Maturation of the Lung
The unique architecture of the lung is largely dependent upon
its ECM and on cell-to-cell and cell-substrate adhesion proper-
ties. Components of the ECM are synthesised by a variety of cell
types and together they provide the structural scaffolding that sup-
ports the lung cells.84 Consequently, the ECM plays an integral
role in lung development from the early in utero stages through
to postnatal life. Indeed, different components of the ECM are
considered to be critically involved with cell migration, branching
morphogenesis, cellular proliferation and cytodifferentiation as
well as determining tissue compliance.85 The ECM of the lungs is
composed of collagen (principally types I, III, V and VI), elastin,
glycoproteins (e.g., fibronectin and laminin) and proteoglycans.85
At the level of the peripheral airways, these components form the
epithelial and endothelial basement membranes and the structural
fibres that course through the interstitium located at the interal-
veolar septa; these structural fibres connect with axial fibres run-
ning in parallel with major conducting airways and blood vessels
and are further braced by fibres projecting in from the pleura.
Versican is one of the most abundant proteoglycans located within
the perisaccular parenchyma of the developing lung.86 Its high
ionic charge density promotes the retention of water within tis-
sue, which contributes to tissue volume and has a major influence
on tissue viscoelastic properties. As versican content decreases in
parallel with the age-related decrease in the volume of tissue in the
peripheral lung during late gestation, a loss of versican from the
perialveolar tissue compartment may contribute to the reduction
in tissue volume and the thinning of interalveolar walls.86 Thus,
major alterations in lung architecture, such as those that occur
during development, likely involve remodelling of the ECM.
The increase in fetal plasma cortisol concentration before par-
turition is thought to play an important role in maturing the lung
by influencing its architecture, its tissue compliance, development
of the vascular bed, differentiation of epithelial cells and the syn-
thesis of surfactant.87 These changes lead to an increase in poten-
tial airspace volume, a reduction in gas-diffusing distance and an
increase in compliance of the air-filled lung. Indeed, over the last
third of gestation in fetal sheep, there is a large increase in lung
luminal volume (see Fig. 11.4), which is associated with increased
alveolar surface area and reduced interalveolar tissue volume.88
The findings that fetal infusions of corticosteroids accelerate
these changes in lung architecture but that fetal adrenalectomy or
hypophysectomy (which removes or reduces the source of endog-
enous fetal corticosteroids) retards them36,37 indicate that cortico-
steroids are intimately involved in the structural modification of
the lung during late gestation. Furthermore, mice with a targeted
disruption of the glucocorticoid receptor gene die at birth because
of respiratory failure89; their lungs are morphologically immature,
and hypercellular with abnormal development of the terminal
airways.90,91 Collectively, these findings indicate that a progres-
sive increase in circulating concentrations of fetal corticosteroids
plays an important role in promoting structural changes within
the lung. This concept is consistent with numerous studies dem-
onstrating that antenatal corticosteroid treatment greatly increases
lung compliance92,93 and ventilatory efficiency94 in prematurely
delivered fetuses.
One mechanism by which corticosteroids stimulate structural
maturation of the fetal lung is by inhibiting the proliferation of
interstitial mesenchymal cells,90,95,96 which promotes thinning of
100
Proportion of alveolar epithelial cells %
Birth Type I AECs
80 Type II AECs
Undifferentiated cells %
60
40
20
0
–20
80 d,100 d,120 d,140 d, 2 wk, 8 wk, 2 yr
Gestational age Postnatal age
• Fig. 11.6  Changes in the relative proportions of undifferentiated (inverted
triangles), type I (blue circles) and type II (yellow circles) alveolar epithe-
lial cells in sheep during the last third of gestation and up to 2 years of
age. (Redrawn from Flecknoe SJ, Wallace MJ, Cock ML, et al. Changes in
alveolar epithelial cell proportions during fetal and postnatal development
in sheep. Am J Physiol Lung Cell Mol Physiol. 2003;285:L664–L670.)
the interstitial tissue and decreases in the thickness of the alveo-
lar wall and the air–blood barrier. Glucocorticoids also promote
structural maturation of the lung via remodelling of the ECM.96
The collagen and elastin contents of the lung parenchyma97,98
increase with the exponential increase in circulating cortisol con-
centrations and lung luminal volumes in the fetal sheep lungs.37
By regulating interstitial cell proliferation and ECM remodelling,
corticosteroids are able to influence structural development of the
lung and thus its ability to function as a gas exchange organ after
birth. 
Epithelial Cell Differentiation
The success of pulmonary gas exchange after birth depends on
many factors. These include a large surface area for gas exchange,
adequate blood flow through alveolar capillaries, a thin air–blood
barrier and a high degree of lung compliance. Many of these fac-
tors are dependent upon the maturation of pulmonary epithelial
cells. During early stages of lung development, epithelial cells are
either columnar (pseudoglandular stage) or cuboidal (canalicular
stage), are unable to synthesise surfactant and form a thick barrier
to gas exchange. During the canalicular stage (by 22–24 weeks),
alveolar epithelial cells (AEC) begin to differentiate into thinner
type I cells, which have a thin attenuated cytoplasm with a large
surface area for gas exchange and cuboidal type II cells which syn-
thesise, store and release pulmonary surfactant.99
During the early stages of lung development, all epithelial cells
within the terminal airways are undifferentiated99,100 and gradu-
ally differentiate into both cell types as development progresses
(Fig. 11.6). Although it is widely considered that both type I and
type II cells arise as daughter cells from proliferating type II cells,
recent data indicate that this concept may not be correct for the
following reasons: (i) in sheep fetuses, type I cells can be detected
at an earlier period in gestation than type II cells,88,100 and (ii)
increased fetal lung expansion induces type II to type I cell transdif-
ferentiation, but reductions in fetal lung expansion induce type I
to type II AEC transdifferentiation81(see Fig. 11.6).
The mechanisms that regulate the differentiation of pulmo-
nary epithelial cells are largely unknown but are influenced by
corticosteroids,91,101 the degree of fetal lung expansion (i.e., tissue

stress),58,80,81 structural development of the ECM102 and endo-
thelial cells.103 Studies in sheep and mice show that the absence
of glucocorticoid signalling increases numbers of type II and
undifferentiated epithelial cells and reduces the number of type
I cells.91,101 Similar changes are observed in fetal sheep studies
after prolonged reductions in lung expansion, which increase
the proportion of type II cells and reduce the proportion of type
I cells.80 Conversely, increasing the degree of lung expansion
reduces the proportion of type II epithelial cells and increases the
proportion of type I cells.80 Similar results have been observed
in  vitro in lung explants and monolayer cultures of pulmonary
epithelial cells exposed to static stretch, which increases markers of
type I cells and decreases markers of type II cells.104,105 Given the
known role of glucocorticoid signalling in structural maturation
of the lungs,89,90,95 it is possible that the absence of glucocorti-
coid signalling alters AEC differentiation secondary to changes in
the structural maturity of the lungs which reduces the ability of
the lungs to expand.91 In addition, differentiation of type I cells
appears to depend on their close proximity to the developing vas-
cular endothelium.103  labour due to postural changes imposed on the fetus causing
Pulmonary Surfactant
Pulmonary surfactant is essential for postnatal lung function
because it stabilises alveoli, making the lung easier to expand,
thereby decreasing the work of breathing and enhancing gas
exchange. Pulmonary surfactant is a complex, surface active mixture
of phospholipids and proteins (∼90% lipid and 10% protein) that
is synthesised within type II epithelial cells.106 After being secreted,
surfactant forms a monolayer of tightly packed lipid molecules at
the air–liquid interface of the liquid film that lines the alveoli. This
monolayer displaces water molecules from the interface, thereby
greatly lowering the surface tension. Surfactant deficiency is com-
mon in preterm infants because of immaturity and reduced num-
bers of type II alveolar epithelial cells. The lack of surfactant can
lead to RDS (or hyaline membrane disease), which is characterised
by laboured breathing and progressive cyanosis because of inad-
equate gas exchange.107,108 In preterm infants, the effects of sur-
factant deficiency may be exacerbated by structural immaturity of
the lungs. Antenatal glucocorticoid treatment enhances surfactant
CHAPTER 11  Lung Growth and Maturation 113
synthesis and structural maturation of the lungs,109 both of which
are essential for normal lung function after birth. 
Conclusions
Survival at birth depends upon the lung being sufficiently large
and structurally mature to enable it to immediately take over the
critical role of gas exchange. The physiological and pathophysi-
ological processes affecting lung growth and development before
birth involve both endocrine and physical factors. The fetal lung
is not collapsed, but the ‘airways’ contain a liquid secreted by the
lung epithelium; the degree to which this liquid expands the lung
is a major determinant of lung tissue growth and maturation. The
level of fetal lung expansion is determined by the lung’s physi-
cal environment, including intrathoracic space, fetal posture and
FBMs. Lung tissue stretch stimulates gene networks, leading to
tissue growth and differentiation. Lung hypoplasia results if the
fetal lung is chronically underexpanded.
• Clearance of liquid from the airways begins with the onset of
lung liquid efflux via the nose and mouth, as well as active liq-
uid reabsorption. Lung liquid remaining after birth is cleared
as a result of the transpulmonary pressure gradient generated
by inspiration.
• PBF is generally low during fetal life but can increase tran-
siently with FBMs. At birth, pulmonary vascular resistance
markedly decreases, permitting the high PBF essential for ade-
quate gas exchange.
• The increase in PBF at birth is triggered by lung aeration,
which in turn underpins the cardiovascular transition at birth.
With the loss of umbilical venous return, the increase in pul-
monary venous return takes over the critical role of supplying
preload blood to the left ventricle.
• Maturation of the lung in preparation for birth involves ECM
remodelling, alveolar epithelial cell differentiation and surfac-
tant production. These changes are driven by mechanical stress
on the lung tissue and corticosteroid signalling.
Access the complete reference list online at ExpertConsult.com.
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References 20. Jakubowska AE, Billings K, Johns DP, et  al.
Respiratory function in lambs after prolonged
to moderate asphyxia in fetal sheep. J Dev
Physiol. 1988;10:473–485.
oligohydramnios during late gestation. Pediatr 40. Hooper SB, Harding R. Changes in lung
1. Burri PH. Fetal and postnatal development of
Res. 1993;34:611–617. liquid dynamics induced by prolonged fetal
the lung. Ann Rev Physiol. 1984;46:617–628.
21. Harding R, Liggins GC. Changes in thoracic hypoxemia. J Appl Physiol. 1990;69:127–135.
2. Jones R, Reid LM. Development of the pul-
dimensions induced by breathing movements in 41. Hooper SB, Wallace MJ, Harding R.
monary vasculature. In: Harding R, Pinkerton
fetal sheep. Reprod Fertil Dev. 1996;8:117–124. Amiloride blocks the inhibition of fetal lung
KE, Plopper CG, eds. The Lung: Development,
22. Patrick J, Natale R, Richardson B. Patterns liquid secretion caused by AVP but not by
Aging and the Environment. London: Elsevier
of human fetal breathing activity at 34 to 35 asphyxia. J Appl Physiol. 1993;74:111–115.
Academic Press; 2004:81.
weeks’ gestational age. Am J Obstet Gynecol. 42. Wallace MJ, Hooper SB, McCrabb GJ,
3. Dodson RB, Galambos C, Abman SH. Devel-
1978;132:507–513. Harding R. Acidaemia enhances the inhibi-
opmental physiology of the pulmonary circu-
23. Bocking AD, Gagnon R. In: Thorburn GD, tory effect of hypoxia on fetal lung liquid
lation. In: Harding R, Pinkerton KE, Plopper
Harding R, eds. Textbook of Fetal Physiology. secretion in sheep. Reprod Fertil Dev. 1996;8:
CG, eds. The Lung: Development, Aging and the
Oxford, United Kingdom: Oxford University 327–333.
Environment. London: Elsevier Academic Press;
Press; 1994:186. 43. Harding R, Hooper SB, Han VK. Abolition
2004:121–136.
24. Hooper SB, Harding R. In: Harding R, of fetal breathing movements by spinal cord
4. Tod ML, Cassin S. In: Crystal RG, West DB,
Bocking AD, eds. Fetal Growth and Develop- transection leads to reductions in fetal lung
Weibel ER, Barnes PJ, eds. The Lung: Scientific
ment. Cambridge, United Kingdom: Cam- liquid volume, lung growth, and IGF-II gene
Foundations. Philadelphia: Lippincott-Raven;
bridge University Press; 2001:114. expression. Pediatr Res. 1993;34:148–153.
1997:2129.
25. Liggins GC, Vilos GA, Campos GA, et  al. 44. Miller AA, Hooper SB, Harding R. Role of
5. Polglase GR, Wallace MJ, Grant DA,
The effect of spinal cord transection on lung fetal breathing movements in control of fetal
Hooper SB. Influence of fetal breathing
development in fetal sheep. J Dev Physiol. lung distension. J Appl Physiol. 1993;75:
movements on pulmonary hemodynamics in
1981;3:267–274. 2711–2717.
fetal sheep. Pediatr Res. 2004;56:932–938.
26. Wigglesworth JS, Desai R. Effect on lung 45. Nardo L, Hooper SB, Harding R. Lung hypo-
6. Hooper SB. Role of luminal volume changes
growth of cervical cord section in the rabbit plasia can be reversed by short-term obstruc-
in the increase in pulmonary blood flow at
fetus. Early Hum Dev. 1979;3:51–65. tion of the trachea in fetal sheep. Pediatr Res.
birth in sheep. Exp Physiol. 1998;83:833–842.
27. Adamson TM, Boyd RD, Platt HS, Strang LB. 1995;38:690–696.
7. Lang JA, Pearson JT, Binder-Hesch C, et  al.
Composition of alveolar liquid in the foetal 46. Perks AM, Cassin S. The rate of production
Increase in pulmonary blood flow at birth:
lamb. J Physiol. 1969;204:159–168. of lung liquid in fetal goats, and the effect
role of oxygen and lung aeration. J Physiol.
28. Olver RE, Strang LB. Ion fluxes across the of expansion of the lungs. J Dev Physiol.
2016;594:1389–1398.
pulmonary epithelium and the secretion 1985;7:149–160.
8. Sobotka KS, Hooper SB, Allison BJ, et al. An
of lung liquid in the foetal lamb. J Physiol. 47. Nardo L, Hooper SB, Harding R. Stimula-
initial sustained inflation improves the respira-
1974;241:327–357. tion of lung growth by tracheal obstruction
tory and cardiovascular transition at birth in
29. Brace RA, Wlodek ME, McCrabb GJ, in fetal sheep: relation to luminal pressure
preterm lambs. Pediatr Res. 2011;70:56–60.
Harding R. Swallowing and urine flow and lung liquid volume. Pediatr Res. 1998;43:
9. Teitel DF, Iwamoto HS, Rudolph AM.
responses of ovine fetuses to 24 h of hypoxia. 184–190.
Changes in the pulmonary circulation dur-
Am J Physiol. 1994;266:R1345–R1352. 48. Lines A, Hooper SB, Harding R. Lung liquid
ing birth-related events. Pediatr Res. 1990;27:
30. Harding R, Sigger JN, Wickham PJ, Bocking production rates and volumes do not decrease
372–378.
AD. The regulation of flow of pulmonary before labor in healthy fetal sheep. J Appl
10. Lang JA, Pearson JT, te Pas AB, et al. Ventila-
fluid in fetal sheep. Respir Physiol. 1984;57: Physiol. 1997;82:927–932.
tion/perfusion mismatch during lung aeration
47–59. 49. Siew ML, Te Pas AB, Wallace MJ, et al. Posi-
at birth. J Appl Physiol. 2014;117:535–543.
31. Hooper SB, Harding R. Fetal lung liquid: a tive end-expiratory pressure enhances devel-
11. Rudolph AM. Distribution and regulation of
major determinant of the growth and func- opment of a functional residual capacity in
blood flow in the fetal and neonatal lamb. Circ
tional development of the fetal lung. Clin Exp preterm rabbits ventilated from birth. J Appl
Res. 1985;57:811–821.
Pharmacol Physiol. 1995;22:235–247. Physiol. 2009;106:1487–1493.
12. Bhatt S, Alison BJ, Wallace EM, et al. Delaying
32. Brown MJ, Olver R, Ramsden CA, et al. Effects 50. Harding R, Bocking AD, Sigger JN. Upper
cord clamping until ventilation onset improves
of adrenaline and of spontaneous labour on airway resistances in fetal sheep: the influence
cardiovascular function at birth in preterm
the secretion and absorption of lung liquid in of breathing activity. J Appl Physiol. 1986;60:
lambs. J Physiol. 2013;591:2113–2126.
the fetal lamb. J Physiol. 1983;344:137–152. 160–165.
13. Crossley KJ, Allison BJ, Polglase GR, et  al.
33. Hooper SB, Harding R. Effect of beta- 51. Olver RE, Ramsden CA, Strang LB, Walters
Dynamic changes in the direction of blood
adrenergic blockade on lung liquid secretion DV. The role of amiloride-blockable sodium
flow through the ductus arteriosus at birth.
during fetal asphyxia. Am J Physiol. 1989;257: transport in adrenaline-induced lung liq-
J Physiol. 2009;587:4695–4704.
R705–R710. uid reabsorption in the fetal lamb. J Physiol.
14. Bocking AD, Gagnon R, White SE, et  al.
34. Wallace MJ, Hooper SB, Harding R. Regu- 1986;376:321–340.
Circulatory responses to prolonged hypox-
lation of lung liquid secretion by arginine 52. Hooper SB, Kitchen MJ, Siew ML, et  al.
emia in fetal sheep. Am J Obstet Gynecol.
vasopressin in fetal sheep. Am J Physiol. Imaging lung aeration and lung liquid clear-
1988;159:1418–1424.
1990;258:R104–R111. ance at birth. FASEB J. 2007;21:3329–3337.
15. Harding R. In: Crystal RG, West DB, Wei-
35. Walters DV, Ramsden CA, Olver RE. Dibu- 53. Harding R, Hooper SB, Dickson KA. A
bel ER, Barnes PJ, eds. The Lung: Scientific
tyryl cAMP induces a gestation-dependent mechanism leading to reduced lung expan-
Foundations. Philadelphia: Lippincott-Raven;
absorption of fetal lung liquid. J Appl Physiol. sion and lung hypoplasia in fetal sheep dur-
1997:2093.
1990;68:2054–2059. ing oligohydramnios. Am J Obstet Gynecol.
16. Harding R, Hooper SB. Regulation of lung
36. Wallace MJ, Hooper SB, Harding R. Effects 1990;163:1904–1913.
expansion and lung growth before birth. J Appl
of elevated fetal cortisol concentrations on 54. Siew ML, Wallace MJ, Kitchen MJ, et  al.
Physiol. 1996;81:209–224.
the volume, secretion, and reabsorption of Inspiration regulates the rate and temporal pat-
17. Moore PJ, Hanson MA. Control of breathing:
lung liquid. Am J Physiol. 1995;269:R881– tern of lung liquid clearance and lung aeration
central influences. In: Hanson MA, Spencer
R887. at birth. J Appl Physiol. 2009;106:1888–1895.
JAD, Rodeck CH, Walters DV, eds. Fetus and
37. Wallace MJ, Hooper SB, Harding R. Role of 55. Miserocchi G, Poskurica BH, Del Fabbro M.
Neonate: Breathing. Cambridge, United King-
the adrenal glands in the maturation of lung Pulmonary interstitial pressure in anesthetized
dom: Cambridge University Press; 1994:109.
liquid secretory mechanisms in fetal sheep. Am paralyzed newborn rabbits. J Appl Physiol.
18. Harding R, Bocking AD, Sigger JN. Influence
J Physiol. 1996;270:R33–R40. 1994;77:2260–2268.
of upper respiratory tract on liquid flow to and
38. Cassin S, Perks AM. Studies of factors which 56. Hooper SB, Wallace MJ. Role of the physico-
from fetal lungs. J Appl Physiol. 1986;61:68–74.
stimulate lung fluid secretion in fetal goats. J Dev chemical environment in lung development.
19. Maloney JE, Adamson TM, Brodecky AV,
Physiol. 1982;4:311–325. Clin Exp Pharmacol Physiol. 2006;33:273–279.
et  al. Diaphragmatic activity and lung liquid
39. Hooper SB, Dickson KA, Harding R. Lung 57. Hooper SB, Han VK, Harding R. Changes
flow in the unanesthetized fetal sheep. J Appl
liquid secretion, flow and volume in response in lung expansion alter pulmonary DNA
Physiol. 1975;39:423–428.

113.e1
113.e2 References
synthesis and IGF-II gene expression in fetal
sheep. Am J Physiol. 1993;265:L403–L409.
58. Alcorn D, Adamson TM, Lambert TF, et  al.
Morphological effects of chronic tracheal liga-
tion and drainage in the fetal lamb lung. J Anat.
1977;123:649–660.
59. Moessinger AC, Harding R, Adamson TM,
et  al. Role of lung fluid volume in growth
and maturation of the fetal sheep lung. J Clin
Invest. 1990;86:1270–1277.
60. Vandenburgh HH. Mechanical forces and
their second messengers in stimulating cell
growth in vitro. Am J Physiol. 1992;262:R350–
R355.
61. Ingber DE. Cellular mechanotransduction:
putting all the pieces together again. FASEB J.
2006;20:811–827.
62. Dahl KN, Ribeiro AJ, Lammerding J. Nuclear
shape, mechanics, and mechanotransduction.
Circ Res. 2008;102:1307–1318.
63. Pajerowski JD, Dahl KN, Zhong FL, et al. Phys-
ical plasticity of the nucleus in stem cell differ-
entiation. Proc Natl Acad Sci U S A. 2007;104:
15619–15624.
64. Lelievre SA. Contributions of extracellular
matrix signaling and tissue architecture to
nuclear mechanisms and spatial organization
of gene expression control. Biochim Biophys
Acta. 2009;1790:925–935.
65. Liu M, Xu J, Liu J, et al. Mechanical strain-
enhanced fetal lung cell proliferation is medi-
ated by phospholipase C and D and protein
kinase C. Am J Physiol. 1995;268:L729–L738.
66. Liu M, Liu J, Buch S, et al. Antisense oligonu-
cleotides for PDGF-B and its receptor inhibit
mechanical strain-induced fetal lung cell
growth. Am J Physiol. 1995;269:L178–L184.
67. Wallace MJ, Thiel AM, Lines AM, et al. Role
of platelet-derived growth factor-B, vascular
endothelial growth factor, insulin-like growth
factor-II, mitogen-activated protein kinase
and transforming growth factor-beta1 in
expansion-induced lung growth in fetal sheep.
Reprod Fertil Dev. 2006;18:655–665.
68. Sozo F, Wallace MJ, Zahra VA, et  al. Gene
expression profiling during increased fetal
lung expansion identifies genes likely to regu-
late development of the distal airways. Physiol
Genomics. 2006;24:105–113.
69. McDougall AR, Hooper SB, Zahra VA, et al.
The oncogene Trop2 regulates fetal lung cell
proliferation. Am J Physiol Lung Cell Mol
Physiol. 2011;301:L478–L489.
70. Harding R, Albuquerque C. In: Gaultier C,
Bourbon J, Post M, eds. Lung Development.
Oxford, United Kingdom: Oxford University
Press; 1999:364–394.
71. Askenazi SS, Perlman M. Pulmonary hypo-
plasia: lung weight and radial alveolar count
as criteria of diagnosis. Arch Dis Child.
1979;54:614–618.
72. Thibeault DW, Beatty Jr EC, Hall RT, et al.
Neonatal pulmonary hypoplasia with prema-
ture rupture of fetal membranes and oligohy-
dramnios. J Pediatrics. 1985;107:273–277.
73. Suzuki K, Hooper SB, Cock ML, Harding R.
Effect of lung hypoplasia on birth-related
changes in the pulmonary circulation in sheep.
Pediatr Res. 2005;57:530–536.
74. Wigglesworth JS. Pathology of the lung in the
fetus and neonate, with particular reference to
problems of growth and maturation. Histopa-
thology. 1987;11:671–689.
75. Moessinger AC, Collins MH, Blanc WA, et al.
Oligohydramnios-induced lung hypoplasia:
the influence of timing and duration in gesta-
tion. Pediatr Res. 1986;20:951–954.
76. Deprest JA, Nicolaides K, Gratacos E. Fetal
surgery for congenital diaphragmatic hernia
is back from never gone. Fetal Diag Ther.
2011;29:6–17.
77. Al-Maary J, Eastwood MP, Russo FM, et  al.
Fetal tracheal occlusion for severe pulmonary
hypoplasia in isolated congenital diaphragmatic
hernia: a systematic review and meta-analysis
of survival. Ann Surg. 2016;264(6):929–933.
78. Ruano R, Ali RA, Patel P, et  al. Fetal endo-
scopic tracheal occlusion for congenital dia-
phragmatic hernia: indications, outcomes,
and future directions. Obstet Gynecol Survey.
2014;69:147–158.
79. Ruano R, da Silva MM, Campos JA, et al. Fetal
pulmonary response after fetoscopic tracheal
occlusion for severe isolated congenital diaphrag-
matic hernia. Obstet Gynecol. 2012;119:93–101.
80. Flecknoe S, Harding R, Maritz G, Hooper SB.
Increased lung expansion alters the propor-
tions of type I and type II alveolar epithelial
cells in fetal sheep. Am J Physiol Lung Cell Mol
Physiol. 2000;278:L1180–L1185.
81. Flecknoe SJ, Wallace MJ, Harding R, Hooper SB.
Determination of alveolar epithelial cell pheno-
types in fetal sheep: evidence for the involvement
of basal lung expansion. J Physiol. 2002;542:
245–253.
82. Lines A, Nardo L, Phillips ID, et  al. Altera-
tions in lung expansion affect surfactant pro-
tein A, B, and C mRNA levels in fetal sheep.
Am J Physiol. 1999;276:L239–L245.
83. Snoek KG, Reiss IK, Greenough A, et  al.
CDH EURO Consortium. Standardized
Postnatal Management of Infants with Con-
genital Diaphragmatic Hernia in Europe: the
CDH EURO Consortium Consensus—2015
update. Neonatology. 2016;110:66–74.
84. Schellenberg JC. In: Johnston BM, Gluckman
PD, eds. Respiratory Control and Lung Devel-
opment in the Fetus and Newborn. Ithaca, NY:
Perinatology Press; 1986:3–62.
85. Mizikova I, Morty RE. The extracellular
matrix in bronchopulmonary dysplasia: target
and source. Front Med (Lausanne). 2015;2:91.
86. Faggian J, Fosang AJ, Zieba M, et al. Changes
in versican and chondroitin sulfate proteo-
glycans during structural development of the
lung. Am J Physiol Regul Integr Comp Physiol.
2007;293:R784–R792.
87. Hillman NH, Kallapur SG, Jobe AH. Physi-
ology of transition from intrauterine to extra-
uterine life. Clin Perinatol. 2012;39:769–783.
88. Alcorn DG, Adamson TM, Maloney J,
Robinson PM. A morphologic and morpho-
metric analysis of fetal lung development in
the sheep. Anat Rec. 1981;201:655–667.
89. Cole TJ, Blendy JA, Monaghan AP, et  al.
Molecular genetic analysis of glucocorticoid
signaling during mouse development. Steroids.
1995;60:93–96.
90. Bird AD, Tan KH, Olsson PF, et al. Identifica-
tion of glucocorticoid-regulated genes that con-
trol cell proliferation during murine respiratory
development. J Physiol. 2007;585:187–201.
91. Cole TJ, Solomon NM, Van Driel R, et  al.
Altered epithelial cell proportions in the fetal
lung of glucocorticoid receptor null mice. Am
J Respir Cell Mol Biol. 2004;30:613–619.
92. Jobe AH, Polk D, Ikegami M, et  al. Lung
responses to ultrasound-guided fetal treat-
ments with corticosteroids in preterm lambs.
J Appl Physiol. 1993;75:2099–2105.
93. Liggins GC, Schellenberg JC, Finberg K, et al.
The effects of ACTH1-24 or cortisol on pul-
monary maturation in the adrenalectomized
ovine fetus. J Dev Physiol. 1985;7:105–111.
94. Polk DH, Ikegami M, Jobe AH, et  al. Post-
natal lung function in preterm lambs: effects
of a single exposure to betamethasone and
thyroid hormones. Am J Obstet Gynecol.
1995;172:872–881.
95. Bird AD, Choo YL, Hooper SB, et  al. Mes-
enchymal glucocorticoid receptor regu-
lates development of multiple cell layers of
the mouse lung. Am J Respir Cell Mol Biol.
2014;50(2):419–428.
96. Habermehl D, Parkitna JR, Kaden S, et  al.
Glucocorticoid activity during lung matu-
ration is essential in mesenchymal and less
in alveolar epithelial cells. Mol Endocrinol.
2011;25:1280–1288.
97. Schellenberg JC, Liggins GC. Elastin and col-
lagen in the fetal sheep lung. I. Ontogenesis.
Pediatr Res. 1987;22:335–338.
98. Shibahara SU, Davidson JM, Smith K,
Crystal RG. Modulation of tropoelastin pro-
duction and elastin messenger ribonucleic acid
activity in developing sheep lung. Biochemistry.
1981;20:6577–6584.
99. Possmayer F. In: Jones CT, ed. The Biochemi-
cal Development of the Fetus and Neonate. St.
Louis: Elsevier; 1982:337–391.
100. Flecknoe SJ, Wallace MJ, Cock ML, et  al.
Changes in alveolar epithelial cell propor-
tions during fetal and postnatal development
in sheep. Am J Physiol Lung Cell Mol Physiol.
2003;285:L664–L670.
101. Crone RK, Davies P, Liggins GC, Reid L. The
effects of hypophysectomy, thyroidectomy,
and postoperative infusion of cortisol or adre-
nocorticotrophin on the structure of the ovine
fetal lung. J Dev Physiol. 1983;5:281–288.
102. Honda T, Ishida K, Hayama M, et al. Type II
pneumocytes are preferentially located along
thick elastic fibers forming the framework of
human alveoli. Anat Rec. 2000;258:34–38.
103. Hermanns MI, Fuchs S, Bock M, et al. Primary
human coculture model of alveolo-capillary
unit to study mechanisms of injury to periph-
eral lung. Cell Tissue Res. 2009;336:91–105.
104. Foster CD, Varghese LS, Skalina RB, et  al.
In  vitro transdifferentiation of human fetal
type II cells toward a type I-like cell. Pediatr
Res. 2007;61:404–409.
105. Gutierrez JA, Ertsey R, Scavo LM, et  al.
Mechanical distention modulates alveolar epi-
thelial cell phenotypic expression by transcrip-
tional regulation. Am J Respir Cell Mol Biol.
1999;21:223–229.
106. Orgeig S, Morrison JL, Sullivan LC, Daniels
CB. In: Harding R, Pinkerton KE, eds. The
Lung: Development, Aging and the Environment.
London: Academic Press; 2015:183–210.
107. Whitsett J. In: Crystal RG, West DB,
Weibel ER, Barnes PJ, eds. The Lung: Scien-
tific Foundations. Philadelphia: Lippincott-
Raven; 1997:2167.
108. Jobe AH, Ikegami M. Lung development and
function in preterm infants in the surfactant treat-
ment era. Ann Rev Physiol. 2000;62:825–846.
109. Liggins GC, Schellenberg JC. In: Johnston BM,
Gluckman PD, eds. Respiratory Control and
Lung Development in the Fetus and Newborn.
Ithaca, NY: Perinatology Press; 1986:107–133.
12
Development of the Kidneys and
Urinary Tract in Relation to Renal
Anomalies
PAUL J.D. WINYARD
KEY POINTS
• D evelopment of the kidneys (nephrogenesis) occurs between
the 5th and 32nd weeks of human gestation when the
ureteric bud interacts with metanephric mesenchyme, which
undergoes mesenchymal–epithelial conversion to form glom-
eruli and tubules and renal stroma, with coordinated vascular
development and signalling being critical.
• Nephron number is the major factor determining long-term
kidney function. The development of the nephrons is final-
ised by the 32nd week; the average nephron number is about
900,000 per kidney; smaller babies with fewer nephrons have
an increased long-term risk for hypertension and kidney
failure.
• There is an increased risk for hypertension in ex-premature
children and young people, with a possible renal link to ste-
roid use.
• Congenital anomalies of the kidney and urinary tract (CA-
KUTs), such as aberrant renal development and urinary tract
obstruction, have the potential to decrease the number of
nephrons, and postnatal processes such as cyst formation,
inflammation or infection can have similar effects on renal
function by destroying mature nephrons.
• There are several known causes of CAKUTs, including genetic
defects; urinary tract obstruction; and maternal environment,
diet and teratogens, although most CAKUT remains unex-
plained.
Introduction
Kidneys that produce urine and a lower urinary tract that permits
urine flow into the amniotic fluid are essential for normal human
in utero development. Kidneys generate urine from around the
12th week of gestation, which comprises the majority of the
amniotic fluid from the second trimester and more than 90% by
late gestation. Failure to either generate enough urine or expel
it into the amniotic sac causes the eponymous ‘Potter sequence’
of severe oligohydramnios with limb and craniofacial malforma-
tions, such as clubbed feet, contractures, a flattened ‘parrot-beak’
nose, a recessed chin and low-set ears, accompanied by pulmonary
114
hypoplasia. Potter described this sequence with bilateral renal
agenesis but other causes include bilateral multicystic, dysplastic
or polycystic kidneys or lower urinary tract obstruction with pos-
terior urethral valves or urethral atresia, all of which represent the
severe end of the spectrum of congenital anomalies of the kidney
and urinary tract (CAKUTs).
Urine is produced in the kidneys by nephrons, with filtration
of blood in the glomerulus, modification of the filtrate as it passes
through tubules, loop of Henle and collecting duct, before transi-
tion through the renal pelvis into the ureters. Nephron number
is determined by the 32nd week of gestation, by which point the
kidneys can regulate fluid balance, electrolytes and acid–base bal-
ance. However, full renal function does not develop until birth,
when renal blood flow increases, and then postnatally as nephrons
elongate and mature. The fetal kidneys only receive around 3% to
5% of cardiac output compared with around 20% for the mature
organs, and nephrons lack many specialised transporters in early
developmental stages. Moreover, only dilute urine can be pro-
duced because the medulla is relatively small, and there is reduced
aquaporin expression, which prevents development of a full med-
ullary osmotic gradient and reabsorption of water, respectively.
Such renal immaturity is unimportant if the mother has normal
renal function because the placenta is an efficient biological dialy-
sis machine to balance fetal biochemistry. This should be taken
into consideration when considering early delivery of fetuses with
renal dysfunction because it is much easier to dialyse a 3-kg rather
than a 1.5-kg baby even without factoring in increased risk for
respiratory and other prematurity-related problems. 
Timeline of Kidney Development
Humans pass through three stages of renal development during
nephrogenesis: the pronephros, mesonephros and metanephros,
which arise sequentially on the dorsal body wall.1 Hence, those
with normal development will have had six distinct kidneys before
birth, with excretory function improving significantly at each stage.
Whereas the pronephros and mesonephros regress and disappear
in the fetus, the metanephros matures into the fully function-
ing definitive kidney. The pronephros is the functioning kidney
of adult hagfish and some amphibians, as is the mesonephros in
adult lampreys, some fishes and amphibians. Conservation of
 CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies
TABLE Comparative Timing of Human and Mouse
12.1 Nephrogenesisa
Human
(Postconception
Days Unless
Stated)
Structure
Pronephros Appears 22
Regresses 25
Mesonephros Appears 24
Regresses 16 wk
Metanephros 32
First glomeruli
8 wk
End of nephrogenesis 32 wk
Length of gestation 40 wk
aRats’
timing is about 1 day longer or later than mice.
  
gene function across species means that valuable information per-
tinent to human development can still be gleaned from these dif-
ferent stages in animals; many recent investigations, for example,
involve functional experiments in zebrafish larvae which have a
pronephros containing just two glomeruli.2
The timing of key events in human kidney development is
outlined in Table 12.1. The equivalent stages are also listed for
mice, the most frequently used models of nephrogenesis. There
is a distinct difference in later stages, however, because murine
nephrogenesis continues after birth; this allows experimental
surgical and pharmacologic interventions, but extrapolation of
results may not always be applicable to humans, in whom nephro-
genesis completes in the protected in utero environment (unless
born prematurely).
The Pronephros
The human pronephros is first visible at the 10-somite stage,
around 22 days postconception, which is morphologically equiva-
lent to E9 in mice.1 It comprises a small group of nephrotomes
with segmental condensations, grooves and vesicles between the
second and sixth somites. Extrapolation from animal studies sug-
gest that the pronephros does filter fluid, although human data
are lacking. The pronephric duct develops from the intermediate
mesoderm lateral to the notochord adjacent to the ninth somite.
The duct elongates caudally and reaches the cloaca by day 26. It
is renamed the mesonephric, or Wolffian duct, as mesonephric
tubules develop. The nephrotomes and pronephric part of the
duct involute and cannot be identified by day 25.  Metanephric kidney development starts by day 28 in humans,
The Mesonephros
In humans, the long sausage-shaped mesonephros develops
from around 24 days postconception with a duct that grows in
a caudal direction connected to adjacent tubules. Mesonephric
tubules originate from intermediate mesoderm medial to the duct
by ‘mesenchymal–epithelial’ transformation, a process which is
subsequently reiterated during nephron formation in metaneph-
ric development. In humans, a total of around 40 mesonephric
115
Mesenchyme
Epithelium
Mouse Mesenchyme
(Postconception Stromal
Days) Collecting ducts differentiation
Early parts
of nephron
9
10
Normal, fully developed kidney
10
14
• Fig. 12.1  Cellular model of normal kidney development. Note that vas-
11.5 cular development occurs concurrently via a combination of migration and
in situ differentiation
14
7 after birth
tubules are produced (several per somite), but the cranial tubules
20 regress at the same time as caudal ones are forming; hence, there
are maximum of around 30 pairs at any time.
Each mesonephric ‘nephron’ has a medial cup-shaped sac
encasing a knot of capillaries, functionally equivalent to Bowman’s
capsule and glomerulus of the mature kidney. This connects to
segments of the tubule that histologically resemble mature proxi-
mal and distal tubules but lack a loop of Henle. The human meso-
nephros is thought to produce small quantities of urine between
weeks 6 and 10 that drains via the mesonephric duct, but again
there is little direct evidence for this and much is extrapolated from
sheep and cattle.3 The mouse metanephros organ is rudimentary
and has poorly differentiated glomeruli. The mesonephros disap-
pears by 16 weeks, except in male fetuses, in whom the proximal
segments of some caudal mesonephric tubules contribute to the
efferent ducts of the epididymis whilst the mesonephric duct is
incorporated into ductular parts of the epididymis, the seminal
vesicle and ejaculatory duct. 
The Metanephros
The adult human kidney develops from the metanephros, which
consists of two major cell types at its inception: the epithelial cells
of the ureteric bud and the mesenchymal cells of the metanephric
mesenchyme. A series of reciprocal interactions between epithelia
and mesenchymal cells cause the ureteric bud to branch sequen-
tially to form the ureter, renal pelvis, calyces and collecting tubules
whilst the mesenchyme has a more varied fate; most focus has been
on the portion of mesenchyme that undergoes epithelial conver-
sion into nephrons, but other mesenchymal cells differentiate into
interstitial cells or stroma in the mature kidney (Fig. 12.1). Three-
way induction between epithelia, tubular- and stromal-progenitor
mesenchyme appears to be of importance in generating a normal
kidney.4,5
when the ureteric bud sprouts from the distal part of the meso-
nephric duct. By day 32, the tip (ampulla) of the bud penetrates
the metanephric blastema, a specialised area of sacral interme-
diate mesenchyme, and this condenses around the growing
ampulla to generate the metanephros. The first glomeruli form
by 8 weeks, and nephrogenesis continues in the outer rim of the
cortex until somewhere between the 32nd and 36th week; this
was originally suggested to continue longer,6 but more recent
studies suggest 32 weeks.7,8 Many of the nephrons generated in
early weeks of nephrogenesis only have transient function before
116 SE C T I O N 3     Fetal Physiology and Pathology
being lost by apoptosis during increase in kidney size9; effec-
tively, their initial ‘cortical’ position is overrun and remodelled
as medulla growth expands outwards. In mice, the ureteric bud
enters the metanephric mesenchyme by embryonic day 10.5, the
first glomeruli form by embryonic day 14 and nephrogenesis
continues for up to 1 week after birth10 (earlier reports incor-
rectly state 14 days).
New nephrons are never generated after completion of
nephrogenesis, although strategies to restart nephron formation
are actively being researched as an obvious treatment for both
developmental and acquired kidney diseases.11 Kidney develop-
ment is not complete at this point, however, because nephron
segments elongate, the medulla expands and segment-specific
differentiation continues, whilst blood supply increases, and glo-
merular filtration rate (GFR) increases over the first 18 months
of life.  around age 40 onwards.19 However, because the kidneys have the
The Ureteric Bud and Collecting Duct Lineage
The ureteric bud grows into the metanephric blastema and the
adjacent mesenchymal cells begin to condense around its tip.
Signalling from the mesenchyme stimulates bifurcation of the
ampulla to form a ‘t-shape’, and this process of growth and branch-
ing occurs repeatedly during nephrogenesis to generate a tree-like
collecting duct system. Around 20 rounds of branching occur in
humans, double that of mice.12 The bud tips connect to the distal
part of developing nephrons while more central parts differentiate
into collecting ducts that drain successively into minor calyces,
major calyces, the renal pelvis and then the ureter.6  Using unbiased stereology, a more accurate range with a mean
Differentiation of the Mesenchyme
Renal stroma differentiates from one subset of the metanephric
mesenchyme whilst nephrons are induced in mesenchymal cells
adjacent to each ampullary tip of the ureteric bud. The mes-
enchyme is initially loosely arranged, but the cells destined to
become nephrons grow closely together and compact or condense
around the bud tips before undergoing phenotypic transforma-
tion into epithelial renal vesicles. Each vesicle elongates to form a
comma shape, which folds back on itself to become an S-shaped
body.13 The proximal S-shape develops into the glomerulus whilst
the distal portion elongates and differentiates into all nephron
segments from proximal convoluted tubule to distal convoluted
tubule. It was suggested that sequential phases of nephron forma-
tion occurred with an initial bud branch–to–nephron ratio of 1
to 1, and then several branches per bud in an arcade extending
outwards as the kidney grows and finally the terminal bud branch
attached to as many as five nephrons.6 This theory was based on
postmortem dissection rather than repeated, quantitative studies,
but it appears plausible with recent mouse work also suggesting
several distinct phases.14  mechanism appears to be epigenetic modification of diverse
Development of the Vasculature
Blood is supplied to the kidneys via the renal arteries, which give
rise to distinct microcirculations in glomerular capillaries, cortical
vessels and vasa rectae which pass alongside loops of Henle into
the medulla. Renal vessels develop from a combination of vas-
culogenesis, in which mesenchyme differentiates in situ to form
capillary endothelia, and angiogenesis, which involves ingrowth of
existing capillaries.15 Renal vascular resistance is high during fetal
life, predominantly controlled via the renin–angiotensin system
(RAS) and renal nerves, so the kidneys only receive 3% to 5% of
cardiac output.16
Measurement of GFR is contentious in neonates but appears
low at birth at around 20 mL/min/1.73 m2 in term infants (or
15 mL/min/1.73 m2 in infants with low birth weight).17 Over-
all renal blood flow rises as systemic blood pressure increases and
renal resistance falls after birth with the kidneys receiving 10%
of the cardiac output by the end of the first postnatal week. GFR
increases two- to threefold during the first month of life but does
not reach adult levels until 18 months to 2 years of age.18 
Final Nephron Number
Final nephron number is important because this determines
renal function into adulthood, before GFR starts to decline from
ability to adapt and compensate, it is possible for individuals with
a wide range of nephron numbers to appear to have the same level
of renal function as assessed by plasma creatinine or estimated
GFR. The search for an accurate estimate of nephron number
has evolved through many different techniques in the past 25
years with a broad range suggested of 0.6 to 1.3 million per kid-
ney.20-22 The current gold-standard counting method is unbiased
stereology, but this is time consuming and expensive and, more
important, only possible when the kidney is dissected.23 Magnetic
resonance imaging-based techniques are being developed for live
estimates and seem to work in mice,24 with prospective clinical
uses planned in future.
of around 900,000 nephrons per kidney has now been reported
across many populations. Strikingly, as a population, Australian
aboriginals have a much lower mean of around 680,00025; this
group has a high prevalence of both renal disease and hyperten-
sion, and similar links between low nephron count and primary
hypertension are reported in other populations.26 Reiterating the
possible high variability within a population without obvious
immediate renal dysfunction, nephron number spanned a 12-fold
range between 210,000 and 2.7 million in a large study of 176
African Americans.27
Two hypotheses outlined by Barker and colleagues28 in
Southampton, United Kingdom, and Brenner in Boston, Mas-
sachusetts, United States, have linked nephron number with in
utero development and associated predisposition to progressive
renal disease. Barker and colleagues28 initially demonstrated an
inverse correlation between systolic blood pressure and birth
weight and proposed that ‘the intrauterine environment influ-
ences blood pressure during adult life’. Their group (and many
others) have subsequently confirmed the link between birth
weight and hypertension but also associated risks of cardiovas-
cular disease, type 2 diabetes and obesity.29-31 An important
tissues, including kidney, liver and pancreas.32,33 Direct proof
linking the ‘Barker hypothesis’ with nephron number comes
from maternal undernutrition or protein restriction and diabetic
experimental models with consequent decreased nephron num-
bers, often linked to hypertension.34-37
Although originally based on the deleterious effects of
increased dietary protein,38 the ‘Brenner hypothesis’ is that glo-
merular hyperfiltration causes progressive glomerular sclerosis,
proteinuria and nephron loss, which then exacerbates hyperfil-
tration in the surviving functional glomeruli, leading to a recur-
ring cycle of nephron dropout and increased stress on remaining
 CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies
Genetic
factors
Impaired
urinary flow, Maternal environment,
obstruction diet, teratogens
Mesenchyme
Epithelium
Mesenchyme
Abnormal branching
to primitive ducts Primitive Increased
nephrons stroma
Cysts
Formation of
Failed nephron metaplastic
Aberrant collecting
differentiation cartilage
system
Nephron deficit
• Fig. 12.2  Conceptual cellular model underlying congenital anomalies of
the kidney and urinary tract.
glomeruli. Hyperfiltration is inevitable when a low nephron
number has been caused by disease or induced by fetal program-
ming39,40; hence, it is important to identify potentially affected
babies and follow up looking for proteinuria and hypertension
so that early treatment can be given.  region and ending between the 12th thoracic and 3rd lumbar
Types of Renal Anomalies
Renal anomalies are often detected on antenatal ultrasound exam-
ination, but many are incidental changes such as minor dilata-
tion of the renal pelvis, usually nonpathological.41 The differential
diagnosis of anomalies is reviewed in Chapter 33, but a brief out-
line is given here in relation to their pathogenesis.
Agenesis, or absent kidney, is often associated with aberrant
or absent ureters; hence, a potential underlying mechanism has
been suggested as early failure of ureteric bud branching. Agen-
esis may be isolated, but it can also occur as part of multiorgan
disorders such as Branchio-Oto-Renal, Kallmann, Fraser and
DiGeorge syndromes.42 Bilateral agenesis has an incidence of 1
in 5,000 to 10,000, whilst unilateral is much commoner at 1 in
1,000.
Dysplasia includes conditions in which the kidney fails to
undergo normal development and differentiation, with abnor-
mal structure and which may contain metaplastic tissues such
as cartilage.43 These pathological processes are depicted sche-
matically in Fig. 12.2, along with normal and abnormal histol-
ogy in Fig. 12.3. There is a spectrum of dysplasia from large
kidneys distended with cysts, such as ‘multicystic dysplastic
kidneys’, which are often attached to atretic ureters, or small
echobright organs with a few rudimentary tubules that resem-
ble ‘frustrated’ ureteric bud branches.6 Dysplasia can occur
as an isolated anomaly or in a multi-organ syndrome, such as
the renal cysts and diabetes (RCAD) or renal-coloboma syn-
dromes.44,45 Around a third have an associated abnormal con-
tralateral kidney, often with vesicoureteric reflux. The incidence
of multicystic dysplastic kidney is around 1 in 2500,46although
117
this may be an underestimate because many involute and can
be completely reabsorbed, which leads to an incorrect diagno-
sis of agenesis when detected later.
Hypoplasia is defined pathologically as a kidney weighing
less than 50% of expected,42 but the term is often used loosely
to imply significantly fewer nephrons than normal. Dysplasia
cannot be present, so all of the nephrons should appear nor-
mal, and undifferentiated tissues are not present. Again, this
appears to represent a spectrum with some kidneys appearing
grossly normal, albeit small on scan, whilst others are tiny. This
raises a frequent question as to whether kidney size correlates
with nephron number. The broad answer is yes, in that, given a
normal radiologic appearance with no cysts or other structural
abnormalities, a large kidney is likely to have more nephrons
than a small one. However, this in itself does not necessar-
ily mean that GFR will be different initially, but the smaller
kidneys are likely to develop more severe kidney disease in the
long term.47,48
Duplex kidneys represent some degree of duplication of the
renal pelvis and ureter. This finding is relatively common, occur-
ring in about 5% of unselected autopsies. The range of anatomy
includes simple bifurcation of the extrarenal renal pelvis through
complete duplication with two distinct (but contiguous) kid-
neys, separate ureters and two ureterovesical openings.49 Many
cases are asymptomatic, although up to half may develop com-
plications requiring treatment; these classically include obstruc-
tion to the upper part and reflux into the lower moiety.
Ectopic kidneys and malrotation represent abnormal renal
position. The kidneys should relatively ‘ascend’ to a progressively
more rostral position during development, starting in the sacral
vertebral bodies. There is also associated rotation such that the
renal pelvis changes from an anterior- to a medial-facing orien-
tation by term. Failure to ascend completely is relatively com-
mon, around 1 in 800 on routine renal imaging, and is usually
associated with retention of a more anterior-facing renal pelvis.
Occasionally, in crossed ectopia, the kidney is on the wrong side
of the body as well as in the wrong position and may be fused
to the contralateral kidney in cross-fused ectopia. Ectopic kid-
neys are often dysplastic and may also be associated with reflux
or hydronephrosis or obstruction because of abnormal ureteric
positioning and length. Kidneys can also be fused in a horseshoe
kidney (1 in 600); these are usually situated lower than nor-
mal and, again, have an increased risk for vesicoureteric reflux
or hydronephrosis. 
Causes of Human Renal Anomalies
In the era of next generation sequencing and rapid gene screen-
ing, there is a temptation to focus on genetic causes of CAKUTs,
but recognised mutations still account for only a small fraction
of cases.50-52 This raises the possibility that most anomalies are a
consequence of polygenic inheritance, perhaps with individually
recessive genes in combination in compound heterozygotes to gen-
erate a CAKUT phenotype.53 Lower urinary tract obstruction and
maternal environment may also cause malformations, both alone
and in combination with genetic factors. Finally, random stochas-
tic events remain likely, but they are almost impossible to prove. A
general cause-and-effect schematic is depicted for dysplastic kid-
neys in Fig. 12.2. This demonstrates that one or multiple pertur-
bations, including genetic, obstructive and environmental factors
have the same net effect by perturbing epithelial–mesenchymal
118 SE C T I O N 3     Fetal Physiology and Pathology

A C

D E
• Fig. 12.3  Histology of developing and dysplastic kidneys. 11-week (A) 12-week (B and C) gestation
normal kidneys. D and E, Dysplastic kidney. A, Low-power view demonstrating ureteric buds radiating out
from the centre (lower right) of the metanephros. Intermediate (B) and higher (C) magnification of nephro-
genic cortex: multiple ureteric buds visible with condensing mesenchyme adjacent to tips. Each deeper
layer shows progressive nephron differentiation from comma-shapes, through early to mature glomeruli.
D, Dysplastic kidney demonstrating lack of normal renal tissues; instead there is stromal proliferation with
a few primitive epithelia tubules and cysts. E, String of tortuous blood vessels (bottom left towards top
right), demonstrating vascular as well as nephron abnormalities in dysplastic kidneys.

interactions leading to abnormal kidney development. This chap-
ter briefly lists a few of the common causes, but readers are referred
to recent reviews for a more detailed account.54-56
ii. PAX–EYA–SIX transcription factor genes. Altered expres-
Genetic Factors
i. TCF2 gene. Although the list of genes linked to kidney mal-
formations is increasing rapidly, there is one that repeatedly
occurs: the TCF2 gene, encoding the transcription factor
hepatocyte nuclear factor-1β (HNF1β). This was originally
described in the RCAD syndrome,57 which accounts for up
to one third of antenatal presentations with ‘bright’ kidneys
and 10% of CAKUTs overall.58,59 The wide spectrum of renal
malformations in RCAD ranges from grossly cystic dysplastic
kidneys through hypoplasia to agenesis.60 Females may also
have uterine abnormalities. An important issue is that both
cysts and diabetes can develop at different ages; hence, repeated
enquiry about new family diagnoses is worthwhile at follow up.
sion of the transcription factor PAX2 has been linked to a spec-
trum from renal agenesis (absent PAX2), through hypoplasia
(reduced levels) to cystogenesis (overexpression).61-63 PAX2
mutations cause the ‘renal-coloboma’ syndrome with optic
nerve colobomas, renal anomalies and vesicoureteric reflux,64
although other genes have now been implicated, too.65 PAX
genes interact with several other transcription factors, includ-
ing members of the SIX and EYA families. Many of these have
been linked to renal malformations, including EYA1 in the
branchio-oto-renal syndrome and SIX1 and SIX2 in familial
CAKUTs.66,67
 CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies
iii. GDNF/RET system. Glial cell line-derived neurotrophic
factor (GDNF) and its receptor, RET, are critical factors
determining initial outgrowth of the ureteric bud from the
mesonephric duct and then in regulating ureteric bud branch-
ing in the metanephros.68 Mutations were originally described
in multiple endocrine neoplasia but are now also recognised
in CAKUTs.69 Because it is important to develop just one
ureteric bud at the right time and in the right place, there
are a number of negative regulators of GDNF/RET signal-
ling, including the SLIT2-ROBO pathway, Semaphorin and
Sprouty genes. Mutations in many of these cause duplex kid-
neys and other CAKUTs in mice, and these are now also being
reported in human malformations.70-72
Common polymorphisms, rather than rare mutations, may
also affect kidney development. Examples include PAX2 and RET
in which smaller newborn kidneys have been reported for cer-
tain common variants, with the presumption that these will have
lower nephron numbers.73,74 Polymorphisms of genes in the renin
angiotensin system (RAS) have also been linked to variation in
kidney size at birth.75  cause of autosomal recessive renal tubular dysgenesis,90 whilst angio-
Impaired Urinary Flow and Obstruction
Dysplastic kidneys represent the largest group cause of chronic
kidney disease in childhood, with urinary tract obstruction a close
second.76,77 Examples include: posterior urethral valves (1 in
2500 boys), urethral atresia, and pelviureteric and ureterovesi-
cal junction obstruction. Moreover, the most severely perturbed
nephrogenesis seen in multicystic dysplastic kidneys is classically
associated with an obstructed ureter. Experimental obstruction
of the developing urinary tract has been known to generate renal
malformations for more than 40 years, and many of the same pat-
terns of perturbed gene expression are seen as in nonobstructed
human CAKUTs.56,78,79
It is worth noting that most of the cases labelled as ‘obstruction’
do not have a complete blockage; rather, they have a narrowing
or restriction of the renal outflow tract that impairs flow. This is
important because a complete block within the tract will cause
severe oligo- or an-hydramnios and Potter sequence. Renal pathol-
ogy can be reduced in animal models of obstruction by treatment
with growth factors such as insulin-like growth factor and epi-
dermal growth factor, but it is currently difficult to determine
how such therapies might be selectively delivered to the kidney in
human fetuses in utero.
There are conflicting data on whether correcting early lower
tract obstruction in utero allows renal function to recover. There
are good results in large animals but poorer outcomes in mice and
rats. The human data are limited to relatively small cases series,
but even the largest clinical trial (PLUTO - percutaneous shunting
in lower urinary tract obstruction) demonstrated poor efficacy of
in utero vesicoamniotic shunt: survival seemed more likely with a
shunt, but the size and direction of effect were uncertain; chances
of surviving with good renal function were low.80  pregnancy-associated diabetes were also associated with CAKUTs,
Maternal Environment, Diet, and Teratogens
An aberrant maternal in utero environment, diet or teratogens can
cause both reduced nephron numbers and structural kidney defects.
The first evidence that maternal diet affected the adult
health of the offspring came from the World War II Dutch
famine cohort; these mothers were grossly malnourished by
limited food access, and a large follow-up study demonstrated
119
both an increased risk for hypertension and decreased glucose
tolerance.81,82 Similar hypertension was noted in births after
the siege of Leningrad and has been replicated in many other
cohorts.83 These effects are now believed to result from mal-
nutrition causing reduced nephron number, with extensive
data in animals implicating protein restriction.34,35,84 Other
maternal factors leading to lower birth weight (e.g., placental
insufficiency, smoking and many chronic conditions) also pre-
dispose to adult hypertension, presumably again via reduced
nephron numbers.85 Being born with such a deficit can then
be exacerbated postnatally by overfeeding; this has been shown
in laboratory models of protein restriction and then excess, but
there is a worry that the same ‘biological experiment’ will be
reiterated with obesogenic diet in humans.86,87
Diverse medication and chemicals have the potential to disrupt
nephrogenesis.88 They include exogenous drugs and endogenous fac-
tors which become pathogenic when present in abnormal quantities.
A good example is the RAS: the RAS is important in normal renal
growth89 and may affect nephron number,75 and mutations are one
tensin-converting enzyme inhibitors and receptor blockers in preg-
nancy can cause fetal tubular dysgenesis (and skull malformations).91
Intriguingly, maternal protein restriction suppresses the neonatal RAS
in rats and predisposes to hypertension, demonstrating how several
factors might act in concert.92
High glucose levels in mothers with diabetes are associated
with an increased incidence of kidney and lower urinary tract
malformations, as well as abnormalities in the nervous, cardio-
vascular and skeletal systems.31,93 This effect may be multifacto-
rial, however, because diabetes is associated with caudal regression
syndrome in fetuses. Furthermore, one should always consider the
possibility that mother and fetus may share HNF1β mutations
that cause CAKUTs in the renal cysts and diabetes syndrome.59
Supplementary vitamins are routinely recommended during
pregnancy but should be taken with caution because too much
can be as deleterious as too little. A classic example is retinoic acid,
a natural metabolite of vitamin A, which perturbs nephrogene-
sis if depleted or in excess.94 A polymorphism of the ALDH1A2
gene involved in retinoic acid metabolism causes a 22% increase
in newborn kidney size95; whether this relates to increased neph-
ron number is unproven. Vitamin D deficiency is very common
worldwide and has been linked to lower birth weight with adverse
pregnancy outcomes.96 Impact on nephrogenesis is less certain,
however, with one study in rats showing that vitamin D deficiency
generated a 20% increase in nephron number.97
A recent study from the Netherlands sought to identify mater-
nal risk factors in 562 children with CAKUTs and 2139 healthy
control participants.98 Specific use of folic acid rather than multi-
vitamins increased the likelihood of CAKUTs, particularly duplex
collecting systems and vesicoureteral reflux, which is interesting
because high-dose folic acid can be used to induce experimen-
tal kidney disease in laboratory mice.99 Maternal obesity and
with the latter particularly linked to posterior urethral valves (odds
ratio, 2.6; 95% confidence interval, 1.1–5.9).98 
Potential Adverse Effects of Prematurity
Based on a study measuring nephron number in 11 human
fetuses between 15 and 40 weeks’ gestation20 (and acknowledg-
ing the small sample size), it appears that less than one third of
the final nephron complement have formed before 24 weeks of
120 SE C T I O N 3     Fetal Physiology and Pathology
gestation. Premature babies born at that gestation have to rely
exclusively on their own kidneys for excretion and electrolyte
homeostasis, and the kidney is suddenly exposed to significantly
increased renal blood flow. This causes hyperfiltration in already
formed nephrons and some disruption of developing nephrons.
Additive deleterious effects would also be predicted from neph-
rotoxic drugs, suboptimal nutrition and possible infections. In
premature babies, therefore, one would expect fewer nephrons
to form, with an increased rate of damage and loss of those that
have already been generated (via mechanism underlying the
Brenner hypothesis).
Kidney function may appear initially normal in premature
infants because urine output and creatinine remain within nor-
mal limits initially. However, these measures are too crude to
accurately predict long-term renal function, and there should
be ongoing screening for hypertension and kidney failure into
adulthood.100-102  Self-assessment questions available at ExpertConsult.com.
Conclusions
Development of the kidneys with concomitant contribution of
urine to the amniotic fluid is essential for normal fetal develop-
ment. Renal anomalies vary from gross structural malformations
such as multicystic dysplastic kidneys through subtle reductions in
nephron number, undetectable at birth but predisposing to hyper-
tension in later life. Many texts focus on genetics as the main cause
of such anomalies, but known mutations account for fewer than
20% of cases at present. Obstruction of the urinary tract, maternal
environment, diet and teratogens may be just as important, along
with chance, stochastic maldevelopment. Children who have, or
are at risk for having, a low nephron number need regular follow
up to identify and treat hypertension early.
Access the complete reference list online at ExpertConsult.com.
References rule. Causes and consequences. Lab Invest.
1999;79(5):515–527.
hemodynamically mediated glomerular injury
in the pathogenesis of progressive glo-
22. Bertram JF, Douglas-Denton RN, Diouf merular sclerosis in aging, renal ablation,
1. Vize PD, Woolf AS, Bard JBL, eds. The Kid-
B, et  al. Human nephron number: implica- and intrinsic renal disease. N Engl J Med.
ney: From Normal Development to Congenital
tions for health and disease. Pediatr Nephrol. 1982;307(11):652–659.
Disease. London, UK: Academic Press; 2003.
2011;26(9):1529–1533. 39. Brenner BM, Garcia DL, Anderson S. Glom-
2. Drummond IA, Davidson AJ. Zebraf-
23. Puelles VG, Bertram JF. Counting glom- eruli and blood pressure. Less of one, more the
ish kidney development. Methods Cell Biol.
eruli and podocytes: rationale and meth- other? Am J Hypertens. 1988;1(4 Pt 1):335–
2010;100:233–260.
odologies. Curr Opin Nephrol Hypertens. 347.
3. Moritz KM, Wintour EM. Functional devel-
2015;24(3):224–230. 40. Luyckx VA, Brenner BM. Birth weight,
opment of the meso- and metanephros. Pedi-
24. Baldelomar EJ, Charlton JR, Beeman SC, malnutrition and kidney-associated out-
atr Nephrol. 1999;13(2):171–178.
et  al. Phenotyping by magnetic resonance comes—a global concern. Nat Rev Nephrol.
4. Hatini V, Huh SO, Herzlinger D, et al. Essen-
imaging nondestructively measures glomeru- 2015;11(3):135–149.
tial role of stromal mesenchyme in kidney
lar number and volume distribution in mice 41. Hothi DK, Wade AS, Gilbert R, Winyard
morphogenesis revealed by targeted disruption
with and without nephron reduction. Kidney PJ. Mild fetal renal pelvis dilatation: much
of Winged Helix transcription factor BF-2.
Int. 2016;89(2):498–505. ado about nothing? Clin J Am Soc Nephrol.
Genes Dev. 1996;10(12):1467–1478.
25. Hoy WE, Hughson MD, Singh GR, et  al. 2009;4(1):168–177.
5. Fetting JL, Guay JA, Karolak MJ, et al. FOXD1
Reduced nephron number and glomerulo- 42. Liapis H, Winyard PJD. Cystic diseases and
promotes nephron progenitor differentiation
megaly in Australian Aborigines: a group at developmental kidney defects. In: Jennette JC,
by repressing decorin in the embryonic kid-
high risk for renal disease and hypertension. Olson JL, Silva FG, D’Agati VD, eds. Heptin-
ney. Development. 2014;141(1):17–27.
Kidney Int. 2006;70(1):104–110. stall’s Pathology of the Kidney. Philadelphia:
6. Potter EL. Normal and Abnormal Development
26. Keller G, Zimmer G, Mall G, et al. Nephron Wolters Kluwer; 2015:119–172.
of the Kidney. Chicago: Year Book Medical
number in patients with primary hyperten- 43. Winyard P, Chitty LS. Dysplastic kidneys.
Publishers; 1972.
sion. N Engl J Med. 2003;348(2):101–108. Semin Fetal Neonatal Med. 2008;13(3):142–
7. Jennette JC, Olson JL, Silva FG, D’Agati VD.
27. Puelles VG, Hoy WE, Hughson MD, et  al. 151.
Heptinstall’s Pathology of the Kidney. Philadelphia:
Glomerular number and size variability and 44. Martinovic-Bouriel J, Benachi A, Bonniere M,
Lippincott Williams & Wilkins (LWW); 2015.
risk for kidney disease. Curr Opin Nephrol et al. PAX2 mutations in fetal renal hypodys-
8. McMahon AP. Development of the mammalian
Hypertens. 2011;20(1):7–15. plasia. Am J Med Genet A. 2010;152A(4):830–
kidney. Curr Topics Dev Biol. 2016;117:31–64.
28. Barker DJ, Osmond C, Golding J, et  al. 835.
9. Winyard PJD, Nauta J, Lirenman DS, et  al.
Growth in utero, blood pressure in childhood 45. Clissold RL, Hamilton AJ, Hattersley AT,
Deregulation of cell survival in cystic and
and adult life, and mortality from cardiovascu- et al. HNF1B-associated renal and extra-renal
dysplastic renal development. Kidney Int.
lar disease. BMJ. 1989;298(6673):564–567. disease-an expanding clinical spectrum. Nat
1996;49(1):135–146.
29. Barker DJ. The origins of the developmental Rev Nephrol. 2015;11(2):102–112.
10. Rumballe BA, Georgas KM, Combes AN,
origins theory. J Intern Med. 2007;261(5):412– 46. Winding L, Loane M, Wellesley D, et al. Pre-
et al. Nephron formation adopts a novel spa-
417. natal diagnosis and epidemiology of multicys-
tial topology at cessation of nephrogenesis.
30. Heindel JJ, Vandenberg LN. Developmental tic kidney dysplasia in Europe. Prenat Diagn.
Dev Biol. 2011;360(1):110–122.
origins of health and disease: a paradigm for 2014;34(11):1093–1098.
11. Little MH, Kairath P. Regenerative medicine
understanding disease cause and prevention. 47. Kandasamy Y, Smith R, Wright IM, Lumbers
in kidney disease. Kidney Int. 2016;90(2):289–
Curr Opin Pediatr. 2015;27(2):248–253. ER. Reduced nephron endowment in the neo-
299.
31. Hokke S, Arias N, Armitage JA, et al. Mater- nates of Indigenous Australian peoples. J Dev
12. Ekblom P. Embryology and prenatal develop-
nal glucose intolerance reduces offspring Orig Health Dis. 2014;5(1):31–35.
ment. In: Holliday MA, Barrett TM, Avner
nephron endowment and increases glomerular 48. Matsell DG, Cojocaru D, Matsell EW,
ED, eds. Pediatric Nephrology. Baltimore:
volume in adult offspring. Diabetes Metab Res Eddy AA. The impact of small kidneys. Pediatr
Williams and Wilkins; 1994:2–18.
Rev. 2016;32(8):816–882. Nephrol. 2015;30(9):1501–1509.
13. Saxen L. Organogenesis of the Kidney. Cam-
32. Godfrey KM, Costello PM, Lillycrop KA. 49. Doery AJ, Ang E, Ditchfield MR. Duplex kid-
bridge, United Kingdom: Cambridge Univer-
Development, epigenetics and metabolic ney: not just a drooping lily. J Med Imaging
sity Press; 1987.
programming. Nestle Nutr Inst Workshop Ser. Radiat Oncol. 2015;59(2):149–153.
14. Short KM, Combes AN, Lefevre J, et  al.
2016;85:71–80. 50. McPherson E. Renal anomalies in families of
Global quantification of tissue dynamics
33. Eriksson JG. Developmental Origins of Health individuals with congenital solitary kidney.
in the developing mouse kidney. Dev Cell.
and Disease—from a small body size at birth Genet Med. 2007;9(5):298–302.
2014;29(2):188–202.
to epigenetics. Ann Med. 2016;48(6):456– 51. Weber S, Moriniere V, Knuppel T, et  al.
15. Woolf AS, Gnudi L, Long DA. Roles of angio-
467. Prevalence of mutations in renal developmen-
poietins in kidney development and disease. J
34. Langley-Evans SC, Welham SJ, Jackson AA. tal genes in children with renal hypodyspla-
Am Soc Nephrol. 2009;20(2):239–244.
Fetal exposure to a maternal low protein diet sia: results of the ESCAPE study. J Am Soc
16. Drukker A, Guignard JP. Renal aspects of the
impairs nephrogenesis and promotes hyper- Nephrol. 2006;17(10):2864–2870.
term and preterm infant: a selective update.
tension in the rat. Life Sci. 1999;64(11):965– 52. Hwang DY, Dworschak GC, Kohl S, et  al.
Curr Opin Pediatr. 2002;14(2):175–182.
974. Mutations in 12 known dominant disease-
17. Filler G, Guerrero-Kanan R, Alvarez-Elias AC.
35. Welham SJ, Wade A, Woolf AS. Protein causing genes clarify many congenital anoma-
Assessment of glomerular filtration rate in the
restriction in pregnancy is associated with lies of the kidney and urinary tract. Kidney Int.
neonate: is creatinine the best tool? Curr Opin
increased apoptosis of mesenchymal cells at 2014;85(6):1429–1433.
Pediatr. 2016;28(2):173–179.
the start of rat metanephrogenesis. Kidney Int. 53. Kohl S, Hwang DY, Dworschak GC,
18. Schwartz GJ, Work DF. Measurement and
2002;61(4):1231–1242. et  al. Mild recessive mutations in six Fraser
estimation of GFR in children and adolescents.
36. Villar-Martini VC, Carvalho JJ, Neves MF, syndrome-related genes cause isolated congen-
Clin J Am Soc Nephrol. 2009;4(11):1832–
et al. Hypertension and kidney alterations in ital anomalies of the kidney and urinary tract.
1843.
rat offspring from low protein pregnancies. J J Am Soc Nephrol. 2014;25(9):1917–1922.
19. Denic A, Glassock RJ, Rule AD. Structural
Hypertens Suppl. 2009;27(6):S47–S51. 54. Rodriguez MM. Congenital anomalies of the
and functional changes with the aging kidney.
37. Watkins AJ, Lucas ES, Torrens C, et  al. kidney and the urinary tract (CAKUT). Fetal
Adv Chronic Kidney Dis. 2016;23(1):19–28.
Maternal low-protein diet during mouse pre- Pediatr Pathol. 2014;33(5-6):293–320.
20. Hinchliffe SA, Sargent PH, Howard CV, et al.
implantation development induces vascular 55. Nicolaou N, Renkema KY, Bongers EM, et al.
Human intrauterine renal growth expressed in
dysfunction and altered renin-angiotensin- Genetic, environmental, and epigenetic fac-
absolute number of glomeruli assessed by the
system homeostasis in the offspring. Br J Nutr. tors involved in CAKUT. Nat Rev Nephrol.
disector method and Cavalieri principle. Lab
2010;103(12):1762–1770. 2015;11(12):720–731.
Invest. 1991;64(6):777–784.
38. Brenner BM, Meyer TW, Hostetter TH. 56. Chevalier RL. Prognostic factors and biomark-
21. Merlet-Benichou C, Gilbert T, Vilar J,
Dietary protein intake and the progres- ers of congenital obstructive nephropathy.
et  al. Nephron number: variability is the
sive nature of kidney disease: the role of Pediatr Nephrol. 2016;31(9):1411–1420.

120.e1
120.e2 References
57. Bohn S, Thomas H, Turan G, et al. Distinct
molecular and morphogenetic properties of
mutations in the human HNF1beta gene that
lead to defective kidney development. J Am
Soc Nephrol. 2003;14(8):2033–2041.
58. Decramer S, Parant O, Beaufils S, et al. Anom-
alies of the TCF2 gene are the main cause of
fetal bilateral hyperechogenic kidneys. J Am
Soc Nephrol. 2007;18(3):923–933.
59. Raaijmakers A, Corveleyn A, Devriendt K,
et al. Criteria for HNF1B analysis in patients
with congenital abnormalities of kidney
and urinary tract. Nephrol Dial Transplant.
2015;30(5):835–842.
60. Bockenhauer D, Jaureguiberry G. HNF1B-
associated clinical phenotypes: the kidney and
beyond. Pediatr Nephrol. 2016;31(5):707–
714.
61. Dressler GR, Wilkinson JE, Rothenpieler
UW, et  al. Deregulation of Pax-2 expression
in transgenic mice generates severe kidney
abnormalities. Nature. 1993;362:65–67.
62. Winyard PJD, Risdon RA, Sams VR, et  al.
The PAX2 transcription factor is expressed in
cystic and hyperproliferative dysplastic epi-
thelia in human kidney malformations. J Clin
Invest. 1996;98:451–459.
63. Sharma R, Sanchez-Ferras O, Bouchard
M. Pax genes in renal development, dis-
ease and regeneration. Semin Cell Dev Biol.
2015;44:97–106.
64. Sanyanusin P, Schimmentl LA, McNoe LA,
et al. Mutations of the PAX2 gene in a fam-
ily with optic nerve colobomas, renal anom-
alies and vesicoureteral reflux. Nat Genet.
1995;9:358–364.
65. Okumura T, Furuichi K, Higashide T,
et  al. Association of PAX2 and other gene
mutations with the clinical manifestations
of renal coloboma syndrome. PLoS One.
2015;10(11):e0142843.
66. Ruf RG, Xu PX, Silvius D, et al. SIX1 muta-
tions cause branchio-oto-renal syndrome by
disruption of EYA1-SIX1-DNA complexes.
Proc Natl Acad Sci U S A. 2004;101(21):8090–
8095.
67. Weber S. Novel genetic aspects of congenital
anomalies of kidney and urinary tract. Curr
Opin Pediatr. 2012;24(2):212–218.
68. Costantini F, Kopan R. Patterning a complex
organ: branching morphogenesis and nephron
segmentation in kidney development. Dev
Cell. 2010;18(5):698–712.
69. Chatterjee R, Ramos E, Hoffman M, et  al.
Traditional and targeted exome sequencing
reveals common, rare and novel functional
deleterious variants in RET-signaling com-
plex in a cohort of living US patients with
urinary tract malformations. Hum Genet.
2012;131(11):1725–1738.
70. Basson MA, Akbulut S, Watson-Johnson J,
et al. Sprouty1 is a critical regulator of GDNF/
RET-mediated kidney induction. Dev Cell.
2005;8(2):229–239.
71. Hwang DY, Kohl S, Fan X, et al. Mutations of
the SLIT2-ROBO2 pathway genes SLIT2 and
SRGAP1 confer risk for congenital anomalies
of the kidney and urinary tract. Hum Genet.
2015;134(8):905–916.
72. Reidy K, Tufro A. Semaphorins in kidney
development and disease: modulators of ure-
teric bud branching, vascular morphogenesis,
and podocyte-endothelial crosstalk. Pediatr
Nephrol. 2011;26(9):1407–1412.
73. Zhang Z, Quinlan J, Hoy W, et  al. A com-
mon RET variant is associated with reduced
newborn kidney size and function. J Am Soc
Nephrol. 2008;19(10):2027–2034.
74. Quinlan J, Lemire M, Hudson T, et  al. A
common variant of the PAX2 gene is associ-
ated with reduced newborn kidney size. J Am
Soc Nephrol. 2007;18(6):1915–1921.
75. Kaczmarczyk M, Kuprjanowicz A, Loniewska B,
et  al. Epistatic interaction between common
AGT G(-6)A (rs5051) and AGTR1 A1166C
(rs5186) variants contributes to variation in
kidney size at birth. Gene. 2015;572(1):72–
78.
76. Pruthi R, O’Brien C, Casula A, et  al. UK
Renal Registry 16th Annual Report: Chapter
7 Demography of the UK Paediatric Renal
Replacement Therapy population in 2012.
Nephron Clin Pract. 2013;125:127–138.
77. Woolf AS, Stuart HM, Newman WG. Genet-
ics of human congenital urinary bladder dis-
ease. Pediatr Nephrol. 2014;29(3):353–360.
78. Yang SP, Woolf AS, Quinn F, Winyard PJD.
Deregulation of renal transforming growth
factor-á1 after experimental short-term ure-
teric obstruction in fetal sheep. Am J Pathol.
2001;159:109–117.
79. Holst BD, Goomer RS, Wood IC, et al. Bind-
ing and activation of the promoter for the
neural cell adhesion molecule by Pax-8. J Biol
Chem. 1994;269(35):22245–22252.
80. Morris RK, Malin GL, Quinlan-Jones E, et al.
Percutaneous vesicoamniotic shunting ver-
sus conservative management for fetal lower
urinary tract obstruction (PLUTO): a ran-
domised trial. Lancet. 2013;382(9903):1496–
1506.
81. Roseboom TJ, van der Meulen JH,
Ravelli AC, et  al. Blood pressure in adults
after prenatal exposure to famine. J Hypertens.
1999;17(3):325–330.
82. Ravelli AC, van der Meulen JH, Michels RP, et al.
Glucose tolerance in adults after prenatal expo-
sure to famine. Lancet. 1998;351(9097):173–
177.
83. Sparen P, Vagero D, Shestov DB, et al. Long
term mortality after severe starvation during
the siege of Leningrad: prospective cohort
study. BMJ. 2004;328(7430):11.
84. Wood-Bradley RJ, Barrand S, Giot A,
Armitage JA. Understanding the role of
maternal diet on kidney development; an
opportunity to improve cardiovascular and
renal health for future generations. Nutrients.
2015;7(3):1881–1905.
85. Law CM, Shiell AW. Is blood pressure
inversely related to birth weight? The strength
of evidence from a systematic review of the lit-
erature. J Hypertens. 1996;14(8):935–941.
86. Yim HE, Yoo KH. Early life obesity and
chronic kidney disease in later life. Pediatr
Nephrol. 2015;30(8):1255–1263.
87. Tsuboi N, Utsunomiya Y, Hosoya T.
Obesity-related glomerulopathy and the
nephron complement. Nephrol Dial Trans-
plant. 2013;28(suppl 4):108–113. iv.
88. Shepard TH. Catalog of Teratogenic Agents.
Baltimore: The Johns Hopkins University
Press; 2010.
89. Pope JC, Nishimura H, Ichikawa I. Role
of angiotensin in the development of
the kidney and urinary tract. Nephrologie.
1998;19(7):433–436.
90. Gribouval O, Gonzales M, Neuhaus T, et al.
Mutations in genes in the renin-angiotensin
system are associated with autosomal reces-
sive renal tubular dysgenesis. Nat Genet.
2005;37(9):964–968.
91. Barr Jr M, Cohen Jr MM. ACE inhibitor
fetopathy and hypocalvaria: the kidney-skull
connection. Teratology. 1991;44(5):485–495.
92. Woods LL, Ingelfinger JR, Nyengaard JR,
Rasch R. Maternal protein restriction sup-
presses the newborn renin-angiotensin system
and programs adult hypertension in rats. Pedi-
atr Res. 2001;49(4):460–467.
93. Woolf AS. Environmental influences on renal
tract development: a focus on maternal diet
and the glucocorticoid hypothesis. Klin Padi-
atr. 2011;223(suppl 1):S10–S17.
94. Lee LM, Leung CY, Tang WW, et  al. A
paradoxical teratogenic mechanism for
retinoic acid. Proc Natl Acad Sci U S A.
2012;109(34):13668–13673.
95. El KR, Manolescu DC, Lakhal-Chaieb L,
et  al. A human ALDH1A2 gene variant is
associated with increased newborn kidney
size and serum retinoic acid. Kidney Int.
2010;78(1):96–102.
96. Reichetzeder C, Chen H, Foller M, et  al.
Maternal vitamin D deficiency and fetal pro-
gramming—lessons learned from humans and
mice. Kidney Blood Press Res. 2014;39(4):315–
329.
97. Maka N, Makrakis J, Parkington HC, et  al.
Vitamin D deficiency during pregnancy and
lactation stimulates nephrogenesis in rat off-
spring. Pediatr Nephrol. 2008;23(1):55–61.
98. Groen In’t Woud S, Renkema KY, Schreuder
MF, et  al. Maternal risk factors involved in
specific congenital anomalies of the kidney and
urinary tract: a case-control study. Birth Defects
Res A Clin Mol Teratol. 2016;106(7):596–603.
99. Kolatsi-Joannou M, Price KL, Winyard PJ,
Long DA. Modified citrus pectin reduces
galectin-3 expression and disease severity in
experimental acute kidney injury. PLoS One.
2011;6(4):e18683.
100. Carmody JB, Charlton JR. Short-term gesta-
tion, long-term risk: prematurity and chronic
kidney disease. Pediatrics. 2013;131(6):1168–
1179.
101. Viswanathan S, Kumar D, Sykes C, et  al.
Making a diagnosis of hypertension and defin-
ing treatment threshold in very low birth
weight infants’ need revision? J Renal Inj Prev.
2016;5(2):55–60.
102. de Jong F, Monuteaux MC, van Elburg RM,
et  al. Systematic review and meta-analysis of
preterm birth and later systolic blood pressure.
Hypertension. 2012;59(2):226–234.
13
The Perinatal Postmortem Examination
J. CIARAN HUTCHINSON, SUSAN C . SHELMERDINE AND NEIL J. SEBIRE
KEY POINTS
• P erinatal autopsy (examination after death) fulfils several
roles, including determination or clarification of the underly-
ing diagnosis, answering specific questions raised by parents
and clinicians, quality assurance, governance and public
health aspects, and improved understanding of disease
mechanisms through research.
• Examination after death may involve a spectrum of inves-
tigations, including placental pathology, genetic testing,
postmortem imaging and internal organ examination or
sampling.
• Parents should be informed regarding their options, with the
extent of investigation determined by parental acceptability
and appropriateness for specific clinical circumstances.
• Examination after death should be individualised according
to the clinical features present and parental consent require-
ments.
• Placental examination should be considered in all cases of
pregnancy complications even if postmortem fetal examina-
tion is declined.
• Future advances in imaging and laboratory medicine, such as
the widespread introduction of various ‘omic’ technologies,
are likely to significantly change the approach to investiga-
tion after death.
Overview
Examination after perinatal death may be a difficult area for obste-
tricians and fetal medicine practitioners, many of whom may oth-
erwise have little interaction with specialist pathology services.
Therefore, the aim of this chapter is not to present detailed find-
ings of issues in this field but rather to provide practical guidance
for interaction of clinicians and pathologists to maximise utility of
the various facets related to perinatal autopsy examination from
consent through to technical aspects of the process itself and likely
future advances in the area. Some aspects of the major categories
of pathology that may be disclosed through postmortem investi-
gations are also covered; detailed explanations of these conditions
can be found in specialist embryology and perinatal pathology
textbooks.  accurately reflect the future of this approach, with personalised
Introduction
Autopsy: Greek: ‘autos’ (self ) + ‘optos’ (seen); or ‘autoptēs’
(eyewitness)
The role of the perinatal autopsy has come under increasing
scrutiny from medical professionals and the public. Advances in
medical imaging, increasing use of antenatal genetic testing and
controversies associated with human tissue retention have com-
bined with shifting population demographics and changing pub-
lic attitudes, resulting in a reduction in acceptability of traditional
autopsy and the encouragement of development of potentially
more acceptable, contemporary approaches.1 Research into paren-
tal attitudes to autopsy has revealed that traditional postmortem
examination is becoming less acceptable,2 especially among cer-
tain ethnic and religious groups,3-5 with autopsy rates falling in
most countries (Table 13.1). In addition to moral or religious
reasons, parents have aversion to large incisions because of percep-
tions that the fetus or infant has ‘suffered enough’. From a clinical
perspective, there is also a perception that autopsy reports vary in
adequacy, alongside perceived difficulties in negotiating a highly
specific informed consent process.6
Although great insights into normal developmental processes
and pathogenesis of congenital anomalies have resulted from
perinatal autopsy findings, contemporary obstetric practice has
changed, with introduction of widespread first and second trimes-
ter antenatal ultrasound screening resulting in accurate antenatal
detection of a wide range of fetal abnormalities.7 Consequently,
the role of examination of after death is also changing, with
detection of unexpected major fetal anomalies now less frequent,
but the range of antenatal fetal interventions and complexity of
associated pathologies increasing. In addition, mechanistic data
derived from many years of autopsy practice, which have provided
improved understanding of numerous obstetric complications, is
decreasingly likely to generate new insights in the absence of the
introduction of novel approaches.
Although the concept of the autopsy examination as a means
of medical audit and governance remains important (e.g., for ter-
minations of pregnancy or after complex medical treatment8),
the additional direct clinical benefit from autopsy examination
of otherwise uncomplicated cases remains uncertain if there are
no specific additional clinical questions to be addressed. The con-
cept of examination after death must therefore develop in parallel
with changes in antenatal care and technologies if it is to con-
tinue to make important contributions to research and clinical
care. The concept of ‘investigation after death’ may therefore more
investigations performed targeted to address the specific issues of
particular cases to improve the quality of information gained and
increase parental acceptability.9
Investigation after death is unusual in that many factors sur-
rounding the decisions made and approaches used are primarily
121
122 SE C T I O N 3     Fetal Physiology and Pathology

TABLE Number of Postmortem Examinations Offered and Consented to by Type of Death (Stillbirth, Neonatal Death,
13.1A Extended Perinatal Death): United Kingdom and Crown Dependencies, for Births in 2014

STILLBIRTHSa NEONATAL DEATHSa EXTENDED PERINATAL DEATHSa

Postmortem Status Number (%) Number (%) Number (%)

Not offered 50 (1.6) 137 (10.0) 187 (4.1)
Not known if offered 67 (2.1) 155 (11.3) 222 (4.8)
Offered but no consent 1503 (46.6) 628 (45.7) 2131 (46.3)
Offered but unknown consent 83 (2.6) 54 (3.9) 137 (3.0)
Offered and limited consent 120 (3.7) 28 (2.0) 148 (3.2)
Offered and full consent 1402 (43.5) 372 (27.1) 1774 (38.6)
aExcluding termination of pregnancy and births <24+0 weeks gestational age.
Reproduced from Manktelow BN, Smith LK, Seaton SE, et al on behalf of the MBRRACE-UK Collaboration. MBRRACE-UK Perinatal Mortality Surveillance Report, UK Perinatal Deaths for Births from January
to December 2014. Leicester: The Infant Mortality and Morbidity Studies, Department of Health Sciences, University of Leicester, 2016, p84.
  

the behest of a legal representative, i.e., the coroner in the United

TABLE Rates of Request and Permission Granted for an
13.1B Autopsy According to Year
Kingdom) and those for which consent is obtained from relatives.
In adult practice, the consented hospital autopsy has largely disap-
Permission Consent Given (%) peared over the past decade because of multiple complex reasons,
Number of Requested (Percentage of which are not covered here. Conversely, in perinatal practice, con-
Year Deaths (%) Permission Requested) sented autopsies are by far the most common investigation since
because primary aim is to provide information for the parents
2003 403 336 (83.4) 198 (58.9) regarding the underlying diagnosis and therefore recurrence rates
2004 432 361 (83.6) 197 (54.6) and implications for future pregnancies. There has, however, also
been a recent decline in the proportion of perinatal deaths under-
2005 443 380 (85.8) 213 (56.1) going consented autopsy investigation, although not as marked as
2006 415 324 (78.1) 195 (60.2) with adult practice but with current overall consent rates of less
than 50% for intrauterine fetal deaths.10
2007 466 359 (77.0) 210 (58.5)
Medicolegal postmortem investigations in the perinatal age
2008 425 381 (89.6) 175 (45.9) range are relatively rare but may be indicated for deaths related to
medical procedures, those in which negligence or criminal activity
2009 481 436 (90.6) 175 (40.1)
may have been contributing factors and for neonates who die sud-
2010 440 414 (94.1) 169 (40.8) denly and unexpectedly for whom a cause of death, and therefore
2011 415 382 (92.0) 146 (38.2)
death certificate, cannot be issued by the clinicians. In such cases,
parental consent is not required, and conduct of the examination
2012 412 386 (93.7) 188 (48.7) is on behalf of the coroner or police with several broad legal and
Total 4332 3759 (86.8) 1866 (49.6) epidemiological purposes:
1. Ascertain a cause, and if necessary, a mode of death.
Reproduced from Kotecha SJ, Rolfe K, Watkins WJ, et al. All Wales Perinatal Survey Commen- 2. Identify evidence of possible unlawful or unnatural causes of
tary 2013: Perinatal and infant autopsy rate in Wales over a ten year period. Cardiff, Wales: death.
Perinatal Survey Office, Department of Child Health, School of Medicine, Cardiff University.
https://awpsonline.uk/awps-commentary-2013.
3. Contribute to statistical information regarding population
mortality.
   These aims overlap with the legal requirements of the coro-
ner, whose remit is to ‘establish the facts’ about the deceased,
based around the wishes and expectations of the parents, and specifically:
pathologists and clinicians alike should work together to shift 1. The identity of the deceased
the emphasis towards personalised investigation by providing the 2. The time and place of death
advantages and disadvantages of all options to address meaningful 3. The cause of death
clinical questions (see Table 13.1).  4. The manner of death
Although unlawful death is uncommon in the perinatal set-
Legal Aspects, Aims and Types of Autopsy ting, occasional cases of baby destruction or infanticide are still
reported.
The precise arrangements for legal frameworks vary by country, but In contemporary practice, the aim of consented perinatal
the principles described in this chapter for the United Kingdom autopsy is primarily to provide potentially clinically important
are generally applicable to varying extents in most geographical information for families regarding the underlying diagnosis or
locations. Autopsies can be broadly divided into those performed cause of death or the risk for recurrence or implications for sib-
for medicolegal reasons (i.e., without parental or family consent at lings, to act as a medical audit tool for fetal medicine specialists

or ultrasonographers, and to provide samples for genetic or other
ancillary studies. Perinatal autopsy findings have historically con-
tributed significantly to the understanding of human fetal develop-
ment, the pathogenesis of fetal abnormalities and the pathogenesis
of obstetric complications and continue to contribute to disease
understanding and therapy development (e.g., descriptions of pla-
cental anatomical features in twin-to-twin transfusion syndrome in
relation to outcomes). It is well established that the yield of clini-
cally useful information is greater when perinatal autopsies are per-
formed by specialist paediatric and perinatal pathologists compared
with general pathologists, and it is therefore recommended that
when possible, all perinatal autopsies should be performed in spe-
cialist centres.11-13 There are published guidelines for performance
of such autopsies and requirements for staffing and training.14,15 
Consent for Investigation After Death
Consent for investigation after death should be viewed as a pro-
cess, for which the ultimate aim is to establish permission to carry
out the appropriate investigations to answer clinical questions
posed and guide management of future pregnancies.16 In some
circumstances, adequate information to answer such questions
may be obtained through noninvasive means such as postmor-
tem imaging and targeted biopsies (e.g., sampling of specific organ
abnormalities such as suspected autosomal recessive polycystic
renal disease), but in other cases, a full invasive autopsy may be
required (e.g., in the setting of a complex postoperative perinatal
death).
A useful set of guiding principles regarding consent includes:
1. Consent should be obtained by an experienced and appropri-
ately trained professional, who should be respectful of the par-
ents’ wishes.
2. The parents should be fully informed regarding all available
options (including more limited approaches if available and
applicable). The parents should be approached at an appropri-
ate time, by a member of staff with whom they have a rapport,
and should not be pressured into making a decision.
3. The parents should understand the specific procedure to which
they are consenting, including current practice and recent
developments. It should be noted that, for many parents, the
possibility to contribute to research and therefore potentially
help others in the future has been reported as a significant fac-
tor in parental decision making.
4. All parties should be made aware of the specific medicolegal
frameworks (e.g., in the United Kingdom under the regula-
tions of the Human Tissue Authority).
If one or more of the parents do not engage in the consent
process or if they only wish for limited information to be given,
this can make the consent procedure more difficult; in these
circumstances, there should be clear documentation of what is
discussed.  compared with standard autopsy across a range of clinical scenar-
The Autopsy Procedure
The traditional perinatal autopsy consists of several distinct com-
ponents, all of which are incorporated into an overall autopsy
report. An overview of the processes involved and the types of
pathologies that may be detected at each stage is provided next.
To consent parents to the appropriate investigations, a working
knowledge of the diagnostic yield provided by specific investiga-
tions is required by the practicing fetal medicine clinician and
consent taker.
CHAPTER 13  The Perinatal Postmortem Examination 123
Clinical Review
After transfer of the appropriate authority to the investigating
pathologist (usually via the consent mechanism), the patholo-
gist should review the clinical case notes, including the findings
of antenatal imaging, other investigations and care provided; the
maternal medical, obstetric and gynaecological history; and the
circumstances of delivery and death. This information enables
the pathologist to target the examination appropriately to bet-
ter address important clinical questions, such as identification of
potential genetic conditions, and may influence the performance
of subsequent aspects of the investigation. 
External Examination
A detailed external examination of the fetus should be carried out
to include identification of subtle dysmorphic features which may
be difficult or impossible to identify sonographically, such as some
types of facial dysmorphism, posterior cleft palate and genital
abnormalities. 
Postmortem Imaging
Either before or after the external examination, a range of post-
mortem imaging investigations may be undertaken. The princi-
ples of postmortem imaging are discussed in more detail later but
can include a combination of plain radiographs, cross-sectional
imaging (computed tomography (CT) and magnetic resonance
imaging (MRI)), ultrasound examination and other novel inves-
tigations such as contrast-enhanced imaging. The precise imaging
modality of choice will depend upon the gestational age, clinical
history and organ or system to be visualised. 
Internal Examination
Traditional autopsy includes systematic examination of all internal
organs, within the limits of the authority provided to the patholo-
gist by the consent. The parents may wish to limit the examina-
tion to specific organs or body cavities if there is a query about a
particular diagnosis or clinical issue. Standard open internal exam-
ination is performed via a large midline incision from the manu-
brium to the pelvis. After removal of the ribcage, the internal
organs are then inspected, examined and removed to be weighed
and dissected. More recently, it has been demonstrated that other
approaches such as endoscopic-assisted techniques can be used, in
conjunction with postmortem imaging, to obtain tissue samples
via a much smaller incision. (A 1- to 2-cm incision permits sam-
pling of all abdominal and thoracic organs.) This approach allows
direct visualisation of fetal organs and permits photography, video
recording and sampling, but its accuracy for specific diagnoses
ios remains to be established, and it is not appropriate for all cases.
Regardless of approach, in situ sampling for microbiology, genetic
studies, virology and histology can be performed to minimise the
invasiveness of the procedure. Unless there is a specific indication
for additional examination, organs are then returned to the body
according to the wishes of the parents.
If formal neuropathological examination is required (e.g.,
after termination of pregnancy for a central nervous system
(CNS) abnormality), then the standard approach is to remove
the brain for a period of fixation before dissection and sampling.
This is especially required for fetal cases in whom the brain
124 SE C T I O N 3     Fetal Physiology and Pathology
• Fig. 13.1  A standard paraffin block and slide demonstrating histology
sampling. The tissue within the block is considerably smaller than the
block itself.
contains relatively little myelin and is therefore extremely friable,
making examination of the unfixed brain effectively impossible.
This period of fixation and subsequent examination may result
in a delay in the final autopsy report completion, and the parents
should be made aware of this when a neuropathological indica-
tion exists. However, for many structural CNS abnormalities,
postmortem imaging approaches provide excellent anatomical
detail, and it is likely that the requirements for brain removal
and formal neuropathological examination in this setting may
reduce in future.  placental tissue samples for molecular analyses. It should be noted
Histologic Examination
Published autopsy guidelines recommend histologic sampling of
most major internal organs for perinatal postmortem examina-
tions regardless of clinical indication.14,15 However, such guide-
lines are based on expert opinion rather than published evidence
of efficacy and are based on an approach designed to minimise
the risk for missing an important diagnosis if autopsies are per-
formed by less experienced practitioners. The primary purpose
of histologic sampling is to provide a morphological diagnosis
of pathology and to exclude or confirm the presence of disease.
Although tissue diagnosis remains critically important in some
scenarios (e.g., diagnosis of subtypes of cystic kidney diseases),
the yield of histologic examination of macroscopically and radio-
logically normal organs for clinically significant abnormalities is
low. It is therefore possible that more individualised and limited
sampling protocols may be indicated in particular cases according
to the clinical features present, without reduction in diagnostic
accuracy.
Currently, tissue samples obtained for microscopic examina-
tion are processed into small paraffin wax blocks and glass micro-
scope slides (Fig. 13.1). The average size of these tissue samples is
less than that of a postage stamp and measures around 3 to 5 mm
in thickness; in small fetuses, the samples are considerably smaller.
The sections obtained are then examined by the pathologist under
a microscope for identification of morphological features of dis-
ease. With technological laboratory advances, it is, however, likely
that in the future, such samples will undergo a range of genomic,
proteomic, metabolomics and other investigations, resulting in
improved diagnostic accuracy from smaller tissue samples, further
reducing sampling requirements.  initial funeral arrangements if required, or the parents may request
Placental Examination
A wide range of placental pathologies are described (Chapter 9).
The placenta should always be submitted for examination with the
fetus or infant for perinatal postmortem examination, particularly
in cases of intrauterine fetal death, in which placental examination
is the single most important investigation to determine the cause
of the loss.17 In this setting, placental examination may reveal dis-
orders with significant recurrence risks in future pregnancies, for
example, chronic histiocytic intervillositis.
The pathologist will make an anatomical appraisal of the pla-
centa, slice it for assessment or parenchymal lesions and obtain
placental tissue samples for histologic examination (Fig. 13.2)
in addition to material for extraction of fetal DNA for genetic
investigations. 
Ancillary Investigations
If samples are required for genetic studies or fibroblast culture, it
is recommended that these are obtained as soon as possible after
delivery (or prenatally) rather than at the time of autopsy to maxi-
mise the chance of successful culture and analysis and minimise
the effects of changes occurring after death or delivery. Samples
may therefore include fetal or umbilical cord blood, fetal skin
biopsy or placental tissue biopsy, all of which may be used for
karyotyping and other molecular genetic examinations. If there
is a prolonged postmortem interval, samples obtained at the time
of autopsy may be suboptimal for use for karyotyping and cul-
ture, but adequate DNA can usually be extracted from fetal or
that if a sample, either fetal or placental, is fixed in formalin or
formaldehyde, it will no longer be suitable for culture or stan-
dard karyotyping, although genetic testing using other techniques
such as polymerase chain reaction may be possible because DNA
extraction is possible.
Other ancillary investigations which are of limited value in
perinatal autopsies but may be indicated in specific circumstances
(e.g., neonatal deaths) include microbiologic and virologic anal-
yses, as well as metabolic studies (e.g., blood and bile spots for
acylcarnitine profiling by tandem mass spectrometry or enzyme
assays using cultured fibroblasts harvested from a postmortem
skin biopsy) and cytogenetic and DNA analysis. For such investi-
gations, it is important that samples are obtained as soon as pos-
sible after death or delivery. 
Retention of Organs
In the vast majority of cases, even standard autopsy examination
can be performed without the need to retain organs and delay
funeral arrangements. However, in some circumstances, it may be
necessary for the pathologist to temporarily retain an organ for
fixation and further detailed examination. This is particularly the
case when considering pathology involving the CNS because the
fetal brain is extremely soft and difficult to assess when fresh, and
important diagnostic information may be lost if it is not thor-
oughly formalin fixed before dissection. Temporarily retaining the
organ will allow for fixation to occur over a period of time (typi-
cally around 1–2 weeks), and after the examination and sampling,
the retained organ can be reunited with the body before funeral
arrangements. If the delay in burial is seen as a problem, the organ
can be released to the parents via an undertaker for burial after the
 CHAPTER 13  The Perinatal Postmortem Examination 125

A B
• Fig. 13.2  Placental examination often yields useful information. A, A bilobed placenta from a singleton
pregnancy with a succenturiate lobe. B, This photomicrograph of a term placenta demonstrates villitis of
unknown aetiology, with multiple villi demonstrating an abundant lymphocytic infiltrate.

that the organ is sensitively disposed of by the hospital or may

donate it for audit, teaching and research. 

The Postmortem Report

It has been suggested that a preliminary autopsy report should be
submitted to the clinician within 24 to 48 hours of the postmortem
examination followed by a final report which incorporates the his-
tologic findings and results of further investigations, usually within
4 to 6 weeks. However, in the setting of a consented examination
(as opposed to an autopsy on behalf of the coroner), it is often most
appropriate to simply provide a complete, final perinatal postmor-
tem report which describes all of the significant macroscopic and
microscopic findings, the results of ancillary investigations and a spe-
cific summary of the findings with clinicopathological correlation.
Although the parents are, of course, entitled to be provided with a
copy of the report, it is generally recommended that postmortem
reports are issued to the referring clinician so that the contents of the
report can be discussed with the parents, preferably in person, because
aspects of technical terminologies used in such reports may be dis-
tressing or confusing. To optimise the reporting process and improve
clinicopathological correlation, multidisciplinary team meetings
should take place to discuss cases so that all relevant antenatal, perina-
tal and autopsy features may be consolidated.  formed for many years but has been generally regarded as a
Effects of Changes After Death
After fetal death, either from natural causes or secondary to feticide,
there may be a period of intrauterine retention for several days, dur-
ing which secondary changes occur to the fetus (maceration). Such
changes can be useful for estimating the interval from intrauterine
death to delivery in cases such as stillbirths, but it can also have an
effect upon the detection of subtle abnormalities at autopsy. The
precise biological mechanisms involved during maceration remain
poorly understood but are thought to involve autolysis and enzyme-
induced degradation of fetal tissues after death. It has been sug-
gested that feticide with injection of potassium chloride may further
contribute to maceration changes.18 In particular, the fetal brain
becomes increasingly friable. In addition to intrauterine retention,
further degenerative changes occur during the postmortem inter-
val from delivery to autopsy examination. If an autopsy is to be
performed, the fetus should be refrigerated in an appropriate bag
or container (not wrapped in absorbent materials, which leads to
• Fig. 13.3  Advanced maceration in a stillbirth case at term. There is skin
slippage (red areas) with discolouration of the soft tissue and skin and dry-
ing of the mucous membranes.
marked fluid loss) and the postmortem examination performed as
soon as reasonably possible after delivery (Fig. 13.3). 
Postmortem Imaging
Postmortem imaging with plain radiography has been per-
minor ancillary investigation to the autopsy itself.19 In recent
years, increasing numbers of parents are refusing traditional
autopsy; as a result, there has been interest in whether alterna-
tive methods which are more acceptable to families may be
possible. Postmortem cross-sectional imaging in particular has
shown promise, especially when used as part of a minimally
invasive approach (Fig. 13.4). Postmortem magnetic resonance
imaging (PMMRI) with placental examination and other
investigations which require no fetal incisions, such as genetic
studies, have demonstrated overall concordance with standard
autopsy of around 95% in fetuses.20 Furthermore, in cases in
which a structural organ abnormality is detected, PMMRI
may be used to guide less invasive organ examination and
tissue sampling in conjunction with postmortem ultrasound
scanning or endoscopic examination.21,22 Although PMMRI
provides excellent anatomical detail in third trimester fetuses
and infants, because of fetal size and limits of resolution, such
methods are less effective below 18 weeks of gestation or a
fetal weight of 300 g.23 In this setting, it is possible that newer
126 SE C T I O N 3     Fetal Physiology and Pathology
• Fig. 13.4  Postmortem magnetic resonance imaging. An image from a
case of spontaneous intrauterine fetal death of a 21-week fetus with no
abnormalities present. (Courtesy Dr Owen Arthurs, Great Ormond Street
Hospital.)
• Fig. 13.5  Microfocus computed tomography slice through a 14-gestational-
week fetus after iodine immersion, at approximately 51-μm resolution. There is
excellent morphological detail of the internal anatomy.
methods, such as microfocus computed tomography (micro-
CT), may provide high-quality fetal imaging, which may even
be superior to traditional approaches, especially for early ges-
tation fetuses24,25 (Fig. 13.5). These alternative imaging-based
investigations will greatly expand the range of postmortem
approaches available to pathologists, clinicians and families,
and ‘investigation after death’ will better serve to represent
this future, with imaging becoming the likely investigation of
choice for all structural abnormalities.  detect such changes, particularly when extensive, on postmortem
Specific Issues Regarding Categories of
Perinatal Autopsy
Details of the main clinical features and genetic or syndromic
associations of fetal anomalies are provided elsewhere; the aim
of this section is to highlight specific postmortem-related issues.
Depending on the clinical indications, the most appropriate
investigations, incisions and approaches to the autopsy will
vary. 
Central Nervous System Anomalies
Most fetal CNS abnormalities are structural defects and as such
are readily evaluated by postmortem MRI if required or available.
However, to determine histologic information, which may provide
additional findings regarding underlying mechanisms or timing of
events in some cases, the brain must be removed for examina-
tion and sampling. The fetal brain is soft and poorly myelinated
and hence requires formalin fixation for a period of days to weeks
before dissection, sampling and return to the body. The parents
should therefore be aware that this process may delay the release of
the body and postmortem report compared with other postmor-
tem cases. Furthermore, it should be recognised that around 10%
to 20% of postmortem fetal CNS examinations are nondiagnos-
tic because of changes of maceration or friability.26,27 In addition,
specific anomalies are associated with specific issues, namely those
discussed next.
Ventriculomegaly
Ventriculomegaly may be difficult to confirm at autopsy, espe-
cially when apparently isolated and mild in degree. It was initially
suggested that this may be a consequence of brain removal and
fixation, but recent data based on PMMRI indicate that in around
50% of cases of terminations of pregnancy for well-documented
ventriculomegaly, the ventricles are of normal size after delivery.28
This is likely a consequence of fluid shifts after fetal death and
delivery, and the parents should be made aware that failure to
confirm mild ventriculomegaly after death does not indicate an
antenatal diagnostic error. Because there are numerous aetiologies
of ventriculomegaly, full neuropathological examination is usually
required. 
Posterior Fossa Abnormalities
Antenatally diagnosed abnormalities of the posterior fossa range
from typical Dandy-Walker malformation through cerebellar ver-
mis hypoplasia to apparently isolated enlarged cisterna magna.
Similar to ventriculomegaly, particularly for more subtle abnor-
malities, there may be relatively poor correlation with autopsy
findings in some cases. In addition, adequate visualisation of the
posterior fossa at autopsy requires a modified, posterior approach
to the opening of the skull; hence, it is important that this infor-
mation is provided to the pathologist. 
Abnormalities of Gyration
Abnormalities such as lissencephaly and polymicrogyria are tra-
ditionally histologic diagnoses, but it is increasingly possible to
imaging. 
Hypoxic Ischaemic Changes
A wide spectrum of hypoxic-ischaemic (HI) changes may affect
the CNS, ranging from those that are readily identifiable by post-
mortem imaging, such as intraventricular or periventricular haem-
orrhage and periventricular leukomalacia or porencephalic cyst
formation, through to subtle HI histologic changes of scattered

TABLE
13.2 Autopsy Approach to Cardiac Examination and Dissection
Feature Autopsy Approach
Right atrium Dissected from the inferior caval orifice across the base of
the atrial appendage
Right ventricle Dissected along the lateral border to the apex following
superior inspection of the tricuspid valve
Pulmonary arte- Dissection of the right ventricle along the ventricular
rial supply septal border, through the pulmonary valve and along
the arterial duct
Pulmonary Identification of root, course and insertion of the pulmo-
venous return nary venous vessels
Left atrium Dissected from the pulmonary venous drainage to the tip
of the atrial appendage
Left ventricle Incised along the lateral border to the apex following
inspection of the mitral valve from above
Aorta Incised using an incision from the apex, along the left
ventricular septal border and through the aortic root
after inspection of the course of the left main coronary
arteries
neurons, which obviously require formal neuropathological exam-
ination. In addition to detection, histologic features may aid in
the temporal sequence of HI events, which may be particularly
useful in potential medicolegal cases.  in detail after a brief period of fixation before return to the
Cardiovascular Anomalies
Cardiovascular abnormalities in fetuses and neonates are rela-
tively common, with an incidence of approximately 9 cases per
1000 live births. Associated morbidity varies from minimal to
life limiting depending on the type, complexity and presence of
associated abnormalities. Fetuses with complex or severe con-
genital heart disease may undergo multiple, complex, high-risk
operations in the perinatal and neonatal period; the practicing
pathologist should therefore have a low threshold for discussing
such cases with the clinical team and specialist cardiovascular
pathologists.
In fetuses, congenital heart disease continues to represent a
common indication for termination of pregnancy. Such anoma-
lies may be seen in isolation, as a result of a teratogen, as part of an
underlying specific genetic syndrome or may indicate a complex
genetic disorder. Counselling for future pregnancies can therefore
be difficult, with more than 30 genes linked to nonsyndromal
congenital heart defects.29 Even when no underlying genetic fac-
tors are identified, recurrence risk for CHD in future pregnancies
is greater than in the background population.30
Because congenital cardiac defects may be associated
with extracardiac anomalies and abnormalities of the major
CHAPTER 13  The Perinatal Postmortem Examination 127
Possible Major Anomalies
Morphological: Isomerism, atrioventricular discordance, atrial septal
defect. Vascular: aberrant systemic venous return, coronary sinus
anomaly. Valvular: Tricuspid atresia or dysplasia, premature foramen
ovale closure, atrial septal defect.
Morphological: Ventriculoarterial or atrioventricular discordance.
Hypoplasia or hypertrophy. Valvular atresia or stenosis. Myocardial:
Ventricular septal defect. Vascular: Transposition of the great vessels.
Pulmonary trunk anomalies.
Stenosis, hypoplasia. Premature closure of the arterial duct. May be
absent in truncus arteriosus.
Aberrancy of pulmonary venous drainage or connection. potentially
involving abrupt ‘J’ or ‘T’ anastomoses with other veins either below or
above the diaphragm.
Morphological: Isomerism, atrioventricular discordance, atrial septal
defect. Vascular: Aberrant pulmonary venous return, persistent left
superior vena cava. Valvular: Mitral atresia or dysplasia.
Morphological: Ventriculoarterial or atrioventricular discordance.
Hypoplasia or hypertrophy. Valvular atresia or stenosis. Myocardial:
Ventricular septal defect. Vascular: Transposition of the great vessels.
Aortic anomalies.
Morphological: Hypoplasia, coarctation, common arterial trunk. Vas-
cular: Aberrant origin or course of the coronary arteries. Valvular:
Bicuspid valve, stenosis or dysplasia.
vessels, detailed cardiac examination generally requires an open
approach in which the heart is dissected in situ. In some cases,
it may also be removed en bloc with the lungs and examined
body. Although the specific approach may require modification,
especially for evaluation of complex congenital cardiac disease
which has undergone surgical repair, in general, the heart is
dissected according to a standard schema known as sequential
segmental analysis, in which each cardiac component is exam-
ined in sequence. Recent evidence suggests that postmortem
cross-sectional imaging may provide adequate cardiac diagnos-
tic information in the majority of cases, and ex vivo specimen
imaging allows three-dimensional reconstruction and detailed
anatomical evaluation.
Table 13.2 provides the autopsy approach to assessment of the
heart and indicates some of the abnormalities that may be seen
(Fig. 13.6). 
Respiratory Anomalies
There are relatively few congenital anomalies affecting fetal
lungs, these being predominantly tumourous lesions such as
congenital pulmonary airway malformation and intrapulmonary
sequestration, both of which are readily identifiable on postmor-
tem imaging but may require histologic confirmation for specific
diagnosis. In contrast to many other systems, however, pulmo-
nary pathology is a major contributor to neonatal morbidity and
mortality, and in this setting, postmortem imaging has extremely
128 SE C T I O N 3     Fetal Physiology and Pathology

poor diagnostic accuracy, and tissue is almost always required for systems, each with variable genetic recurrence risks, making spe-
histologic diagnosis and ancillary investigations (Table 13.3).  cific diagnosis important. Traditionally, in addition to autopsy
to detect associated features, such as cystic renal disease, bone
Gastrointestinal Anomalies histology was a mainstay of diagnosis. Whilst for some specific
disorders, histology remains useful, in the majority of cases,
Congenital intestinal disorders such as bowel atresia, volvu- postmortem imaging can often both detect associated structural
lus and malrotation usually require open autopsy and detailed abnormalities and provide a likely diagnosis based on radio-
inspection to identify the affected segment and provide histo- graphic features. The molecular basis of such disorders is also
logic confirmation of the lesion. Similarly, in the neonatal setting increasingly understood, with specific genetic testing providing
with necrotising enterocolitis, direct inspection and sampling is a definitive diagnosis in many cases, and the combination of
required to determine the extent of involvement (Table 13.4).  postmortem imaging and genetic testing is likely to become the
standard approach. 
Genitourinary Anomalies
Multicystic or polycystic kidney diseases (Fig. 13.7) represent
categories of structural malformations which are relatively easy
to identify on antenatal sonography but with imaging appear-
ances which may represent a wide range of underlying con-
ditions, with differing implications and recurrence risks. The
diagnosis of renal cystic disease can be easily confirmed by
postmortem imaging, and with advances in this area, specific
diagnosis may be possible in the future. In the meantime, all
such cases require tissue sampling from the kidneys and liver
(for associated ductal plate malformation) for histologic exami-
nation and determination of the specific type of cystic disease.
Tissue should also be obtained for genetic testing. Finally, it
should be noted that complex urogenital and anorectal mal-
formations often exhibit unusual anatomy, and definitive diag-
nosis of such anatomical features often requires a standard
autopsy approach (Table 13.5). 

Musculoskeletal Anomalies
The main group of fetal musculoskeletal disorders for which
autopsy is indicated are the congenital skeletal dysplasias (Fig.
13.8), of which there numerous with several classification
• Fig. 13.6  Microfocus computed tomography rendering of a heart and
lung block with from an 18-week fetus with hypoplastic right heart syn-
drome; resolution approximately 29 μm. Virtual dissection facilitates dis-
cussion and documentation of findings in technically challenging cases.

TABLE
13.3 Autopsy Approach to Examination of the Respiratory System
Feature Autopsy Approach Possible Major Anomalies
Upper aerodi- Careful inspection of the hard and soft palate using a probe and torch, probing Choanal atresia, cleft lip, cleft palate
gestive tract of the nasal choanae.
Lung lobation Assessed in situ before the removal of the thoracic organ block. Isomerism. Pulmonary agenesis (usually unilateral)
Major airways After removal of the thoracic organ block, the larynx and oesophagus are Laryngeal atresia, clefting, stenosis or obstruc-
probed to assess patency before opening the oesophagus using a fine pair of tion. Laryngeal cysts. Laryngomalacia. Tracheal
scissors. The mucosa is then inspected for abnormalities, including tracheo- agenesis, stenosis or fistula. Bronchial atresia or
esophageal fistula. stenosis. Bronchial isomerism.
Small airways The cut surface of the lungs is assessed macroscopically. Histology is also Congenital pulmonary adenomatoid malformation
required for a complete assessment. (CPAM), congenital lobar emphysema, alveolar
capillary dysplasia with misalignment of the
pulmonary veins, pulmonary sequestration,
pulmonary hypoplasia, infection
Pulmonary Major connections assessed during cardiac examination (see previous section). See cardiac section.
vasculature
Diaphragm Inspected from above or below after removal of the thoracic or abdominal Congenital diaphragmatic hernia, eventration
organs, respectively.

  
 CHAPTER 13  The Perinatal Postmortem Examination 129

Postfetal Intervention
When specific in utero fetal interventions have been performed,
close correlation with the pathologist is indicated both for
clinical management and for governance and evaluation of
potential complications. Techniques in which specific clinical
questions and anatomical features are present include tracheal
occlusion for fetal diaphragmatic hernia, fetoscopic ablation of
fetal tumours and in utero surgery for open neural tube defects
or urinary tract obstruction. The specific postmortem investi-
gations and approaches in such cases should be individualised
according to the clinical circumstances. In addition, placental

TABLE Autopsy Approach to Examination of the TABLE Autopsy Approach to Examination of the
13.4 Gastrointestinal System 13.5 Genitourinary System
Possible Major Possible Major
Feature Autopsy Approach Anomalies Feature Autopsy Approach Anomalies
Tongue Visual inspection at Macroglossia Kidneys Inspected in situ after removal Renal cystic disease,
autopsy of the intestines, pancreas hydronephrosis,
Upper aerodi- See respiratory See respiratory section. and spleen. The ureters renal dysplasia,
gestive tract section. and vascular connec- ectopia, agenesis,
tions are inspected and fusion, hypoplasia
Oesophagus Probed after removal Atresia, fistula dissected. The kidneys are and supernumerary
of the thoracic then removed and bivalved kidneys
block before before histologic sampling.
opening with fine Ureters Inspected in situ; then dis- Hydroureter or dilation,
scissors. sected out along their ureterocele, ectopia,
Stomach May be opened Pyloric atresia, pyloric course duplication
along the stenosis (infants), Bladder Inspected in situ. If required, it Megacystis, exstrophy,
greater or lesser microgastria can be removed by dividing cloacal malforma-
curve. Contents the anterior pelvic fascia. tions, outflow
and mucosa obstruction (see
inspected. urethra)
Intestines Inspected in situ, Abnormal rotation, atresia Urethra May be removed along with Hypospadias, posterior
appendix located, of any segment, fistula, the bladder valves (may cause
removed from the herniation, enteric obstruction), atresia,
rectum towards duplication, enteric cysts, utricular cyst (may
the ileum and necrotising enterocolitis, cause obstruction),
inspected for meconium peritonitis, duplication
anomalies. cloacal malformations,
anorectal malformations. Repro- Inspected in the context of Gonadal dysgenesis,
Exomphalos and gas- ductive the gestational age and ovotestis, uterus
troschisis will be evident organs external genitalia. Sampled didelphis, uterus
externally. for histologic analysis. bicornis

     

• Fig. 13.7  Microfocus computed tomography image of autosomal reces- • Fig. 13.8  Microfocus computed tomography image of a femur from
sive polycystic kidney disease from the kidney of a 34-week-old fetus, a second trimester fetus with osteogenesis imperfect type 2b. There is
demonstrating radially aligned oblong cysts. Scale bar = 6.5 mm. severe deformity with a large fracture. Scale bar = 2.5 mm.
130 SE C T I O N 3     Fetal Physiology and Pathology
examination for the presence of residual anastomoses in twin-
to-twin transfusion syndrome after in utero laser ablation
may be required, for which a range of specialist techniques is
available.  Less invasive autopsy approaches may be more accepted by par-
Conclusion
Examination after death remains important in certain circum-
stances for guiding the management of future pregnancies
and as a means of medical audit and governance. Although
invasive autopsy techniques are in decline, advances are being
made in other approaches, specifically incorporating imag-
ing (e.g., ultrasound, cross-sectional imaging and micro-CT)
in combination with targeted tissue sampling. When there is
a clinical history of intrauterine fetal death, examination of
the placenta by a specialist pathologist is important because
placental abnormalities account for a significant proportion of
deaths in this context, with some findings directly affecting
future management.
ents and yield data which can be digitally stored, reanalysed and
shared with the clinical team to improve patient care and research
in this field. Fetal medicine specialists represent an important pro-
fessional group in communicating options to parents and obtain-
ing consent for investigation after death. Future developments
are likely to be based around novel investigations using molecular
techniques, such as genomics and proteomics, which may eluci-
date mechanisms of intrauterine fetal death and placental dys-
function that are currently unexplained.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References
1. Cannie M, Votino C, Moerman P, et  al.
Acceptance, reliability and confidence of
diagnosis of fetal and neonatal virtuopsy
compared with conventional autopsy: a pro-
spective study. Ultrasound Obstet Gynecol.
2012;39(6):659–665.
2. Heazell AE, McLaughlin MJ, Schmidt EB,
et  al. A difficult conversation? The views
and experiences of parents and profes-
sionals on the consent process for peri-
natal postmortem after stillbirth. BJOG.
2012;119(8):987–997.
3. Kang X, Cos T, Guizani M, et  al. Paren-
tal acceptance of minimally invasive fetal
and neonatal autopsy compared with
conventional autopsy. Prenat Diagn.
2014;34(11):1106–1110.
4. Mohammed M, Kharoshah MA. Autopsy in
Islam and current practice in Arab Muslim
countries. J Forensic Leg Med. 2014;23:80–
83.
5. Rutty J. Religious attitudes to death and
post-mortem examinations. In: Burton J,
Rutty G, Hodder Arnold, eds. The Hospital
Autopsy: A Manual of Fundamental Autopsy
Practice. Florida: CRC Press; 2010:39–58. po.
6. Horowitz RE, Naritoku WY. The autopsy
as a performance measure and teaching
tool. Hum Pathol. 2007;38(5):688–695.
7. Syngelaki A, Chelemen T, Dagklis T, et al.
Challenges in the diagnosis of fetal non-
chromosomal abnormalities at 11-13 weeks.
Prenat Diagn. 2011;31(1):90–102.
8. Hickey L, Murphy A, Devaney D, et  al.
The value of neonatal autopsy. Neonatology.
2012;101(1):68–73.
9. Hutchinson JC, Arthurs OJ, Sebire NJ.
Postmortem research: innovations and
future directions for the perinatal and paedi-
atric autopsy. Arch Dis Child Educ Pract Ed.
2016;101(1):54–56.
10. Manktelow B, et al. MBRRACE-UK Perina-
tal Mortality Surveillance Report UK Perina-
tal Death for Births from January to December
2013—Supplementary Report: UK Trusts
and Health Boards. University of Leicester:
Leicester: The Infant Mortality and Morbid-
ity Studies Group; 2015.
11. Ernst LM. A pathologist’s perspective on
the perinatal autopsy. Semin Perinatol.
2015;39(1):55–63.
12. Taylor GP, Faye-Petersen OM, Ernst L,
et  al. Small patients, complex challenging
cases: a reappraisal of the professional efforts
in perinatal autopsies. Arch Pathol Lab Med.
2014;138(7):865–868.
13. Rushton DI. Should perinatal post mortems
be carried out by specialist pathologists? Br J
Obstet Gynaecol. 1995;102(3):182–185.
14. Royal College of Obstetricians & Gynae-
cologists. Green–Top Guideline No. 55: Late
Intrauterine Fetal Death and Stillbirth. Royal
College of Obstetricians & Gynaecologists;
2010.
15.  The Royal College of Obstetricians and
Gynaecologists and The Royal College of
Pathologists. Fetal and Perinatal Pathology
Report of a Joint Working Party. 2001.
16. Judge-Kronis L, Hutchinson JC, Sebire NJ,
et  al. Consent for paediatric and perinatal
postmortem investigations: implications of
less invasive autopsy. J Forensic Radiol Imag-
ing. 2016;4:7–11.
17. Heazell AE, Martindale EA. Can post-
mortem examination of the placenta help
determine the cause of stillbirth? J Obstet
Gynaecol. 2009;29(3):225–228.
18. Hern W. Laminaria, induced fetal demise
and misoprostol in late abortion. Int J Gyn-
aecol Obstet. 2001;75:279–286.
19. Arthurs OJ, Calder AD, Kiho L, et  al.
Routine perinatal and paediatric post-
mortem radiography: detection rates and
implications for practice. Pediatr Radiol.
2014;44(3):252–257.
20. Thayyil S, Sebire NJ, Chitty LS, MARIAS
collaborative group, et al. Post-mortem MRI
versus conventional autopsy in fetuses and
children: a prospective validation study.
Lancet. 2013;382(9888):223–233.
21. Sebire NJ, Weber MA, Thayyil S, et  al.
Minimally invasive perinatal autopsies using
magnetic resonance imaging and endoscopic
postmortem examination (‘keyhole autopsy’):
feasibility and initial experience. J Matern
Fetal Neonatal Med. 2012;25(5):513–518.
22. Cox J, Lukande RL, Kalungi S, et al. Prac-
tice of percutaneous needle autopsy; a
descriptive study reporting experiences from
Uganda. BMC Clin Pathol. 2014;14:44.
23. Jawad N, ebire NJ, Wade A, et  al. Body
weight lower limits of fetal postmortem
MRI at 1.5 T. Ultrasound Obstet Gynecol.
2016;48(1):92–97.
24. Lombardi CM, Zambelli V, Botta G, et al.
Postmortem microcomputed tomography
(micro-CT) of small fetuses and hearts.
Ultrasound Obstet Gynecol. 2014;44(5):600–
609.
25. Hutchinson JC, Arthurs OJ, Ashworth
MT, et  al. Clinical utility of postmortem
microcomputed tomography of the fetal
heart: diagnostic imaging vs macroscopic
dissection. Ultrasound Obstet Gynecol.
2016;47(1):58–64.
26. Piercecchi-Marti MD, Liprandi A, Sigaudy
S, et al. Value of fetal autopsy after medical
termination of pregnancy. Forensic Sci Int.
2004;144(1):7–10.
27. Arthurs OJ, Thayyil S, Pauliah SS, Mag-
netic Resonance Imaging Autopsy Study
(MaRIAS) Collaborative Group, et  al.
Diagnostic accuracy and limitations of
post-mortem MRI for neurological abnor-
malities in fetuses and children. Clin Radiol.
2015;70(8):872–880.
28. Sebire NJ, Miller S, Jacques TS, et  al.
Post-mortem apparent resolution of fetal
ventriculomegaly: evidence from mag-
netic resonance imaging. Prenat Diagn.
2013;33(4):360–364.
29. Blue GM, Kirk EP, Sholler GF, et al. Con-
genital heart disease: current knowledge
about causes and inheritance. Med J Aust.
2012;197(3):155–159.
30. Calcagni G, Digilio MC, Sarkozy A, et  al.
Familial recurrence of congenital heart dis-
ease: an overview and review of the litera-
ture. Eur J Pediatr. 2007;166(2):111–116.
130.e1
14
Epidemiologic and Research Methods in
Fetal Medicine
CANDE V. ANANTH AND ALAN T. TITA
KEY POINTS
• T he randomised controlled trial (RCT) is the least biased
method of assessing the effectiveness of clinical interven-
tions. It has been little used in fetal medicine, but reports are
increasing.
• The details of good clinical trial methodology are now well
established. It is critically important to avoid selection bias by
ensuring allocation concealment at randomisation.
• Research synthesis allows the reader to review the totality of
relevant evidence on a particular topic. Systematic reviews
can be performed of RCTs (‘reviews of effectiveness’),
screening and diagnostic tests, or other types of scientific
literature. Meta-analysis may or may not be a component of a
systematic review.
• The Cochrane Database of Systematic Reviews is the largest
source of high-quality systematic reviews of health care
interventions.
• The likelihood ratio describes the usefulness of a screening or
diagnostic test.
• Routinely collected perinatal datasets can generate useful
information and important hypotheses (e.g., the ‘Barker
hypothesis’) as long as the quality of data is sound.
Introduction
Epidemiology–the science of the study of the distribution and
causes of diseases in populations–has produced a rich set of tools
that are being increasingly applied in clinical research. This grow-
ing specialty has led to birth of a subspecialty that is termed ‘clini-
cal epidemiology’. This chapter explores these tools and the
concepts that underpin them and illustrates their application with
reference to diagnostic and screening tests and therapeutic inter-
ventions in fetal and perinatal medicine. Fetal medicine is itself a
young speciality, and its short history and rapid progress have
inevitably resulted in some errors and blind alleys. The method-
ological concepts presented here hopefully will help obstetricians
caring for fetuses–‘maternal-fetal medicine’ specialists in North
America and ‘fetal medicine’ specialists in Europe–learn from past
mistakes and provide introduction to scientific research founda-
tions crucial to ensure that the application of fetal medicine con-
tributes more good than harm.
132
Care during pregnancy and childbirth has been among the
vanguard areas of clinical activity in moving towards ‘evidence-
based’ clinical practice. The basis of this process–the production of
systematic reviews of scientifically rigorous studies–has been lik-
ened in scale and importance to the Human Genome Project.1
This chapter is organised under two themes. We begin with a
description of epidemiologic methods necessary to understand
and make meaningful interpretations of research findings in fetal
medicine, and part two is devoted to principles and concepts in
general research methods. Empirical applications of methods
relating to fetal medicine are infused throughout to facilitate an
easier grasp of the concepts. 
Epidemiologic Study Designs
Epidemiologic study designs fall under two broad categories:
experimental design and observational designs. Randomised con-
trolled trials (RCTs) are experimental designs (discussed later).
Observational designs can be classified as analytical or descriptive.
The most common analytical study designs include prospective
(including the longitudinal design), retrospective (including the
case-control design) and cross-sectional study designs. Descriptive
studies include meta-analysis (both aggregate and individual
patient-level meta-analysis) and case series. Interested readers are
referred to the large body of literature on the topic of epidemio-
logical study designs.2
Randomised Controlled Trials
Not all interventions can be evaluated by randomised trials; one
cannot foresee, for example, randomised trials of fetal transfusion
for severe fetal anaemia or of immediate versus delayed delivery
for prolonged fetal bradycardia in labour. However, different
methods of fetal transfusion or of techniques of caesarean delivery
would be obvious candidates for further evaluation.
Randomisation. The RCT is a simple but powerful method of
avoiding systematic errors, or bias, by ensuring that experimental
(study) and control groups are comparable in all important
respects other than in their exposure to the intervention being
tested. By random allocation, the investigator accounts not only
for known confounding variables but also for factors that are
unknown but are also potentially important determinants of final
outcome. Random allocation depends on allocation solely on the
basis of chance–metaphorically, on the basis of the flip of a coin.
 CHAPTER 14  Epidemiologic and Research Methods in Fetal Medicine
The essence of secure randomisation requires that those
involved in the study cannot know in advance to which group a
particular woman will be allocated (i.e., concealment of alloca-
tion). Thus the use of hospital case numbers, alternate days or date
of birth will not adequately conceal the direction of allocation.
This prevents clinicians having preconceptions about the effective-
ness of the two treatment options from selectively enrolling
patients based on the next treatment assignment. These methods
of participant allocation to the study groups are sometimes called
‘quasi-random’ and with current concepts of good trial methodol-
ogy should not be used.3
Even apparently robust methods of random allocation, such as
the commonly used sealed opaque envelope to be opened only
after the woman has consented to entering the trial, have been
known to be abused on occasion. The gold-standard methods,
used now in large trials, include computerised online or web and
telephone randomisation in which someone based at a remote site
gives randomisation instructions only after basic descriptive data
about the woman and confirmation of eligibility have been
recorded. Electronic communication may be particularly difficult
in parts of the developing world, and randomised trials may be
particularly important in such settings because rates of both
maternal and fetal mortality are high. The Collaborative Eclamp-
sia Trial,4 which for the first time demonstrated the indisputable
preeminence of magnesium sulphate as the anticonvulsant of
choice for eclampsia, took place mainly in developing countries.
This trial used identical boxes containing magnesium sulphate,
diazepam or phenytoin, which were opened only when a woman
had an eclamptic seizure. Increasingly, web-based randomisation
procedures are used.  Data Monitoring
Explanatory Versus Pragmatic Trials. There are two types of
randomised trials; both are valid, and the appropriate trial design
depends on the underlying research question to be answered. The
explanatory trial assesses efficacy, or the performance of the inter-
vention under ideal circumstances; the pragmatic trial assesses
effectiveness, or performance under what may be less than
optimal, but real life, circumstances.
The Term Breech Trial5 was a pragmatic RCT that compared
the outcome of term fetuses presenting in labour in the breech
position after either a planned caesarean section or a planned vagi-
nal delivery. Clinicians were required to consider themselves
‘skilled’ at vaginal breech delivery, with confirmation by their head
of department. Although study sites were located worldwide, ran-
domisation was controlled in Toronto, Canada, with a computer-
ised system accessed by touch tone telephone. Because this was a
pragmatic trial evaluating the safety of breech deliveries in real-
world practice, 90% of women in the planned caesarean group
delivered by caesarean section, and 57% in the vaginal delivery
group delivered vaginally. The trial showed a considerable advan-
tage to babies in the planned caesarean group (perinatal mortality
or neonatal mortality or serious neonatal morbidity: 1.6% vs 5%;
relative risk 0.33; 95% confidence interval, 0.19–0.56). There
were no differences in maternal mortality or serious maternal mor-
bidity, nor were there differences in neurodevelopmental delay
among newborns followed until 2 years of age.6
The Term Breech Trial has had a major impact on clinical prac-
tice in many countries but has also generated considerable contro-
versy,7,8 mainly around the issue of generalisability of the findings
(external vs internal validity). The true results obtained in one
population (internal validity) may not necessarily be the same in
another population (external validity). Can the results of a large
trial performed in diverse settings with differing levels of facility
133
and expertise be applied to other institutions and practices? This is
a necessary consideration with any trial. Investigators of the Term
Breech Trial were well aware of this.9 
Sample Size Calculations
All statistical tests are subject to forms of two errors. A type I error,
denoted as α, occurs when the results of a trial suggest a difference
when, in fact, none exists. In contrast, a type II error, denoted as
β, occurs when the results do not suggest a difference, although
one does, in fact, exist. The principal protection against both types
of errors lies in planning, in advance, an adequate sample size
predicated on knowledge of the baseline incidence of the primary
outcome and a realistic judgment on what would prove to be a
clinically useful change secondary to the new treatment. Another
way to describe the importance of prespecified sample size calcula-
tions is to compare the clinical value of a study that has confirmed
a predicted reduction in perinatal mortality after an intervention
with a study that has observed a difference between two groups
when looking at the data in retrospect. Clearly, the former should
carry more weight.
An excellent online (free) resource to estimate sample size
for common study designs including randomised trials, cohort
studies, and case-control studies can be found at http://www.o
penepi.com/Menu/OE_Menu.htm. Although not ideal,
underpowered trials may still be useful, and the results can be
included in meta-analysis, as long as they are of sound
methodology.10 
It is also a principle of good clinical trial design and execution to
ensure that an independent panel of experts will have access to
interim results to advise whether or not a trial should continue. A
charter now exists to guide the workings of data monitoring com-
mittees.11 Advice may be given to abort a trial early if there is
overwhelming evidence that either the treatment group or the
control group is at significant advantage or disadvantage on the
basis of treatment. Thus in both the Term Breech Trial5 and the
Magpie Trial12 (magnesium sulphate vs placebo for preeclampsia
prevention), recruitment was stopped earlier than planned on the
recommendation of the Data Monitoring Committees because of
large, clinically and statistically significant differences in the pri-
mary outcomes between the randomised groups.
Data monitoring committees may also have to decide if further
recruitment to a trial can be ethically justified in a pursuit of pre-
specified sample size, in which the tested intervention is clearly
ineffective. Such a trial may be abandoned on grounds of futility.
It is bad practice for the researchers themselves to continually
monitor the results because of the possibility of stopping a trial
after a ‘statistically significant’ result is obtained because this is
likely to produce a type I error. 
Evaluation of Screening and Diagnostic Tests
Before delving into an understanding of the evaluation of tests, it
is imperative to highlight a clear distinction between screening
and diagnostic tests (terms that are confusing and often used
interchangeably in practice; see Chapter 16). Screening tests are
those that are applicable to large populations to help screen and
identify clinically unsuspected disease. In contrast, a diagnostic
test is one that is usually more complex, expensive and precise and
134 SE C T I O N 4   Epidemiology

TABLE Layout of a 2 × 2 Table Illustrating the Cross- TABLE

14.1a Classification of the Disease State Based on the 14.1b Characteristics for Evaluating a Screening Test
Results of a Screening Test Mathematical Alternate
DISEASE STATE Test Characteristic Formulation Formulation
Test Result Present Absent Total Sensitivity (Se) a TP
Se = TPR =
Test a b a+b a+c TP + FN
positive True positive False positive Total test positive
(TP) (FP) Specificity (Sp) d TN
Sp = TNR =
b+d TN + FP
Test c d c+d
negative False negative True negative Total test negative Positive predictive a TP
PPV = PPV =
(FN) (TN) value (PPV) a+b TP + FP
Total a+c b+d a+b+c+d=N
Negative predictive d TN
Total diseased Total disease Total subjects NPV = NPV =
value (NPV) c+d TN + FN
free

   Likelihood ratio, a Se
a+c LR + =
positive test (LR+) LR + = 1 ‐ Sp
1‐ d
b+d
is applied to make a specific diagnosis of a disease, particularly in a
Likelihood ratio, 1‐ 1 ‐ Se
high-risk populations. a+c LR ‐ =
negative test (LR–) LR ‐ = Sp
The advent of a variety of screening and diagnostic tests in d
obstetrics and fetal medicine has led to substantial and impres- b+d
sive reductions in fetal and maternal conditions. Importantly, Odds ratio (OR; Se LR +
antepartum and intrapartum fetal surveillance, including ultra- diagnostic test) 1 ‐ Sp OR =
OR =
1 ‐ Se LR ‐
sound and Doppler velocimetry, nonstress test, contraction
stress test and fetal biophysical profile, have not only enabled the Sp
early identification of ‘at-risk’ fetuses but have paved ways to
FN, False negative; FP, false positive; TN, true negative; TNR, true negative rate; TP, true
reduce the burden of fetal deaths. The primary purpose of fetal positive; TPR, true positive rate.
surveillance is to identify fetuses with impending asphyxia early   
in the course of disease to time their delivery to prevent fetal or
neonatal demise or long-term damage. A secondary purpose is to
avoid neonatal complications arising from asphyxia-related
causes. Predictive values of a test are influenced by the prevalence of
An ideal test would predict the occurrence or absence of a con- the condition being studied in the population. Thus the positive
dition with perfect accuracy.13 Unfortunately, no such diagnostic predictive value of an elevated maternal serum α-fetoprotein result
test exists for fetal surveillance. The optimal screening test maxi- to identify a fetus with a neural tube defect, for example, will be
mises prediction of ‘sick’ fetuses with the lowest occurrence of greater in a Celtic population with a higher a priori incidence of
false-positive findings. We highlight a set of epidemiologic meth- neural tube defects. For populations with similar background
ods to evaluate the effectiveness of a screening test. Consider a 2 × risks, the more sensitive a test is, the better is the negative predic-
2 table (Table 14.1a). The two columns denote the true disease tive value; the more specific a test is, the better is the positive
states, and the two rows denote the results of the test. Cross-clas- predictive value.
sification of the true disease state with the result of the test yields The LR is another measure of the value of a screening test that
four subpopulations: (i) diseased fetuses with positive test results, seems to be more intuitive for clinicians. It combines the sensitiv-
represented as ‘cell a’; (ii) nondiseased fetuses with positive test ity and specificity of a test and expresses it as a ratio representing
results, represented as ‘cell b’; (iii) diseased fetuses with negative the increase or decrease in the probability a fetus has the disease
test results, represented as ‘cell c’; and (iv) nondiseased fetuses with based on the test result. In other words, how much more likely it
negative test results, represented as ‘cell d’. is that the disease is present when the test result is positive and
The most useful characteristics for evaluating a screening how much less likely it is after a negative test result. If a test has a
test include sensitivity, specificity, positive and negative pre- LR of 1, the pretest probability of the disease has not changed
dictive values, and likelihood ratios (LRs) for a positive and despite the test result, and hence the test is of little value. There is
negative test result. These characteristics are summarised in no magic LR cut-off above or below which a test should be con-
Table 14.1b. Sensitivity specifies the probability that a test will sidered clinically useful. Sometimes a simple test (e.g., a question)
classify subjects as being diseased when, in fact, they are dis- with a relatively low LR may be clinically more appropriate than
eased, and specificity is the probability that a test will classify an invasive and expensive diagnostic test with a higher LR.
subjects as being nondiseased when, in fact, they are nondis- The relationship between sensitivity and specificity can be dis-
eased. An optimal test is one that will maximise both sensitiv- played figuratively in receiver operator curves (ROCs), the term
ity and specificity (cells ‘a’ and ‘d’ denote the disease states that being derived from radar technology during the Second World
are classified correctly by the test). The power of a test (1-β) to War. ROCs are useful in identifying the optimal tradeoff between
detect a difference in the probability of detection is the sensi- sensitivity and specificity for a given test so as to pinpoint the best
tivity of the test. cutoff to differentiate ‘normal’ from ‘abnormal’ test results in a
 CHAPTER 14  Epidemiologic and Research Methods in Fetal Medicine 135

l. Derivation ll. External validation lll. Impact analysis

Original population Related populations Related population

Temporal Geographical Fully Implementation samples

Derivation sample
samples samples independent
samples

Model Internal
Adjustment or reestimation Cluster randomised trial
building validation

Apparent
performance Overfitting Generalisability Effectiveness

Original model Updated model Implemented model

• Fig. 14.1  Research phases for the establishment of a clinical prediction model. I, Derivation and internal
validation; II, external validation; and III, impact analysis. (Reproduced with permission from Labarere J,
Renaud B, Fine MJ. How to derive and validate clinical prediction models for use in intensive care medi-
cine. Intensive Care Med 40(4):513–527, 2014.)

screening program. It should be noted that in screening tests, ‘nor-
mal’ results do not represent the absence of disease; rather, they
separate high-risk results in which further testing or intervention
is recommended from low-risk groups in which the likelihood of
disease is low. This forms the underlying basis for the development
of a diagnostic test.
Criteria for the Evaluation of a Screening Test
There are four essential, but often overlooked, criteria for a good
screening test. These include the following:
1. An objective diagnostic test or intervention exists (‘gold stan-
dard’) and is reproducible.
2. The prevalence of the disease (outcome) in the general popula-
tion must be known.
3. Management will be modified based on results of the screening
test.
4. The screening test should be safe, and the secondary or adverse
effects of additional testing or intervention should fall within
acceptable standards.  Analysing Databases and Survey Data
Prediction Models in Fetal Medicine
Prediction models are statistical models that are developed to pre-
dict outcomes. An implicit goal of such models is to come up with
reliable predictions. Prediction models that are developed to pre-
dict the probability of future occurrence of a disease or ones devel-
oped to predict existing disease are termed ‘prognostic’ or
‘diagnostic’ prediction models, respectively. Prediction models
have an important role in fetal medicine.14,15 Before attempting to
develop prediction models, it is important to first understand
their purpose (i.e., diagnostic or prognostic purposes) and the
underlying study designs for each approach. Whereas diagnostic
prediction models are usually developed from cross-sectional data,
prognostic models are derived from prospectively ascertained
data.16
Methods to derive such prediction models involve a series of
steps. The overall process for establishing prediction models is illus-
trated in Fig. 14.1 and involves three phases: model derivation,
external validation of the derived model and an impact analysis.16
Each of these three phases has unique and distinct requirements.
For instance, the model derivation state requires splitting the data
in two parts: model building (or ‘testing’) and model validation (or
‘internal validation’), with the derived model being refined and
updated in the second phase. The third phase–an often-neglected
step in the pursuit of the development of prediction models–is the
‘impact analysis’ phase. This phase involves testing the robustness
of the model in real-life scenarios to ascertain its performance,
often through the implementation of randomised controlled trial.
Some of the best available resources to understand prediction and
prognostic models can be found elsewhere.17-21 As an example of
an application of prediction models in fetal medicine, a middle
cerebral artery flow velocity above 1.5 multiples of the median has
been shown to be predictive of fetal anaemia (see Chapter 40).
Previously, perinatal epidemiologists concentrated on analysing
large existing datasets either collected routinely or assembled for a
specific research project. Although important insights have been
derived from such studies, misleading evidence also emerged from
the ‘data dredging’ or ‘fishing expeditions’ implemented on such
resources. Contemporary perinatal epidemiologists are more likely
to address hypothesis-driven questions, ideally through imple-
menting RCTs or by studying the results of a number of different
randomised trials focusing on the same question(s), through sys-
tematic reviews or meta-analyses, to reach the most informative
and least biased conclusion. When existing databases and surveys
are used, focused questions, prespecified outcomes and attention
to sources of error, including selection bias, information bias, con-
founding and inadequate power, are crucial. The types of studies
derived from databases, and surveys are typically retrospective
136 SE C T I O N 4   Epidemiology
cohort, case-control, cross sectional and descriptive observational
studies. The STROBE (STrengthening the Reporting of OBserva-
tional studies in Epidemiology) statement provides internationally
agreed upon guidelines for strengthening the reporting of these
studies (http://www.strobe-statement.org).
Studies of routinely collected administrative and clinical out-
comes data of maternal deaths and perinatal deaths have been use-
ful in tracking the frequency of these events, elucidating risk
factors and designing relevant interventions to address them.
Datasets such as the US National Vital Statistics Databases and
hospital perinatal databases continue to prove useful in a number
of ways (e.g., generating hypotheses, some of which are best tack-
led definitively by an RCT), identifying patterns of disease distri-
bution in populations and identifying rare but serious problems.
Thus US National Vital Statistics data have been instrumental in
quantifying the frequency, trends and risk factors of important
perinatal outcomes, including preterm births, caesarean delivery
and maternal and perinatal deaths. The ‘Barker hypothesis’ linking
undernutrition at critical stages of fetal life with chronic adult dis-
eases, including coronary artery disease,22 was generated by the
study of routinely collected data on birth weights, placental
weights and neonatal measurements and linking these findings
with health and disease in later adult life. Routinely collected
maternity records in Iceland have proven valuable for population
studies of inheritance of genetic predisposition to preeclamp-
sia.23,24 Records in Scotland have provided important insights
into twin births,25 recurrent pregnancy complications26 and fertil-
ity after a caesarean delivery in the previous pregnancy.27  understand the variance and source of variance in the associa-
Systematic and Other Reviews
The value of reviews to inform busy clinicians and the develop-
ment of clinical guidelines is increasingly recognised to address
the huge amount of primary medical literature and the informa-
tion available in specialised areas of clinical or scientific interest
that practitioners have to keep up with. These reviews include
conventional narrative or expert reviews and the more rigorous
systematic reviews (with or without meta-analyses). Because con-
ventional or expert reviews often are subject to less rigorous cri-
teria, different experts may reach entirely different conclusions
after reviewing the same topic; readers often are not informed
about how the reviewer chose to select certain studies and ignore
others. A review may be out of date at the time of publication if
the topic is progressing rapidly. Frequently used expert reviews
include guidelines from professional organisations such as the
American Congress of Obstetricians and Gynecologists (ACOG)
and the Royal College of Obstetricians and Gynaecologists
(RCOG) and electronic resources such as UpToDate. The Grad-
ing Recommendations Assessment, Development and Evalua-
tion (GRADE) criteria are available to help ensure that
recommendations from such reviews reflect the quality of the
evidence (http://www.gradeworkinggroup.org).
Systematic reviews such as the Cochrane Reviews, by contrast,
are based on an explicit and rigorous process that includes:
• A clear description of the objectives
• Explicit criteria for including studies
• An attempt to identify all relevant studies, whether published
or not
• Explicit description of why apparently relevant studies have
not been considered
• Extraction of data
• Pooling of data from similar studies (meta-analysis)
• D escription of results
• Drawing appropriate conclusions and discussing implications
for clinical practice and future research
The ‘Preferred Reporting Items for Systematic Reviews and
Meta-analyses’ (PRISMA) resource provides guidelines for trans-
parent reporting (http://prisma-statement.org). 
Meta-analysis
Meta-analysis is a technique applicable to systematic reviews
to address important research questions about rare but impor-
tant outcomes (e.g., fetal death). Such questions could be
addressed by very large studies or alternatively by (i) pooling
data from a number of different studies of similar structure
and purpose (i.e., meta-analysis) and (ii) reanalysing individual
patient-level data across multiple studies (i.e., individual,
patient-level meta-analysis [IPD]). The IPD meta-analysis is
one when the analyst gathers individual patient-level data on
the exposure(s) and the outcome(s) as well as data on related
confounders and stratification variables from every individual
study considered for inclusion in the pooling of data. These
data are then analysed as a single study, paying careful atten-
tion to heterogeneity. The IPD analysis provides a more
nuanced approach to address biases due to confounding, effect
measure modification (interaction) and vastly improved statis-
tical power than a regular meta-analysis. Perhaps the overarch-
ing benefit of an IPD analysis is the ability to characterise and
tion being reported.
There are now several examples of both approaches to meta-
analyses providing clear guidance about the value of interventions
during pregnancy to optimise fetal or maternal outcome, (e.g.,
corticosteroid treatment before likely preterm delivery).28 There
are ongoing debates about whether large single trials are preferable
to the meta-analysis of results from several smaller trials. Results
from both approaches tend to agree most of the time, and plausi-
ble explanations can be identified for any clinically important dif-
ferences between them.29
Some of the important considerations in reporting results from
a meta-analysis include the following:
• Develop a protocol before implementing a meta-analysis. It is of
paramount importance to first develop a clean protocol that
describes the purpose of meta-analysis as detailed as possible.
This includes providing the hypothesis, specific aims, back-
ground review of the literature, defining the primary and sec-
ondary end points, study selection and review criteria, plans for
statistical analysis and dissemination of results. A recent initia-
tive to strengthen the reporting of a meta-analysis is the
requirement of registration of such planned analysis through
PROSPERO (https://www.crd.york.ac.uk/PROSPERO).
• Predetermine the eligibility criteria. Accurate identification of
appropriate studies, ensuring that studies are not ‘missed’ from
inclusion (a thorough literature search preferably undertaken
by two independent authors and their findings corroborated).
• Standardised data collection. Establish a standardised data col-
lection protocol that can be used to abstract data from every
eligible study.
• Evaluate bias. One of the initiatives to ensure a successful
meta-analysis is to grade every eligible study in a systematic
framework regarding potential biases. These biases include
selection bias, attrition bias, performance bias, detection
bias), as well as an assessment of issues related to the
 CHAPTER 14  Epidemiologic and Research Methods in Fetal Medicine
randomisation process (blinding, loss to follow-up or attri-
tion). Considered together, these biases provide qualitative
evidence regarding the extent to which the meta-analysis pro-
vides internal validity (minimisation of bias) and external
validity (generalisability of findings).
• Calculate pooled summary effects. Before data are pooled across
studies, it is important to ascertain the presence of (statistical)
heterogeneity in studies. This heterogeneity is denoted through
an I2 statistic (expressed as a percent), with a high I2 value
(generally ≥50%). Through statistical analysis, data can be
pooled to estimate a single summary measure. This pooled esti-
mate can be estimated from fitting either a fixed-effects or a
random-effects models. Whereas the fixed-effects approach is
valid when the pooled data do not show statistical heterogene-
ity, the random-effects analysis has better statistical properties
for pooling the data in the presence of statistical heterogeneity.
• Plotting the data. The forest plot and funnel plot are two impor-
tant graphical tools that remain important for communicating
the results of a meta-analysis. The forest plot is effectively a plot
of the summary measure (e.g., risk ratio or a risk difference) for
every study included in the meta-analysis, as well as the pooled
summary effect measure. Other attributes of the forest plot
include the statistical weight assigned for each study in the
meta-analysis, and a statistical analysis of heterogeneity. The
funnel plot shows the distribution of the effect measure (on the
x-axis) against the standard error of the effect measure of each
study (on the y-axis). This method is used to evaluate the pres-
ence of publication bias in the meta-analysis.  high-quality service. Essential steps in the practice of evidence-
The Cochrane Collaboration
The Cochrane Collaboration is an international network of indi-
viduals and institutions committed to producing up-to-date sys-
tematic reviews of the effectiveness of health care measures.30
Cochrane reviews tend to be of better quality than other system-
atic reviews.31 The Collaboration is based on the principles of
genuine collaboration, equity and inclusiveness, and it consists of
four dimensions–review groups, centres, fields, and methodology
and software development groups. The centres are scattered
around the world and provide support for review groups based
within their geographical area of responsibility; each centre also
has responsibility for some strategic activity for the collaboration
(e.g., trial registration, training of reviewers, software production).
The ‘fields’ deal with large generic issues that transcend the inter-
ests of any one review group (e.g., children, older adults, people
living in developing countries). The review groups produce the
systematic reviews, which have expanded greatly from their origin
in perinatal medicine (pregnancy and childbirth).32 There are
now, for example, productive review groups in the fields of stroke,
infectious diseases, schizophrenia and menstrual disorders and
subfertility.
137
Cochrane systematic reviews have focused primarily on RCTs
because of the scientific strength of this method of assessing clini-
cal interventions and because of the methodological difficulties of
dealing with other types of scientific data (e.g., qualitative research
data). Systematic reviews of screening and diagnostic tests and
reviews of non-RCTs of relevance to perinatal medicine specialists
have been published by others and include the prognosis for twins
after intrauterine death of a co-twin33 and the value of fetal urine
analysis in urinary tract obstruction.34
The main product of the Cochrane Collaboration is the
Cochrane Database of Systematic Reviews, which is published in
electronic form at 3-monthly intervals within the Cochrane
Library, together with databases of clinical trials, methodological
papers and abstracts of other systematic reviews. Published preg-
nancy and childbirth reviews have been all aggregate reviews based
on published reports. IPD meta-analyses are increasingly being
undertaken and are much more resource intensive but allow much
more sophisticated exploration of subgroup analyses. The first
perinatal IPD review focused on low-dose aspirin.35 
Evidence-Based Medicine
Evidence-based medicine is the conscientious, explicit and judi-
cious use of the current best evidence in making decisions about
patients. The limited resources of all health care systems increases
the pressure to deliver care according to clinical effectiveness. This
should also be the goal for all clinicians who want to provide a
based medicine include asking appropriate questions; determining
what information is required to answer the question, conducting
a literary search, selecting best studies, critically assessing the evi-
dence for validity and extracting the message and applying it to
clinical problems.
As increasingly more interventions are evaluated using good
clinical trials, more topics will be reviewed systematically to iden-
tify treatments that are effective and which should be used widely;
ineffective or harmful interventions should be abandoned, and
those of uncertain effectiveness should be considered for future
research. 
Conclusion
In summary, epidemiology has produced a rich set of tools that are
being increasingly applied in clinical research. Concepts that
underpin study designs and research methods remain critical to
interpretation of research studies in fetal medicine. Any successful
research rests on carefully designed study, proper implementation,
accurate data analysis and sound interpretation.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 13. Ananth CV, Smulian JC, Vintzileos AM. Epi-
demiology of antepartum fetal testing. Curr
24. Arngrimsson R, Sigurard S, Frigge ML, et al. A
genome-wide scan reveals a maternal suscepti-
Opin Obstet Gynecol. 1997;9(2):101–106. bility locus for pre-eclampsia on chromosome
1. Naylor CD. Grey zones of clinical practice:
14. Chavez MR, Ananth CV, Smulian JC, Vintz- 2p13. Hum Mol Genet. 1999;8(9):1799–1805.
some limits to evidence-based medicine. Lan-
ileos AM. Fetal transcerebellar diameter mea- 25. Smith GC, Shah I, White IR, et  al. Mode
cet. 1995;345(8953):840–842.
surement for prediction of gestational age at of delivery and the risk of delivery-related
2. Rothman KJ, Lash TL, Greenland S: Modern
the extremes of fetal growth. J Ultrasound Med. perinatal death among twins at term: a retro-
Epidemiology, 3rd ed, New York, NY: Lippin-
2007;26(9):1167–1171. quiz 1173–1164. spective cohort study of 8073 births. BJOG.
cott Williams & Wilkins; 2008.
15. Smulian JC, Ananth CV, Vintzileos AM, 2005;112(8):1139–1144.
3. Schulz KF, Grimes DA. Allocation conceal-
Guzman ER. Revisiting sonographic abdomi- 26. Smith GC, Shah I, White IR, et  al. Previous
ment in randomised trials: defending against
nal circumference measurements: a comparison preeclampsia, preterm delivery, and delivery of
deciphering. Lancet. 2002;359(9306):614–
of outer centiles with established nomograms. a small for gestational age infant and the risk of
618.
Ultrasound Obstet Gynecol. 2001;18(3): unexplained stillbirth in the second pregnancy:
4. Which anticonvulsant for women with eclamp-
237–243. a retrospective cohort study, Scotland, 1992-
sia? Evidence from the Collaborative Eclampsia
16. Labarere J, Renaud B, Fine MJ. How to derive 2001. Am J Epidemiol. 2007;165(2):194–202.
Trial. Lancet. 1995;345(8963):1463.
and validate clinical prediction models for use 27. Smith GC, Wood AM, Pell JP, Dobbie R. First
5. Hannah ME, Hannah WJ, Hewson SA, et al.
in intensive care medicine. Intensive Care Med. cesarean birth and subsequent fertility. Ferti
Planned caesarean section versus planned
2014;40(4):513–527. Steril. 2006;85(1):90–95.
vaginal birth for breech presentation at
17. Harrell Jr FE, Lee KL, Califf RM, et al. Regres- 28. Roberts D, Dalziel S. Antenatal corticoste-
term: a randomised multicentre trial. Term
sion modelling strategies for improved prognos- roids for accelerating fetal lung maturation
Breech Trial Collaborative Group. Lancet.
tic prediction. Stat Med. 1984;3(2):143–152. for women at risk of preterm birth. Cochrane
2000;356(9239):1375–1383.
18. Harrell Jr FE, Lee KL, Mark DB. Multivari- Database Syst Rev (3). 2006:CD004454.
6. Hannah ME, Whyte H, Hannah WJ, et  al.
able prognostic models: issues in developing 29. Cappelleri JC, Ioannidis JP, Schmid CH,
Maternal outcomes at 2 years after planned
models, evaluating assumptions and adequacy, et  al. Large trials vs meta-analysis of smaller
cesarean section versus planned vaginal birth
and measuring and reducing errors. Stat Med. trials: how do their results compare? JAMA.
for breech presentation at term: the interna-
1996;15(4):361–387. 1996;276(16):1332–1338.
tional randomized Term Breech Trial. Am J
19. Harrell Jr FE, Lee KL, Matchar DB, 30. Chalmers I, Dickersin K, Chalmers TC. Get-
Obstet Gynecol. 2004;191(3):917–927.
Reichert TA. Regression models for prog- ting to grips with Archie Cochrane’s agenda.
7. Glezerman M. Five years to the term
nostic prediction: advantages, problems, BMJ. 1992;305(6857):786–788.
breech trial: the rise and fall of a random-
and suggested solutions. Cancer Treat Rep. 31. Sheikh L, Johnston S, Thangaratinam S, et al.
ized controlled trial. Am J Obstet Gynecol.
1985;69(10):1071–1077. A review of the methodological features of sys-
2006;194(1):20–25.
20. Harrell Jr FE, Lee KL, Pollock BG. Regression tematic reviews in maternal medicine. BMC
8. Kotaska A. Inappropriate use of randomised
models in clinical studies: determining rela- Med. 2007;5:10.
trials to evaluate complex phenomena:
tionships between predictors and response. J 32. Dodd JM, Crowther CA. Cochrane reviews
case study of vaginal breech delivery. BMJ.
Natl Cancer Inst. 1988;80(15):1198–1202. in pregnancy: the role of perinatal random-
2004;329(7473):1039–1042.
21. Harrell Jr FE, Margolis PA, Gove S, et  al. ized trials and systematic reviews in estab-
9. Hofmeyr J, Hannah M. Five years to the
Development of a clinical prediction model for lishing evidence. Semin Fetal Neonatal Med.
Term Breech Trial: the rise and fall of a ran-
an ordinal outcome: the World Health Orga- 2006;11(2):97–103.
domized controlled trial. Am J Obstet Gynecol.
nization Multicentre Study of Clinical Signs 33. Ong SS, Zamora J, Khan KS, Kilby MD.
2006;195(6):e22; author reply e23.
and Etiological agents of Pneumonia, Sepsis Prognosis for the co-twin following single-
10. Schulz KF, Grimes DA. Sample size calcula-
and Meningitis in Young Infants. WHO/ARI twin death: a systematic review. BJOG.
tions in randomised trials: mandatory and
Young Infant Multicentre Study Group. Stat 2006;113(9):992–998.
mystical. Lancet. 2005;365(9467):1348–1353.
Med. 1998;17(8):909–944. 34. Morris RK, Quinlan-Jones E, Kilby MD, Khan
11. Damocles Study Group NHSHTAP. A pro-
22. Barker DJ, Osmond C, Forsen TJ, et al. Mater- KS. Systematic review of accuracy of fetal urine
posed charter for clinical trial data monitoring
nal and social origins of hypertension. Hyper- analysis to predict poor postnatal renal function
committees: helping them to do their job well.
tension. 2007;50(3):565–571. in cases of congenital urinary tract obstruction.
Lancet. 2005;365(9460):711–722.
23. Arngrimsson R, Bjornsson S, Geirsson RT, Prenat Diagn. 2007;27(10):900–911.
12. Altman D, Carroli G, Duley L, et  al. Do
et  al. Genetic and familial predisposition to 35. Askie LM, Duley L, Henderson-Smart DJ,
women with pre-eclampsia, and their babies,
eclampsia and pre-eclampsia in a defined popu- et al. Antiplatelet agents for prevention of pre-
benefit from magnesium sulphate? The Magpie
lation. Br J Obstet Gynaecol. 1990;97(9):762– eclampsia: a meta-analysis of individual patient
Trial: a randomised placebo-controlled trial.
769. data. Lancet. 2007;369(9575):1791–1798.
Lancet. 2002;359(9321):1877–1890.

137.e1
15
Ethical Issues in Maternal-Fetal
Medicine
WYBO J. DONDORP AND GUIDO M. de WERT
KEY POINTS
• A fetus can be treated but is not a patient in the normative
sense of the term.
• Because a fetus can only be treated via the body of a preg-
nant woman, fetal treatment always makes her a patient and
requires her informed consent.
• Regardless of whether a fetus has a high or low moral status,
the interests of the future child are relevant for decision mak-
ing about fetal treatment.
• Without clear benefits compared with nontreatment or
postnatal treatment, there can be no justification for fetal
treatment.
• Avoiding therapeutic misconception is an important chal-
lenge for the ethical conduct of fetal treatment trials.
• Fetal treatment should not be presented as a morally pre-
ferred alternative to a termination of pregnancy.
Introduction
This chapter discusses ethical issues in maternal-fetal medicine,
focusing on questions arising with the development of options for
fetal treatment. This is a very broad field ranging from open surgery
to pharmacotherapy, from experimental procedures to accepted
treatment and from interventions aimed at saving fetuses from in
utero or perinatal death to treatments with a rationale of improv-
ing long-term health outcomes. Although specific ethical issues
arise with each distinct form of fetal treatment, this chapter will
inevitably remain on a more general level, referring to treatments
for concrete disorders only by way of illustration. What makes fetal
treatment challenging from an ethical point of view is that a fetus
can only be treated via the body of a pregnant woman. Fetal treat-
ment is therefore always maternal-fetal treatment, which means that
the relevant procedures require her informed consent. However, as
elsewhere in medicine, consent is not enough to render a treat-
ment offer ethical. Clinicians offering treatment have a professional
responsibility not to expose their patients to disproportionally high
risks. What this means is often difficult to determine but even more
so in this field in which pregnant women are offered potentially
risky treatment to benefit not themselves (at least not directly) but
the fetus or the child that the fetus may grow into.
In this chapter, we will first discuss the fundamental issue
whether or not in addition to the pregnant woman in the care of
a medical doctor, the fetus (or fetuses) she is carrying should be
regarded as a patient (or patients). Next, we will address the dif-
ferent ethical challenges arising with the two distinct aims of fetal
treatment for lethal and nonlethal conditions: saving the life of the
fetus (or the neonate) versus improving the quality of life of the
future child. Finally, we will explore the implications for prenatal
decision making of the availability of experimental and established
fetal treatment options.
Two further preliminary remarks need to be made. First,
although interventions in a fetus always also involve treatment of
a pregnant woman and although we are aware that language is
important, we will for convenience sake use the term ‘fetal treat-
ment’ in this chapter. Second, when discussing the challenges of
counselling and decision making about fetal treatment, we do
as if this only concerns the clinician and the woman. Of course,
we are aware that these decisions are often shared by pregnant
women with their partners and that these (mostly the biologi-
cal father-to-be) do have an interest in these choices as well. And
clearly, with her authorisation, it is only appropriate for profes-
sionals to address and share information with the prospective par-
ents as a couple. However, precisely because the fetus can only be
approached through the body of the pregnant woman and because
any treatment decisions will make her a patient, it is only on the
basis of her voluntary and autonomous consent (or dissent) that
such decisions should be taken.1 
The Fetus as a Patient?
The term ‘patient’ as used to designate the fetus need not imply
more than that, as a matter of fact, health problems in a fetus can
increasingly be treated. But ‘patient’ is also a social role-related
concept with normative implications. Being a patient means being
in a relationship with a doctor that entails a claim to medical con-
sideration. Whether the fetus should be regarded as a patient in
this sense is far from obvious. What should be avoided is that mix-
ing up these two uses of the term suggests that because fetuses can
be referred to as patients in the former sense, they are also patients
in the latter, thus preempting questions about responsibilities and
obligations that clinicians may have toward the fetus and about
how these relate to those owed to the pregnant woman. From the
mere fact that fetuses can be treated, nothing as yet follows about
whether they should and if so, on what conditions.
139
140 SE C T I O N 5    Ethics
The Debate About Moral Status
On what basis can we establish whether professionals (especially
obstetricians and maternal-fetal medicine specialists) do have
responsibilities and obligations toward a fetus independently of
what they owe to the pregnant woman? It would seem that this
requires a prior answer as to what the fetus is, in moral terms.2
This leads, however, into a conundrum of ethical debate. What
meaning should be given to both the continuity (fetuses are begin-
ning forms of human life) and the discontinuity (they still lack
most of the defining characteristics of human beings)?
Very different answers to this question have been given. For
instance, according to the Roman Catholic Church, the conti-
nuity is the morally decisive factor: being destined to become
fully developed human beings (or persons) is what gives human
embryos and fetuses the same high moral status as should be
accorded to all human beings.3 As ‘potential persons’ (in this
strong sense of the term), they deserve full protection as from con-
ception. Although basically following the same reasoning, Judaism
and Islam differ from Roman Catholic teachings in taking the
moment of ‘ensoulment’ (following Aristotle, this is often set at
40 days for male fetuses) as the starting point of a human life with
full moral status.4,5
By contrast, secular philosophers and ethicists have tended
to stress the discontinuity. What makes human beings especially
worthy of respect and protection is that we are persons in the sense
of beings with capacities for the ‘complex forms of consciousness’6
that allow us to be ‘self-interpreting animals’.7 Clearly, this does
not hold for embryos or fetuses. Although late-gestation (sentient)
fetuses may well have interests, for instance, in being protected
from pain and perhaps even a (weak) interest in continued exis-
tence, they are not persons and therefore do not commend the
same level of respect and protection.8 Still, according to many
authors, the capacity to develop into a human person is morally
relevant and gives embryos and fetuses a certain moral standing,
although lower than that of persons. This is often thought of as
increasing with fetal development, referring to the development of
the necessary conditions for later personhood (e.g., brain develop-
ment capable of sustaining consciousness).
A challenge for this reasoning is to explain why birth should
make such a difference. Clearly, looking at how personhood is
defined, not only fetuses but also infants seem to fall short. Still,
they are mostly regarded as sharing the full moral status of per-
sons. And indeed, even prematurely born infants are regarded as
such, whereas (on the present reasoning) more fully developed
near-term fetuses are not. Building on the work of Joel Feinberg
and others, Carson Strong argues that infants have a ‘conferred’
moral status, ascribed for social reasons to ‘near persons’ on the
basis of similarity to the paradigm.2 This, he says, would apply
to infants and to a lesser degree also to late-gestation fetuses: ‘We
might say that advanced fetuses have a conferred right to life, but
one that is not as strong as that of infants’.2 However, others have
defended biting the bullet that both fetuses and infants fall below
the threshold of respect for persons.8,9
Because this is a longstanding and ongoing debate between
positions that depend on diverse and often irreconcilable (reli-
gious and secular) worldviews, it seems unlikely that consensus
can ever be reached about what, if anything, is owed to a fetus at
what precise stage of its development. What does that mean for
the normative framework for maternal-fetal medicine? As a way
out, ethicist Laurence McCullough and obstetrician Frank Cher-
venak have suggested that we can simply bypass this whole intrac-
table debate and still regard a fetus a patient also in a normative
sense of that term.10 Because they have built an influential theory
on the idea that clinicians in this field have responsibilities to both
their pregnant and fetal patients,11 we will discuss their view in
more detail. 
The McCullough and Chervenak Model
According to McCullough and Chervenak, a fetus need not have
a prior moral status to be regarded as a patient, but conversely: if
a fetus can benefit from medical treatment, it is a patient and as a
patient it has a ‘dependent moral status’, dependent, that is, upon
the social role of being a patient.10 The crucial step is that the
fetus is presented for medical care that can reasonably be expected
to benefit from it. This notion of fetal benefit, as McCullough
and Chervenak explain, requires the existence of ‘links’ connect-
ing the fetus to the person with independent moral status it may
later become. The reasoning here is that ‘achieving independent
moral status is among the goods that humans value. . . . As a con-
sequence, the fetus reliably linked to later achieving independent
moral status has present interests in the . . . necessary and . . .
sufficient conditions for later achieving [that] status’.10 Viability,
they say, is one such link, given that from this stage onwards, the
fetus can survive into the neonatal period with adequate techno-
logical support. With regard to previable fetuses, the existence of
the required link would depend on whether the pregnant woman
intends to carry the pregnancy to term and makes the fetus a
patient by presenting it into the care of a professional.
McCullough and Chervenak use the term ‘beneficence-
based’ obligations to distinguish what obstetricians owe to their
fetal patients from the ‘autonomy-based’ obligations that can
only be owed to (competent) persons. Obstetricians, they say,
have autonomy-based as well as beneficence-based obligations
to pregnant women in their care, but with regard to fetuses,
their obligations are beneficence based only, as is the case with
regard to neonates or young children. Because a fetus can only
be treated through the woman’s body, her consent will, of course,
be needed. Although the authors acknowledge that the pregnant
woman may have legitimate interests ‘not to be obligated to take
unreasonable health risks’ to allow the fetus to be treated and
that clinicians have autonomy-based obligations to respect the
woman’s choices in this regard,10 the qualifier ‘unreasonable’
indicates that there may be cases in which the balance is such
that pregnant women can be morally expected to accept reason-
able burdens and risks.
The reasoning behind this claim is that whereas with regard to
a previable fetus it is entirely up to the woman to autonomously
decide whether or not the fetus should be presented for medical
treatment, her freedom to make the same decision with regard
to a viable fetus is limited by the fact that a viable fetus with the
capacity to later become a person has an independent interest in
the fulfilment of the conditions for this achievement. According
to McCullough and Chervenak, this would create a moral obliga-
tion for the pregnant woman to present the fetus for medical treat-
ment and for the clinician to propose and provide such treatment
whenever there is an intervention that would clearly promote and
protect the interests of the fetus without disproportionately put-
ting the woman at risk. In such cases, clinicians should not simply
take an informed refusal as the end of the story but may need to
move from information to negotiation and ‘respectful persuasion’,
if necessary involving the aid of an ethics committee, to try to
convince the woman that she has a moral obligation to allow the
fetal patient to be treated.12 

The Fetus as a Patient: Where Does That Leave
the Pregnant Woman?
A recurrent theme in the (feminist) critique of the fetal patient
terminology is that it conceptually separates the fetus from the
woman in a way that is not only at odds with the reality of preg-
nancy but also threatens the position of the pregnant woman as a
patient in her own right. In a commentary on an early legal case
in which criminal charges were filed against a woman who, after
having neglected medical advice, gave birth to a severely brain-
damaged child, George Annas has minted the ‘fetal container’
metaphor to powerfully illustrate this concern: ‘Favouring the
fetus radically devalues the pregnant woman and treats her like an
inert incubator, or a culture medium for the fetus’.13 In a classi-
cal sociological study of the early history of fetal surgery, Monica
Casper observes that this is not far apart from the language actu-
ally used by some of the pioneering surgeons. In this field, she
says, ‘the interests of the fetal patient are regarded as paramount
and pregnant women are conceptualised either as inert tools for
enhancing fetal access or, conversely, as barriers restricting fetal
access. Pregnant women’s autonomy in such a framing may be
severely diminished’.14
Of course, there is nothing in the notion of the fetus as a
patient that would necessitate this problematic framing in which
the pregnant woman either eclipses behind the fetus or is expected
to assume the role of a self-sacrificing ‘heroic mom’.14 However,
putting a second patient next to her inevitably leads to the ques-
tion whose interests are to be regarded as overriding in cases of
conflict. A radical stance is taken by Elselijn Kingma, who argues
that because no firm physical boundaries can be drawn between
the pregnant woman and the fetus, the fetus should be understood
‘as part of the pregnant organism’. As she says, there is ‘one organ-
ism throughout the pregnancy and only at birth does this organ-
ism split and becomes two organisms’.15 In this view, anything
that affects the fetus, be it the woman’s behaviour or medical treat-
ment, affects the ‘pregnant organism’ as a whole. Accordingly, it
would be impossible to say that clinicians have distinct obligations
towards the pregnant woman and her fetus.
In an insightful earlier discussion, Susan Mattingly has made
clear that the alternate view argued for by Kingma is in fact the
understanding that was dominant in obstetrics in the era preceding
the advent of high-resolution ultrasonography: ‘Unable to interact
with the fetus in clear distinction from its host, physicians concep-
tualised the maternal-fetal dyad as one complex patient, the gravid
female, of which the fetus was an integral part’.16 Mattingly does
not suggest that also in the present era of the transparent womb,
this traditional one-patient understanding should still guide our
ethical thinking. However, she points out that ‘Ironically, when
the fetus is construed as a second independent patient, physicians’
prerogatives to act as fetal advocates are actually diminished’. This
is because ethics codes relevant to the doctor–patient relation-
ship consistently rule out counselling patients to accept treatment
against their will to benefit another patient. Think of living tissue
donation as a context to illustrate this claim.
Others have similarly argued that a ‘two-patient view’ seems
difficult to reconcile with what patienthood as a normative con-
cept entails in terms of professional duties, as in cases of conflict,
it may be impossible for the clinician to fulfil his or her obliga-
tions (including nonabandonment) to both patients.17 Anne
Drapkin Lyerly and colleagues use the example of tragic cases in
which a continued pregnancy would entail a significant threat to
maternal life, to bring out what they call the inevitable ‘normative
CHAPTER 15  Ethical Issues in Maternal-Fetal Medicine 141
asymmetry’ between the fetus and the pregnant woman.18 If the
fetus were a patient in the same sense as the woman (normative
symmetry), it would not be obvious what clinicians in such cases
should recommend. Here again, the concern of these authors is
that with the fetus understood as a second patient, doctors may
feel justified to pressure women to allow them to treat that patient
through their body.
McCullough and Chervenak may respond – as they have
done19 – that precisely because the fetal patient is not separate
from the pregnant patient, normative symmetry between two
independent patients is not implied in their framework. They do
not say that the interests of the fetus should be of equal weight
as those of the pregnant woman or that her reasons for reject-
ing a proposed treatment should not count. However, the point
remains that in their view, the clinician’s understanding of the
relative force of his or her obligations to both patients determines
what the outcome of a possible conflict should be.11 
Why the McCullough and Chervenak Model Is
Flawed
The fact that the McCullough and Chervenak model captures the
widely held moral intuition among professionals in the field that
they do indeed have to deal with two patients, while allegedly
steering free from contestable moral presuppositions about the
status of the fetus, may explain its unabided influence in the field
of maternal-fetal medicine and the relevant literature.20 However,
the model has invited fundamental philosophical criticism.2,21,22
As Joan Callahan has made clear in her review of McCullough
and Chervenak’s book Ethics in Obstetrics and Gynecology, their
reasoning is flawed precisely on the point where they suggest to
be able to circumvent the intractable philosophical debate about
moral status.23
Callahan observes that the only way to make sense of the state-
ment that a viable fetus has interests deriving from its capacity
to later become a person is to understand it as a version of ‘the
argument from potential personhood’, which is in fact a position
in the very debate that McCullough and Chervenak wanted us to
forget about. A problematic version to be sure, as there is no good
reason why the capacity to later become a person and insofar to be
able to benefit from treatment would depend on viability.21,23 But
more fundamentally, what becomes clear at this juncture is that
notwithstanding their repeated claims to the contrary, underlying
their theory is an account of fetal interests that deserve protection
by the pregnant woman and her clinician. Strong makes the same
point when saying that McCullough and Chervenak simply ‘beg
the question’ when speaking about a woman’s obligation to pres-
ent her viable fetus for medical care.2 Obviously, it is as a poten-
tial person that the fetus deserves to be treated as a patient. In
other words: moral status precedes patient status, rather than the
other way around. How indeed could it be otherwise? To quote
Stephan Brown: ‘Anyone who does not already believe that fetuses
deserve protection (except as requested by the parents) will reject
the authors’ view that the clinician has duties of beneficence to
it. The proposal to designate the fetus a “patient” would change
nothing’.24
Why is this important? It is important because, as the
McCullough and Chervenak model remains influential in the
field, it may be used to constrain women’s right to autonomously
control what happens to their bodies for reasons resting on a ques-
tionable footing.21 Precisely because McCullough and Chervenak
are right when saying that the debate about the status of the fetus
142 SE C T I O N 5    Ethics

is heavily loaded with religious and other worldview-dependent TABLE Implications of Core Concepts of Two
presumptions, the fact that their model does not succeed in side- 15.1 Approaches to the Ethics of Fetal Therapy
stepping that debate renders it a problematic basis for defining
the moral duties of the parties involved. If accounts of fetal moral The Concept of the Fetus as a The Concept of the Future
status are fundamentally contestable, the same goes for norma- Patient Person
tive accounts of fetal patienthood that explicitly or (as in the case Captures moral intuition that also Qualifies this moral intuition as
of the McCullough and Chervenak model) tacitly depend on it. the interests of the unborn about future interests that will
From the fact that the clinician may hold views about the status should count, next to those of emerge when (and if) the child
of the fetus that require him or her to propose medical treatment the pregnant woman is born
to benefit it or to save its life, it does not follow that the woman
can be expected to agree with those views and to concur that she At odds with what it means to be There is only one patient, norma-
a patient, normatively speaking tively speaking: the pregnant
is under an obligation to allow the proposed treatment to proceed woman
through her body. Neither does it follow that an ethical case can
be made for ‘respectfully persuading’ her towards the clinician’s Inevitably accords moral status to Whether or not the fetus has inter-
point of view, let alone for coercing her through a court order.  the fetus, thus rendering the ests, those of the future person
concept ethically contested can be affected before birth

The Interests of the Future Child
What follows from this? Should we say that in maternal-fetal med-
icine, there are no clear moral interests at stake that we can agree
about beyond those of the woman? And that the relevant choices
for clinicians should therefore be framed in terms of what they
owe to their pregnant patients only, both in terms of beneficence
and respect for patient autonomy? Although that is what some
women’s rights advocates would defend, it ignores the perspec-
tive of parental and professional responsibilities towards the future
child. For if the pregnancy leads to a child, this will be a person
whose interests can be undermined or promoted by what happens
to the fetus during pregnancy.21 As has been argued by several phi-
losophers, including Joel Feinberg and Thomas Murray, the fact
that those future persons are not yet born is irrelevant: the point
is that if they are allowed to be born, they will then have interests
that should already count during pregnancy.25-27 Importantly, the
moral responsibilities (of both pregnant women and professionals)
connecting to those interests do not depend on gestational age;
the interests of the future child count as much in early pregnancy
as they do after viability. Concerns that acknowledging this would
undermine women’s right to have a termination are mistaken. For
if she has an abortion, there will be no child whose interests can be
harmed or promoted by her choices or those of the clinician. But
if she decides to carry the pregnancy to term, the interests of what
Murray calls ‘the not-yet-born-child’ may provide an independent
reason for her obstetrician to propose and for herself to consider
(established forms of ) fetal treatment, apart from how she would
define her own interests in this regard.28
Compared with accounts based on fetal interests and fetal
moral status, an important difference in terms of implications for
obstetric practice is that reasoning from the interests of the future
child does not lead to a moral imperative to try to save the life of
the fetus for its own sake. This is because the death of the fetus
does not affect the interests of the child. Of course, professionals
can still be morally required to do what is reasonably possible to
save the life of the fetus for the sake of the woman if that is her
informed and well-considered choice (Table 15.1).
This is not to say that the interests of the fetus-as-fetus should
not count for what they are. The observation of a stress response
to potentially noxious stimuli in midgestation fetuses suggests that
these fetuses may perhaps feel pain. It is therefore only appropriate
that potentially ‘painful’ surgical procedures are accompanied by pain
relief measures.29 Because there is evidence that experiences of stress
or pain will be ‘remembered’ by the nervous system in later life,30
taking these measures also serves the interest of the future child.  with only a small poor prognosis group remaining eligible for
Distinguishes between interests The interests of the future person
of viable and nonviable fetuses do not depend on viability or
gestational age
Two-patients view invites a prob- Moral duty to protect the future
lematic ‘normative symmetry’ person’s interests is limited by
perspective for balancing considerations of proportionality
maternal and fetal interests
May require saving the life of No such requirement. Here the
the fetus into a poor-quality question is rather if doing so
postnatal existence is morally acceptable. It is, as
long as the future person’s life
is expected to be worth living.
  
Aims of Fetal Treatment: From Saving Lives
to Improving Health
General criteria for offering fetal treatment include that such
interventions should only be considered if (i) the diagnosis of
the condition is certain, (ii) the natural history of the disorder is
clearly understood and (iii) there is no equally effective postnatal
therapy.29 These criteria are crucial as without clear benefits that
may not also be obtained postnatally, there can be no justification
for making the pregnant woman a patient to treat the fetus.
Rationale of Offering Fetal Treatment
The ‘no equally effective postnatal treatment’ criterion can be met
in two types of cases, reflecting two different reasons for offering
fetal treatment:
1. Lethal conditions that without in utero interventions lead to
high rates of fetal or perinatal mortality. Accepted examples
include intrauterine blood transfusion for fetal anaemia,31 tho-
racoamniotic shunting in fetuses with thoracic lesions compli-
cated by hydrops,32 laser coagulation in case of twin-to-twin
transfusion syndrome33 and administration of corticosteroids
before imminent preterm birth.34 Other treatments are con-
sidered experimental, such as vesicoamniotic shunting for fetal
obstructive uropathy.35 For some prenatal interventions that
were developed in the early days of the field, improved survival
after postnatal treatment has led to making this a better alter-
native. For instance, fetal surgery for congenital hernia dia-
phragmatica (CDH) has been replaced by postnatal correction,

experimental fetal treatment in the form of fetoscopic trachea
occlusion.36-38 Improvements in critical neonatal care have
made it possible in certain conditions to consider the option
of inducing premature birth so as to allow lifesaving postnatal
treatment.39 The latter approach has the benefit of avoiding
maternal complications, but fetal treatment may avoid some
of the otherwise unavoidable prematurity and its effects on the
prognosis for the surviving children.
2. Nonlethal conditions in which prenatal treatment leads to bet-
ter health and quality-of-life outcomes for the future child than
can be expected without treatment or with postnatal interven-
tion. An example is in utero repair of spina bifida so as to avoid
the effects of prolonged exposure of neuronal tissue to amni-
otic fluid.40 A similar rationale of timely preventing irreversible
health effects is behind other still experimental interventions,
such as dietary treatment with the aim of avoiding restricted
prenatal brain development in fetuses with 3-phosphoglycerate-
dehydrogenase deficiency, a rare metabolic disorder.41,42 Further
examples on the brink of clinical studies include in utero stem
cell therapy for osteogenesis imperfecta, building on the concept
of intervening in the still preimmune fetus, before permanent
damage is done,43 and pharmacotherapeutic treatment aimed
at timely reversing abnormal brain development in fetuses with
Down syndrome.44 A possible future development is in utero
gene therapy, for instance, for haemophilia A.45  procedure?50 Clearly, on one of the ‘moral status’ views briefly
Giving the Fetus ‘a Chance for Life’?
An ethical pitfall of the first type of fetal treatment (aimed at
avoiding in utero or perinatal death) is that it invites the accep-
tance of any possible risks if there is no other way to save the
life of the fetus or the neonate.46 This ‘ultima ratio’ reasoning
was pervasive in the pioneering phase of open fetal surgery in
the early 1990s, driven both by the belief of the clinicians that
it was their medical duty to use these techniques to save the life
of the endangered ‘fetal patient’ and the desperation of preg-
nant women feeling a strong need to have done all they could to
protect the fetus.14 This constellation may have led clinicians to
propose disproportionally risky procedures and their patients to
accept them on the basis of what may have been less than fully
informed consent.46,47 It also did not promote a commitment to
evaluate the benefits and risks for those ‘innovative’ procedures
in proper clinical research.48 The concern about this was again
raised in the 2011 recommendations of the American College
of Obstetricians and Gynecologists (ACOG) together with the
Academy of Pediatrics (AAP), stating: ‘Although the first few
uses of a new intervention may be motivated by a desire to help
particular fetuses, once feasibility and potential benefit have
been identified, innovations should be subjected to systematic
formal research as soon as feasible’.1 The document stresses that
a comprehensive evidence-based approach to offering prenatal
treatment is essential to enable pregnant women to make truly
informed decisions and to help them overcome the misguided
belief ‘that there are only two possible results: success (fetal cure)
or failure (fetal death)’.1
To avoid this one-dimensionality, women should be prop-
erly informed about what is known (or still unclear) about all
possible outcomes. First, this includes the risks to themselves.
Depending on the nature of the intervention, these range from
small (as in the case of a needle for transfusion) to serious (as in
the case of open surgery), possibly also affecting future pregnan-
cies (as in the case of hysterotomy scars). Second, prospective
CHAPTER 15  Ethical Issues in Maternal-Fetal Medicine 143
parents confronted with a diagnosis of a lethal disorder should
be aware that the option of lifesaving fetal treatment may come
at the price of giving them a child with possibly severe health
problems or disabilities. These may either result from the disor-
der that was the reason for the intervention or be caused by the
procedure, for instance, when very premature birth leads to neu-
rodevelopmental disabilities. In either case, the outcome for the
parents is a child with a compromised quality of life that without
the intervention either might not have been born or might not
have survived beyond the neonatal period. As Ray Noble and
Charles Rodeck remark: ‘It is tempting to conclude that a fetus
should be given a “chance for life”. . . . This runs on the concept
that any chance of life is better than none. However, a partial
success in such cases might lead to greater prolonged suffering in
the offspring and greater psychological and socioeconomic bur-
dens on the parents’.49 
Saving the Fetus at What Price to the Child?
What about the perspective of the interests of the future child
in such cases? Suppose that fetal treatment for a lethal condi-
tion comes at the price of a severely disabled child? Should one
not say that in such cases, the child has been harmed by the
intervention? How would this affect the acceptability of the
referred to at the beginning of this chapter, saving the fetus
equals saving the child. This means that if unavoidable, even
serious remaining or procedure-related health problems or dis-
abilities will be outweighed by the child’s survival, provided the
child’s life is still worth living. But what about the alternative
view that children and adults are persons but fetuses are not?
Here saving the fetus equals bringing a person into the world
who otherwise would never have existed. There is, of course,
no moral requirement to do so. Here the question is rather if
it is morally acceptable. Isn’t it morally problematic to save the
life of the fetus if the unavoidable result is a child with a poor
quality of life? Depending on how one thinks about the moral
status of infants, the same question may also emerge if fetal
treatment brings about neonatal survival leading to a life with
compromised health prospects. For instance, vesicoamniotic
shunting for fetal obstructive uropathy rescues the child into a
life of often severe renal insufficiency.35
This question connects to a more general debate about the
welfare of the child in (assisted) reproduction. In their paper
‘When is birth unfair to the child?’, Bonnie Steinbock and Ron
McClamrock have argued that the principle of parental responsi-
bility requires refraining from reproduction unless one’s children
will at least be able to have a minimally decent life.51 Taking the
same perspective, guidance documents for professionals working
in assisted reproduction state that they have a responsibility to
take account of the welfare of the children that they cause to
exist.52,53 This captures a widely shared intuition about respon-
sible reproduction that also seems to be behind the concern in
our debate about whether saving the fetus may entail harming
the child.
However, it is not obvious that a child can be harmed by
being brought into existence if that is the only existence she or
he could possibly have.54 The point here is that any conceivable
better life would be the life of a different child. It is still possible
that the child has been harmed by those responsible for bringing
him or her in a ‘harmful state’ but only if his or her life was so
horrible that any rational being would rather not have existed at
144 SE C T I O N 5    Ethics
all.25,55 According to Feinberg, one should think here of rare cases
in which because of very severe abnormalities, all the child’s pos-
sible interests are ‘doomed to defeat’.25 Above this ‘subzero quality
of life’ bar, bringing a child into existence with significant health
problems or disabilities does not entail harming the child.55
The upshot of this is that regardless of whether we regard the
child as having his or her life saved by fetal treatment or being
brought into existence as a result of it, the child’s interests count
against the intervention only if any unavoidable health problems
or disabilities are so serious as to make his or her life not worth liv-
ing. This means that it will only rarely be the case that unavoidable
high offspring risks outweigh the woman’s self-declared interests
in saving the fetus and having a living child even with serious
disabilities. Those who regard this as counterintuitive must ask
themselves if they would also be willing to consider terminating
the pregnancy to protect the child from a poor but above ‘subzero’
health outcome.56
Of course, the above is not a justification for accepting avoid-
able offspring risks. There can be no debate that it does harm a
child to be brought into existence with health problems or dis-
abilities if they could well have been avoided through more careful
treatment. For in this case, there would have been the possibility
of a less compromised existence for this child.  this context, the argument is used to question the acceptability
Giving the Child a Better Life
The broadening of the field to also include nonlethal conditions
requires evidence that choosing fetal treatment instead of inter-
vening (or not intervening) after birth gives the child a better
life without exposing the pregnant woman to disproportionally
high risks. This requires a long-term evaluation perspective that
reflects the field’s commitment to evidence-based fetal treatment.
An important milestone in this connection is the Management of
Myelomeningocele Study (MOMS). This multicentre randomised
controlled trial (RCT) of open fetal surgery for spina bifida has
clearly established the value of this approach over postnatal surgery
in terms of a reduced need for a hydrocephalus shunt by the age of
12 months and significantly improved mental and motor function
scores at 30 months of age.40 Preliminary follow-up data suggest
improved ambulatory status also at 10 years of age.57 However, the
MOMS trial also confirms that fetal surgery comes with high rates
both of serious maternal complications and preterm birth (13%
before 30 weeks).40,58 Although minimally invasive endoscopic
surgery would theoretically seem to promise lower complication
risks, a recent meta-analysis found evidence for the opposite.59
Based on the MOMS trial findings, the ACOG has recommended
that women with fetuses meeting the inclusion criteria of the trial
should be ‘made aware of the findings’ and counselled accordingly,
while stressing ‘the potential for significant morbidity and possibly
mortality, even in the best and most experienced hands’.60 The
ongoing MOMS 2 study compares the long-term effects of prena-
tal and postnatal surgery both on the child’s health and well-being
and on the future reproductive health of the mother.
Before the MOMS trial, the idea of fetal treatment for nonle-
thal conditions such as spina bifida has invited two kinds of ethi-
cal criticism. First, it has been argued that the proportionality of
surgical interventions would be more problematic in the sense of
higher order risk profiles being less easily outweighed by the sup-
posedly lower benefits of such treatment.48 As stated by Lyerly
and colleagues, ‘it is difficult to justify risking maternal or fetal
morbidity or fetal demise in attempts to reduce the chances of
a woman having a child with a disability’.46 The trial has only
confirmed the concerns of these authors about the high risks of
open fetal surgery. In the light of the findings and the need for
more follow-up data, it is certainly not obvious that the procedure
can be regarded as proportional. But why would this be a more
problematic state of affairs when the aim is to give the child a bet-
ter life rather than if (with other forms of fetal surgery) it is to save
the life of the fetus? If the fetus is not a person but the future child
clearly is, it would seem that the opposite may well be defended.
However, precisely from the perspective of the interests of the
future child, there is still a reason for considering the proportion-
ality balance as more precarious in fetal treatment for nonlethal
disorders. Whereas with regard to lethal disorders, the burdens of
any unavoidable iatrogenic health problems or disabilities will be
outweighed by the value for the child of a life still worth living (see
above), here such outcomes must be outweighed by an overall bet-
ter quality of life. If not, fetal treatment actually harms the child.
Second, it has been argued that the option of fetal treatment
for conditions involving a disability may be offending to people
living with these conditions, sending the ‘derogatory message’ that
their lives are of insufficient quality. This ‘expressivist argument’
plays an important role in the so-called ‘disability rights critique’
of prenatal diagnosis and screening for fetal abnormalities.61 In
of prenatal testing to enable selective termination of fetuses with
abnormalities such as spina bifida or Down syndrome. In the con-
text of fetal treatment, the argument is directed against what is felt
as an only conditional (parental or societal) acceptance of a child
with a disabling condition. For instance, Lyerly and colleagues
have suggested that instead of proposing fetal surgery, profes-
sionals should help women ‘understand that carrying a child with
spina bifida to term is a possible and acceptable option’.46
Underlying the disability rights critique is what has been called
the social model of disability, which focuses on sociocultural barri-
ers to equal participation of people with impairments.62,63 Follow-
ing this view, if anything is in need of change, it is not people with
disabilities but society’s failure to properly support them. Although
the argument has been invoked with regard to fetal surgery for
spina bifida, it is difficult to see how it would apply here without
also problematising standard postnatal surgery for this condition.
And indeed, the suggestion that to fully accept their disabled chil-
dren, parents should reject accepted medical treatment that will
improve their health and well-being is simply absurd. But it is not
unreasonable to expect that the disability rights critique will play a
role in the societal debate about the prospect of fetal treatment for
Down syndrome aimed at improving neurocognitive outcome, for
which the first trial has started.64
The rationale for fetal neurocognitive treatment for Down syn-
drome consists of two claims: first that interventions leading to
a higher intellectual quotient (IQ) in children with Down syn-
drome will benefit these children and their families and second
that such interventions will be most successful if performed pre-
natally. With regard to the first claim, although abnormalities in
virtually every organ system, except the brain, are routinely treated
in children with Down syndrome, intellectual disability remains a
key aspect of the condition. In a recent study, parents of children
with Down syndrome were found to have mixed feelings about
the hypothetical scenario of a (postnatal) treatment that would
mitigate or completely reverse intellectual disability in their child.
Many respondents rejected the idea that a ‘cure’ for Down syn-
drome would be desirable, arguing that the problem is with soci-
etal acceptance of diversity rather than with their child’s level of
functioning. Several parents also objected that neurocognitive

treatment would change their child’s personality. However, many
others welcomed the promise of greater independence for their
child and the resulting improvement of the quality of life both for
the child and the family.65
It is certainly true that society should be more inclusive of peo-
ple with disabilities such as Down syndrome. However, the social
model of disability is one sided in that it reduces their difficulties
to prejudice and exclusion. It is problematic when this leads to
denying people the opportunity of profiting from treatment that
will enhance their autonomy by giving them greater control over
their lives and improve their well-being also by diminishing the
impact of a constant source of frustration.66
Given that there are sufficient ethical considerations in favour
of developing neurocognitive treatment for people with Down
syndrome,67 the argument for fetal treatment (the second claim)
is that better results are to be expected than postnatally. Based on
animal research, it is expected that in utero drug therapy may lead
to a timely reversal of the development of abnormal embryonic
brain phenotype in fetuses with Down syndrome.68,69 Current
research aims to identify safe candidate drugs.70 This is expected
to lead to (further) human clinical trials in the coming years.
In addition to safety and effectiveness studies, long-term follow
up will be needed to confirm that, on balance, the availability of
fetal treatment for Down syndrome is indeed beneficial for those
concerned. Clearly, the ‘change of personality’ objection would
not seem to apply to fetal therapy. In fact, several respondents in
the quoted parental attitude study said they might have consid-
ered treatment at or before birth, when their child’s personality
was still to be formed, but not later in its life.65 But other con-
cerns connect to the fact that even with a significant improvement
of neurocognitive functioning, a comprehensive ‘cure’ for Down
syndrome will not be achieved.67 For instance, it may be that a
partial improvement in cognitive functioning will make persons
with Down syndrome only more aware of not being able to fully
participate in society or to realise the professional and reproduc-
tive options open to others. Moreover, to the extent that fetal
therapy for Down syndrome would bring the IQ of some people
with Down syndrome into the typical range, they would still be
physically recognisable. How would this affect their social func-
tioning and acceptance?65  fetal surgery’.75 Smajdor dismisses the idea that pregnant women’s
Fetal Treatment and Prenatal Decision
Making
Whereas fetal ‘treatment’ or ‘therapy’ may suggest established treat-
ments, in fact, many prenatal interventions are still experimental
or investigational. It is important that women are informed about
the absence of evidence and to avoid misunderstanding, these
labels should be used with caution. There is currently much sup-
port in the field for introducing new fetal treatments in a clini-
cal research setting, ideally using RCTs, based on prior preclinical
evidence from animal studies regarding initial safety and efficacy.
There are several ethical challenges in this connection.
Ethical Challenges of Fetal Therapy Research
First, it can be too early and too late for an RCT. As long as lit-
tle is known about the safety and efficacy of a new treatment, it
is not acceptable to withhold the standard treatment from trial
subjects.49,71 But if the pre-RCT period lasts too long, clinicians
who have come to believe in the benefits of a specific form of fetal
CHAPTER 15  Ethical Issues in Maternal-Fetal Medicine 145
treatment may find it difficult to enroll patients into trials where
randomisation means a 50% chance of not being given the treat-
ment. Pregnant women often share a similar preference. These
preferences then make it difficult to still conduct an RCT even
though the situation remains one of ‘clinical equipoise’.72 Whereas
the MOMS trial could only be conducted on the basis of a gen-
eral agreement not to perform the surgery outside the trial, there
is debate about whether such ‘closing of the back door’ is ethical.
Building on their experience with the European Tracheal Occlusion
To Accelerate Lung growth (TOTAL) trial (comparing tracheal
occlusion with watchful waiting in a selected population of fetuses
with severe CDH), Catharina Rodriques and colleagues describe
how in a situation between what they refer to as remaining clinical
and lost patient or physician equipoise, only suboptimal solutions
are possible.73 They conclude that the ‘perhaps ungrateful task of
knowing when to stop experimenting’ is essential to responsible
innovation in this area.
Second, although ‘therapeutic misconception’ is a general
problem in clinical research, in this context, there is a concern
that it would interfere with decisions about whether or not to ter-
minate the pregnancy after a bleak prenatal diagnosis.74 Whereas
women may hope that with treatment, the outcome will be better
than without, the reverse may well be the case, and when finding
this out, termination may no longer be possible. The outcome
may be that whereas otherwise they might have chosen termina-
tion, they now have to adjust their lives to care for a child with
severe disabilities or health problems.43 This may be a reason to
only propose participation in a fetal treatment trial to women who
already on independent grounds have made the decision not to
choose a termination.29 Of course, the message should not be that
trial participation commits them to continue the pregnancy. The
loss of data that a termination would entail is not a justification
for any form of pressure in this regard.49
Third, there is a more general concern that pregnant women
asked to participate in fetal treatment research will accept any pos-
sible risk to themselves to benefit their unborn child. As Anna
Smajdor has put it: ‘Even if the risks . . . were still greater, the
benefits more marginal, and women had access to every possible
relevant fact and statistic, they might still be willing to undergo
often self-effacing choices for fetal therapy cannot be regarded as
well-considered and insofar truly autonomous. In her view, the
real problem lies behind women’s choices in the societal expecta-
tions that frame the context for their decision making. This makes
it an issue of social criticism rather than medical ethics. Although
agreeing that pregnant women generally are able to make autono-
mous choices, Maria Sheppard has argued that a specific group
should be regarded as vulnerable research subjects, namely those
who have just been found to carry a fetus with a disorder for which
the only option apart from watchful waiting is enrolment in a clin-
ical trial.76 For these women, special safeguards would be needed
to ensure that their consent to participate in a trial is informed and
voluntary. As with other vulnerable research subjects, more than
usual care should be taken to ascertain that women to whom this
applies do indeed fully understand the nature, aims, burdens and
risks of the study. The ACOG and the AAP have also proposed
protective measures to this end, for instance, the appointment of
a ‘research subject advocate’.1
Finally, funding problems and restrictive regulations, for
instance, with regard to excluding pregnant women from clini-
cal trials involving drug research stand in the way of furthering
evidence-based fetal therapy. Lack of funding is also seriously
146 SE C T I O N 5    Ethics
hampering essential long-term follow-up studies. There is still a
dearth of research looking into long-term effects on the (reproduc-
tive) health and psychosocial well-being of women who under-
went prenatal treatment to benefit their unborn children.  give their children an optimal start in life.75,81 However, there are
Fetal Treatment as an Additional Option for
Reproductive Choice
In the last parts of this section, we assume the increasing availabil-
ity of forms of fetal treatment that are proven to be safe and effec-
tive. This creates an alternative option for reproductive choice for
women found to be carrying a fetus with a serious (lethal or non-
lethal) condition. Women who without this option might have
considered asking for a termination may now decide to continue
the pregnancy. This development may lead to two moral pitfalls
at opposite ends of the ‘pro-life’ versus ‘pro-choice’ debate that
should both be avoided.
Jérôme Lejeune, the French paediatrician and geneticist tradi-
tionally credited with the discovery of the chromosomal basis of
Down syndrome, has always regarded the use of prenatal diagnosis
for terminations as a morally problematic form of medicine that
should ideally be replaced by facilitating treatment.77 Others have
also stated that the development of safe and effective fetal therapy
belongs to the ‘ultimate goals’ of prenatal diagnosis.78 As such, this
seems an ideal that no one could reasonably criticise. However,
the notion that fetal treatment makes terminations ‘unnecessary’
may connect to a pro-life agenda that denies women the right
to abortion. Although it should be welcomed that fetal therapy
gives women a further choice, presenting this as the morally pre-
ferred option would be ethically problematic, given the contested
nature of the underlying view of the status of the fetus (see earlier
discussion).
The opposite moral pitfall is to dismiss the option of fetal
therapy as ‘unnecessary’. For instance, in a recent paper on the
ethics of fetal surgery, a clinician was quoted as saying that she did
not understand ‘why anyone would go to such lengths when they
could simply abort and start over’.20 Although this is anecdotal
and one would not find this view defended in the literature, it may
be quite influential. Ethically, this view is problematic because it
betrays a motivation that is either eugenic or paternalistic – or
both. It fails to acknowledge that for quite some women or cou-
ples, abortion is simply not acceptable and that for many of those
who are not categorically opposed to it, deciding to end a wanted
pregnancy remains an extremely difficult choice that may have
lifelong psychosocial consequences. Moreover, in many parts of
the world, abortion is illegal.  this example the reasoning does not depend on a specific position
Reproductive Autonomy and Parental
Responsibility
The reasoning that fetal therapy creates an alternative choice that
should neither be imposed upon pregnant women nor withheld
from them reflects the ethos of professional nondirectiveness and
respect for reproductive autonomy that also determines the widely
accepted normative framework for prenatal diagnosis and screen-
ing.79,80 However, when women decide not to have a termination,
the moral landscape changes because the interests of the future
child need to be taken into account (see earlier). Parental responsi-
bility does not only start at birth, and although the fetus is not (in
a normative sense) a patient, professionals also have a responsibil-
ity to consider how their acts or omissions affect the health and
well-being of the child to be.
As several authors have remarked, this is not something most
pregnant women need to be reminded of. Many tend to go to great
lengths to safeguard the healthy development of their fetuses and to
exceptions to this picture. For instance, Mark Evans and colleagues
remark that they ‘have occasionally been surprised and frustrated by
the refusal of a few . . . patients to attempt even innocuous thera-
pies’,81 and recently, the ACOG has issued an Ethics Committee
statement on how to deal with refusals of medically recommended
treatment during pregnancy. According to the ACOG, directive
counselling may well be appropriate in such situations because non-
coercive recommendations ‘do not violate but rather enhance the
requirements of informed-consent’.82 This connects with another
relevant ACOG document in which the question is raised how
autonomy can be protected ‘if it is addressed in a vacuum, apart
from an individual’s concrete roles and relationships’.83 We agree
with this: in the context of a pregnancy that is intended to be car-
ried to term, addressing women in their parental role need not be at
odds with respecting them as autonomous persons. Given a shared
responsibility to protect the health and well-being of the future
child, professionals may well be justified in going beyond merely
informing women about offspring risks. One may think here of
directive counselling of competent pregnant women with regard to
modification of lifestyle patterns that are known to be harmful to
the future child or with regard to having a caesarean section in cases
when it would be necessary to avoid the birth of a child with seri-
ous disabilities.84 The same reasoning would then also apply to rec-
ommending or encouraging pregnant women to undergo safe and
effective forms of fetal therapy in the interest of their future child.
The classic example here is intrauterine blood transfusion for
fetal anaemia. When left untreated, this condition may lead to fetal
or neonatal death and to neurologic damage leading to cerebral
palsy or mental retardation in surviving children.85 The procedure
itself is considered safe for the woman but comes with a small
risk for fetal complications.31 In its report on ‘Critical care deci-
sions in fetal and neonatal medicine: ethical issues’, the British
Nuffield Council on Bioethics discusses a hypothetical case of a
woman refusing recommended transfusion for fetal anaemia out
of fear of losing the pregnancy. The Council concludes that efforts
to persuade her to reconsider her position would be justified given
that ‘her interest to proceed as she thinks is best appears to be
less important’.28 Although the report construes this as a conflict
between the interests of the woman and those of her 26-week-
old fetus, the reasoning would be more convincing if argued in
terms of the undeniable interests of the future child, because in
in the debate on moral status of the fetus.84
Of course, one should only think here of established and gen-
erally accessible forms of fetal treatment that would evidently
protect the future child from significant and irreversible harm
without exposing the pregnant woman to a serious risk. Many
present forms of fetal therapy do not fulfil these conditions. They
are experimental or investigational or come with significant risk
profiles. For instance, even with further evidence of long-term
benefit for the future child, it is hard to see how directive counsel-
ling for open fetal surgery for spina bifida could ever be justified.
Current research may lead to new forms of fetal treatment
with a favourable balance of benefits and risks. The development
of neurocognitive fetal treatment for Down syndrome is only one
possible fruit of what Diana Bianchi has referred to as the emerg-
ing field of ‘fetal personalized medicine’, in which the integration
of several kinds of -omics data may lead to many more drug-based

fetal treatment opportunities.86 If it can be shown that these are
safe and effective and lead to better outcomes compared with no
or postnatal treatment and if such treatments would be available
via the health care system or otherwise not be too costly, it would
then follow that women known to be carrying an affected fetus
and willing to continue the pregnancy should be counselled to at
least seriously consider such treatment.
This development may also affect the ethics of prenatal screen-
ing for fetal abnormalities. As said, the aim of this screening is
to provide women with options for autonomous reproductive
choice. It is therefore regarded as essential that the screening offer
is provided in a nondirective way. However, if the scenario of ‘fetal
personalised medicine’ becomes a reality and prenatal screening
will not only lead to termination options but also to treatment
opportunities for affected pregnancies that will not be terminated,
it is no longer obvious that the screening offer should indeed be
nondirective. This requires a rethinking of the normative frame-
work for prenatal screening.87  possible risk to their offspring at any possible costs to themselves
Can Coerced Fetal Treatment Ever Be Justified?
Would it ever be acceptable to move beyond directive counsel-
ling to forced treatment in rare cases of continued refusal of safe
and effective fetal treatment by a competent pregnant woman?
This raises ethical and legal red flags because it entails an evident
infringement not only of the pregnant woman’s autonomy but
also a violation of her bodily integrity. For these reasons, many
commentators and committees strongly reject all forms of coer-
cion in the care of pregnant women. For instance, the Nuffield
Council on Bioethics states that ‘although in moral terms [the
pregnant woman] acts wrongly in harming her future child, it
would be wrong to force her to behave rightly’.28 Similarly, the
ACOG ‘opposes the use of coerced medical interventions for preg-
nant women, including the use of the courts to mandate medical
interventions for unwilling patients.’82
In many European countries, the law would not allow such
coercive measures because it does not recognise fetuses as bearers
of legal rights. Moreover, after the 2004 Vo vs France ruling of the
European Court of Human Rights, it is clear that Article 2 of the
European Convention of Human Rights (stating that ‘Everyone’s
right to life shall be protected by law’) does not require member
states to protect the life of the unborn. Whereas some commenta-
tors have called for a greater legal protection of unborn children,88
an effort clearly linked with a pro-life position in the fetal status
debate, others have argued that it is strange that the interests of
future children are not given their due in these legal debates.27 The
fact that these right bearers do not yet exist is not a good reason
for failing to protect their interests. To make this point, Govert
den Hartogh borrows Feinberg’s example of a terrorist placing a
bomb near a kindergarten, set to explode in 6 years’ time.25,89 The
children who will be killed or maimed by the blast are not even
conceived yet. Of course, ‘one cannot say that this prejudices the
interests in life and health of those presently non-existing beings’,
but ‘what we can and must say is that the interests and rights of
the children who will be killed by it are harmed by the placing of
the bomb’. Den Hartogh concludes that the rights of the future
CHAPTER 15  Ethical Issues in Maternal-Fetal Medicine 147
child fully counts already before its birth but adds that nothing
as yet follows about the justification of coerced fetal treatment in
concrete cases. As this entails overriding the pregnant woman’s
own rights, including the especially weighty right to bodily integ-
rity, he leaves this a question for further ethical and legal analysis.
John Robertson, the current chair of the Ethics Committee
of the American Society for Reproductive Medicine, follows the
same reasoning in a recent contribution to this debate.90 Like
Feinberg and Den Hartogh, he says it is confusing to frame the
debate in terms of protecting the fetus with its unclear status and
contestable interests when the issue is about ‘protecting intended
children from harmful prenatal conduct’. Robertson suggests that
if we agree about the relevance of that perspective, the debate
becomes one of policy (determining under which conditions of
proportionality coerced treatment might perhaps be acceptable in
rare cases) rather than principle. Concerns that accepting so much
will lead on to a justification of coercing women to avoid any
are unconvincing, as slippery slope arguments almost always are.90
As said, situations in which pregnant women refuse medically
indicated treatment that would benefit their children are rare. It
has been rightly pointed out that adequate and timely information
and communication may help further avoid such difficult con-
flicts arising. These aspects should therefore be given due attention
also as a form of ‘preventive ethics’ in obstetric practice.10 
Conclusion
In this chapter, we have argued that there is only one patient in
fetal treatment, namely, the pregnant woman. Fetal patient talk
tacitly rests on a position in the debate about the moral status of
fetuses. This is problematic as a basis for defining the responsibili-
ties of professionals and pregnant women. However, regardless of
whether the fetus has a high or low moral status, the interests
of the future child are relevant for decision making about fetal
treatment.
With regard to fetal treatment for lethal conditions, the view
that any possible risks are justified if there is no other way to save
a life ignores that this may lead to worse outcomes than fetal or
neonatal death even though the child her- or himself need not be
harmed by being brought into a compromised existence. What
makes fetal treatment for nonlethal conditions more difficult to
justify is not a less important aim but the fact that evidence of
benefit requires a long-term comparative perspective.
Challenges of fetal treatment research include the need to
timely set up well-designed clinical trials, the need to avoid ter-
mination decisions being affected by a therapeutic misconception
and the need to safeguard the interests of vulnerable research sub-
jects. When safe and effective treatment is available and accessible,
a woman who decides not to choose a termination after a positive
prenatal diagnosis may have a moral duty to undergo fetal treat-
ment for that condition in the interest of the future child.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References patient’ in maternal-fetal surgery. J Med Ethics.
2013;39(4):219–223.
44. Guedj F, Bianchi DW, Delabar JM. Prenatal
treatment of Down syndrome: a reality? Curr
23. Callahan J. First steps in preventive ethics. Opin Obstet Gynecol. 2014;26(2):92–103.
1. American College of Obstetricians and Gyne-
Hastings Cent Rep. 1996;26(2):45–46. 45. Almeida-Porada G, Atala A, Porada CD. In
cologists, Committee on Ethics; American
24. Brown SD. The ‘fetus as patient’: a critique. Am utero stem cell transplantation and gene ther-
Academy of Pediatrics, Committee on Bioeth-
J Bioeth. 2008;8(7):47–50;discussion W4–W6. apy: rationale, history, and recent advances
ics. Maternal-fetal intervention and fetal care
25. Feinberg J. Harm to Others. The Moral Limits of toward clinical application. Mol Ther Methods
centers. Pediatrics. 2011;128(2):e473–e478.
the Criminal Law. New York: Oxford Univer- Clin Dev. 2016;5:16020.
2. Strong C. Ethics in Reproductive and Perinatal
sity Press; 1984. 46. Lyerly AD, Mahowald MB. Maternal-fetal sur-
Medicine. A New Framework. New Haven, CT:
26. Murray TH. Moral obligations to the not- gery: the fallacy of abstraction and the prob-
Yale University Press; 1997.
yet born: the fetus as patient. Clin Perinatol. lem of equipoise. Health Care Anal. 2001;9(2):
3. Lanzone A. Ethical issues in human reproduc-
1987;14(2):329–343. 151–165.
tion: catholic perspectives. Gynecol Endocrinol.
27. Savulescu J. Future people, involuntary medi- 47. Doyal L, Ward C. Fetal surgery: ethical and
2013;29(11):953–954.
cal treatment in pregnancy and the duty of easy legal issues. Semin Neonatol. 1998;3:255–265.
4. Schenker JG. Human reproduction: Jewish
rescue. Utilitas. 2007;19(1):1–20. 48. Reitsma AM, Moreno JD. Maternal-fetal
perspectives. Gynecol Endocrinol. 2013;29(11):
28. Nuffield Council on Bioethics. Critical Care research and human research protections pol-
945–948.
Decisions in Fetal and Neonatal Medicine: Ethical icy. Clin Perinatol. 2003;30(1):141–153.
5. Serour GI. Islamic perspectives in human repro-
Issues. London: Nuffield Council on Bioethics; 49. Noble R, Rodeck CH. Ethical considerations
duction. Reprod Biomed Online. 2008;17(suppl
2006. of fetal therapy. Best Pract Res Clin Obstet Gyn-
3):34–38.
29. Deprest J, Toelen J, Debyser Z, et al. The fetal aecol. 2008;22(1):219–231.
6. DeGrazia D. Human Identity and Bioethics.
patient—ethical aspects of fetal therapy. Facts 50. Sheppard M, Spencer RN, Ashcroft R, et  al.
Cambridge, United Kingdom: Cambridge
Views Vis Obgyn. 2011;3(3):221–227. Ethics and social acceptability of a proposed
University Press; 2005.
30. Anand KJ. Pain, plasticity, and premature clinical trial using maternal gene therapy to treat
7. Taylor C. Self-interpreting animals. Philosophi-
birth: a prescription for permanent suffering? severe early-onset fetal growth restriction. Ultra-
cal Papers. 1985;1:45–76.
Nat Med. 2000;6(9):971–973. sound Obstet Gynecol. 2016;47(4):484–491.
8. McMahan J. The Ethics of Killing. Ethics at the
31. Lindenburg IT, van Kamp IL, Oepkes D. 51. Steinbock B, McClamrock R. When is
Margins of Life. Oxford, United Kingdom:
Intrauterine blood transfusion: current indi- birth unfair to the child? Hastings Cent Rep.
Oxford University Press; 2002.
cations and associated risks. Fetal Diagn Ther. 1994;24(6):15–21.
9. Kuhse H, Singer P. Should the Baby Live?
2014;36(4):263–271. 52. Pennings G, de Wert G, Shenfield F, et  al.
Oxford, United Kingdom: Oxford University
32. Grethel EJ, Wagner AJ, Clifton MS, et  al. ESHRE Task Force on Ethics and Law 13: the
Press; 1985.
Fetal intervention for mass lesions and hydrops welfare of the child in medically assisted repro-
10. McCullough LB, Chervenak FA. Ethics in
improves outcome: a 15–year experience. J duction. Hum Reprod. 2007;22(10):2585–2588.
Obstetrics and Gynecology. New York: Oxford
Pediatr Surg. 2007;42(1):117–123. 53. Human Fertility and Embryology Authority.
University Press; 1994.
33. Johnson A. Diagnosis and management of Code of Practice. Section 8. Welfare of the Child.
11. Chervenak FA, McCullough LB, Brent RL.
twin-twin transfusion syndrome. Clin Obstet 8th ed. London: Human Fertility and Embry-
The professional responsibility model of
Gynecol. 2015;58(3):611–631. ology Authority; 2016.
obstetrical ethics: avoiding the perils of clash-
34. Azad K, Mathews J. Preventing newborn 54. Parfit D. Reasons and Persons. Oxford, United
ing rights. Am J Obstet Gynecol. 2011;205(4):
deaths due to prematurity. Best Pract Res Clin Kingdom: Clarendon Press; 1984.
315.e1–5.
Obstet Gynaecol. 2016;36:131–144. 55. Wilkinson S. Choosing Tomorrow’s Children.
12. Chervenak FA, McCullough LB. Ethical issues
35. Mann S, Johnson MP, Wilson RD. Fetal tho- The Ethics of Selective Reproduction. Oxford,
in recommending and offering fetal therapy.
racic and bladder shunts. Semin Fetal Neonatal United Kingdom: Clarendon Press; 2010.
West J Med. 1993;159(3):369–399.
Med. 2010;15(1):28–33. 56. Clarkeburn H. Parental duties and untreat-
13. Annas GJ. Pregnant women as fetal containers.
36. Harrison MR, Keller RL, Hawgood SB, able genetic conditions. J Med Ethics.
Hastings Cent Rep. 1986;16(6):13–14.
et  al. A randomized trial of fetal endoscopic 2000;26(5):400–403.
14. Casper MJ. The Making of the Unborn Patient.
tracheal occlusion for severe fetal congeni- 57. Danzer E, Thomas NH, Thomas A, et  al.
A Social Anatomy of Fetal Surgery. New Bruns-
tal diaphragmatic hernia. N Engl J Med. Long-term neurofunctional outcome, execu-
wick, NJ: Rutgers University Press; 1998.
2003;349(20):1916–1924. tive functioning, and behavioral adaptive skills
15. Gelonesi J. The metaphysics of pregnancy.
37. Deprest J, De Coppi P. Antenatal management following fetal myelomeningocele surgery. Am
Interview with philosopher Elselijn Kingma:
of isolated congenital diaphragmatic hernia J Obstet Gynecol. 2016;214(2):269.e1–269.e 8.
ABC Radio National Australia; 2014. http://
today and tomorrow: ongoing collaborative 58. Danzer E, Johnson MP. Fetal surgery for neu-
www.abc.net.au/radionational/programs/
research and development. J Pediatr Surg. ral tube defects. Semin Fetal Neonatal Med.
philosopherszone/the-metaphysics-of-preg-
2012;47(2):282–290. 2014;19(1):2–8.
nancy/5950930 (last accessed 20 July 2018).
38. Grivell RM, Andersen C, Dodd JM. Prenatal 59. Araujo Junior E, Eggink AJ, van den Dobbel-
16. Mattingly SS. The maternal-fetal dyad. Explor-
interventions for congenital diaphragmatic steen J, et al. Procedure-related complications
ing the two-patient obstetric model. Hastings
hernia for improving outcomes. Cochrane of open vs endoscopic fetal surgery for treat-
Cent Rep. 1992;22(1):13–18.
Database Syst Rev. 2015;(11):CD008925. ment of spina bifida in an era of intrauterine
17. Fletcher JC. The fetus as patient: ethical issues.
39. Oepkes D. Fetal Therapy. Update on the Current myelomeningocele repair: systematic review
JAMA. 1981;246(7):772–773.
Level of Knowledge; 2008. The Hague. and meta-analysis. Ultrasound Obstet Gynecol.
18.  Lyerly AD, Little MO, Faden RR. A cri-
40. Adzick NS, Thom EA, Spong CY, et  al. A 2016;48(2):151–160.
tique of the ‘fetus as patient’. Am J Bioeth.
randomized trial of prenatal versus postnatal 60. American College of Obstetricians and Gyne-
2008;8(7):42–44;discussion W4–W6.
repair of myelomeningocele. N Engl J Med. cologists, Committee on Obstetric Practice.
19. McCullough LB, Chervenak FA. Response
2011;364(11):993–1004. ACOG Committee opinion no. 550: maternal-
to commentaries on ‘a critical analysis of the
41. de Koning TJ, Klomp LW, van Oppen AC, fetal surgery for myelomeningocele. Obstet
concept and discourse of “unborn child”’. Am J
et al. Prenatal and early postnatal treatment in Gynecol. 2013;121(1):218–219.
Bioeth. 2008;8(7):W4–W6.
3–phosphoglycerate-dehydrogenase deficiency. 61. Parens E, Asch A. Disability rights critique of
20. Antiel RM. Ethical challenges in the new world
Lancet. 2004;364(9452):2221–2222. prenatal genetic testing: reflections and recom-
of maternal-fetal surgery. Semin Perinatol.
42. van der Crabben SN, Verhoeven-Duif mendations. Ment Retard Dev Disabil Res Rev.
2016;40(4):227–233.
NM, Brilstra EH, et  al. An update on ser- 2003;9(1):40–47.
21. Bewley S. Ethical issues in maternal-fetal medi-
ine deficiency disorders. J Inherit Metab Dis. 62. Shakespeare T. Disability Rights and Wrongs
cine. In: Rodeck CH, Whittle MJ, eds. Fetal
2013;36(4):613–619. Revisited. 2nd ed. London: Routledge; 2014.
Medicine: Basic Science and Clinical Practice.
43. Westgren M, Gotherstrom C. Stem cell trans- 63. Davis DS. Genetic Dilemmas: Reproductive
2nd ed. London: Elsevier; 2009:206–221.
plantation before birth - a realistic option for Technology, Parental Choices, and Children’s
22. Rodrigues HC, van den Berg PP, Duwell
treatment of osteogenesis imperfecta? Prenat Futures. 2nd ed. Oxford, United Kingdom:
M. Dotting the I’s and crossing the T’s:
Diagn. 2015;35(9):827–832. Oxford University Press; 2010.
autonomy and/or beneficence? The ‘fetus as a

147.e1
147.e2 References
64. Rochman B. Parents turn to Prozac to treat
Down syndrome. MOT Technol Rev. Janu-
ary 12 2016. https://www.technologyreview.
com/s/545191/parents-turn-to-prozac-to-treat-
down-syndrome/ (last accessed 20 July 2018).
65. Inglis A, Lohn Z, Austin JC, et al. A ‘cure’ for
Down syndrome: what do parents want? Clin
Genet. 2014;86(4):310–317.
66. Solomon A. Far From the Tree. Parents, Children
and the Search for Identity. New York: Scribner;
2012.
67. de Wert G, Dondorp W, Bianchi DW. Fetal
therapy for Down syndrome: an ethical explo-
ration. Prenat Diagn. 2017;37(3):222–228.
68. Guedj F, Bianchi DW. Noninvasive prenatal
testing creates an opportunity for antenatal
treatment of Down syndrome. Prenat Diagn.
2013;33(6):614–618.
69. Stagni F, Giacomini A, Guidi S, et al. Timing
of therapies for Down syndrome: the sooner,
the better. Front Behav Neurosci. 2015;9:
265.
70. Guedj F, Pennings JL, Massingham LJ, et  al.
An integrated human/murine transcriptome
and pathway approach to identify prena-
tal treatments for Down syndrome. Sci Rep.
2016;6:32353.
71. Chervenak FA, McCullough LB, Birnbach DJ.
Ethical issues in fetal surgery research. Best Pract
Res Clin Anaesthesiol. 2004;18(2):221–230.
72. Ashton CM, Wray NP, Jarman AF, et al. Eth-
ics and methods in surgical trials. J Med Ethics.
2009;35(9):579–583.
73. Rodrigues HC, Deprest J, v d Berg PP. When
referring physicians and researchers disagree on
equipoise: the TOTAL trial experience. Prenat
Diagn. 2011;31(6):589–594.
74. Verweij EJ. Ethics of involving pregnant women
in fetal therapy trials. In: Schmitz D, Clarke A,
Dondorp W, eds. The fetus as a patient. A con-
tested concept and its normative implications.
Abingdon/New York: Routledge: 2018:133–143.
75. Smajdor A. Ethical challenges in fetal surgery. J
Med Ethics. 2011;37(2):88–91.
76. Sheppard MK. Vulnerability, therapeutic mis-
conception and informed consent: is there a
need for special treatment of pregnant women
in fetus-regarding clinical trials? J Med Ethics.
2016;42(2):127–131.
77. Neri G, Opitz JM. Down syndrome: com-
ments and reflections on the 50th anniversary
of Lejeune’s discovery. Am J Med Genet A.
2009;149A(12):2647–2654.
78. Epstein CJ, Cox DR, Schonberg SA, et  al.
Recent developments in the prenatal diagnosis
of genetic diseases and birth defects. Annu Rev
Genet. 1983;17:49–83.
79. de Jong A, de Wert GM. Prenatal screening:
an ethical agenda for the near future. Bioethics.
2015;29(1):46–55.
80. Dondorp W, de Wert G, Bombard Y, et  al.
Non-invasive prenatal testing for aneuploidy
and beyond: challenges of responsible innova-
tion in prenatal screening. Eur J Hum Genet.
2015;23(11):1438–1450.
81. Evans MI, Johnson MP, Isada NB, et al. Coer-
cion for fetal therapy? In: Beller FK, Weir RF,
eds. The Beginning of Human Life. Dordrecht,
The Netherlands: Kluwer Academic Publishers;
1994:319–324.
82. American College of Obstetricians and Gyne-
cologists, Committee on Ethics. Commit-
tee Opinion No. 664: Refusal of Medically
Recommended Treatment During Pregnancy.
Obstet Gynecol. 2016;127(6):e175–e182.
83. American College of Obstetricians and Gyne-
cologists, Committee on Ethics. Committee
Opinion No 439, Informed Consent. Obstet
Gynecol. 2009;114(2 Pt 1):401–408.
84. Health Council of the Netherlands. Care for the
Unborn Child. Ethical and Legal Aspects of Fetal
Therapy. The Hague, The Netherlands: Health
Council of the Netherlands; 2009.
85. Lindenburg IT, van Klink JM, Smits-Wintjens
VE, et  al. Long-term neurodevelopmental
and cardiovascular outcome after intrauterine
transfusions for fetal anaemia: a review. Prenat
Diagn. 2013;33(9):815–822.
86. Bianchi DW. From prenatal genomic diagno-
sis to fetal personalized medicine: progress and
challenges. Nat Med. 2012;18(7):1041–1051.
87. Dondorp W, De Wert G. The ‘normalization’ of
prenatal screening. Prevention as prenatal benefi-
cence. In: Schmitz D, Clarke A, Dondorp W, eds.
The fetus as a patient. A contested concept and
its normative implications. Abingdon/New York:
Routledge: 2018:144–153.
88. Dorscheidt JH. Developments in legal and
medical practice regarding the unborn child
and the need to expand prenatal legal protec-
tion. Eur J Health Law. 2010;17(5):433–454.
89.  Den Hartogh G. Prenatale en postmortale
schade. Temporele grenzen van rechtssub-
jectiviteit (Dutch). Nederlands Juristenblad.
2010;645:778–783.
90. Robertson J. Protecting intended children
from harmful prenatal conduct. Am J Bioeth.
2016;16(2):14–15.
16
Principles of Screening
JOSHUA I. ROSENBLOOM AND GEORGE A. MACONES

KEY POINTS healthy and asymptomatic people. Prenatal screening should be

• Screening is the identification of unrecognised disease or
differentiated from prenatal diagnosis, in which a definitive diag-
defect found by testing an asymptomatic population.
nosis is made.
• Prenatal screening detects conditions that are deleterious to
Prenatal diagnosis first became available in the 1960s with the
the mother, fetus or both.
introduction of amniocentesis for Down syndrome. At that time,
• Prenatal screening allows for diagnostic testing and sub-
the only screen was maternal age; patients with advanced maternal
sequent pregnancy options, including termination of the
age were offered amniocentesis as a diagnostic test. In the 1970s,
pregnancy, preparation for the birth of a child with chronic or
the first maternal serum screen became available with the discov-
fatal illness or the use of advanced reproductive technology
ery of differences in maternal serum alpha-fetoprotein (AFP) with
to avoid carrying a fetus with the disease in question.
neural tube defects.2 In 1984, associations between reduced serum
• The validity of a screening test is described by its sensitivity,
AFP and Down syndrome were recognised, and screening with
specificity, and positive and negative predictive values.
this test was introduced shortly thereafter. Since then, the field has
• Likelihood ratios allow the calculation of posttest odds based
progressed rapidly to include advanced ultrasound and noninva-
on pretest odds and test results.
sive prenatal testing (NIPT) using cell-free fetal DNA.3 
• To set cutoffs for tests with continuous results, a receiver
operator characteristic curve can be used. Goal and Scope of Prenatal Screening
• Pursuing multiple tests in sequence raises specificity while
sacrificing sensitivity; conversely testing in parallel improves
The goal of prenatal screening is to detect conditions that can be
sensitivity at the expense of specificity.
deleterious to the mother, fetus or both. Prenatal screening includes
• An effective screening test must have excellent specificity
both maternal (and occasionally paternal) and fetal screening. In
and sensitivity, must be acceptable to the population, must
the course of routine prenatal care, mothers are screened for a
screen for a prevalent and clinically important disease, must
number of conditions such as sexually transmitted diseases and
offer potential for diagnostic testing and intervention in the
gestational diabetes that can affect both the mother and fetus.
natural course of the disease and must be cost effective.
Patients can be screened for carrying genetic diseases such as cys-
• Harms of screening include psychological distress and false-
tic fibrosis, haemoglobin S trait and Tay Sachs disease. Based on
positive results as well as harms resulting from subsequent
these results, further testing such as invasive fetal testing or pater-
diagnostic testing.
nal genetic testing can be recommended. Finally, fetal screening
• Patients often do not fully understand the testing being of-
focuses on screening the fetus for conditions such as aneuploidy
fered to them.
or congenital defects, and this can be accomplished either through
maternal blood testing or fetal ultrasound. The results of prenatal
screening and subsequent diagnostic testing may be used to make
a decision to terminate a pregnancy, to prepare for the birth of a
Definition and Brief History child with chronic or fatal illness or to use advanced reproductive
technology to avoid carrying a fetus with the disease in question in
Screening was first formally defined in 1951 by the United States a subsequent pregnancy. The purpose of this chapter is to explore
Commission of Chronic Illness as: the basic principles underlying all of these screening tests. 

. . . the presumptive identification of unrecognised disease or defect

by the application of tests, examinations, or other procedures which
can be applied rapidly. Screening tests sort out apparently well per-
sons who probably have a disease from those who probably do not.
A screening test is not intended to be diagnostic. Persons with posi-
tive or suspicious findings must be referred to their physicians for
diagnosis and necessary treatment.1
Put simply, the purpose of screening is to identify patients at
high risk for a specific condition within a group of apparently
Basic Parameters of Diagnostic and
Screening Tests
To be useful, a screening test must be valid. The validity of a
screening test is defined as its ability to distinguish between those
who have a disease and those who do not. This is further broken
down into sensitivity and specificity. Sensitivity is the ability of a
test to correctly identify those who have a disease. Specificity is the
ability of the test to identify those who do not have the disease in

149
150 SE C T I O N 6     Prenatal Screening and Diagnosis
TABLE
16.1 Possible Test Results
Does Not Nave Down
Has Down Syndrome Syndrome
Test positive True positive False positive
Test negative False negative True negative
  
TABLE Results From Down Syndrome Screen in 1000
16.2 Women: Prevalence of a Down Syndrome
Pregnancy Is 1 in 10
Has Down Syndrome Does Not Nave Down
(n = 100) Syndrome (n = 900)
Test positive 70 100
Test negative 30 800
  
question.4 Accuracy is another important aspect of a test and refers
to the ‘closeness of the measured value to the correct value’.5
Imagine a population of 1000 gravidas. One hundred of them
are carrying a fetus with Down syndrome, and the rest are not.
Table 16.1 illustrates the four possibilities of a screening test in
this population. Sensitivity is defined as True positives/(True posi-
tives + False negatives). Specificity is defined as True negatives/
(True negatives + False positives). If we put numbers into our table
(Table 16.2), we can calculate the sensitivity and specificity of the
screening test. In this case, the sensitivity is 70/(70 + 30) = 70%,
and the specificity is 800/(800 + 100) = 88.9%.
Predictive Values
The concepts of sensitivity and specificity are properties of the test
itself. The real clinical question is: ‘In this patient with a positive
test result, what is the chance that her fetus really has Down syn-
drome’? To answer this question, we must look at the test’s positive
predictive value (PPV) and negative predictive value (NPV).
Going back to Table 16.1, PPV is the chance that a patient
with a positive test result really has the disease in question: (True
positive)/(True positive + False positive), which in this case is 70/
(70 + 100) = 41%. The NPV is the chance that a patient with a
negative test result truly does not have the disease, or (True Nega-
tive)/(True Negative + False Negative) which in our example is
800/(800+30) = 96%.
Unlike sensitivity and specificity, which are test characteristics
without any relationship to the population under study, the posi-
tive and NPVs depend on the prevalence of the disease in a popu-
lation. In a rare disease, the specificity also greatly impacts the PPV
and NPV. Table 16.3 shows the same specificity and sensitivity as
shown in Table 16.2 but now in a population with a prevalence
of Down syndrome of only 5%. The sensitivity and specificity are
unchanged at 70% and 88.9%, respectively; however, the PPV is
now only 25%, and the NPV is now 98%.
A general rule of thumb regarding the relationship between
prevalence and predictive values is the following: given the same
sensitivity and specificity, as the prevalence rises, the PPV increases,
and the NPV decreases. Likewise, given the same sensitivity
TABLE Results From Down Syndrome Screen in 1000
16.3 Women: Prevalence of a Down Syndrome
Pregnancy Is 1 in 20
Does Not Nave
Has Down Down Syndrome (n
Syndrome (n = 50) = 950)
Test positive 35 105
Test negative 15 845
and specificity, as the prevalence of disease decreases, the PPV
decreases, and the NPV increases. Therefore an important prin-
ciple of screening is that the condition in question must have a
reasonably high prevalence so that the tests will have acceptable
PPV and NPV. This concept can be difficult for both patients and
clinicians to grasp.
A recent illustration of this principle is in regards to NIPT
with cell-free fetal DNA. Although the sensitivity and specific-
ity of the various commercially available tests are high, the PPV
is quite different depending upon the woman’s basic risk level,
particularly in regards to age. For example, the sensitivity and
specificity for detecting Down syndrome are greater than 99%,
but in a 25-year-old woman (in whom the prevalence of Down
syndrome is 0.7–0.9/1000), the PPV is 33%, but in a 40-year old
woman (baseline prevalence of Down syndrome, 8.5–13.7/1000),
the PPV is 87%.6 Because of this, there has been hesitancy on the
part of perinatologists to extend NIPT to ‘low-risk’ women.7
There is one particularly interesting aspect to the history of pre-
natal screening and diagnosis that deserves mention. Specifically,
in the history of research on prenatal screening and diagnosis, new
tests are usually evaluated in ‘high-risk’ populations first. When
acceptable sensitivities and specificities are seen in these initial high-
risk population studies, there is a call from the prenatal diagnosis
community for additional studies in low-risk populations to assess
the validity of the screening test in a lower risk population before
implementation in the general population. This line of thought is
incorrect. As was mentioned previously, sensitivity and specificity
are characteristics of the test itself, and those characteristics do not
change in a different population. The predictive values will change,
however, as the prevalence changes. Thus the constant call for vali-
dation of new tests in low-risk populations is improper because the
sensitivity and specificity will obviously be the same regardless of
the population tested. This has been borne out most recently in
studies of NIPT, in which, as expected, the sensitivity and specificity
were the same in low- and high-risk studies.8,9 This flawed thought
process has likely led to a significant delay in offering new screening
tests to the general population. When considering extending a new
screening test to a ‘low-risk’ population, the question should not be,
‘Will this test be valid in the low-risk population?’ but rather, ‘What
are the costs and benefits of using this test given that the PPV will
inherently be very low?’ 
Likelihood Ratios
A different method of analysing a screening test is the concept of
likelihood ratio (LR). A LR is the ‘ratio of the likelihood of that
result in someone with the disease to the likelihood of that result
in someone without the disease’.10 In other words, the LR repre-
sents the probability of having a certain test result in a patient with

disease divided by the probability of having that ratio in someone
without the disease.
A positive LR (LR+) is the ratio of the proportion of diseased
individuals who test positive (sensitivity) divided by the propor-
tion of nondiseased people who test positive (1 – specificity). The
negative LR (LR–) is the ratio of the proportion of diseased people
who test negative (1 – sensitivity) divided by the proportion of
nondiseased people who test negative (specificity). To use our
example from Table 16.2, the LR+ is (0.7/[1 – 0.889]) = 6.3. The
LR– is ([1 – 0.7]/0.889) = 0.33. The higher the LR+ and the lower
the LR–, the better the test. We can express the LR+ in words by
saying that a positive test result is about six times more likely to
be found in a patient whose fetus has Down syndrome than in a
patient whose fetus does not.
One advantage of LRs is that they can be applied to individual
patients to determine the likelihood of that specific patient having
the disease in question. To do this, the LR is multiplied by the pre-
test odds of the patient having the disease; this calculation yields
the posttest odds of the patient having the disease. Thus if the
pretest odds are high and the LR+ is high, the posttest odds will
be higher still, which is another way of saying that if the disease
is prevalent in a certain population, the PPV is higher than if the
disease is less prevalent, as discussed earlier.
We can illustrate the practical use of the LR with our Down
syndrome example. The posttest odds of the fetus having Down
syndrome will be equal to (pretest odds × LR+). We calculated the
LR+ above as 6.3. Odds are defined as (probability/[1 – probabil-
ity]). In our case, assume a pretest probability of Down syndrome
of 10%, which was the prevalence of the syndrome in our ficti-
tious population. Therefore the pretest odds are (0.1/0.9) = 0.11.
Thus our posttest odds are 0.11 × 6.3 = 0.693. We can covert the
posttest odds back into a probability using the formula (probabil-
ity = odds/[1 + odds]), which in our case is (P = .693/1.693) =
0.41. We can explain this to a patient by saying that before the test
there was a 10% chance of carrying a fetus with Down syndrome;
now that we have a positive result, the chance is 41%. However,
one of the benefits of LRs is that they can be applied sequentially.
If we took this example further, suppose the patient now under-
went a second, perhaps more invasive, screening test with an LR+
of 10 and had a positive result. In this situation, the pretest odds
for her were 0.693, so the posttests odds are now 0.693 × 10 =
6.93, which corresponds to a probability of 0.87 or 87%. This
sequential testing is the basis of the genetic sonogram in which
the baseline pretest odds (usually based on age) can be multiplied
by the LRs for any positive findings to compute a posttest odds.
However, it is important to also account for negative findings by
using the LR– as well. In our earlier example, assume that the
second test has a LR– of 0.3 and that the patient has a negative
result on this test. Therefore her posttest odds will be 0.693 × 0.3
= 0.21 for a posttest probability of 0.17 or 17%. It is important
to note that such sequential use of the LR assumes independence
of each test, which may not be a valid assumption depending on
the circumstances. Although the LR on its own can be a difficult
concept for a patient to grasp, applying the LR clinically can help
personalise results for an individual patient.  sensitivity and specificity and is represented by the upper left cor-
Receiver Operator Characteristic Curves
The previous discussion is predicated on having a test that gives
a binary response (test positive or test negative). However, the
results of most tests are not binary but rather are continuous, and
CHAPTER 16  Principles of Screening 151
Ideal diagnostic test
1.0
B
A
0.75
Sensitivity
0.5
Useless
diagnostic
test
0.25 C
0.0
0.0 0.25 0.5 0.75 1.0
1–Specificity
• Fig. 16.1  A sample receiver operating characteristic curve. (From Board-
man LA, Peipert JF. Screening and diagnostic testing. Clin Obstet Gynecol
41(2):267–274, 1998.)
therefore the test designers must decide the appropriate cut points
to define positive and negative results. The placement of these cut
points will greatly influence the test performance, specifically the
sensitivity and specificity of the test.
A classic example of cut points in prenatal care is testing for
gestational diabetes. The usual approach in the United States is
a screening test with a serum glucose level obtained 1 hour after
the patient drinks a 50-g glucose load (glucose challenge test
(GCT)). Various studies have attempted to determine the opti-
mal cut point to differentiate between those who screen positive
and require a confirmatory diagnostic glucose tolerance test ver-
sus those who screen negative. In a recent paper, Rebarber and
colleagues11 investigated the sensitivity and specificity of a cutoff
of 130 mg/dL, 135 mg/dL or 140 mg/dL in diagnosing patients
with GDM in twin gestations and found that a GCT cutoff of
135 mg/dL or greater was 100% sensitive and 76.4% specific for
gestational diabetes, and a cutoff of 130 mg/dL or greater was
still 100% sensitive but only 69.8% specific. On the other hand,
a cutoff of 140 mg/dL or greater was only 93.5% sensitive, but it
was 81.5% specific.11 As this illustrates, the assignment of a cutoff
in any continuous scale determines the sensitivity and specificity
of the test. Furthermore, there is usually a tradeoff between the
two values: a higher specificity is coupled with a lower sensitivity.
To maximise sensitivity and specificity as much as possible, the
designers of a test may use a receiver operator characteristic (ROC)
curve to find the ideal cutoff for a diagnostic test. To do this, the
sensitivity for multiple cut points is plotted on the y-axis of a graph
as a function of 1 – specificity on the x-axis. An example of an ROC
curve is shown in Fig. 16.1. A number of points can be made about
the figure. First, as described previously, the y-axis is the sensitivity,
and the x-axis is 1 – specificity. The ideal diagnostic test maximises
ner, where the sensitivity and specificity are both 100%. The dashed
diagonal line represents a ‘useless’ diagnostic test because any gain in
specificity is directly lost in sensitivity; for example, where sensitiv-
ity is 100%, specificity is 0. Another way to understand this ‘useless’
line is that it represents a test with 50% sensitivity and 50% specific-
ity, which is no better than flipping a coin.
152 SE C T I O N 6     Prenatal Screening and Diagnosis
Although the ideal cutoff point is the upper left corner,
in real life, this cannot be achieved. Therefore the best cutoff
for this hypothetical test is the one at point A or inflection
point, which is closest to this ideal point. This point has the
maximum sensitivity and specificity achievable in the test. It is
worth noting that depending on the disease condition, a dif-
ferent point may be preferable. For instance, in a fatal but cur-
able disease, it may be prudent to maximise sensitivity at the
cost of specificity so no cases are missed (as few false negatives
as possible). In Fig. 16.1, this is seen at point B, which has a
sensitivity of nearly 100% but a specificity of only 25%. Point
C represents a cutoff value with high specificity but only 25%
sensitivity.
Another important aspect of the ROC curve is the area under
the curve (AUC). The AUC represents the overall accuracy of the
test. For example, an AUC of 1.0 represents a perfect test, and if
the AUC is 0.5, then the test is no better than chance. The AUC
allows comparison of ROC curves of different tests to determine
which test is most accurate.12  10. Case-finding should be a continuing process and not a ‘once
Approaches to Screening
When multiple screening tests for the same condition are avail-
able, the clinician is faced with the decision on how many, and
in what order, to use the relevant tests. In the area of prena-
tal screening, this issue is found especially with screening for
Down syndrome. Clinicians are faced with a battery of options
regarding first trimester and second trimester tests, which can be
used either sequentially or in parallel. In contingent sequential
screening, a first trimester test is used. The results can be posi-
tive, negative or intermediate. Patients with intermediate results
continue on to have second trimester screening.13 It can be
shown that this sequential screening strategy results in a loss of
net sensitivity but an increase in net specificity; that is, there are
fewer false-positive results than with either test alone.4 In con-
trast, in parallel screening, in which two tests are used at once,
there is a loss of specificity but a gain in sensitivity.4 Another
approach to screening is to use multiple independent screening
tests and interpret each one separately. For instance, a first tri-
mester screen can be ordered and interpreted, and then a second
trimester quadruple screen could be ordered and interpreted
entirely independently. Depending on whether the patient pro-
ceeds with diagnostic testing after a positive first trimester result,
this approach will alter the overall sensitivity and specificity. For
example, deferring diagnostic testing after a positive first trimes-
ter screen until the second trimester results are evaluated could
reduce sensitivity because true positives in the first trimester may
become false negatives in the second trimester screen. Alterna-
tively, for patients with unaffected pregnancies, this approach
will lead to a lower PPV of second trimester screening because
the pretest risk is reduced by the negative first trimester results.
For these reasons, independent screening is not recommended in
most circumstances.  A common way of assessing cost in medicine is through the
Characteristics of a Good Screening Test
Now that we have defined the basic parameters of diagnostic and
screening tests, we can discuss the characteristics of a good screen-
ing test. For instance, to take an extreme example, what makes
nuchal translucency a better screening test for aneuploidy com-
pared with maternal age?
In a classic paper from 1968, The World Health Organization
laid out 10 principles of a good screening test:14
1. The condition sought should be an important health prob-
lem.
2. There should be an accepted treatment for patients with rec-
ognised disease.
3. Facilities for diagnosis and treatment should be available.
4. There should be a recognisable latent or early symptomatic
stage.
5. There should be a suitable test or examination.
6. The test should be acceptable to the population.
7. The natural history of the condition, including development
from latent to declared disease, should be adequately under-
stood.
8. There should be an agreed policy on whom to treat as patients.
9. The cost of case finding (including diagnosis and treatment
of patients diagnosed) should be economically balanced in
relation to possible expenditure on medical care as a whole.
and for all’ project.
Not all of these principles are applicable to the specific case
of prenatal screening, but most are. Consider screening for aneu-
ploidy. This is considered to be a major public health issue which
burdens families and affected individuals (criterion 1). Although
there is no treatment, further diagnostic testing with amniocen-
tesis or chorionic villus sampling can be pursued, termination of
pregnancy is an option and these are widely available to patients
(criteria 2 and 3). Criterion 5 above relates to the statistical prop-
erties of screening tests discussed earlier; that is, there should be a
test with adequate sensitivity and specificity. The tests (ultrasound
and blood tests) are generally acceptable to obstetric patients (cri-
terion 6). Criteria 4, 7 and 10 relate more to chronic diseases and
are less applicable here. There is general agreement that patients
with aneuploidy should be offered diagnosis and treatment (crite-
rion 8). We will discuss cost effectiveness more later (criterion 9).
Returning to our question, age alone as well as nuchal trans-
lucency fit all of the criteria except, crucially, number 5. For pur-
poses of screening, a suitable test is one with a high sensitivity and
a reasonably high specificity. This is not the case with age alone,
but it is with more advanced screening modalities for aneuploidy.
It is also important that the disease have a relatively high preva-
lence to ensure that the PPV of the test is acceptable. 
Cost Effectiveness of Prenatal Screening
Another important consideration in prenatal screening is cost.
When thinking about cost, we have to think from the perspective
of what is best for our society rather than thinking narrowly about
our own clinical interests. Put another way, every million dollars
spent trying to diagnose every single case of a rare disorder such as
Pena Shokeir syndrome will be 1 million dollars less that can be
used for free school breakfasts for underserved children.
use of cost-effectiveness analysis. These analyses can be difficult to
undertake because of the inherent difficulties in measuring both
costs and effectiveness. Although the actual cost of a particular
serum screening test is not hard to measure, the overall cost of test-
ing incorporates the comprehensive costs, including costs associ-
ated with the test, costs of genetic counselling, costs of subsequent
or confirmatory testing and costs of downstream interventions.
Furthermore, effectiveness must be measured in some way, which

can be challenging. For example, Odibo and colleagues15 investi-
gated the cost effectiveness of nine different strategies for Down
syndrome screening, finding that integrated serum screening was
most cost effective. On the other hand, some diseases are so rare,
or the screening so expensive, that screening may not be cost effec-
tive. For instance, although universal screening for spinal muscu-
lar atrophy (SMA) is recommended by the American Colleges of
Obstetrics and Gynecology and of Medical Genetics and Genom-
ics, a recent cost-effectiveness study found that universal screening
for SMA costs $4.9 million per quality-adjusted life year and costs
$5.0 million per case averted.16-18  As with any medical procedure, prenatal screening requires
Risks and Benefits of Screening
Prenatal screening offers benefits to prospective mothers and, less
often, to the fetus. For the mother, prenatal screening allows the
mother to know if her fetus has a certain condition, and if the
fetus does, to prepare for the birth of the child, if required to
deliver in a hospital with appropriate neonatal facilities or to ter-
minate her pregnancy. Fetal benefits of screening are less common,
although in the case of certain conditions such as fetal anaemia,
in utero treatment is available.19 However, there are many risks
associated with screening as well.
As discussed earlier, every screening test has false positives and
false negatives. False positives may lead to invasive diagnostic pro-
cedures, which have their own risks to the mother and fetus. False
negatives give false reassurance to a mother. Multiple studies have
shown that positive results in general, whether false or true, cause
increased anxiety and distress to parents.20-22 Therefore it is of the
utmost importance that screening tests with excellent sensitivity
and specificity be chosen.
An interesting example of the harms of screening relates to soft
markers for Down syndrome. Soft markers are sonographic signs
that have little intrinsic significance but that are associated with
aneuploidy.23 Examples include echogenic intracardiac focus, cho-
roid plexus cysts and echogenic bowel. Although soft markers are
associated with aneuploidy, the majority of fetuses with soft mark-
ers will have a normal karyotype. A recent study demonstrated
that mother–infant bonding was negatively affected by the finding
CHAPTER 16  Principles of Screening 153
of soft markers prenatally, even when the infant had no actual
abnormalities. Mothers of fetuses with soft markers had higher
infant avoidance and lower maternal sensitivity.24 This study illus-
trates that even benign findings in prenatal screening may have
unforeseen consequences. 
Informed Consent and Counselling of
Patients
informed consent. The concept of informed consent is predi-
cated on two components: comprehension and free consent.25
However, despite the ubiquity of prenatal screening, studies have
shown barriers to informed consent for this testing.26 These bar-
riers relate largely to knowledge deficits regarding the conditions
being screened for, the technology used to screen and the implica-
tions of screening results. In addition, patients often do not have a
good understanding of the statistical concepts underlying screen-
ing and can be falsely reassured. For instance, in a study from
the Netherlands in 2006, only 51% of patients made informed
choices regarding prenatal screening.27 
Conclusions
In this chapter, we have discussed principles of screening. We have
shown that an effective screening test must have excellent specificity
and sensitivity, must be acceptable to the population, must screen
for a prevalent and clinically important disease, must offer poten-
tial for diagnostic testing and intervention in the natural course of
the disease and must be cost effective. We have discussed the risks
of screening tests, including false negatives and false positives, psy-
chological distress, information overload and the fact that many
patients do not give true informed consent for prenatal screening.
To understand the prenatal screening tests available, it is important
to have a basic grasp on the principles outlined in this chapter.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 10. Fletcher RH, Fletcher SW. Clinical epidemiol-
ogy. 4th ed. Baltimore: Lippincott Williams &
screening for fetal abnormality: a prospective
study. Prenat Diagn. 1992;12(3):205–214.
Wilkins; 2005. 21. Kaasen A, Helbig A, Malt UF, et  al. Pater-
1. Commission on Chronic Illness. Chronic ill-
11. Rebarber A, Dolin C, Fields JC, et al. Screening nal psychological response after ultrasono-
ness in the United States. In: Prevention of
approach for gestational diabetes in twin preg- graphic detection of structural fetal anomalies
chronic illness. Vol. I. Cambridge, MA: Har-
nancies. Am J Obstet Gynecol. 2014;211(6):639. with a comparison to maternal response: a
vard University Press; 1957:1–45. From Mora-
e1–e5. cohort study. BMC Pregnancy Childbirth.
bia A, Zhang FF. History of medical screening:
12. Goetzinger KR, Odibo AO. Statistical analysis 2013;12(13):147.
from concepts to action. Postgrad Med J.
and interpretation of prenatal diagnostic imag- 22. Leithner K, Maar A, Fischer-Kern M, et  al.
80(946):463–469, 2004.
ing studies, Part 1: evaluating the efficiency of Affective state of women following a prenatal
2. Cuckle H, Maymon R. Development of pre-
screening and diagnostic tests. J Ultrasound diagnosis: predictors of a negative psycho-
natal screening-A historical overview. Semin
Med. 2011;30(8):1121–1127. logical outcome. Ultrasound Obstet Gynecol.
Perinatol. 2016;40(1):12–22.
13. Wright D, Bradbury I, Benn P, et al. Contin- 2004;23(3):240–246.
3. Palomaki GE. Screening for Down’s syndrome.
gent screening for Down syndrome is an effi- 23. Ahman A, Axelsson O, Maras G, et al. Ultraso-
N Engl J Med. 1995;333(8):532.
cient alternative to non-disclosure sequential nographic fetal soft markers in a low-risk pop-
4. Gordis L. Epidemiology. 5th ed. Philadelphia:
screening. Prenat Diagn. 2004;24(10):762–766. ulation: prevalence, association with trisomies
Saunders; 2014.
14. Wilson JMG, Jungner G. Principles and prac- and invasive tests. Acta Obstet Gynecol Scand.
5. Trajković G. Measurement: accuracy and pre-
tice of screening for disease. Geneva, Switzerland: 2014;93(4):367–373.
cision, reliability and validity. In: Kirch W,
World Health Organization; 1968. 24. Viaux-Savelon S, Dommergues M, Rosenblum
ed. Encyclopedia of public health. New York:
15. Odibo AO, Stamilio DM, Nelson DB, et al. A O, et  al. Prenatal ultrasound screening: false
Springer; 2008.
cost-effectiveness analysis of prenatal screening positive soft markers may alter maternal repre-
6. Hook EB. Rates of chromosome abnormali-
strategies for Down syndrome. Obstet Gynecol. sentations and mother-infant interaction. PLoS
ties at different maternal ages. Obstet Gynecol.
2005;106(3):562–568. One. 2012;7(1):e30935.
1981;58(3):282–285.
16. Prior TW; Professional Practice and Guidelines 25. ACOG Committee on Ethics. ACOG Com-
7. Committee Opinion No. 640: cell-free DNA
Committee. Carrier screening for spinal muscu- mittee Opinion No. 439: informed consent.
screening for fetal aneuploidy. Obstet Gynecol.
lar atrophy. Genet Med. 2008;10(11):840–842. Obstet Gynecol. 2009;114(2 Pt 1):401–408.
2015;126(3):e31–e37.
17. Little SE, Janakiraman V, Kaimal A, et  al. 26. Green JM, Hewison J, Bekker HL, et  al.
8. Gil MM, Quezada MS, Revello R, et  al.
The cost-effectiveness of prenatal screening for Psychosocial aspects of genetic screening of
Analysis of cell-free DNA in maternal blood
spinal muscular atrophy. Am J Obstet Gynecol. pregnant women and newborns: a systematic
in screening for fetal aneuploidies: updated
2010;202(3):253.e1–e7. review. Health Technol Assess. 2004;8(33). iii,
meta-analysis. Ultrasound Obstet Gynecol.
18. ACOG Committee on Genetics. ACOG com- ix–x, 1–109.
2015;45(3):249–266.
mittee opinion No. 432: spinal muscular atro- 27. van den Berg M, Timmermans DR, ten Kate
9. Nicolaides KH, Syngelaki A, Ashoor G, et  al.
phy. Obstet Gynecol. 2009;113(5):1194–1196. LP, et al. Informed decision making in the con-
Noninvasive prenatal testing for fetal trisomies
19. Gates EA. Ethical considerations in prenatal text of prenatal screening. Patient Educ Couns.
in a routinely screened first-trimester popula-
diagnosis. West J Med. 1993;159(3):391–395. 2006;63(1-2):110–117.
tion. Am J Obstet Gynecol. 2012;207(5):374.
20. Marteau TM, Cook R, Kidd J, et al. The psycho-
e1–e6.
logical effects of false-positive results in prenatal

153.e1
17
Conveying Information About Screening
and Diagnosis
JENNY HEWISON, LOUISE D. BRYANT
AND JANE FISHER
KEY POINTS
• Good practice in information giving is essential as choosing
to have a prenatal screening test can have far-reaching conse-
quences.
• To be of good quality, information must be up to date and
evidence-based. It should as a minimum include the purpose
of the test; information about the tested-for condition(s),
what the test procedure involves, any risks associated with
the test, implications of the possible test results and the dif-
ference between screening and diagnosis.
• Information-giving alone is not sufficient to ensure a deci-
sion based on personal values. Clinicians can be required to
actively support parental decision making.
• Each significant change in testing technology brings new
informational challenges. The limitations of new technologies
are not always apparent to women (e.g., the fact that those
who receive a positive noninvasive prenatal testing result for
a chromosomal anomaly will still need invasive testing for a
definitive diagnosis).
• Regardless of gestation or the severity of the condition, the
emotional impact of a diagnosis is usually profound. Clini-
cians play an important role at this highly difficult time, and a
positive experience of care makes an important contribution
to how parents cope.
This chapter aims to provide clinicians with the key points rel-
evant to high-quality communication and information provi-
sion in prenatal screening and diagnostic practice. Specifically,
it will:
• Define informed decision making and explain why good
practice in information giving is such an important part
of delivering high-quality care within the prenatal testing
pathway.
• Identify the essential components of good-quality informa-
tion based on research with women and their partners. Dis-
cuss the importance of providing accurate, up-to-date and
balanced information about the conditions being tested for.
• Explain why information giving alone is not sufficient to
ensure an informed decision takes place and the role of clini-
cians in helping make choices that are relevant to their per-
sonal values and circumstances.
154
• Discuss the specific issues associated with different testing
technologies, including the reason why noninvasive prenatal
testing (NIPT) is unlikely to replace combined screening or
invasive diagnostic testing in the foreseeable future.
• Demonstrate that the psychological impact of a prenatal diag-
nosis on parents is significant and long-lasting. Highlight the
importance of an individualised approach to delivering difficult
news.
• Set out reasons why after a prenatal diagnosis, parents need
well-coordinated care and clear information about all their
pregnancy management options and support following their
decision.
Why Good Practice in Information-Giving Is
so Important
Choosing to have a prenatal screening test can have far-reaching
consequences. High levels of anxiety are frequently associated with
receiving a positive screening result–anxiety that may dissipate but
is never forgotten.1-3 Most prenatal diagnostic testing procedures
are associated with a risk for miscarriage, and the period spent
waiting for the results is often characterised by acute anxiety.4
This anxiety is partly about pregnancy loss, but, especially if the
test was carried out after a positive screening result, worry about
fetal abnormality.5 In most cases, the only intervention on offer
after a diagnosis of anomaly is termination of pregnancy. For most
women, choosing to end a wanted pregnancy is painful, and for
some women, it is emotionally devastating.6 If the limitations of
screening are not understood, parents may find it harder to adjust
if a disabled child is subsequently born.7 For all these reasons, pre-
natal screening and testing decisions should reflect the values of
the individual woman. There are a number of definitions of what
constitutes an informed decision, however, the following works
well within the prenatal testing context:
An informed decision occurs when an individual understands the
nature of the disease or condition being addressed; understands the
clinical service and its likely consequences, including risks, limita-
tions, benefits, alternatives, and uncertainties; has considered [her]
preferences as appropriate; has participated in decision making at
a personally desirable level; and makes a decision consistent with
[her] preferences and values.8 
 Chapter 17  Conveying Information About Screening and Diagnosis
The Information Needs of Women and Their
Partners
First and foremost, women want timely, up-to-date, accurate
information about testing delivered by a source they trust, in a
format that is understandable.1 There are a number of informa-
tion components that are considered essential to convey and as a
minimum information (verbal or written) given before the offer
of any prenatal test should make clear:
• The purpose of the test (e.g., what it is screening for and in
certain circumstances what it does not screen for)
• Information about the tested-for condition(s)
• What the procedures for testing involve
• Any maternal or fetal risks associated with the test
• The implications of the possible test results, including anxiety,
uncertain results, decisions about further tests and termination
of pregnancy
• The difference between screening and diagnosis
Individual women will have different information preferences
and requirements, and an important factor in women’s decisions
about prenatal diagnosis and termination of pregnancy is their
perception about the quality of life for, and with, a child with
a tested-for condition.9 A particular concern is whether or not a
child would experience pain or other forms of ‘suffering’ as a result
of the condition or its medical treatment.10 Knowledge of even
relatively common conditions such as Down syndrome has been
shown to be low because many people have little personal experi-
ence of interacting with disabled people and their families.11,12
It is essential, therefore, that balanced information based on the
lived experience of families and individuals with a tested-for con-
dition is made available throughout the testing pathway. Public
Health England’s Screening Tests for You and Your Baby booklet,
which is given to all pregnant women in England and Wales, has
addressed this need to some degree, providing information on the
main conditions covered by the Fetal Anomaly Screening Pro-
gramme: sickle cell and thalassaemia, Down syndrome, Edwards
syndrome and Patau syndrome, developed in consultation with
parent support organisations.
It has sometimes been assumed that certain ethnic or religious
groups, particularly Muslim women, would not use prenatal test-
ing or terminate an affected pregnancy.13 However, although reli-
gion is a factor in reproductive decision making for many people,
it is not necessarily the most important one. Women across a
range of ethnicities and religions make testing and termination
decisions based on their own values and beliefs, which are influ-
enced by personal experiences as well as information provided by
clinicians.14 It is crucial to recognise individual diversity in beliefs
and preferences to ensure equality of access to prenatal testing
services. This includes access to information; for example, in the
United Kingdom, the Screening Tests for You and Your Baby booklet
has been translated from English into 12 other languages.  to make difficult decisions more manageable.
Is Information-Giving Sufficient for Informed
Decision Making?
Good-quality information is necessary for an informed decision,
but in many cases, it will not be sufficient. In particular, women
who have lower levels of literacy or for whom English is not their
first language are not well served by written information. Not all
women wish to make choices ‘on their own’ but instead value
155
professional input to the decision-making process.14,15 However,
not all clinicians are comfortable with helping a woman make the
decision because of fears of being directive and so prefer to act
as information providers only.16 Helping a woman and her part-
ner to understand, for example, the limitations of screening and
facilitating a discussion about potential use of test results should
be considered an integral part of the role of clinicians delivering
prenatal care. 
Conveying Information About Risk
Practitioners are often concerned about how to convey risk infor-
mation so that women can use this information to understand test
results and make further informed decisions. However, the math-
ematical concepts of population screening that underlie screening
tests are not ones that most people deal with during their everyday
lives. Research shows that many women struggle to understand
risk in relation to prenatal tests, as do some clinicians.1,17 In some
countries, including the United Kingdom, the offer of further
testing is made only to women whose screening risk exceeds a
predetermined cutoff value (currently 1 in 150), which reflects
policy decisions about the optimum tradeoff between detection
and safety for the screened population as a whole. The rationale
behind the choosing of a specific risk cutoff for offering further
(potentially invasive) tests is probably understood by only a tiny
percentage of those involved.
There is some evidence that understanding of residual risk in
‘screen negatives’ can be enhanced by presenting screening test
results in numbers (e.g., 1 in 800) rather than in nonspecific word
terms, such as a ‘low-risk’ result.18 Some have also recommended
providing multiple formats of the same information, for exam-
ple, a 1 per 100 chance of an affected pregnancy should also be
presented as a 1% chance of the baby being affected and a 99%
chance that the baby will not be affected.19 Providing alternative
framings of the same risk information may also help to reduce
certain decision-making biases associated with ‘negative’ and ‘posi-
tive’ presentations.
Severely anxious reactions to screen positive results have often
been attributed to women misunderstanding the meaning of a
screening test risk result.20 This has partly driven the search to
find the optimal way to deliver risk information so that women
can understand risk appropriately. The assumption has been
that correct understanding of risk will reduce anxiety and ensure
that women realise the residual risk inherent in a screen negative
result. However, although conveying risk information appropri-
ately is important, understanding risk may not necessarily alle-
viate anxiety associated with screening results. Women need to
be informed about the potential for unwanted, inconclusive or
unexpected results, even if information alone does not adequately
prepare them. ‘Misunderstandings’, as judged by clinicians, may
also reflect a need for people to simplify risk-related information
Women offered an invasive test after a screen positive result
can, of course, decline it, and the lower the individual woman’s
screening risk, the more likely she is to decline further testing.21
This suggests that even if women do not understand the math-
ematics of screening, they do use risk information in a more basic
sense when making decisions about invasive testing. Some have
interpreted this as showing that women consider safety to be more
important than detection, but such a simple conclusion would
be unjustified. The views of women not currently offered inva-
sive testing because their screening risk is considered too low to
156 SE C T I O N 6     Prenatal Screening and Diagnosis
justify the associated miscarriage risk must also be examined if a
full picture is to be obtained. A number of hypothetical studies
have shown ‘unmet demand’ for diagnostic testing amongst such
women, and this has been supported more by studies of actual
uptake rates.21-24 Therefore, for some women in some circum-
stances, detection is more important than a miscarriage risk of
around 1%. The logical conclusion of this body of work is that
all women, not just those deemed to be at high risk, should be
given information about their individual risk and be supported in
deciding whether or not to have further tests, including an inva-
sive test. In the United States, for example, it is recommended that
all women are offered the option of an invasive test as well as the
option of doing nothing or having screening.25
Technology-Specific Considerations
Although many of the information needs associated with screen-
ing and diagnostic tests apply across all protocols, advances
in technology have modified some of these and added others.
Increasingly, for many women and their partners, the choice is not
only whether or not to have a screening test but also ‘which test
to have’. Practitioners are familiar with the idea that women must
be helped to find their own balance between safety and certainty,
but that simple tension has been superseded by a much more com-
plicated set of costs and benefits arising from the proliferation of
different testing protocols.
Some of the ways in which screening protocols differ are the
result of intentional additions to the options being made avail-
able to women, such as offering screening for different conditions,
and others reflect different ways of trading off safety and certainty,
such as introducing a noninvasive option after an initial screen
positive result. Both are relevant to decision making, adding to the
debate about how much information should be provided about
screening tests and what aspects of information are most impor-
tant to convey. The most salient dimensions on which current
screening protocols differ and what relevance these may have for
information provision are now discussed.  a great deal to understanding how NIPT may work in UK clinical
Noninvasive Prenatal Testing Using Cell-Free
DNA
This important new technology changes both the means and the
ends of screening–the means because the safety versus certainty
tradeoff is altered and the ends because the range of potential
tested-for conditions could more easily be extended.
It was initially hoped that NIPT would completely resolve
the safety versus certainty dilemma, but at least in respect of the
most common prenatal testing programmes, that simple, easy-to-
explain goal has not been achieved. In some circumstances, the
information-giving challenge may even have been increased. In
part, this is because of the oversimplified and misleading media
accounts which need rebuttal; however, it is also because the
degree of certainty about the meaning of an NIPT result is not
constant and varies in a number of ways which have important
practical implications. Perhaps most important, and despite the
original labelling of the technology as noninvasive prenatal diag-
nosis (NIPD), the sampled DNA does not come from the fetus as
was first supposed (cell-free fetal DNA [cffDNA]) but from the
genetically distinct placenta (cell free DNA [cfDNA]). As a result
of this and other factors, such as a vanishing twin, or maternal
cancer, NIPT does have a small but nonzero false-positive rate.
This means that women with positive NIPT results still need
to undergo an invasive diagnostic procedure–with its attendant
miscarriage risk–to obtain definitive information that the baby
is affected. All women contemplating NIPT need to know this,
but the awareness is particularly important for those who are con-
sidering terminating a pregnancy after a positive diagnosis. The
likelihood that a positive test result is a false positive depends on
the condition (test performance being better for Down syndrome
than for the other trisomies) and on the woman’s prior risk status,
so it is important to know whether a positive NIPT result came
from a test delivered in a private health care setting to a low-risk
woman or from a test taken on a contingent basis as part of a
screening programme. For women with positive NIPT results, the
old safety versus certainty dilemma remains. Work is needed to
find out how best to provide feedback and further information in
these circumstances, in particular, how should the residual degree
of uncertainty be conveyed, without creating false expectations
in either direction? Clinicians used to conveying results from less
accurate screening tests will need help in striking the right balance.
The uncertainty about the meaning of a negative NIPT result
for Down syndrome is much less than a positive result.26,27 However,
it must always be remembered that although the detection rate is
very high, it is not 100%, and there will still be a small number
of missed cases. Clinicians need to know that this apparent asym-
metry exists (although fewer may want to know why) to enable
them to give the appropriate degree of reassurance to women.
It is through this ‘ruling out’ mechanism that the real benefit of
NIPT for Down syndrome lies because it reduces the numbers of
women having to face the invasive testing dilemma. The picture
is less clear for other chromosomal conditions detected by current
screening programmes, particularly when test failures or incon-
clusive results from NIPT are taken into account.27,28 The need
for more comprehensive performance data has been identified.29
Noninvasive prenatal testing is widely available internationally.
In the United Kingdom, it can readily be obtained privately, and
starting in 2018, it is also available through the National Health
Service. Two substantial applied research projects have contributed
practice,30,31 but decisions have yet to be made about where in the
current test pathway NIPT might be offered, about risk thresholds
and about the choices available to women at each stage. Efforts
to refine pathway components also continue.32,33 The most likely
plan for England is that NIPT would be offered as a second-line,
or contingent, test to women identified as at increased risk by cur-
rent methods of combined testing, using the same 1 in 150 risk
threshold as at present.34 As described earlier, NIPT used in a con-
tingent role can provide reassurance to many women without the
need for invasive testing–it can reduce, although not eliminate,
the programme’s false-positive rate. However, it also follows logi-
cally that unless initial risk thresholds are changed, contingent use
of NIPT cannot identify any affected pregnancies ‘missed’ by the
first-round screen. Therefore, in test performance terms, this form
of contingent NIPT cannot increase detection rates because screen
negative women would not be offered the contingent test. If, how-
ever, the availability of contingent NIPT leads to an increase in
the numbers of women choosing to be tested, then at programme
level, more affected pregnancies may be identified. Pathways of
care where the options available are contingent on previous events
can be confusing, and achieving the right balance between techni-
cal correctness and oversimplification is no easy task.
Contingent use of NIPT will also increase the length of time
taken for a woman who tests positive on the initial screen and
again on NIPT to obtain a definitive result: all women need to
 Chapter 17  Conveying Information About Screening and Diagnosis
understand this and to understand that NIPT has a failure rate of
about 3%.28,30 However, the extra time is likely to be particularly
salient for some groups of women. In Islam, for example, termi-
nation of pregnancy for a condition such as Down syndrome is
regarded by religious authorities as permissible but only before
‘breathing the soul’ takes place at 120 days of gestation.35 Muslim
women, like all women, hold their own individual opinions about
termination for congenital conditions, but many do (or would)
choose termination if circumstances allow.
The reproductive options offered by the current first trimester
pathway are known to be valued by many women, and plans for
introducing NIPT have specifically taken that into account. Even
within the planned timetable, however, it is recognised that some
women will seek early certainty,30,31 so in a future UK programme,
invasive diagnostic testing would also be offered as an alternative
to NIPT to women screening positive on the combined test. Some
women will be clear which of the available options is right for
them, but others may need support in choosing between them.
In some respects, NIPT will add to the requirement to develop
information that meets the needs of different women. Amongst
those considering screening but previously deterred by miscarriage
risk will be women who are clear they would like information
about the baby if it could be obtained safely. There will also be
women who have in effect ‘sheltered’36 behind the miscarriage
risk and not asked themselves what they would do if safer means
of obtaining information were available. Concerns have been
voiced in terms of needing to ensure that the option of ‘just hav-
ing another blood test’ does not create pressures on women to be
tested or more generally undermine informed choice.37 There is
also concern that the potential for NIPT is to screen for other
chromosomal anomalies, microdeletions or duplications and
eventually the full fetal genome will result in generating incidental
findings and identify variants of unknown significance will add to
the information burden placed on women.38
The more complex the pathway and the more that provision
is made for individual values and preferences, the greater is the
need for information to support women’s decision making. There
is evidence from research39 that NIPT is welcomed by women,
but that a significant proportion of those at elevated risk will pre-
fer invasive testing.29,30 If contingent NIPT is introduced in the
National Health Service, the impact on women’s decisions across
the pathway will not be known for some time; behind the figures,
dilemmas for some will decrease, but they may increase for others.  The United Kingdom also has an antenatal screening pro-
Ultrasound in Screening
The increasing use of ultrasound technology in screening brings
with it additional challenges in relation to information provision.
Scanning is very popular with women and their partners because
of the opportunity it affords of ‘seeing the baby’ and in the event
most scans are reassuring experiences that parents remember with
pleasure. In the United Kingdom, scans are offered at two points
in the screening programme: the early pregnancy scan at 11 to 14
weeks (the nuchal translucency [NT] scan), as one component
of combined testing in the first trimester, and the fetal anomaly
scan in the second trimester (18+0-20+6 weeks). In both cases,
the main information challenge is to convey that the true purpose
of scanning is to look for signs of problems. Women undergoing
ultrasound testing need to be prepared for such an outcome even
though the warning may be unwelcome and the information itself
a possible cause of disquiet. For the early pregnancy scan, the sec-
ond challenge is to convey that the stand-alone value of the NT
157
measurement is limited; its value lies in its contribution to the
combined test, so it is important that women who want screening
complete both parts of the combined test protocol.
For the fetal anomaly scan, the challenge is how to convey that,
unlike screening for a specific condition, scanning is a very open-
ended investigation with many different possible outcomes and
that the problems identified can be serious or minor, common or
rare. It also needs to be explained that although a scan can some-
times function as a diagnostic test and identify some problems
with certainty, in many other instances, the scan only functions
as a screening test, and furthermore, probably invasive, diagnostic
testing may be indicated. The more skilled the sonographer, the
more likely it becomes that minor variations will be identified.
Once called ‘soft markers’, these used to be routinely reported to
women, causing unnecessary anxiety–some of it long lasting–and
leading to an inappropriate use of invasive testing.40 In the UK
screening programme, the policy now is not to report or act upon
the presence of any marker not previously established as of prog-
nostic significance. 
Screening for Multiple Conditions
A related but distinct issue from ultrasound scanning is how to
ensure informed choice when screening (of any form) is being
explicitly offered for multiple conditions. It cannot be assumed
that an individual woman’s views about testing and termination
will be the same for all conditions,41 so the information provided
must as far as possible support separate decision making for each
condition.
In the United Kingdom, the Down’s Syndrome Screening Pro-
gramme has recently been extended to include the offer of first
trimester screening for two other trisomies, Edwards and Patau
syndromes. The evidence suggests that many people see a clear
distinction between the different conditions, the disabling effects
of Edwards and Patau being regarded as much more serious and
extensive. Some people will, of course, not want screening for any
of these conditions, and some will want screening for all three,
but it also seems likely that a proportion of those who see no rea-
son to screen for Down syndrome will take a different view about
Edwards and Patau syndromes. A small minority may draw the
opposite conclusion. Information is being provided to support
each of the possible choices.
gramme for haemoglobinopathies, and it is clear that offering
separate choices about each tested for condition will eventually
become an unsustainable strategy. The performance of tests,
including NIPT, varies by condition,33,42 and whole-genome
sequencing with NIPT is technically some way off in terms of
clinical implementation at least. A cautious, evaluative approach
to new indications has been advocated.43 In the meantime, a new
balance between supported choice and information overload will
need to be found. The revealed choices of women in different parts
of implemented screening programmes will be informative here,
but a major consultation about future directions and about means
and ends is also likely to be needed. 
Prenatal Diagnosis of Fetal Anomaly
However ‘prepared’ parents might be for the possibility of the
diagnosis of fetal anomaly, perhaps due to a screen positive result,
when the problem is confirmed, the psychological impact is likely
to be significant.44 This means how information is communicated
158 SE C T I O N 6     Prenatal Screening and Diagnosis
about the findings is important, and this can be challenging for
clinicians.45 It is more difficult to assimilate information when
distressed, so consideration should be given to how to most effec-
tively convey what parents need to know. Sensitive, individualised
care from the point of diagnosis that is coordinated and combined
with good communication can help parents take some positive
memories from a difficult experience and will avoid adding to
existing distress.46
In the context of a wanted pregnancy, prenatal diagnosis of
fetal anomaly requires parents to confront the loss of the ‘healthy’
baby they had previously conceptualised and built their hopes
around. They may need time to accept the reality of the diagnosis
and its possible implications. It may be difficult for them to fully
grasp the potential outcomes in the space of a single consultation.
Some parents may feel the need to have the anomaly confirmed
by a second opinion and are likely to want further consultations.
This should not be seen as undermining the initial clinical judge-
ment. For some parents, it is an important part of the process of
accepting the reality of the situation and understanding what it
may mean for them.47
Effective communication from clinicians involves more than
choosing the right words. When shock means assimilation of
information is hard, a clear, carefully paced explanation of the
anomaly and the predicted prognosis is crucial. The clinician will
need to gauge the response of parents and tailor the communi-
cation accordingly. Only by actively listening and responding to
parents and involving them in discussions will it be possible to
assess the meaning the diagnosis has for them and come to an
agreement on the most appropriate plan for management. As a
result, explanations need to be as jargon free as possible and logi-
cally sequenced with pausing to check that parents are able to take
in the most essential information. It can be useful for them to
have written information to take away to consolidate what has
been said, as well as details of someone they can contact between
appointments with any concerns. They may appreciate being sign-
posted to reliable sources of information outside their health care
providers to gain as much information as possible about the impli-
cations of the diagnosis for them.
It is helpful if parents experience good continuity of care and
consistent information from all involved in their medical care.
This is best achieved by ensuring that there is good multidisci-
plinary coordination and clear channels of communication among
all clinicians working across regional and specialist centres.  using more sensitive testing than conventional karyotyping, such
Diagnosis From Ultrasound
The psychosocial significance of prenatal ultrasound for expectant
parents is well established.48 We know that parents are never psy-
chologically prepared for difficult news from a scan even if given
information about the purpose of the examination.49 The distress
can be particularly acute if the scan brings the first indication of
a potential problem. Communication of findings in this situa-
tion can be particularly challenging because of the necessity of
delivering the difficult news in real time, giving the clinician little
opportunity to prepare. Therefore clear and concise explanation
will be essential, along with due regard to the capacity of the par-
ents to take in what they need to know. Along with clarity, there
is evidence that parents value empathy and compassion from their
providers.48,50
The ultrasound room is not the ideal setting to deliver difficult
and often unexpected news about a pregnancy. The woman will be
in a prone and vulnerable position, especially if she is undergoing
a transvaginal scan. However, it should not be assumed that news
must be deferred until the woman can adjust her clothing and sit
up. Some women appreciate the opportunity to see the scan find-
ing, and this in turn may aid understanding. Others may feel the
need to feel more ‘dignified’. The only way to know is to ask the
woman for her preference.51 Although correct medical terminol-
ogy will, of course, be required, every attempt should be made to
pitch the information to make it comprehensible. Furthermore,
parents appreciate the findings described respectfully and sensi-
tively. This includes taking the lead from them in determining
whether to use the term ‘baby’ rather than ‘fetus’ and being sensi-
tive when describing certain ultrasound markers such as ‘straw-
berry signs’, ‘lemon-shaped head’ ‘Swiss-cheese pattern’. 
Diagnosis From Genetic Testing
Diagnosis of most genetic conditions requires a woman to undergo
invasive procedures such as chorionic villus sampling (CVS) or
amniocentesis. Agreeing to have either procedure involves her
acceptance of the possibility that her quest for a diagnosis may
lead to a procedure-related miscarriage. Although the miscarriage
risk may have led to deliberation and difficulty in taking up the
testing, it does not mean she has made the psychological leap to
the actuality of a confirmed diagnosis and is committed to a par-
ticular course of action in this instance.52 In other words, it is
important that clinicians do not make assumptions about how
news from a CVS or amniocentesis result will be received or that
intentions expressed in the abstract will be retained in the face of
the reality of a diagnosis.
There is little research data to suggest that the timing of the
delivery of test results (i.e., at an agreed time or as soon as results
available) or method of communication (i.e., face to face, tele-
phone or email) reduces anxiety levels.53 We do know that par-
ents are likely to be anxious about the result, so the waiting time
should be minimised, and how they might prefer the results to be
delivered should be discussed with them beforehand. Although
some results will give a clear diagnosis, in the case of common
trisomies and well-described genetic conditions, some parents will
face chromosomal changes or genetic variants about which little
or nothing is known. (Even in the case of well-described genetic
conditions, there will be a level of uncertainty which must be
acknowledged.) This is becoming more common with the move to
as microarray.54 Whole fetal genome sequencing is also on the
horizon.55 Potentially complex scenarios arising from the detec-
tion of genetic variants of uncertain or unknown significance will
require a close collaborative relationship between fetal medicine
and clinical genetics.56 Furthermore, the challenge for parents in
dealing with results that represent a very uncertain prognosis post-
natally must be acknowledged and access to genetic counselling
provided when appropriate.57 
Pregnancy Management After Diagnosis
Postdiagnosis pregnancy management options available to parents
will most often depend on the nature of the anomaly and the legal
framework relating to abortion. In a limited number of circum-
stances, in utero interventions may be possible, such as fetal sur-
gery for diaphragmatic hernia, catheters used to drain excess fluid
from fetal organs or laser ablation for twin-to-twin transfusion.
Here, particularly for fetal medicine specialists, a conflict can arise
between the perceived best interests of two ‘patients’, the mother
 Chapter 17  Conveying Information About Screening and Diagnosis
and the fetus. It is therefore essential to present a clear picture
and discuss the risks and benefits of any possible interventions,
not just what might apply to the fetus but also the potential for
maternal morbidity.58
There is scarce evidence on exactly how parents make decisions
about continuing or ending a pregnancy after diagnosis of fetal
anomaly. Clearly, there are practical and ethical constraints to
obtaining this information concurrently with the decision mak-
ing. The existing literature attests to the immense difficulty in
making the decision even for parents who know what they want
to do.59 What emerges from published accounts is the way par-
ents weigh the impact of the anomaly on the child when born, on
themselves and on other immediate family members (including
existing and potential siblings) in the context of any attitudes and
beliefs they may hold about abortion. Such data as available sug-
gest that few parents regret their decisions, although this work is
mostly in the context of a decision to terminate.60
In the absence of a firm evidence base, the question arises as
to how clinicians might best support parents so they are enabled
to make a decision that they can best live with. It is often a psy-
chologically complex situation for parents who are desperate to
make the ‘right’ decision.61 In view of this, some parents will look
to their clinicians for direction or advice. When they ask ques-
tions such as, ‘What do you think I should do?’ or ‘What would
you do?’, parents are often seeking acknowledgement of the mag-
nitude of their dilemma or acceptance of the decision they have
tentatively made. There has been ongoing debate about whether
the principle of ‘nondirective counselling’ is achievable or even
desirable in these situations.62 Some argue that parents use advice
from a clinician along with many other contributory factors in
their decision making and are not unduly influenced.63 However,
clinicians may be on safer ground empathising with the difficulty
the parents face and helping them to weigh the competing benefits
and harms in their unique circumstances (particularly when most
often, there will be some measure of uncertainty about potential
outcomes).
As first trimester screening for fetal anomalies and genetic
conditions has become the norm in most developed countries,
more parents are faced with decisions at an earlier stage of preg-
nancy. It is unwarranted to assume that an earlier diagnosis is
always psychologically easier to cope with.64 If ending the preg-
nancy, parents still have to come to terms with the loss of, what
is in most cases, a much desired pregnancy. Also, they may have
less recourse to external support because many couples will have
delayed announcement of the pregnancy until the first trimester
is over. If continuing, they will have longer to adjust to a differ-
ent reality but also longer to cope with the anxieties around the
potential outcomes.  who remains a pregnant woman but who can appear to be forgotten
Information Needs When Parents Decide to
Terminate an Affected Pregnancy
In jurisdictions where termination is an option, it is unhelpful
to assume that the diagnosis of a definitely lethal or severely life-
limiting condition will always make a decision about terminating
the pregnancy more psychologically tolerable.65 Parents have to
contend with the devastating news that their baby will not survive,
coupled with the fact that they can choose the timing of their
baby’s death.
Some clinicians working in fetal medicine may believe their
responsibility stops at the offer of termination. However, parents
often look to the clinician who has provided the diagnosis to
159
provide information and guidance on what the termination pro-
cess will involve. It has been shown that information about what
might be ahead is valued by parents and can be empowering at
a time of crisis.66 Their information needs will include options
for method of termination (surgical or medical) and what both
procedures involve, whether feticide will be offered and whether
postmortem investigations are likely to provide information that
will have implications for future pregnancies. It is important that
all such information is evidence-based and takes account of the
woman’s preferences and individual coping strategies.67 
Information Needs When Parents Decide to
Continue With an Affected Pregnancy
Research into the impact of continuing a pregnancy after a con-
firmed prenatal diagnosis was, until recently, relatively rare com-
pared with that on the experiences of termination.44 Much research
focused on when a lethal abnormality had been discovered, and
there was some suggestion that in this situation, the decision not
to terminate may be better for a woman’s emotional well-being
because women who continue a pregnancy could avoid the guilt
they might have felt had they terminated. For many women, how-
ever, the thought of continuing a pregnancy with a baby who they
know will die is as unthinkable as termination is for others.68 The
important issue is that parents are enabled to make the right deci-
sion for them. Care for parents continuing a pregnancy has been
reported as poor during the remainder of the pregnancy.69 The
development of planned palliative care programmes for women,
with intense pregnancy support70 and clear planning around the
birth of the baby and for the neonatal period is clearly to be wel-
comed for the women who choose this option or cannot access
termination. Although evidence suggests that when termination
is available in this circumstance, most women will take up this
option, this must not preclude carefully coordinated and support-
ive care for women continuing their pregnancies.
There are very many parents who continue pregnancies after the
diagnosis of one of the many anomalies that are not lethal and in
which termination might not be offered, such as cleft lip or talipes.
Sometimes prenatally diagnosed conditions require ongoing moni-
toring; sometimes there will be treatment available, and other times
there will be no treatment. Whatever the prognosis, the diagnosis
means that parents have lost the healthy baby they had expected
and will be adjusting to the uncertainty of the remainder of the
pregnancy and after the baby is born and will be experiencing many
complex emotions.71 In a growing base of qualitative data, research-
ers have explored the nature and experience of an ongoing preg-
nancy after a prenatal diagnosis, as well as how to care for the mother
when antenatal care focuses on the health of the baby.68 Issues of
shock at diagnosis, grief for the loss of the expected healthy baby
and isolation from family and clinicians after the decision to con-
tinue are highlighted.72,73 Even in the Republic of Ireland, a coun-
try where prenatal diagnosis is undertaken but until very recently
termination of pregnancy was not legal, except in very restricted
circumstances, recent data suggest that systems of care for parents
continuing pregnancies are not ideal to meet women’s needs.46
Partners. Care in the context of prenatal diagnosis tends to
be concentrated on the mother. Although such focus is inevitable
and right, we know fathers or partners most often have signifi-
cant engagement in a wanted pregnancy.74 There has been little
research undertaken with fathers.60 The limited research we have
suggests fathers will admit to neglecting or denying their own
160 SE C T I O N 6     Prenatal Screening and Diagnosis
needs as they try to take on a supportive or practical role. It must
be remembered that they too have had their expectations of a
healthy baby shattered, evoking feelings that are often in danger
of being subsumed in the needs of the mother. For men raised in
cultures where males are not encouraged to express emotions, it
can be especially difficult to articulate their feelings and needs. It
is therefore incumbent upon clinicians caring for the couple make
every effort to include the father at all times and to encourage him
to seek support if appropriate.  develop the requisite skills to provide the very best individualised
Subsequent Pregnancies
After the diagnosis of fetal anomaly and particularly it has led to
pregnancy loss, the perception of the pregnancy experience will
change for most women.75 This means that subsequent pregnan-
cies (not just the one after the affected pregnancy) can be anxiety
laden for parents, and it will be important that all involved in
their care are cognisant of and sensitive to their history. Many
women will want to be seen again by the clinician who diagnosed
the previous anomaly, but others may wish to avoid those who are
associated with previous anguish.
Anxieties in a subsequent pregnancy do not necessarily subside
completely when the moment of the previous diagnosis has passed
or when all possible testing has revealed no major problems. Those
who had a termination or miscarriage after diagnosis can find the
gestational weeks after the time the previous loss took place dif-
ficult because it is ‘uncharted water’ or evokes guilt because the
previous baby did not make it this far. Others, having had a bad
experience in pregnancy, find it almost impossible to believe they
will have a happy outcome. For many, even the birth of a healthy
child is tinged with sadness because it serves as a reminder of ‘what
should have been’ in the previous pregnancy. In short, the major-
ity of parents in the pregnancy after a loss will need extra support
and carefully coordinated care from their obstetric team.  that parents’ ability to assimilate information is impeded and that
‘Human Aspects of Care’
No matter how skilled or experienced a clinician may be, working
with distressed parents will take its personal toll.76 It is also sig-
nificant that in the context of a diagnosis of serious fetal anomaly,
there are few interventions that can be offered prenatally to guar-
antee a positive outcome for fetus and mother. This can evoke
feelings of failure on the part of the clinician. Indeed, whatever
strategies clinicians use to mitigate against the inevitable distress
that comes with the territory, if they find themselves able to cut
off completely from the human sadness of the situation of prenatal
diagnosis, it will be necessary to reflect on the quality of care they
are able to offer. The majority of women who see obstetricians
have good outcomes to their pregnancies; this can make the occa-
sions when fetal abnormality occurs harder to manage. We argue
for the provision of specialised training for clinicians to help them
care, along with appropriate support to ensure they can manage
the emotional dimension without becoming inured to the impact
on parents. 
Conclusions
This chapter has demonstrated that although the provision of
good-quality information is essential in the prenatal testing path-
way, facilitating an informed decision goes beyond the delivery
of information. The needs and preferences of women and their
partners are diverse, as are the experiences and values they bring
to the clinic. Conveying complex information about multiple
tests and conditions in a way that women can understand is a
challenge for clinicians. As testing options increase and testing
pathways become more complex, new demands are placed on
information providers, and the dilemmas for women are altered
rather than being resolved. NIPT technologies are making a valu-
able contribution to women’s testing options but are unlikely to
replace combined screening or invasive diagnostic testing in the
foreseeable future. The development of high-quality information
along with staff training therefore remains a priority. However, it
is recognised that conveying a prenatal diagnosis of fetal anomaly
to parents will always be challenging because of the psychological
reaction it is likely to evoke. At this distressing time, we know
they appreciate clarity in the communication of the information
they need to know. They also value sensitivity and understanding
from the clinician about what the diagnosis might mean to them
as individuals. As well as information on their options, parents
need well-coordinated care to support them through the ensuing
decision-making process and subsequent outcomes.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Bramwell R, West H, Salmon P. Health pro-
fessionals’ and service users’ interpretation of
33. Kagan KO, Wright D, Nicolaides K. First-
trimester contingent screening for trisomies
screening test results: experimental study. BMJ. 21, 18 and 13 by fetal nuchal translucency and
1. Green JM, Hewison J, Bekker HL, et  al.
2006;333(7562):284. ductus venosus flow and maternal blood cell-
Psychosocial aspects of genetic screening of
18. Marteau TM, Saidi G, Goodburn S, et al. Num- free DNA testing. Ultrasound Obstet Gynecol.
pregnant women and newborns: a systematic
bers or words? A randomized controlled trial of 2015;45(1):42–47.
review. Health Technol Assess. 2004;8(33).
presenting screen negative results to pregnant 34. UK National Screening Committee. The UK
2. Harris JM, Franck L, Michie S. Assessing
women. Prenat Diagn. 2000;20(9):714–718. NSC recommendation on fetal anomaly screening
the psychological effects of prenatal screen-
19. Sullivan A. Involving parents: information in pregnancy. 2016. https://legacyscreening.
ing tests for maternal and foetal conditions:
and informed decisions. In: Sullivan A, Kean phe.org.uk/fetalanomalies.
a systematic review. J Reprod Infant Psychol.
L, Cryer A, eds. Midwife’s Guide to Antena- 35. Abdel Haleem MAS. Medical ethics in Islam. In:
2012;30(3):222–2246.
tal Investigations. Edinburgh: Elsevier; 2006: Grubb A, ed. Choices and Decisions in Health-
3. Lou S, Mikkelsen L, Hvidman L, et  al. Does
17–29. care. Chichester, West Sussex, England: Wiley;
screening for Down’s syndrome cause anxiety
20. Heyman B, Hundt G, Sandall J, et  al. On 1993:1–20.
in pregnant women? A systematic review. Acta
being at higher risk: a qualitative study of pre- 36. Hewison J. Psychological aspects of individual-
Obstet Gynecol Scand. 2015;94(1):15–27.
natal screening for chromosomal anomalies. ized choice and reproductive autonomy in pre-
4. Hewison J, Nixon J, Fountain J, et al. A ran-
Soc Sci Med. 2006;62:2360–2372. natal screening. Bioethics. 2015;29(1):9–18.
domised trial of two methods of issuing pre-
21. Nicolaides KM, Chervenak FA, McCullough 37. Hill M, Wright D, Daley R, et  al. Evalua-
natal test results: the ARIA (Amniocentesis
LB, et  al. Evidence-based obstetric ethics and tion of non-invasive prenatal testing (NIPT)
Results: Investigation of Anxiety) trial. BJOG.
informed decision-making by pregnant women for aneuploidy in an NHS setting: a reli-
2007;114(4):462–468.
about invasive diagnosis after first-trimester able accurate prenatal non-invasive diagnosis
5. Košec V, Nakić Radoš S, Gall V. Develop-
assessment of risk for trisomy 21. Am J Obstet (RAPID) protocol. BMC Pregnancy Childbirth.
ment and validation of the Prenatal Diagnos-
Gynecol. 2005;193(2):322–326. 2014;14(1):229.
tic Procedures Anxiety Scale. Prenat Diagn.
22. Caughey AB, Washington AE, Gildengorin V, 38. Allyse M, Minear MA, Berson E, et  al. Non-
2014;34(8):770–777.
Kuppermann M. Assessment of demand for invasive prenatal testing: a review of interna-
6. Lafarge C, Mitchell K, Fox P. Women’s expe-
prenatal diagnostic testing using willingness to tional implementation and challenges. Int J
riences of coping with pregnancy termina-
pay. Obstet Gynecol. 2004;103(3):539–545. Women’s Health. 2015;7:113–126.
tion for fetal abnormality. Qual Health Res.
23. Marini T, Sullivan J, Naeem R. Decisions 39. Lewis C, Hill M, Chitty LS. Women’s experi-
2013;23(7):924–936.
about amniocentesis by advanced maternal ences and preferences for service delivery of
7. Hassan M, Chitty L, Reardon H. Wrong-
age patients following maternal serum screen- non-invasive prenatal testing for aneuploidy in
ful birth: clinical settings and legal implica-
ing may not always correlate clinically with a public health setting: a mixed methods study.
tions. Semin Fetal Neonatal Med. 2014;19(5):
screening results: need for improvement in PloS One. 2016;11(4):e0153147.
312–316.
informed consent process. Am J Med Genet. 40. Getz L, Kirkengan AL. Ultrasound screen-
8. Briss P, Rimer B, Reilley B, et  al. Promoting
2002;109:171–175. ing in pregnancy: advancing technology, soft
informed decisions about cancer screening in
24. Mueller VM, Huang T, Summers AM, Winsor markers for fetal chromosomal aberrations,
communities and healthcare systems. Am J Prev
SHM. The influence of risk estimates obtained and unacknowledged ethical dilemmas. Soc Sci
Med. 2004;26(1):67–80.
from maternal serum screening on amniocen- Med. 2003;56:2045–2057.
9. Lawson KL, Pierson RA. Maternal decisions
tesis rates. Prenat Diagn. 2005;25:1253–1257. 41. Hewison J, Green JM, Ahmed S, et  al. Atti-
regarding prenatal diagnosis: rational choices
25. American College of Obstetricians and Gyne- tudes to prenatal testing and termination of
or sensible decisions. J Obstet Gynaecol Can.
cologists. Screening for Fetal Aneuploidy. pregnancy for fetal abnormality: a comparison
2007;29(3):240–246.
Practice Bulletin Number 163 Obstet Gynecol. of white and Pakistani women in the UK. Pre-
10. Ahmed S, Hewison J, Green JM, et  al. Deci-
2016;2016(127):e123–e137. nat Diagn. 2007;27:419–430.
sions about testing and termination of
26. Lutgendorf MA, Stoll KA. Why 99% may not 42. Lo KK, Karampetsou E, Boustred C, et  al.
pregnancy for different fetal conditions: a qual-
be as good as you think it is: limitations of Limited clinical utility of non-invasive prenatal
itative study of European white and Pakistani
screening for rare diseases. J Matern Fetal Neo- testing for subchromosomal abnormalities. Am
mothers of affected children. J Genet Couns.
natal Med. 2016;29(7):1187–1189. J Hum Genet. 2016;98(1):34–44.
2008;17(6):560–572.
27. Norton ME, Baer RJ, Wapner RJ, et al. Cell- 43. Dondorp W, de Wert G, Bombard Y, et  al.
11. Bryant LD, Green JM, Hewison J. The role
free DNA vs sequential screening for the detec- Non-invasive prenatal testing for aneuploidy
of attitudes towards the targets of behaviour
tion of fetal chromosomal abnormalities. Am J and beyond: challenges of responsible innova-
in predicting and informing prenatal test-
Obstet Gynecol. 2016;214(6):727.e1–e6. tion in prenatal screening. Eur J Hum Genet.
ing choices. Psychol Health. 2010;25(10):
28. Norton ME, Jacobsson B, Swamy GK, 2015;23(11):1438–1150.
1175–1194.
et  al. Cell-free DNA analysis for noninva- 44. Statham H, Solomou W, Chitty L. Prenatal
12. Lawson KL. Expectations of the parenting
sive examination of trisomy. N Engl J Med. diagnosis of fetal abnormality: psychologi-
experience and willingness to consider selec-
2015;372(17):1589–1597. cal effects on women in low-risk pregnancies.
tive termination for Down Syndrome. J Reprod
29. Biggio JRJ. Prenatal screening for fetal aneu- Baillieres Best Pract Res Clin Obstet Gynaecol.
Infant Psychol. 2006;24:43–59.
ploidy: time to examine where we are and 2000;14(4):731–747.
13. Atkin K, Ahmed S, Hewison J, Green JM.
where we are going. Am J Obstet Gynecol. 45. Guerra FAR, Mirlesse V, Baião AER. Breaking
Decision-making and ante-natal screening for
2016;214(6):A1–A12. bad news during prenatal care: a challenge to be
sickle cell and thalassaemia disorders—to what
30. Gil M, Revello R, Poon L, et  al. Clinical tackled. Cien Saude Colet. 2011;15(5):2361–
extent do faith and religious identity mediate
implementation of routine screening for fetal 2367.
choice? Curr Sociol. 2008;58(1):77–98.
trisomies in the UK NHS: cell-free DNA 46. Lalor JG, Devane D, Begley CM. Unex-
14. Ahmed S, Bryant LD, Tizro Z, Shickle D.
test contingent on results from first-trimester pected diagnosis of fetal abnormality:
Interpretations of informed choice in antena-
combined test. Ultrasound Obstet Gynecol. women’s encounters with caregivers. Birth.
tal screening: a cross-cultural, Q-methodology
2016;47(1):45–52. 2007;34(1):80–88.
study. Social Sci Med. 2012;74(7):997–1004.
31. Chitty LS, Wright D, Hill M, et  al. Uptake, 47. Aite L, Trucchi A, Nahom A, et al. Antenatal
15. Ahmed S, Bryant LD, Tizro Z, Shickle D.
outcomes, and costs of implementing non- diagnosis of surgically correctable anomalies:
Is advice incompatible with autonomous
invasive prenatal testing for Down’s syndrome effects of repeated consultations on parental
informed choice? Women’s perceptions of
into NHS maternity care: prospective cohort anxiety. J Perinatol. 2003;23(8):652–654.
advice in the context of antenatal screen-
study in eight diverse maternity units. BMJ. 48. McCoyd JL. Authoritative knowledge,
ing: a qualitative study. Health Expect.
2016;354:i3426. the technological imperative and women’s
2014;17(4):555–564.
32. Persico N, Boito S, Ischia B, et  al. Cell-free responses to prenatal diagnostic technologies.
16. Ahmed S, Bryant LD, Cole P. Midwives’ per-
DNA testing in the maternal blood in high- Cult Med Psychiatry. 2010;34(4):590–614.
ceptions of their role as facilitators of informed
risk pregnancies after first-trimester combined
choice in antenatal screening. Midwifery.
screening. Prenat Diagn. 2016;36(3):232–236.
2013;29(7):745–750.

160.e1
160.e2 References
49. Garcia J, Bricker L, Henderson J, et al. Wom-
en’s views of pregnancy ultrasound: a system-
atic review. Birth. 2002;29(4):225–250.
50. Denney-Koelsch EM, Côté-Arsenault D,
Lemcke-Berno E. Parents’ experiences with
ultrasound during pregnancy with a lethal
fetal diagnosis. Glob Qual Nurs Res. 2015;2:
2333393615587888.
51. Alkazaleh F, Thomas M, Grebenyuk J, et  al.
What women want: women’s preferences of
caregiver behavior when prenatal sonography
findings are abnormal. Ultrasound Obstet Gyne-
col. 2004;23(1):56–62.
52. Hunt LM, de Voogd KB, Castañeda H. The
routine and the traumatic in prenatal genetic
diagnosis: does clinical information inform
patient decision-making? Patient Edu Couns.
2005;56(3):302–312.
53. Mujezinovic F, Prosnik A, Alfirevic Z. Differ-
ent communication strategies for disclosing
results of diagnostic prenatal testing. Cochrane
Database Syst Rev. 2010;(11):CD007750.
54. Wapner RJ, Martin CL, Levy B, et  al.
Chromosomal microarray versus karyotyp-
ing for prenatal diagnosis. N Engl J Med.
2012;367(23):2175–2184.
55. Talkowski ME, Ordulu Z, Pillalamarri V,
et  al. Clinical diagnosis by whole-genome
sequencing of a prenatal sample. N Engl J Med.
2012;367(23):2226–2232.
56. Bernhardt BA, Soucier D, Hanson K, et  al.
Women’s experiences receiving abnormal pre-
natal chromosomal microarray testing results.
Genet Med. 2012;15(2):139–145.
57. Westerfield L, Darilek S, van den Veyver IB.
Counseling challenges with variants of uncer-
tain significance and incidental findings in
prenatal genetic screening and diagnosis. J Clin
Med. 2014;3(3):1018–1032.
58. Williams C. Dilemmas in fetal medicine: pre-
mature application of technology or respond-
ing to women’s choice? Sociol Health Illness.
2006;28(1):1–20.
59. Sandelowski M, Barroso J. The travesty of choos-
ing after positive prenatal diagnosis. J Obstet
Gynecol Neonatal Nurs. 2005;34(3):307–318.
60. Korenromp MJ, Christiaens G, Van den Bout
J, et al. Long-term psychological consequences
of pregnancy termination for fetal abnormal-
ity: a cross-sectional study. Prenat Diagn.
2005;25(3):253–260.
61. van Berkel D, van der Weele C. Norms and
prenorms on prenatal diagnosis: new ways to
deal with morality in counseling. Patient Educ
Couns. 1999;37(2):153–163.
62. Clarke A. Is non-directive genetic counselling
possible? Lancet. 1991;338:998–1001.
63. Baylis F, Downie J. Professional recommenda-
tions: disclosing facts and values. J Med Ethics.
2001;27(1):20–24.
64. Fisher J. First-trimester screening: dealing with
the fall-out. Prenat Diagn. 2011;31(1):46–49.
65. Bijma HH, Van der Heide A, Wildschut HI.
Decision-making after ultrasound diagnosis
of fetal abnormality. Reprod Health Matters.
2008;16(31):82–89.
66. Fisher J, Lafarge C. Women’s experience of care
when undergoing termination of pregnancy for
fetal anomaly in England. J Reprod Infant Psy-
chol. 2015;33(1):69–87.
67. Kerns J, Vanjani R, Freedman L, et  al.
Women’s decision making regarding choice
of second trimester termination method for
pregnancy complications. Int J Gynecol Obstet.
2012;116(3):244–248.
68. Statham H, Solomou W, Green JM. Commu-
nication of prenatal screening and diagnosis
results to primary-care health professionals.
Public Health. 2003;117(5):348–357.
69. Chitty LS, Barnes CA, Berry C. Continuing with
pregnancy after a diagnosis of lethal abnormality:
experience of five couples and recommendations
for management. BMJ. 1996;313(7055):478.
70. Breeze AC, Lees CC, Kumar A, et  al. Pal-
liative care for prenatally diagnosed lethal fetal
abnormality. Arch Dis Child Fetal Neonatal Ed.
2007;92(1):F56–F58.
71. Jones S, Statham H, Solomou W. When
expectant mothers know their baby has a fetal
abnormality: exploring a crisis of motherhood
through qualitative data-mining. J Social Work
Res Eval. 2005;6(2):195–206.
72. Edwins J. From a different planet: women
who choose to continue their pregnancy after a
diagnosis of Down’s syndrome. Pract Midwife.
2000;3:21–24.
73. Redlinger-Grosse K, Bernhardt BA, Berg K,
et al. The decision to continue: the experiences
and needs of parents who receive a prenatal
diagnosis of holoprosencephaly. Am J Med
Genet. 2002;112(4):369–378.
74. Robson FM. ‘Yes!—a chance to tell my side
of the story’: a case study of a male partner
of a woman undergoing termination of preg-
nancy for foetal abnormality. J Health Psychol.
2002;7(2):183–193.
75. Wollenschein M, Gustke M, Woopen C, Rohde
A. A subsequent pregnancy after a termination
of pregnancy because of fetal anomaly—all for-
gotten and a new beginning? Prax Kinderpsy-
chol Kinderpsychiatr. 2006;56(9):741–757.
76. Menezes MA, Hodgson JM, Sahhar M,
Metcalfe SA. ‘Taking its toll’: the chal-
lenges of working in fetal medicine. Birth.
2013;40(1):52–60.
18
Ultrasound and Biochemical Screening
for Fetal Aneuploidy
HOWARD CUCKLE AND RAN NEIGER
KEY POINTS
• First trimester screening for Down syndrome (DS) with a
combination of ultrasound, nuchal translucency and two
maternal serum markers (combined test) has a much better
performance than second trimester screening with four
serum markers (quad test).
• Combined test performance can be improved by the addition
of serum markers such as placental growth factor (PlGF) and
ultrasound markers such as nasal bone determination.
• Quad test can be improved by the addition of ultrasound
markers such as those based on the facial profile.
• A late second trimester anatomical scan can be used ad hoc
to reinterpret a DS screening result provided ‘soft’ marker–
specific likelihood ratios are applied to a posttest risk; how-
ever, a policy of routine screening with this scan would have
poor performance.
• Sequential protocols using markers in both trimesters have
the best performance; stepwise sequential and contingent
tests perform as well as or better than the integrated test.
• A large proportion of Edwards syndrome (ES) cases are
detected incidentally in pregnancies in which the combined
test is positive for DS; these proportions are much lower for
the quad test, and a separate ES cutoff is needed (similarly for
Patau syndrome).
• Combined test can be extended to screen for adverse preg-
nancy outcome such as preeclampsia, particularly when PlGF
is used.
• Cell-free DNA screening has much better performance
than conventional screening; however, currently the cost is
too high for a policy of primary screening in public health
settings. Instead, secondary and contingent protocols are
recommended.
Historical Perspective
Fetal aneuploidy is a common cause of congenital abnormality; it
is associated with intrauterine fatality and, among those surviving
to term, moderate to severe intellectual impairment, morbidity
and increased mortality. In addition to the learning and health
implications, the birth of an affected infant can have a negative
long-term impact on the parents and their families.
Prenatal diagnosis of aneuploidy requires testing of fetal
material obtained by invasive procedures such as chorionic vil-
lus sampling (CVS) and amniocentesis, which have risks and are
expensive. Depending on the health care system in a given locality,
women are offered these procedures either unselectively, as in the
United States; on the basis of prior risk factors; such as advanced
maternal age (AMA) or family history; or after routine screening.
After invasive prenatal diagnosis, the option of termination of
affected pregnancies is taken up by most of those with severe forms
of aneuploidy, but some are prepared to continue the pregnancy.
The earlier in pregnancy that prenatal diagnosis can be achieved,
the more time there is to counsel parents regarding the potential
impact of the diagnosis. If the decision is made not to terminate
the pregnancy, the additional ‘lead time’ can be used to prepare the
parents for the birth of the child.
The modern era of antenatal screening began in the mid-1970s
with the discovery that maternal serum α-fetoprotein (AFP) lev-
els are increased, on average, in pregnancies affected by fetal neu-
ral tube defects (NTDs).1 At that time, the analyte had not been
standardised, leading to large between-laboratory differences, and
the fact that levels increase rapidly with gestation, doubling about
every 5 weeks, was not taken into account. To overcome this,
results are currently expressed as multiples of the normal median
(MoMs) for unaffected pregnancies of the same gestation for each
laboratory.
α-Fetoprotein screening for NTDs became routine, and as a
consequence, it was incidentally noted, about a decade later, that
levels were relatively low in some cases of aneuploidy.2 Because
fetal loss is also associated with low AFP, this observation might
have been accounted for by nonviable types of aneuploidy. How-
ever, it was soon established that levels were reduced on average
in Down syndrome (DS, trisomy 21) cases which are viable.3 Ini-
tially, women were selected for invasive prenatal diagnosis on the
basis of a low AFP, but it was shown to be more efficient to com-
bine information on maternal age, family history and MoM into
a patient-specific DS risk.
Subsequently, additional ‘markers’ of aneuploidy were added,
both maternal serum analytes and ultrasound markers. Using
multimarker profiles to calculate risk resulted in better screening
performance measured in terms of detection rate (DR, proportion
of affected pregnancies referred for prenatal diagnosis) and false-
positive rate (FPR, proportion of unaffected pregnancies referred).
The risk can be considered as a composite marker, and the result is
classified as ‘positive’ and ‘negative’ by comparison with a fixed risk
161
162 SE C T I O N 6     Prenatal Screening and Diagnosis
cutoff. First trimester multimarker protocols yielded a higher DR
for a fixed FPR compared with the second trimester. In addition
to this improvement, the introduction of first trimester screening
methods enabled earlier detection, earlier reassurance and safer
termination of pregnancy when requested. Sequential protocols
testing in both trimesters had even better screening performance,
although they sacrificed early detection.
For some time, the main focus of aneuploidy screening was
the detection of DS, but gradually centres included separate risks
for Edwards syndrome (ES, trisomy 18) and Patau syndrome (PS,
trisomy13). For some screening protocols, even when only a DS
risk was given, there was a high ‘incidental’ detection of ES, PS
and other forms of aneuploidy because of advanced age and hav-
ing marker profiles similar to DS.
Recently, a completely different screening modality has become
available: noninvasive prenatal testing based on the determination
of cell-free DNA (cfDNA) in maternal plasma. This substan-
tially increases DR and vastly reduces FPR. However, because
cfDNA still has false-positive and false-negative results, it is not
a substitute for invasive testing. Nevertheless, professional bodies
such as the American College of Obstetricians and Gynecologists
(ACOG).4 and the International Society for Prenatal Diagnosis
(ISPD).5 recommend that cfDNA be used as a secondary screen-
ing test after a positive result by conventional screening protocols.
An abnormal cfDNA finding requires confirmation by the gold
standard of invasive testing.
Despite the superior performance, routine cfDNA screening
is unlikely to replace established protocols in the short term. An
important rate limiting step is cost; current prices preclude rou-
tine testing in most localities. A compromise approach is ‘con-
tingent cfDNA’ screening whereby 10% to 30% of women with
the highest risks based on established screening tests are selected
for cfDNA. This approach also allows the use of biochemical and
ultrasound markers to screen for adverse outcomes of pregnancy
and congenital abnormalities other than aneuploidy.  Prevalence According to Gestational Age
Aneuploidy
Fetal aneuploidy results in a wide spectrum of phenotypes, with
viability and clinical outcome varying according to the genotype.
Severity ranges from lethal (e.g. triploidy) to the relatively benign
Turner syndrome (TS, monosomy X) and other sex chromosome
abnormalities (SCAs). Most severely affected embryos abort spon-
taneously early in the first trimester, sometimes even before there
are clinical signs of pregnancy. Among those who survive the first
trimester, there remains a high rate of intrauterine mortality and
an increased risk of infant death. By far, DS is the most frequent
aneuploidy sufficiently viable to survive to term in relatively large
numbers and amenable to screening, with a birth prevalence (in
the absence of prenatal diagnosis and therapeutic abortion) of 1 to
2 per 1000. ES and PS have respectively about one 10th and one
20th the birth prevalence of DS.6
Maternal Age Distribution
In a particular locality, the birth prevalence of DS, ES and PS
will vary according to the maternal age distribution of the local
population. DS prevalence can be estimated by multiplying the
proportion of pregnancies at each single year of age by the mater-
nal age-specific prevalence based on published regression curves.
The best available curves are derived by a meta-analysis of pub-
lished birth prevalence rates for individual years of age determined
before prenatal diagnosis became clinically established. Four meta-
analyses have been published based on 11 different maternal age-
specific birth prevalence series.7-10 In one of these meta-analyses,
all eight series published at that time were included with a total
of 4500 DS births and more than 5 million unaffected births.7
For each year of age, data were pooled by taking the average birth
prevalence rate across the series weighted by the number of births.
The best-fitting curve was ‘additive-exponential’ in which there
are two components: a background prevalence independent of age
and an exponential increase with age. 
Standard Age Distribution
The relative advantages of competing screening protocols can
be demonstrated either directly or by statistical modelling. For
a direct comparison, a very large study will be required in which
there are a substantial number of affected pregnancies tested by
more than one protocol and there is no intervention. The reason
for nonintervention is to avoid ‘viability’ bias because of the high
rate of intrauterine fatality. This bias arises because a proportion of
affected pregnancies terminated after invasive prenatal diagnosis
carried out because of a positive result from one of the protocols
would have ended in fetal loss. Because the equivalent propor-
tion among those with negative results will not be identified, the
observed DR will necessarily be inflated.
Modelling yields more robust estimates and a more realistic
comparison among protocols. The model components include
parameters of the marker distributions and the maternal age dis-
tribution. The latter could be an observed distribution in some
locality, but for protocol comparison purposes, it can also be mod-
elled. Many such modelling exercises, including this chapter, use a
Gaussian age distribution with mean of 27 and a standard devia-
tion (SD) of 5.5 years–the ‘standard’ distribution.11 
Studies of prenatal diagnosis can be used to estimate DS preva-
lence at late first trimester, when CVS is generally performed,
and midsecond trimester when amniocentesis is done. Intrauter-
ine loss rates of DS from the time of prenatal diagnosis until
term are calculated either by comparing the observed number
of cases diagnosed at prenatal diagnosis with that expected from
birth prevalence, given the maternal age distribution or by fol-
low up of individuals declining termination of a DS pregnancies
using direct or actuarial survival analysis. Published prevalence
studies include a total of 341 DS cases diagnosed by CVS and
1159 following amniocentesis.12 There are three published
follow-up series including 110 DS cases after at amniocente-
sis13 and a series of 126 DS cases from the UK National Down
Syndrome Cytogenetic Register (NDSCR), a very complete
national database, which were analysed according to the gesta-
tional age at prenatal diagnosis.14 However, the Register study
was biased as there were miscarriages that occurred in women
who did intend to have pregnancy termination, thus inflating
the rates. An actuarial survival analysis of the Register data was
carried out which overcame this bias and was more data efficient
because all cases contributed to the estimate, not just those in
which pregnancy termination was refused.15 Actual and poten-
tial heterogeneity between the various studies precludes a grand
meta-analysis to estimate the fetal loss rates, but an informal
synthesis concluded that approximately one-half of DS preg-
nancies are lost after first trimester CVS and one quarter after
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy 163

midtrimester amniocentesis.16 Formulae have been published TABLE Down Syndrome: Average Multiples of the
from a large series of more than 57,000 women having inva- 18.1 Normal Median (MoM) and Mahalanobis
sive prenatal diagnosis based only on advanced maternal age.17
Distance for Each Marker, According to
These calculations assumed that fetal loss rates did not vary with
maternal age.18; however, the studies used to calculate the overall Gestationa
rates were largely based on women older than 35 years of age, Mahalanobis
so this could not be readily analysed. Because fetal loss rate in Marker Gestation (wk) Average MoM Distanceb
general increases markedly with maternal age,19 it is likely that
this occurs in DS pregnancies as well, as confirmed in a NDSCR NT 11 2.30 2.02
actuarial survival analysis based on 5116 registered DS pregnan- 12 2.10 1.87
cies, of which 271 ended in a live birth and 149 in fetal loss;
13 1.92 1.65
the remainder were terminated.20 The overall estimated fetal loss
rates from the time of CVS and amniocentesis were similar to PAPP-A 10 0.40 1.31
previous reports, but these rates increased steadily with maternal
11 0.45 1.14
age: from 23% and 19% at age 25 years to 44% and 33% at
age 45 years. One caveat, though, was that the observed mater- 12 0.53 0.90
nal age effect was confounded by differences in marker levels. 13 0.65 0.61
A large proportion of the prenatally diagnosed cases were detected
because of a positive result after routine antenatal screening. Free β-hCG 10 1.66 0.76
The marker distribution in screen positives varied according to 11 1.86 0.94
maternal age; however, marker profile in young women tended
to be extreme, but some older women, even those with moderate 12 2.01 1.05
profiles, had a screen-positive result because of the contribution 13 2.09 1.11
of their advanced age to the risk. 
14–18 2.30 1.33

Screening and Prenatal Diagnosis hCG 10 1.03 0.05

11 1.18 0.32
There is a fundamental difference between screening and diag-
nostic tests despite the use of the same terms to describe their 12 1.41 0.68
respective results: ‘true positive’, ‘false positive’, ‘true negative’ and
13 1.77 1.14
‘false negative’. The aim of ultrasound and biochemical screen-
ing is limited to the identification from among apparently healthy 14–18 2.02 1.15
pregnancies of those that are at high enough risk for a chromo-
AFP 14–18 0.73 0.79
somal abnormality to warrant the use of an invasive diagnostic
test. Thus screening for aneuploidy does not aim to make a diag- uE3 14–18 0.73 0.83
nosis but to ration the use of diagnostic procedures and tests that, Inhibin A 14–18 1.85 1.12
without prior selection, would be more hazardous or expensive.
aBased on meta-analyses.6

Evaluating the Efficacy of Screening Markers

The potential utility in screening of a given marker depends on the
extent of separation between the marker distributions in affected
and normal populations. This can be expressed as the absolute
difference between the distribution means divided by the average
SD for the two distributions, a form of Mahalanobis distance. For
continuous variables, the choice of a cutoff level that determines
whether a value is positive or negative is arbitrary because there is
no intrinsic division between the distributions. The choice is influ-
enced by the perceived relative importance of three factors: DR,
FPR and the positive predictive value (PPV), that is, the chance
of being affected given that the screening result is positive. The
prior risk in those screened influences the PPV. (See Chapter 16
for further discussion of PPV.)
All serum markers used in aneuploidy screening are continuous
variables whose distribution of values is higher or lower on aver-
age in affected pregnancies. Typically, these markers have consid-
erable overlap in the distribution of results between affected and
unaffected individuals. In contrast, the distribution of values for
variables used in diagnosis has essentially no overlap. Most of the
principal ultrasound markers are also continuous variables with
overlapping distributions. There are also some dichotomous ultra-
sound markers, which present difficulties of quality assessment.  but the Mahalanobis distance declines rapidly with increasing
bLog (average MoM)/((SD in DS + SD in unaffected pregnancies)/2), expressed as a positive
number, where SD is the standard deviation of log (MoM).
AFP, α-Fetoprotein; DS, Down syndrome; hCG, human chorionic gonadotrophin; PAPP-A,
pregnancy-associated plasma protein A; uE3, unconjugated estriol.
  
Principal Down Syndrome Markers
Of the more than 50 maternal blood, urine and ultrasound mark-
ers of DS, seven are widely used in routine multimarker screening.
These are maternal serum AFP, human chorionic gonadotrophin
(hCG), the free-β subunit of hCG, unconjugated estriol (uE3),
inhibin A and pregnancy-associated plasma protein (PAPP)-A and
ultrasound nuchal translucency (NT). Of these markers, PAPP-A
and NT are only used in the first trimester; the remainder can be
used in the first or second trimester, but generally AFP, uE3 and
inhibin A are used in the second.
Table 18.1 shows the average MoM in DS pregnancies and the
Mahalanobis distance for each of the principal markers. For com-
parison AFP as a marker of NTDs has a distance exceeding 3, and
maternal age as a ‘marker’ of DS would have a distance of about 1.
Nuchal translucency is by far the single best individual marker.
Among the serum markers, PAPP-A is the most discriminatory,
164 SE C T I O N 6     Prenatal Screening and Diagnosis
gestation. Free β-hCG is more discriminatory at 14 to 18 weeks
than at 10 to 13 weeks, although there is a gradual change in
Mahalanobis distance between 10 and 18 weeks. At 14 to 18
weeks’ gestation, hCG is less discriminatory than free β-hCG, and
before 13 weeks, it is a poor marker. At 14 to 18 weeks, inhibin A
is of comparable discriminatory power to hCG. AFP and uE3 are
not very discriminatory markers.  specific birth prevalence with an additive component because of
Multimarker Testing Strategies
A large number of marker combinations have been evaluated.
Many of them are in use today and are recommended by ISPD.5
Among the optimal strategies, those yielding the highest DR for a
given FPR, there are just six in widespread use:
Combined test. This first-trimester strategy is a combination
of two serum markers, PAPP-A and either hCG or free β-hCG,
together with ultrasound measurement of NT. The blood sample
can be taken from 10 to 13 weeks, although some laboratories
accept an earlier sample. However, the NT has a narrower accept-
able range of 11 to 13 weeks or an ultrasound crown–rump length
(CRL) of 45 to 85 mm.  is used. The model parameters are the log-transformed means, SD
Quadruple test. A second trimester serum-only strategy com-
bining AFP, hCG or free β-hCG, uE3 and inhibin A. To use the
AFP level for both NTD and aneuploidy screening, the test has to
be carried out at 16 to 18 or at least 15 to 19 weeks’ gestation, the
window for optimal NTD detection.  Parameters are best derived by meta-analysis, excluding the viabil-
Integrated test. This strategy combines markers in both tri-
mesters. PAPP-A and NT are determined in the first trimester, but
hCG or free β-hCG measurement is delayed until the second tri-
mester when it is measured together with AFP, uE3 and inhibin.21
The protocol requires nondisclosure of risk based on the PAPP-
A and NT levels. Some regard the nondisclosure to be unethi-
cal or at least impractical because of the difficulty for the health
professional not to act on first trimester findings which would of
themselves be abnormal, particularly the NT. The increase in DR
offered by this approach is offset by the delayed early diagnosis
and reassurance that a first trimester test offers.  mined. This form of risk calculation assumes that the marker
Serum integrated test. This is a serum only version of the Inte-
grated test.  that the marker levels are unrelated to the probability of intra-
Stepwise sequential test. The first stage uses the combined test
markers and women with very high risks–much higher than for
a combined test per se–are immediately offered invasive testing.
Those with risks below this cutoff are offered the quad test mark-
ers with their final risk based on all markers.22 
Contingent testing. Contingent testing is performed as with
the stepwise sequential test except that second trimester marker
testing is contingent on the first trimester results. In this approach,
two first trimester cutoffs are used. Very-high-risk patients are
referred for diagnostic testing, and low-risk patients only have
first trimester screening performed. Intermediate first trimester
values have second trimester serum screening. Only 10% to 20%
of women with borderline high-risk results are offered the second
trimester stage.23  a variable volume in the maternal unit. However, this cannot be
Risk Screening
Prior Risk
The prior, or pretest, DS risk can be estimated from the maternal
age and family history. It can be expressed either as the chance of
having a term pregnancy with the disorder or the chance of the
fetus being affected at the time of testing. In so far as the aim of
screening is to reduce the prevalence at birth, the former is most
appropriate. Because screening is also about providing women
with information on which to base an informed choice about
prenatal diagnosis, it can be argued that the latter is more rel-
evant. If term risks are used, they can be estimated from the age-
family history.6 If first or second trimester risks are used they
can be estimated by applying the intrauterine loss rates to the
prevalence. 
Likelihood Ratio
Statistically, the optimal way of interpreting the multimarker pro-
file is to estimate the DS risk from the individual marker levels.24
This is done by modifying the prior risk by calculating a patient
specific likelihood ratio (LR) derived from the patient’s marker
profile and a model of marker distributions. Because all the prin-
cipal markers follow an approximately log Gaussian distribution
over most of the MoM range, a multivariate log-Gaussian model
and correlation coefficients in affected and unaffected pregnan-
cies. There will be MoM values beyond which there is substantial
deviation from the model. Values beyond these ‘truncation limits’
are assumed to be at the nearest limit for risk calculation purposes.
ity bias that occurs in prospective intervention studies or at least
adjusting for bias.
The LR for a single marker is calculated as the ratio of the
heights of the two overlapping ‘bell-shaped’ distributions at the
specific level. For two markers, the overlapping bivariate distri-
butions can be represented as ‘football shaped’ mountains, and
the heights are determined at the longitude and latitude of the
two specific levels. When more than two markers are included,
the multivariate distributions are difficult to visualise, but the
principle is the same whereby the ratio of ‘heights’ is deter-
levels and maternal age are independent determinants of risk and
uterine survival. Although there is evidence that extreme values
of biochemical and ultrasound markers can be associated with
increased fetal demise,6 values within the truncation limits will
not be affected. 
Covariables
More precise LRs can be estimated when the MoMs and the dis-
tribution parameters take account of covariables such as mater-
nal weight, smoking status and ethnicity. The serum markers,
when expressed in MoMs, are negatively correlated with mater-
nal weight. This is usually explained in terms of dilution: a fixed
mass of chemical produced in the fetoplacental unit is diluted by
the only factor involved because the extent of correlation differs
between the markers (e.g. the correlation is almost twice as great
for PAPP-A than AFP or hCG; inhibin has a weaker correlation
than these two; and for uE3, there is hardly any association at all,
particularly in the first trimester). It is standard practice to adjust
all serum marker levels for the individual’s weight, dividing the
observed MoM by the expected value for the weight derived by
regression. A regression formula using 1/weight is more accurate
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy
for very large and very small women than simple linear regres-
sion25 and should be derived from a local population.
On average, smokers have reduced levels of PAPP-A, free β-
hCG and second trimester hCG but an increased inhibin level. In
women of Afro-Caribbean origin or African Americans, on aver-
age, AFP, intact hCG and second trimester free β-hCG levels are
increased, and inhibin A is decreased. In women of South Asian
origin, uE3 and total hCG levels appear to be somewhat increased.
As with maternal weight, adjustment can be carried out by divid-
ing the observed MoM by the average value in the local popu-
lation according to smoking status and ethnicity. The correction
factors used for different ethnic groups appear to differ according
to gestational age.26,27  recommended that a standardised technique is adopted for mea-
Gestational Dating
Accurate determination of gestational age is a key to both timing
of the screening test and for MoM calculation. In general obstet-
rics, the gestational age based on the time since the last menstrual
period (LMP) should only be modified by ultrasound findings
if there is a large discrepancy. In early pregnancy, a difference
between LMP- and CRL-based estimates greater than 3 days is
regarded as large and should lead to a change in gestational age
based on the CRL. As pregnancy continues, fetal measurements
are somewhat less precise and a difference of more than 7 days is
required. In practice, screening performance will be improved if in
every pregnancy in which ultrasound dating is available, it is used
for MoM calculation instead of the LMP.  may be the same mechanism that leads to low levels of PAPP-A in
Evaluating the Performance of Multimarker
Screening
Two widely used methods of estimating DR and FPR are numeri-
cal integration and Monte-Carlo simulation. Numerical integra-
tion is based on the same model of the log Gaussian distributions
of each marker in DS and unaffected pregnancies used for risk
calculation together with a maternal age distribution.24 The theo-
retical range of the markers (plus to minus 3 SDs) across both
outcomes is divided into a number of equal sections, thus forming
a ‘grid’ in multidimensional space. The Gaussian distributions are
then used to calculate for each section (square for two markers,
cube for three and so on) the proportion of DS and unaffected
pregnancies in the section and the LR. It is then a matter of apply-
ing these values to the maternal age distribution. At each maternal
age, the number of DS and unaffected pregnancies is estimated
from the age-specific risk curve. The distributions of DS risks are
then calculated from the grid values. Monte-Carlo stimulation
also uses the Gaussian distributions, but instead of rigid summa-
tion over a fixed grid, it uses a random sample of points in multi-
dimensional space to simulate the outcome of a population being
screened.
When assessing the relative benefits of different policies, it is
best to either fix the FPR (e.g. 1% or 5%) and compare the DRs
or fix the DR (e.g. 75% or 85%) and compare the FPRs. How-
ever, when changing policy, it would be confusing to alter the cut-
off risk to maintain the DR or FPR. In practice, it is common to
retain the cutoff (e.g. 1 in 250 at term or 1 in 270 at midtrimester)
and allow both DR and FPR to vary. In this chapter, performance
is presented using all three methods, and model predictions are
based on Gaussian distributions with parameters derived by meta-
analysis and use the standard maternal age distribution.  and a smaller increase in α-hCG mRNA, suggesting that one of
165
First Trimester Screening
Markers in More Detail
Nuchal translucency. Fetal subcutaneous oedema in the area
of the posterior neck region is associated with DS.28 as well as
other chromosomal and congenital abnormalities. The reasons
for oedema in DS fetuses are not known, but the most plausible
explanations are altered composition of the cellular matrix,29
abnormal or delayed development of the lymphatic system or
cardiac insufficiency.
Because NT measurements are frequently submitted to vari-
ous laboratories that may not have their own normal values, it is
surement.30 Both the Fetal Medicine Foundation in London and
the Nuchal Translucency Quality Review (NTQR) program in the
United States have detailed descriptions of appropriate measure-
ment techniques. The best results are obtained when MoMs are
based on normal medians calculated to the day of gestation using
regression; some practitioners use centre- or operator-specific
curves.31 
PAPP-A. This is a protease for insulinlike growth factor binding
protein 4 and may therefore play a role in regulating fetal growth
and trophoblast proliferation. PAPP-A levels are reduced in first
trimester DS pregnancies.32 This reduction diminishes as preg-
nancy progresses, and there is little or no difference by the second
trimester. The reason for the low levels in DS pregnancies is not
known but is probably associated with placental insufficiency; it
nonviable pregnancies.33 
hCG and free β-hCG. This is a glycoprotein hormone normally
found in blood and urine only during pregnancy. This hormone
is composed of two nonidentical noncovalently linked subunits,
α and β, that exist either free or bound to each other and is
produced by the syncytiotrophoblast cells of the placenta. The
α-subunit is also shared by three other glycoprotein hormones:
lutenising hormone, follicle-stimulating hormone (FSH), and
thyroid-stimulating hormone. The β-subunit is unique and dis-
tinguishes hCG from the other glycoprotein hormones. Five
hCG-related molecules are present in maternal serum: non-nicked
hCG, which represents the active hormone; nicked hCG; free
α-subunit; free β-subunit; and nicked free β subunit.34 Of the
hCG species circulating in maternal serum, β-hCG corresponds to
only about 0.3% to 4%35; β-hCG lacks hCG activity, but several
lines of study indicated that it exerts growth-promoting activity.
For the initiation and maintenance of pregnancy, hCG mediates
multiple placental, uterine and fetal functions. Some of these
include development of syncytiotrophoblast cells, mitotic growth
and differentiation of the endometrium, localised suppression of
the maternal immune system, modulation of uterine morphology
and gene expression and coordination of intricate signal transduc-
tion between the endometrium.36 Not all the factors involved in
hCG secretion are known, but cyclic adenosine monophosphate
(cAMP), prolactin, corticosteroids and gonadoliberin influence
release, and polyamines, estradiol and progesterone inhibit release.
Maternal serum hCG peaks at 8 to 10 weeks and then declines
to reach a plateau at 18 to 20 weeks of gestation and remains
relatively constant until term. Abnormality in cytotrophoblast dif-
ferentiation may be the basis of the elevated hCG levels in DS
pregnancies.37 Molecular biology studies demonstrated that tri-
somy 21 trophoblasts show a marked increase in β-hCG mRNA
166 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Combined Test: Model Predicted Detection Rate (DR) and False-Positive Rate (FPR) for Down Syndrome (DS),
18.2 Without and With Maternal Serum Placental Growth Factor and Ultrasound Nasal Bone (NB), According to
Gestational Age (GA)a
DR FOR FPR FPR FOR DR DR AND FPR FOR FINAL CUTOFF RISK
GA 1% 5% 75% 85% Term: 1 in 250 Midtrimester: 1 in 270
Combined test
Using free β-hCG 11 74% 87% 1.2% 3.8% 81% and 2.4% 84% and 3.3%
13 66% 80% 2.9% 8.8% 75% and 2.8% 78% and 4.0%
Using hCG 11 71% 84% 1.6% 5.3% 79% and 2.5% 82% and 3.5%
13 67% 81% 2.4% 7.4% 76% and 2.7% 79% and 3.8%
Combined test and PlGF
Using free β-hCG 11 76% 89% 0.9% 3.0% 83% and 2.3% 86% and 2.2%
13 70% 84% 1.9% 5.9% 78% and 2.7% 81% and 3.8%
Using hCG 11 73% 86% 1.3% 4.3% 81% and 2.5% 83% and 3.4%
13 71% 85% 1.6% 5.2% 79% and 2.6% 82% and 3.6%
Combined and routine NB
Using free β-hCG 11 88% 95% 0.2% 0.6% 90% and 1.4% 91% and 1.8%
13 85% 93% 0.3% 1.0% 88% and 1.6% 89% and 2.1%
Using hCG 11 87% 94% 0.2% 0.8% 89% and 1.5% 91% and 2.0%
13 86% 93% 0.2% 0.9% 88% and 1.6% 90% and 2.0%
Combined and contingent NBb
Using free β-hCG 11 86% 91% 0.3% 0.8% 86% and 0.9% 87% and 1.3%
13 82% 87% 0.5% 2.8% 82% and 1.0% 83% and 1.4%
Using hCG 11 84% 89% 0.4% 1.2% 84% and 1.0% 85% and 1.3%
13 83% 88% 0.4% 2.1% 83% and 1.0% 84% and 1.3%
aModel parameters based on meta-analyses6,39,40 and a standard maternal age distribution with a mean of 27 and standard deviation of 5.5 years.
bNBonly measured if combined risk 1 in 50 to 1500 (term, 1 in 38–1200 at midtrimester). Final risk cutoffs for the second stage of the combined and contingent NB test based on the revised risk.
hCG, Human chorionic gonadotrophin; PlGF, placental growth factor.
  

the causes of high hCG levels in maternal serum is the increased
hCG production and secretion by the placenta.38  possible to install equipment close to the ultrasound unit that will
Performance: Combined Test
Table 18.2 shows the model-predicted performance when both the
biochemical and ultrasound stages are carried out at 11 or 13 weeks’
gestation and when the two isoforms of hCG are used. The DR for a
5% FPR is 80% to 87% compared with 71% to 77% for NT alone
and 53% to 57% for the two serum markers alone. The table shows
that performance is better earlier than later in the 11- to 13-week
window. Slightly better results are achieved when the biochemistry
is done at 10 weeks and NT at 11 to 13 weeks. Moreover, this over-
comes a practical constraint of the combined test because the result
of the NT scan can be reported to the patient immediately, but
a serum sample, tested in batches and assayed overnight, will not
usually yield a result for several days. In general, one way of dealing
with this is to draw a blood sample a few days before the scheduled
ultrasound appointment and ensure that the serum MoMs are avail-
able for risk calculation as soon as the NT is measured (sometimes
known as IRA-instant risk assessment). Alternatively, it is now
assay a single sample economically and within an hour (sometimes
known as OSCAR or one-stop risk assessment).41
Modelling indicates that the use of additional serum mark-
ers will increase detection. The first candidate would be placental
growth factor (PlGF). This hormone has a Mahalanobis distance
for DS of 0.936 and is currently used in first trimester screening for
preeclampsia (see later). Modelling predicts that this would increase
the DR for a 5% FPR to 84% to 89%, an additional 2% to 4% (see
Table 18.2). Adding other candidates such as AFP, uE3 and inhibin
A would each lead to a further 1% to 2% increase in detection. 
For Localities with High-Quality Ultrasound
Several additional ultrasound markers of DS can be evaluated at
the time of NT measurement. In centres with sufficient expertise
one such marker, assessing the presence or absence of the fetal
nasal bone (NB), is now being routinely determined during the
NT scan.
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy
The NB is a dichotomous marker, so there are just two LRs,
one for absence and the other for presence. On the basis of
a meta-analysis of nine studies,40 these LRs are 49 and 0.31,
respectively. However, quality assurance is problematic with
dichotomous variables such as NB; absence is a relatively rare
event, so the frequency with which the operator misclassifies it
as present, or vice versa, cannot be easily determined. This sug-
gests another protocol, whereby women with borderline risks
based on the combined test are referred to a centre that spe-
cialises in using the more advanced markers. This first-trimester
form of the contingent test would obviate the several weeks’
delay associated with a standard contingent test. Table 18.2
shows the model predictions when NB is added to the com-
bined test routinely or contingently. With routine use of the NB
the DR shows a substantial increase to 93% to 95% for a 5%
FPR. This improvement is so large that it reduces the relative
benefits of different isoforms of hCG and gestation. This model-
ling assumes that NB is uncorrelated with the serum markers.
A small reduction in PAPP-A and an increase in free β-hCG have
been reported in affected pregnancies with absent NB compared
with other DS cases in which the NB could be seen, but these
differences were not statistically significant.40,42 A contingent
use of NB achieves reasonably high detection; the DR is 87% to
91% for a 5% FPR.
Other sonographic markers that could be used during the
assessment of fetal NT are abnormal blood flow in the ductus
venosus (DV) and tricuspid regurgitation (TR). Both require
recognition of the embryonic anatomy and proficiency in using
pulse-wave Doppler. Flow in the DV is a continuous variable,
usually reported as either normal or abnormal, the latter mean-
ing absent or reversed flow, with LRs of 22 and 0.35.43 Similarly,
TR values are dichotomised with LRs of 56 and 0.44. Another
sonographic marker is the frontal-maxillary facial (FMF)
angle.44,45 The FMF angle is a continuous variable and changes
with gestation. It is usually dichotomised to above and below
the 95th centile with LRs of 45 and 0.57.43 Using DV, TR or
FMF instead of NB, performance is comparable but slightly less
discriminatory.  and genitourinary anomalies (34%).53 This casts some doubt
For Localities with Limited or No Ultrasound
Nuchal Translucency Provision
In situations in which NT measurement is not available because
of limited equipment or operators adequately trained in NT mea-
surement, a serum-only first trimester protocol may be considered,
particularly if additional serum markers are used. A first trimester
serum-only quad test has been considered adding PlGF and AFP
to PAPP-A and free β-hCG (or hCG). In one study, using param-
eters from a prospective study which may have been subject to
viability bias, there was a model predicted DR of 82% for a 5%
FPR compared with 87% for the combined test. A serum-only
quintuple test might also be considered using uE3 or inhibin A.
Modelling also predicts a high DR for PAPP-A, hCG and free β-
hCG despite the high correlation between the two hCG species.46
Another option is to carry out the serum screening stage of a
combined test on all women but restrict the NT stage to those
who have relatively high DS risks after serum testing.47 Model-
ling predicts that testing for PAPP-A and free β-hCG at 10 weeks
and contingently measuring NT at 11 weeks on the one-third of
women with the highest risks would only reduce the DR from
87% to 82% for a 5% FPR, still considerably more than the
71% provided by a quad test. Raising the cutoff so that NT was
167
offered to the one-fifth with high risks would yield a 77% DR.
Another issue is timing of the first-trimester biochemical markers:
because PAPP-A is more discriminatory for DS at 8 weeks than at
10 weeks and hCG species are better markers at 13 weeks, it has
been suggested that better detection can be achieved by separating
the collection of these markers.48,49 However, there are practical
implications to such a protocol. 
Early Anatomy Scan to Determine Ultrasound
Markers of Down Syndrome
Cystic hygroma is defined as an enlarged hypoechoic space at
the back of the fetal neck, extending along the length of the fetal
back, and in which septations are clearly visible. It is associated
with a very high risk of aneuploidy and a general poor prognosis.
In the First and Second Trimester Evaluation of Risk (FaSTER)
trial, this sonographic finding was sufficient to offer immediate
invasive diagnostic testing rather than awaiting the results of a
combined or integrated test. This variant on standard testing pro-
tocols probably has little effect on DS detection because in most
cases (although not all), the NT is high, and most affected women
would have had screen-positive results. In one series of 42 cystic
hygroma cases found in nearly 7000 routine first trimester scans,
35 had an NT of 3 mm or more.50
Although several other structural malformations can be
detected during NT measurement, a full fetal anatomical sur-
vey is usually performed toward the end of the second trimester
(see later). However, the recent development of high-frequency
transvaginal ultrasound transducers combined with the improved
technology of sonographic equipment has led to vastly enhanced
ultrasound resolution and improved visualisation of fetal anat-
omy earlier in pregnancy. Many studies have reported DRs of
major fetal anomalies by first trimester ultrasound studies that
were comparable with those achieved in the second trimester
anatomy scan.51,52 However, a meta-analysis of 19 studies includ-
ing a total of 78,002 fetuses, 996 malformed, yielded an overall
51% DR, ranging from 92% for neck anomalies to 34% for face
as to the utility of the first trimester survey in identifying ‘soft’
(mainly qualitative) markers of aneuploidy seen in a late second-
trimester anatomy scan, which might be incorporated into the
combined test. 
Detection of Trisomy 18 and 13
The same markers that are used for DS screening can be used
for trisomy 18 screening. Based on meta-analysis, the means for
NT, PAPP-A and hCG or free β-hCG were 2.77, 0.14 and 0.31
MoM respectively.6 Table 18.3 shows the predicted DRs for tri-
somy 18 using standard DS screening protocols compared with
protocols which explicitly include risk for trisomy 18. The DR
is particularly high in the first trimester even without explicit
screening because most cases are associated with an increased
NT.
It has been suggested that screening be extended to include
trisomy 13.54 The markers profile is more extreme than trisomies
21 and 18 in the first trimester, with extremely high NT cou-
pled with very low free β-hCG and AFP.55 The proposed algo-
rithm uses one set of parameters to calculate the combined risk
of trisomy 18 and 13. Model predictions are that 95% of cases
with one or the other type of aneuploidy can be detected for a
0.3% FPR. 
168 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Combined Test and Quad Test: Model Predicted Detection Rate (DR) and False-Positive Rate (FPR) for Trisomy
18.3 18, Using Down Syndrome (DS) Risk Cutoff Alone and with an Explicit Trisomy 18 Risk Cutoffa
DS CUTOFF ONLY DS/TRISOMY 18 CUTOFF
Midtrimester: 1 Term: 1 in Midtrimester: 1 Term: 1 in Mid-Trimester:
GA Term: 1 in 250 in 270 250/50 in 270/50 250/100 1 in 270/100
Combined test
Using free β-hCG 11 81% 83% 81% 83% 81% 83%
13 80% 82% 81% 83% 82% 84%
Using hCG 11 87% 88% 87% 89% 87% 89%
13 78% 80% 81% 83% 83% 85%
Quad test
Using free β-hCG 31% 36% 48% 53% 52% 55%
Using hCG 29% 35% 32% 38% 39% 45%
aModel parameters based on meta-analyses6 and a standard maternal age distribution with a mean of 27 and a standard deviation of 5.5 years.
hCG, Human chorionic gonadotrophin.
  

Second Trimester Screening
Serum Markers
AFP. This is a fetal-specific globulin similar to albumin in
molecular weight (about 69 kD) but with different primary
structure and antigenically quite distinct. In the first trimester,
AFP is predominantly of fetal yolk sac origin. The reason for
decreased AFP synthesis in DS fetuses is unknown, but in the
second trimester it may reflect hepatic immaturity.  useful. They are nuchal skinfold thickness (NF), femur length
Unconjugated estriol. Maternal urine total estriol excretion
in the third trimester of DS pregnancies was found to be lower
than in unaffected pregnancies,56 and subsequently, the level of
maternal serum uE3 was also found to be lower than average in
both the first and second trimesters.57 In a fetus with DS, there
is adrenal hypoplasia; therefore there is a decreased production
of dehydroepiandrosterone sulphate (DHEAS), a hormone that
the fetal liver hydroxylates and is then transported to the placenta
where it undergoes desulphation and aromatisation into estriol
before entering the maternal circulation.  or FL,65 which in a meta-analysis of five studies had a mean
Inhibin. Levels have been shown to be increased in DS pregnancies
using assays that detect all species58 and those specific for
inhibin-A.59 Inhibin is considered to have a role in the regulation
of gonadotropin biosynthesis and secretion, ovarian and placental
steroidogenesis, and oocyte maturation. Inhibin is regarded as a
member of the transforming growth factor super family and is
characterised by its ability to suppress FSH secretion.  been proposed. These markers include prenasal thickness (PT),
Performance: Quad Test
Table 18.4 shows that the model predicted performance of the
quad test is considerably poorer than for the combined test with a
DR of 67% to 71% for a 5% FPR. This can be improved by the
routine simultaneous determination of ultrasound markers.  ised by reduced NBL rather than an absent NB, and it is a
Detection of Trisomy 18 and 13
Based on meta-analysis, in trisomy 18 pregnancies, the second tri-
mester means for hCG or free β-hCG, AFP, uE3 and inhibin were
0.31, 0.68, 0.44 and 0.81 MoM, respectively.6 In contrast to the
first trimester, explicit risk cutoffs are needed for trisomy 18 to
achieve a reasonably high DR (see Table 18.3). In trisomy 13, the
second trimester uE3 levels are low,60 and inhibin is increased,61
leading to some incidental diagnosis in DS protocols. 
Ultrasound Markers
Nuchal skinfold and long bones. Three markers that can
be determined in the early second trimester are potentially
(FL) and humerus length (HL). An increase in NF among DS
pregnancies was first shown more than 30 years ago,62 but
most studies have not reported values in MoMs. A meta-
analysis of five studies in which MoMs were available showed
the average NF in affected pregnancies was 1.45 MoM, the
Mahalanobis distance was about 1.0, and it was predicted that
adding NF to serum free β-hCG and AFP would increase the
DR for a 5% FPR by 12%.63 There have also been proposals
to incorporate into serum screening protocols either HL64
of 0.94 MoM in affected pregnancies, with a Mahalanobis
distance of 0.80.6 It has been predicted that adding NF, HL
and FL would increase the quad test DR for a 5% FPR by
15%.65 
Facial profile. Rather than using FL and HL as further
ultrasound markers, using quantitative facial profile markers
that are imaged in the same midsagittal plane as the NF has
which is described as the shortest distance between the ante-
rior edge of the lowest part of the frontal bone and the skin
anteriorly and nasal bone length (NBL).66 In a meta-analysis
of 5 studies, the mean PT was 1.33 MoM, with a Mahalanobis
distance of 0.80.67 In the second trimester, DS is character-
weaker marker.68,69 Modelling predicts that a quad test com-
bined with NF, PT and NBL would have a 92% to 93% DR
for a 5% FPR (see Table 18.4). Additional facial markers have
been investigated that could increase detection even further.
These relate to the smaller size and dorsal displacement of the
maxilla in DS, such as the FMF angle70-72 and the prefrontal
space ratio.72,73 
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy 169

TABLE Quad Test: Model Predicted Detection Rate (DR) and False-Positive Rate (FPR) for DS, Without and With
18.4 Ultrasound Nuchal Skinfold Thickness (NF), Nasal Bone Length (NBL) and Prenasal Thickness (PT)a
DR FOR FPR FPR FOR DR DR AND FPR FOR CUTOFF RISK
1% 5% 75% 85% Term: 1 in 250 Midtrimester: 1 in 270
Quad alone
Using free β-hCG 50% 71% 6.9% 15% 68% and 4.2% 73% and 5.9%
Using hCG 46% 67% 9.3% 20% 64% and 4.3% 69% and 6.0%
Quad and NF
Using free β-hCG 64% 80% 3.0% 8.4% 75% and 2.9% 78% and 4.1%
Using hCG 62% 78% 3.7% 10% 73% and 3.0% 76% and 4.2%
Quad, NF and NBL
Using free β-hCG 69% 84% 1.8% 5.5% 78% and 2.6% 81% and 3.5%
Using hCG 68% 83% 2.2% 6.7% 77% and 2.6% 80% and 3.7%
Quad, NF, NBL and PT
Using free β-hCG 83% 93% 0.3% 1.3% 88% and 1.9% 90% and 2.6%
Using hCG 81% 92% 0.4% 1.5% 87% and 2.0% 89% and 2.7%
aModel parameters based on meta-analyses6,66 and a standard maternal age distribution with a mean of 27 and a standard deviation of 5.5 years.
hCG, Human chorionic gonadotrophin.
  

Second Trimester Anatomy Scan Sequential Screening

The sonographic assessment of fetal anatomy, traditionally per-
formed in the late second trimester, is sometimes referred to as the
‘genetic’ sonogram because it is used not only to evaluate the fetus
for structural malformations but also to search for sonographic
markers of genetic disorders. The finding of a major fetal anomaly
is considered a risk factor for aneuploidy. In addition, various soft
sonographic markers that may be detected during the sonographic
study have been identified; the presence of one or more such
markers suggests an increased risk for aneuploidy. These mark-
ers include increased NF, short femur and humerus lengths, renal
pyelectasis, an echogenic intraventricular cardiac focus, echogenic
bowel and an aberrant right subclavian artery (ARSA).
A recent meta-analysis summarised the accumulated data on the
screening performance of second-trimester sonographic markers for
DS.74 Forty-eight studies were included in the analysis. Two LRs
were estimated for each marker–one to be used when the marker
is present and another when absent. They were 5.8 and 0.80 for
intracardiac echogenic focus, 28 and 0.94 for ventriculomegaly, 23
and 0.80 for increased NF, 11 and 0.90 for hyperechogenic bowel,
7.6 and 0.92 for pyelectasis, 3.7 and 0.80 for short FL, 4.8 and
0.74 for short HL, 21 and 0.71 for ARSA and 23 and 0.46 for
absent or hypoplastic NB. The combined negative LR, obtained
by multiplying the values of individual markers, was 0.13 when
FL but not HL was included and 0.12 using HL but not FL. For
most isolated markers, there was only a small effect on DS risk, but
with isolated ventriculomegaly, NF and ARSA there was a three- to
fourfold increase in risk and with hypoplastic NB six- to sevenfold.
Women who do not present until the late second trimester
could be screened using the anomaly scan. The best estimate of DR
is from the FaSTER study group that used modelling to predict a
DR of 59% for an FPR of 3%.75 FaSTER also assessed the pos-
sibility of improving the quad test by routinely using anomaly scan
markers. The model predicted DR was 80% for an FPR of 3%.  the original risk.
The model predicted performance of the four protocols using
both first and second trimester markers is shown in Table 18.5.
The serum integrated test has a performance worse than the first
trimester combined test but better than the second trimester quad
test. The predicted DR is 73% to 78% for a 5% FPR. The full
integrated test would increase detection over a combined test–
a DR 89% to 93% for a 5% FPR. However, both the stepwise
sequential and contingent tests have a performance comparable
with the integrated test–91% to 94% and 88% to 92%, respec-
tively, for a 5% FPR. Given the human and practical benefits and
lower costs, the contingent test should be the sequential strategy
of choice.
Anomaly Scan Results
It is common for a woman who had a borderline positive com-
bined test to delay a decision over invasive prenatal diagnosis
until the second trimester ultrasound examination has been
carried out. Some women with borderline negative results may
require ultrasound assurance. Often the scan is interpreted sim-
plistically, whereby the presence of a major anomaly or soft
marker associated with DS is taken to be sufficient to tip the
balance in favour of invasive testing and the absence of signs
is sufficient to contraindicate testing. This is no longer accept-
able; instead, the risk from the combined test should be for-
mally revised using LRs relating to each marker seen or absent
as described earlier.
Similarly, most women with negative combined test results
eventually have a routine anatomy scan. The detection of an
anomaly or a soft marker generates considerable anxiety, despite
the normal result of the combined test. Often this may only be
resolved by invasive testing, but again, it is best to formally revise
170 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Sequential Screening Protocols: Model-Predicted Detection Rate (DR) and False-Positive Rate (FPR) for DS,
18.5 According to Gestational Age (GA)a
DR FOR FPR FPR FOR DR DR AND FPR FOR FINAL CUTOFF RISK

Protocolb GA 1% 5% 75% 85% Term: 1 in 250 Midtrimester: 1 in 270

Integrated
Full protocol 11 85% 93% 0.3% 1.1% 87% and 1.6% 89% and 2.1%
13 79% 89% 0.6% 2.5% 84% and 2.0% 86% and 2.7%
Serum only 11 61% 78% 3.7% 10% 74% and 3.2% 77% and 4.5%
13 55% 73% 5.7% 14% 70% and 3.7% 74% and 5.2%
Stepwise sequential
Using free β-hCG 11 85% 94% 0.4% 1.0% 89% and 1.7% 91% and 2.2%
13 80% 91% 0.6% 1.9% 86% and 2.1% 88% and 2.8%
Using hCG 11 86% 94% 0.4% 0.9% 89% and 1.6% 90% and 2.1%
13 80% 91% 0.6% 1.9% 85% and 2.0% 87% and 2.6%
Contingent
Using free β-hCG 11 85% 92% 0.4% 1.0% 88% and 1.6% 89% and 2.0%
13 79% 88% 0.7% 2.3% 84% and 1.9% 85% and 2.4%
Using hCG 11 84% 90% 0.4% 1.2% 86% and 1.4% 87% and 1.8%
13 79% 88% 0.6% 2.5% 83% and 1.8% 85% and 2.3%
aModel parameters based on meta-analyses6 and a standard maternal age distribution with a mean of 27 and a standard deviation of 5.5 years
bIntegrated: first stage pregnancy-associated plasma protein A and nuchal translucency, routine second-stage α-fetoprotein, free β human chorionic gonadotrophin(β-hCG), unconjugated estriol and inhibin.

Stepwise sequential: first stage also includes hCG or free β-hCG; 1 in 50 cutoff (term risk, equivalent to 1 in 38 at midtrimester); second stage if negative. Contingent: as stepwise but second stage only if risk
above 1 in 1500 (1 in 1200 at midtrimester). Final risk cutoffs for the Integrated test, the second stage of the stepwise sequential and contingent tests based on all first and second trimester markers included.
hCG, Human chorionic gonadotrophin.
  

Although there may be clinical utility in these ad hoc uses of
the anatomy scan to modify combined test results, it would not
be an effective protocol if adopted routinely. Using data from the
FaSTER trial, it was shown that replacing the quad markers in a
contingent test by ultrasound reduced detection.75
Another group also evaluated the sequential use of a combined
test and the anatomy scan.76 In this simulation study, combined
screening alone had a DR of 88% with an FPR of 4.2%. A fol-
low-up second trimester ultrasound examination in which only
one sonographic marker was found and previous results were not
taken into account would detect an additional 8% DS cases but at
the cost of an additional 13% false positives. However, using indi-
vidual LRs to modify the combined test risk in screen-negative
patients, the overall DR would be 95% and FPR 5.4%. In a con-
tingent protocol, in which the anatomy scan was performed only
for patients with a first trimester risk of 1 in 300 to 2500, DR was
93%, and FPR was 4.9%. 
Special Considerations
Insulin-Dependent Diabetes Mellitus
In the past, women with insulin-dependent diabetes were found
in several series to have reduced second trimester maternal serum
AFP levels, by about 20% on average.77 In more recent series,
the effect was much smaller,78 possibly because of better diabetic
control,79 and some authors have cautioned against adjustment.80
In a meta-analysis, second trimester serum uE3 and hCG values
were also altered to a small extent.78 Several studies have shown
lower first trimester PAPP-A levels in patients with diabetes, but
it is unclear whether this is confined to those with type 2 diabe-
tes81,82 or whether type 1 is also involved.83 Some studies suggest
that low PAPPA could be predictive of gestational diabetes later in
pregnancy.84 There is also inconsistent data on first trimester hCG
levels in patients with diabetes.82,85 Based on the magnitude and
consistency of the findings, we suggest adjusting second trimes-
ter AFP for pregestational insulin-dependent diabetic patients (or
those receiving oral hypoglycaemic agents). Furthermore, adjust-
ment for first trimester PAPPA in pregestational type 2 diabetic
patients would seem to be appropriate. More data are needed for
other markers, and there is also a need for further clarification for
various subgroups of women based on their type of diabetes and
various therapies. 
Renal Transplant
Very high hCG values have been reported in women undergoing
dialysis,86 and somewhat elevated values have also been observed
in women who received a renal transplant in whom some degree
of renal impairment may persist.87,88 For women whose renal
function may be impaired, the serum markers, particularly hCG,
should be interpreted cautiously, and greater reliance on ultra-
sound markers is appropriate. A correction of hCG concentration
based on serum creatine has been proposed.6 
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy
Multiple Pregnancies
Biochemical markers have a poorer discriminatory power in
dichorionic twins (DC) than in singleton pregnancies or mono-
chorionic (MC) twins. This is because in twins discordant for
DS, normal fetoplacental products from the unaffected fetus can
mask the abnormal levels produced by the affected fetus. Con-
sequently, in twins shown to be dichorionic by the presence of a
placental ‘lamba’ sign on ultrasound, many centres use only NT
for screening, ideally allowing for the correlation between NT
measurements between the fetuses.89,90 Alternatively, we believe
that although the combined test is less effective than in single-
tons, it can be used in DC twins to estimate fetus-specific risks,
provided the parameters are adjusted accordingly. Multimarker
screening protocols for MC twins can be used and do not differ
from singletons except that the serum marker parameters require
adjustment.6
In DC twins, whether or not a full combined test is performed,
the predictive value of a negative result is relatively low, so there
is much to be gained by reassessing the risk using first and second
trimester ultrasound markers. The reliability of such risk revision
in twins is not known, but data on FPRs suggest that for some
first trimester markers, it should not differ markedly from sin-
gletons. Large series of twins have been reported showing similar
FPRs compared with singletons for first trimester absent NB91
and abnormal DV.92 Second trimester soft marker LRs have not
been published for twins, but the assumption is that those for
singletons are reliable.
For triplets, regardless of chorionicity, screening must only be
based on ultrasound markers. If NT is used, as with twins, the
most accurate risks will take account of the pairwise correlation in
MoMs between the fetuses.93
Fetal demise of a co-twin (i.e. spontaneous reduction to a
singleton pregnancy or ‘vanishing twin’) is common, particularly
early in pregnancy. Increased marker concentrations could arise
if there is residual trophoblast activity from the deceased twin or
slow clearance of the proteins from the maternal circulation. Fur-
thermore, the deceased twin may well have had a chromosome
abnormality, given the very high aneuploidy rate in early singleton
miscarriages. The question that therefore arises is whether serum
screening markers can reliably be used to assess risk. In one study,
first trimester serum markers were measured in cases in which the
demise was thought to have occurred within 4 weeks of testing.
PAPP-A and free β-hCG were both significantly elevated relative
to singleton pregnancies.94 However, another study failed to iden-
tify significant differences in serum marker levels in cases with a
vanishing twin.95 Because of the uncertainty, it is probably pru-
dent not to use serum markers and consider additional ultrasound
markers to evaluate risk.  that DS screening preferentially identifies SCAs.109 However, the
Assisted Reproduction Technologies
When a nonspontaneous pregnancy has been achieved in a sub-
fertile couple, often after a long waiting period and with some
difficulty, there is additional reason to avoid invasive prenatal diag-
nosis. Such couples need to have the maximum number of markers
tested to best assess the risk of DS. When calculating risk, special
care is needed in determining maternal age. If a donor egg was
used, risk is calculated using the age of the donor at the time of
collection. Similarly, if the woman’s own egg was collected and fro-
zen or fertilised and then frozen, maternal age is based on the date
of freezing. The most consistent finding for first trimester serum
171
markers for women who have used assisted reproductive technolo-
gies (ARTs) is a depression in PAPP-A levels; hCG and free β-hCG
levels may also be altered.6,96,97 Second trimester markers may also
differ from those seen in naturally conceived pregnancies.98 How-
ever, there is considerable heterogeneity among the studies, possi-
bly because of the method of gestational age assessment, the actual
gestational age at screening, the cause of infertility or the type of
treatment. Consequently, most programs currently do not adjust
markers for ART. 
Previous Test Result
For each of the serum markers, there is a positive correlation
between the values measured in consecutive pregnancies. In theory,
taking into account the results from a previous pregnancy could
therefore improve screening efficacy.99-102 However, implementa-
tion into clinical practice would be difficult because it requires
linkage of records and detailed information about the outcome of
the previous pregnancy to ensure that the initial results were not
due to abnormality, pregnancy complications, bad dating, twins
and other covariables. 
Incidental Diagnoses
A wide range of abnormalities are detected incidentally with
extremely high or low marker levels; most fall into five groups.
Triploidy
Two distinct types of second trimester marker profiles are asso-
ciated with a triploid fetus: (i) highly elevated AFP, hCG and
inhibin and low to normal uE3 and (ii) very low hCG, uE3 and
inhibin with low to normal AFP.103 Similarly, in the first trimester,
type 1 shows increased NT, but in type 2, PAPP-A is very low.104 
Turner Syndrome
There are also two distinct patterns seen on ultrasound with and
without hydrops; both types have reduced uE3 but hCG levels
are increased with hydropic disease and reduced when there is no
hydrops.105-107 Generally, PAPP-A levels are low, and NT levels
are very high when Turner Syndrome is present.107 
Other Sex Chromosome Abnormalities
In one meta-analysis of incidental diagnosis, DS represented only
58% of aneuploidies detected and even all three common triso-
mies together only accounted for 76%.108 It has been suggested
results are subject to a strong bias because these cases were gener-
ally diagnosed after a diagnostic procedure was carried out because
of abnormal screening results, but screen-negative cases would
remain unrecognised. 
Other Adverse Outcomes
Smith-Lemli-Opitz Syndrome
This is a rare autosomal recessive disorder with an incidence of
about 1 per 20,000 to 40,000 births. Clinical features include
mental retardation and skeletal, genital, cardiac, pulmonary and
renal malformations. There is considerable clinical variability.
172 SE C T I O N 6     Prenatal Screening and Diagnosis
The disorder is caused by a defect in cholesterol biosynthesis,
and because cholesterol is a precursor of estriol, affected preg-
nancies are characterised by low second trimester maternal
serum uE3.110 AFP and hCG also appear to be reduced. Using
these findings, an algorithm for detection of Smith-Lemli-
Opitz syndrome (SLOS) has been proposed for second trimester
screening.111 In a prospective trial involving more than 1 mil-
lion pregnancies, using a second trimester risk cutoff of 1 in
50, 0.29% of women were screen-positive for SLOS, but just
0.07% were positive for only this disorder (i.e. were negative for
aneuploidy or NTD screening).112 Many of the false-positives
are attributable to aneuploidy, fetal death and a variety of other
fetal abnormalities. The DR is unknown because only a small
proportion of cases were identified (five severely affected among
the screen positives and only one among the screen negative).
The quad protocol of the statewide California Screening Pro-
gram includes SLOS.  prostacyclin with consequent platelet aggregation. Low-dose
X-Linked Ichthyosis
This X-linked disorder, characterised by scaly dark skin on the
scalp, trunk and limbs, occurs in approximately 1 in 2000 male
births. It is caused by deficiency of steroid sulfatase, with con-
sequent abnormal estriol biosynthesis, resulting in extremely low
maternal serum uE3 levels. In one series of nine pregnancies with
low or absent second trimester serum uE3 levels, six were found to
have a complete and one a partial deletion of the steroid sulfatase
deficiency gene.113 In rare cases, the gene deletion involves adja-
cent DNA sequences and can result in Kallmann syndrome, men-
tal retardation and other abnormalities, depending on the genes
lost. Laboratories that screen for SLOS will identify many cases of
X-linked ichthyosis among the positive test results.  forms of preeclampsia: one associated with maternal mortality
Fetal Demise
As previously noted, trisomies 21, 18 and 13 are all associated
with high rates of in utero death, and it is likely that cases with
the most extreme marker levels are at the highest risk for demise.
The same patterns for the markers can also be seen for euploid
fetuses when there is an impending or actual fetal loss. In the first
trimester, low PAPP-A, low or high free β-hCG and very high NT
levels (or cystic hygroma) are associated with fetal loss. Extremely
low PAPP-A may also be caused by an ectopic pregnancy. In the
second trimester, fetal loss is associated with high or low AFP, low
uE3, high hCG and high inhibin-A. Combinations of abnormal
markers can confer even greater risks of fetal demise, for example,
high AFP and low uE3, high AFP and hCG, and high AFP and
inhibin A.
Essentially undetectable AFP and uE3 levels may indicate
a complete hydatidiform molar pregnancy (because there is
no fetus), and these cases may also show very high hCG and
inhibin A.114  treatment reduced the risk of growth restriction; pregnancies
Cardiac Abnormalities
Increased NT appears to be associated with an extremely broad
range of fetal abnormalities. Particularly noteworthy is the asso-
ciation with major cardiac abnormalities; a meta-analysis of 20
studies found a DR of 44% for a FPR of 5.5%.115 Because of these
rates, it is suggested to offer fetal echocardiography to women with
increased NT, regardless of serum marker results.116  (ASPRE) has been carried out to directly assess the efficacy of
Maternal-Fetal Conditions
Nicolaides has proposed that when women undergo the com-
bined test they can also be assessed for their risk for common
maternal-fetal conditions that usually appear during the third
trimester, such as preeclampsia, growth restriction, preterm
delivery and fetal macrosomia.117 Early detection of high risk
may facilitate primary prevention. This concept has been most
fully developed for preeclampsia, a complex disorder with vari-
able presentation and outcome that starts when the uterine
spiral arteries fail to remodel in early pregnancy. In normal
pregnancies, trophoblasts invade the spiral arteries, replacing
the smooth muscle and endothelium. The remodelled arter-
ies have reduced resistance and therefore increased blood flow
into the placenta. When this process fails, there are inadequate
perfusion, placental damage and imbalance of thromboxane–
aspirin has been shown to reduce the uterine artery Doppler
pulsatility index, indicating improved blood flow118, but until
recently, the consensus was that it does not prevent preeclamp-
sia. However, a meta-analysis of nine published trials, in which
treatment began before 16 weeks, has led to a shift in the
consensus.119 The trials included 116 women at risk for pre-
eclampsia, and the incidence in those assigned to aspirin was
significantly reduced (relative risk (RR), 0.47; 95% confidence
interval (CI), 0.34–0.65).
As with DS, screening would modify the prior risk, based on
body mass index, parity and preeclampsia in a previous pregnancy
or in a close family member, by a LR based on a multimarker
profile. Those who were screen positive would receive prophylactic
low-dose aspirin. However, unlike aneuploidy, there are distinct
and morbidity, prematurity, growth restriction and fetal death
and another developing around the time of delivery, more often
having a benign course, and not associated with prematurity. One
operational definition used in many screening studies is to classify
cases as ‘early’, requiring delivery before 34 weeks gestation, ‘very
early’ before 32 weeks, ‘preterm’ before 37 weeks, and ‘term’. Clas-
sifications based on gestation at delivery are more objective than
gestation of onset or severity.
The best estimate of screening performance can be derived
from the large population-based study at Kings College Hos-
pital, London. Women were recruited at 11 to 13 weeks, a
maternal history was taken, mean arterial pressure and uterine
artery Doppler PI were determined and PAPP-A was measured
as part of aneuploidy screening. Additionally, blood samples
were stored and retrospectively tested for PlGF. For a 5% FPR,
modelling predicts a DR for early preeclampsia of 93%.120
Although the meta-analysis was not able to separate early onset
preeclampsia, the RR of severe disease after early treatment
was 0.09. The aspirin meta-analysis also showed that early
at risk for this disorder, in the absence of preeclampsia, were
identified using all the aneuploidy and preeclampsia markers
in combination, including NT and hCG.121 Hence, there is
evidence for extending first trimester aneuploidy screening to
include placental vascular disease. Furthermore, this could also
improve aneuploidy detection because PlGF is also a marker
of aneuploidy.122
Recently, an international multicentre randomised trial
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy 173

TABLE Cell-free DNA (cfDNA) Strategies: Model Predicted Detection Rate (DR), False-Positive Rate (FPR) and Positive
18.6 Predictive Value (PPV) for Down Syndrome (DS)a
Selected for cfDNA DR FPR PPVb
Secondary cfDNA (after combined test at 12 wk)
5% positives 83.9% 0.006% 95%
3% positives 79.9% 0.003% 97%
1% positives 71.2% 0.001% 99%
Primary cfDNA
100% 99.3% 0.11% 54%
Contingent cfDNA (using combined test at 12 wk)
20% with highest risk 93.6% 0.02% 85%
Except 0.5% with very high risk have invasive PND 94.1% 0.52% 19%
20% with highest risk using additionally PlGF and AFP 95.6% 0.02% 85%
aModel parameters based on meta-analyses6,39,124 and a standard maternal age distribution with a mean of 27 and a standard deviation of 5.5 years and a meta-analysis for cfDNA.
bCalculated at term rather than the time of the test.
AFP, α-Fetoprotein; hCG, human chorionic gonadotrophin; PlGF, placental growth factor; PND, prenatal diagnosis.
  

low-dose aspirin in women selected by routine multimarker Cost Effectiveness

screening.123 A total of 1776 with the highest risk after screen-
ing were randomised to 150 mg/night or placebo. The incidences
of preterm preeclampsia were 1.6% and 4.3%, respectively (RR,
0.38; 95% CI, 0.20–0.74; P < .005).  of each additional affected birth avoided by a cfDNA strategy over
Integration of Combined Screening and Cell-
Free DNA Screening
Aneuploid screening using cfDNA is described in detail in
Chapter 21. In this section, we will discuss the relative advan-
tages and disadvantages of each approach and the impact on
overall screening performance. At the present time, both com-
bined screening and cfDNA screening are available, and policies
on their use differ by locality.
Cell-Free DNA Screening Strategies
Table 18.6 shows the model predicted performance of different
strategies incorporating cfDNA with the combined test. Second-
ary cfDNA after a positive combined test result rather than per-
forming a diagnostic test will substantially reduce the number
of amniocenteses or CVS procedures performed, thereby reduc-
ing iatrogenic fetal losses, but will also lead to a small reduc-
tion in detection. Contingent cfDNA maintains a reasonably
high DR at a vastly lower cost in comparison with a policy of
routinely performing cfDNA as the primary screening test for
everyone (‘primary cfDNA’). An approach similar to stepwise
sequential screening whereby those with very high combined
risks are directly offered invasive testing has a slightly higher
DR and despite a much higher FPR is considered worthwhile
because about two-thirds of affected pregnancies are in the very-
high-risk group, and the delay in waiting for cfDNA results is
avoided.  primary cfDNA.
The most useful measure of cost effectiveness of cfDNA is the
‘marginal’ cost or the incremental cost ratio (ICR). This is the cost
and above those avoided by, for example, the combined test. This
can be combined with the lifetime cost of an affected individual,
restricted to the direct medical, educational and social service
costs or including indirect societal costs such as loss of income.
In some cost-effectiveness studies, the lifetime cost is included in
the ICR, which is then compared with some general measure of
affordability, a function of the amount of resources available in a
country such as the gross domestic product per capita. In addition
to the marginal cost, public health planners need to consider the
total cost of changing to the new strategy or the average cost per
woman screened.
Secondary cfDNA strategies are generally cost neutral or lead
to cost savings because the unit cost of the cfDNA test is of the
same order of magnitude or cheaper than the unit cost of invasive
prenatal diagnosis. A more detailed cost-effectiveness analysis is
required to draw conclusions about the primary cfDNA and con-
tingent cfDNA strategies. In its most simple form, the inputs are
the unit costs of the conventional screening test, of cfDNA and
of invasive prenatal diagnosis, as well as uptake rates. These vary
considerably among countries as do lifetime costs.
In a review of published cost analyses,125 seven concluded
that primary cfDNA is too expensive, five based the incremen-
tal cost per DS detected in relation to an existing conventional
screening protocol and two based their conclusion on the
average cost per woman screened. A single analysis found the
strategy to be cost effective but only from a societal perspec-
tive, when the indirect as well as direct lifetime care costs for
individuals with DS were included. Five analyses demonstrated
that contingent cfDNA is considerably more cost effective than
 
174 SE C T I O N 6     Prenatal Screening and Diagnosis
Conventional Markers in the Cell-Free DNA Era
If the unit cost of a cfDNA test falls substantially, primary cfDNA
will be affordable in public health systems. At that point, health
planners may consider abandonment of the first trimester ultra-
sound NT scan, perhaps replacing it with a simpler and less
expensive dating scan. However, such action will not be prudent
because a large NT is associated with an increased risk of struc-
tural abnormalities and genetic syndromes even in the absence of
aneuploidy. Among the former are major cardiac defects, and this
aspect alone may be sufficient to justify retaining the scan.
A case may also be made for retaining some first trimester
biochemical markers, specifically for use in screening for adverse
pregnancy outcomes such as preeclampsia and growth restriction.
These outcomes are much more common than all aneuploidies
combined, and a large proportion can be prevented through first
trimester screening followed by daily low-dose soluble aspirin in
screen-positive women.  increasingly problematic when a large number of markers are used
Planning a Program
Early screening and diagnosis is highly advantageous because it
provides earlier reassurance, and if termination of pregnancy is
chosen, it can be completed earlier in pregnancy when the pro-
cedure is clinically easier and safer126; it is also emotionally easier
for patients who undergo termination before fetal movements are
felt. The early termination of DS pregnancies that are destined
to miscarry is an advantage because it removes the trauma of late
miscarriage. It also potentially provides information on recurrence
risk. Optimal design of a new screening program therefore focuses
on early testing whenever possible. Although the emphasis is on
early screening and diagnosis, it is recognised that not all patients
will present for prenatal care early enough, and some tests (e.g.
NTD detection by AFP testing or ultrasound) are still optimal
later in pregnancy. Considerable flexibility in the design of pro-
grams is therefore required. Until recently, the design of a new
prenatal screening program was largely focused on the constraints
of invasive testing, specifically, minimising the number of invasive
procedure-related fetal losses and ensuring the costs of the inva-
sive testing were manageable. There were a wide range of serum
and ultrasound markers available, and the design of the screening
was therefore determined largely by the availability of sonogra-
phers who were proficient in biometry, gestational age of women
referred for screening, and other practical considerations such as
costs and individual patient preferences.
The availability of cfDNA testing opens up the possibility
of changing the design. Because cfDNA screening has a much
higher PPV than any of the conventional screening tests and
can be offered earlier, per se, this would be the screening test of
choice. However, currently, the cost of the cfDNA test is too high
to consider using it to replace conventional screening approaches
for all women. Moreover, because it is not fully diagnostic, there
is still a need to provide confirmatory diagnostic invasive testing
for women with positive cfDNA results. Conventional screening,
notably protocols that include first trimester ultrasound and NT
measurement, help identify a broad range of fetal abnormalities
currently not detectable through cfDNA testing. Based on these
considerations, ISPD recommended that cfDNA testing be used
contingently.
The increasing range of screening strategies and the differ-
ences in the scope of disorders they may identify place additional
requirements on health care providers to adequately counsel their
patients about the hazards and benefits of the protocols. Guide-
lines from the ACOG,127 the American College of Medical Genet-
ics128 and ISPD5 emphasise patient autonomy in decision-making
during the provision of prenatal screening and diagnosis. Prenatal
screening and diagnostic programs therefore need to have access to
counsellors experienced in the clinical and social aspects.
Quality Control and Quality Assurance
The importance of accuracy in NT measurement and other fetal
biometry is widely recognised. There are defined criteria for accu-
rate NT measurement129 together with requirements for ongo-
ing evaluation for quality assurance.30 Similar attention to quality
control and quality assurance is needed for the laboratory tests.
Modelling the impact of bias in first trimester or second trimester
screening results shows that relatively small differences in analyte
MoMs can materially affect DRs and FPRs.130,131 This becomes
because, as tests are added, there are more sources of error. Labo-
ratories’ quality control includes attention to specimen adequacy
and integrity, evaluation of new test kits before clinical use and
careful review of control materials included in each test run before
the release of results. Retrospective quality assurance measures
include periodic review of control data, continuous monitoring
of MoM values, screen-positive rates and, when possible, collec-
tion of pregnancy outcome data. Quality requirements for cfDNA
testing have not yet been developed. 
Risk Calculation Software
Commercial software is available to calculate risks, but users
should be aware of choices the provider may have made in con-
structing the algorithm:
• Time point: DS risk can be calculated for maternal age at the
estimated date of delivery or another defined point during the
late first trimester or mid second trimester or at the actual week
of the test.6
• Distribution parameters: Multivariate log Gaussian distribu-
tions are generally used, but the parameters could be derived
from a published meta-analysis or from a single large study. It
is unlikely that means of the local distributions will differ from
the published studies, but the SDs may be different. Compo-
nents of variance which differ locally will include analytic pre-
cision (assay performance varies) and gestational error which
is largely dependent on the extent to which ultrasound dating
is carried out before testing. For NT, some software uses two
sets of DS distributions whose proportions differ according to
gestational age (the so-called ‘mixture’ model).132
• Truncation: Most providers use truncation limits for individ-
ual markers derived from the literature (e.g. see21). Some soft-
ware truncates the LR to avoid extreme risks and some software
rounds the calculated risk.
• Risk reversal: For NT, the SD in DS is about twice as wide as
in unaffected pregnancies. Consequently, although risk gener-
ally increases as NT increases, below a certain level, it increases
as NT reduces. Some software regards this ‘risk reversal’ phe-
nomenon as an artefact and truncates the NT accordingly. To
some extent, the phenomenon also occurs with other markers
but is not as obvious.
• Cutoff risk: The choice of a risk cutoff level that determines
whether the result is screen positive or screen negative is arbi-
trary. It is influenced by the perceived relative importance of
 CHAPTER 18  Ultrasound and Biochemical Screening for Fetal Aneuploidy 175

DR, FPR and PPV. In practice, the cutoff is usually determined reductions and technical improvements will make this testing
by the recommendation of national or international bodies.  increasingly important. There is a growing recognition that con-
ventional prenatal screening tests have an important role in the
Conclusions identification of nonchromosomal fetal disorders and pregnancy
complications. Women will require additional prenatal counsel-
Prenatal screening has advanced rapidly in recent years. Both the ling as these advances are introduced into routine prenatal care.
PPV of the tests offered and the range of disorders identifiable
has expanded. Although cfDNA testing is still too expensive to Access the complete reference list online at ExpertConsult.com.
be considered for routine application for entire populations, cost Self-assessment questions available at ExpertConsult.com.
References 15. Morris JK, Wald NJ, Watt HC. Fetal loss in
Down syndrome pregnancies. Prenat Diagn.
Chard T, eds. The Embryo: Normal and Abnor-
mal Development and Growth. New York:
1999;19:142–145. Springer-Verlag; 1991:181–194.
1. Wald NJ, Cuckle H. UK Collaborative AFP
16. Cuckle H. Down syndrome fetal loss rate in 33. Westergaard JG, Sinosich MJ, Bugge M, et al.
Study. Maternal serum alpha fetoprotein mea-
early pregnancy. Prenat Diagn. 1999;19:1177– Pregnancy-associated plasma protein a in the
surement in antenatal screening for anenceph-
1179. prediction of early pregnancy failure. Am J
aly and spina bifida in early pregnancy. Lancet.
17. Snijders RJ, Sundberg K, Holzgreve W, et al. Obstet Gynecol. 1983;145:67–69.
1977:1323–1332, i.
Maternal age- and gestation-specific risk 34. Trenti T, Aloe R, Cervellin G, Lippi G. Human
2. Merkatz IR, Nitowsky HM, Macri JN, John-
for trisomy 21. Ultrasound Obstet Gynecol. chorionic gonadotropin in pregnancy diagnos-
son WE. An association between low maternal
1999;13:167–170. tic. Clin Chim Acta. 2011;412:1515–1520.
serum alpha-fetoprotein and fetal chromo-
18. Cuckle H. Potential biases in Down syndrome 35. Fernando MR, Donato D, Felice P. Predictive
some abnormalities. Am J Obstet Gynecol.
birth prevalence estimation. J Med Screen. value of hormone measurements in maternal
1984;148:886–894.
2002;9:192. and fetal complications of pregnancy. Endo-
3. Cuckle HS, Wald NJ, Lindenbaum RH.
19. Nybo Andersen AM, Wohlfahrt J, Christens crine. 2006;23:230–257.
Maternal serum alpha-fetoprotein measure-
P, et  al. Maternal age and fetal loss, popula- 36. Banerjee P, Fazleabas AT. Extragonadal
ment: a screening test for Down syndrome.
tion based register linkage study. Br Med J. actions of chorionic gonadotropin. Rev Endocr
Lancet. 1984;1:926–929.
2000;320:1708–1712. Metab Disord. 2011;12:323–332.
4. American College of Obstetricians and Gyne-
20. Savva GM, Morris JK, Mutton DE, et  al. 37. 
Wright A, Zhou Y, Weier JF, et al. Trisomy 21
cologists. Noninvasive prenatal testing for fetal
Maternal age-specific fetal loss rates in is associated with variable defects in cytotro-
aneuploidy. Committee opinion No. 545.
Down syndrome pregnancies. Prenat Diagn. phoblast differentiation along the invasive path-
Obstet Gynecol. 2012;120:1532–1534.
2006;26:499–504. way. Am J Med Genet. 2004;130A:354–364.
5. Benn P, Borell A, Chiu R, et al. Position state-
21. Wald NJ, Rodeck C, Hackshaw AK, et  al. 38. Eldar-Geva T, Hochberg A, deGroot N,
ment from the aneuploidy screening commit-
First and second trimester antenatal screen- Weinstein D. High maternal serum chori-
tee on behalf of the board of the international
ing for Down’s syndrome: the results of onic gonadotropin level in Downs’ syndrome
society for prenatal diagnosis. Prenat Diagn.
the serum, urine and ultrasound screen- pregnancies is caused by elevation of both
2013;33:622–629.
ing study (SURUSS). Health Technol Assess. subunits messenger ribonucleic acid level
6. Cuckle HS, Pergament E, Benn P. Multiana-
2003;7(11):1–88. in trophoblasts. J Clin Endocrinol Metab.
lyte maternal serum screening for chromo-
22. Malone FD, Canick JA, Ball RH, et al. First- 1995;80:3528–3531.
somal abnormalities and neural tube defects.
and second-trimester evaluation of risk (Faster) 39. Huang T, Dennis A, Meschino WS, et  al.
In: Milunsky A, Milunsky JM, eds. Genetic
research consortium. First trimester or second- First trimester screening for Down syndrome
Disorders and the Fetus: Diagnosis, Prevention
trimester screening, or both, for Down’s syn- using nuchal translucency, maternal serum
and Treatment. 7th ed. Hoboken, NJ: Wiley-
drome. N Engl J Med. 2005;353:2001–2011. pregnancy-associated plasma protein A, free-β
Blackwell; 2015.
23. Wright D, Bradbury I, Benn P, et  al. Con- human chorionic gonadotrophin, placen-
7. Cuckle HS, Wald NJ, Thompson SG. Esti-
tingent screening for Down’s syndrome is tal growth factor and α-fetoprotein. Prenat
mating a woman’s risk of having a pregnancy
an efficient alternative to non-disclosure Diagn. 2015;35(7):709–716.
associated with Down’s syndrome using her age
sequential screening. Prenat Diagn. 2004;24: 40. Cicero S, Rembouskos G, Vandecruys H, et al.
and serum alpha-fetoprotein level. Br J Obstet
762–766. Likelihood ratio for trisomy 21 in fetuses with
Gynaecol. 1987;94:387–402.
24. Royston P, Thompson SG. Model-based absent nasal bone at the 11-14-week scan.
8. Hecht CA, Hook EB. The imprecision in
screening by risk with application to Down’s Ultrasound Obstet Gynecol. 2004;23:218–223.
rates of Down syndrome by 1-year mater-
syndrome. Stats Med. 1992;11:257–268. 41. Bindra R, Heath V, Liao A, et  al. One-stop
nal age intervals: a critical analysis of rates
25.  Neveux LM, Palomaki GE, Larrivee DA, et al. clinic for assessment of risk for trisomy 21
used in biochemical screening. Prenat Diagn.
Refinements in managing maternal weight at 11-14 weeks: a prospective study of 15
1994;14:729–738.
adjustment for interpreting maternal screening 030 pregnancies. Ultrasound Obstet Gynecol.
9. Hecht CA, Hook EB. Rates of Down syn-
results. Prenat Diagn. 1996;16:1115–1119. 2002;20(3):219–225.
drome at livebirth by one-year maternal age
26. Benn PA, Clive JM, Collins R. Medi- 42. Zoppi MA, Ibba RM, Axiana C, et  al.
intervals in studies with apparent close to
ans for second-trimester maternal serum Absence of fetal nasal bone and aneuploidies
complete ascertainment in populations of
α-fetoprotein, human chorionic gonadotro- at first-trimester nuchal translucency screen-
European origin: a proposed rate schedule for
pin, and unconjugated estriol; differences ing in unselected pregnancies. Prenat Diagn.
use in biochemical screening. Am J Med Genet.
between races or ethnic groups. Clin Chem. 2003;23:496–500.
1996;62:376–385.
1997;43:333–337. 43. Sonek J, Nicolaides K. Additional first-trimester
10. Bray I, Wright DE, Davies CJ, Hook EB. Joint
27.  Cowans NJ, Spencer K. Effect of gestational markers. Clin Lab Med. 2010;30:573–592.
estimation of Down syndrome risk and ascer-
age on first trimester maternal serum prenatal 44. Borenstein M1, Persico N, Kagan KO, et al.
tainment rates: a meta-analysis of nine pub-
screening correction factors for ethnicity and Frontomaxillary facial angle in screening for
lished data sets. Prenat Diagn. 1998;18:9–20.
IVF conception. Prenat Diagn. 2013;33:56–60. trisomy 21 at 11 + 0 to 13 + 6 weeks. Ultra-
11. Cuckle H, Aitken D, Goodburn S, et  al.
28. Nicolaides KH, Azar G, Byrne D, et al. Fetal sound Obstet Gynecol. 2008;32:5–11.
UK national Down’s syndrome screening
nuchal translucency: ultrasound screening for 45. Hsiao CH, Liu WH, Cen RC, Chu WC. The
programme, laboratory advisory group. Age-
­
chromosomal defects in first trimester of preg- fetal frontomaxillary facial angle in normal
standardisation when target setting and auditing
nancy. Br Med J. 1992;304:867–869. and trisomy 21 ultrasounds at 11-13+6 weeks
performance of Down syndrome screening pro-
29. von Kaisenberg CS, Krenn V, Ludwig M, of gestation: findings among the ethnic Chi-
grammes. Prenat Diagn. 2004;24(11):851–856.
et  al. Morphological classification of nuchal nese compared with caucasian. Prenat Diagn.
12. Bray IC, Wright DE. Estimating the sponta-
skin in human fetuses with trisomy 21, 18, 2013;33:1–5.
neous loss of Down syndrome fetuses between
and 13 at 12–18 weeks and in a trisomy 16 46. Wright D, Spencer K, Nix B. First trimester
the time of chorionic villus sampling and live-
mouse. Anat Embryol. 1998;197:105–124. screening for down syndrome using free β
birth. Prenat Diagn. 1998;18:1045–1054.
30. Snijders RJ, Thom EA, Zachary JM, et  al. hCG, total hCG and PAPP-A: an exploratory
13. Hook EB, Topol BB, Cross PK. The natural
First-trimester trisomy screening: nuchal study. Prenat Diagn. 2007;27:1118–1122.
history of cytogenetically abnormal fetuses
translucency measurement training and qual- 47. Christiansen M, Larsen O. An increase in
detected at midtrimester amniocentesis which
ity assurance to correct and unify technique. cost-effectiveness of first trimester maternal
are not terminated electively: new data and
Ultrasound Obstet Gynecol. 2002;19:353–359. screening programmes for fetal chromosome
estimates of the excess and relative risk of late
31.  Logghe H, Cuckle H, Sehmi I. Center-specific anomalies is obtained by contingent testing.
fetal death associated with 47,+21 and some
ultrasound nuchal translucency medians needed Prenat Diagn. 2002;22:482–486.
other abnormal karyotypes. Am J Hum Genet.
for Down’s syndrome screening. Prenat Diagn. 48. Wright D, Spencer K, Kagan KK, et al. First-
1989;45:855–861.
1995;23:389–392. trimester combined screening for trisomy 21
14. Hook EB, Mutton DE, Ide R, et al. The natu-
32. Brambati B, Lanzani A, Tului L. Ultrasound at 7-14 weeks’ gestation. Ultrasound Obstet
ral history of Down syndrome conceptuses
and biochemical assessment of first trimester Gynecol. 2010;36:404–411.
diagnosed prenatally that are not electively ter-
pregnancy. In: Chapman M, Grudzinskas G,
minated. Am J Hum Genet. 1995;57:875–881.

175.e1
175.e2 References
49.  Koster MP, Wortelboer EJ, Stoutenbeek P,
et al. Modeling Down syndrome screening per-
formance using first-trimester serum markers.
Ultrasound Obstet Gynecol. 2011;38:134–139.
50. Kharrat R, Yamamoto M, Roume J, et  al.
Karyotype and outcome of fetuses diagnosed
with cystic hygroma in the first trimester in
relation to nuchal translucency thickness. Pre-
nat Diagn. 2006;26:369–372.
51. Monteagudo A, Timor-Tritsch IE. First tri-
mester anatomy scan: pushing the limits.
What can we see now? Curr Opin Obstet Gyne-
col. 2003;15:131–141.
52. Timor-Tritsch IE, Fuchs KM, Monteagudo A,
D’alton ME. Performing a fetal anatomy scan
at the time of first-trimester screening. Obstet
Gynecol. 2009;113:402–407.
53. Rossi AC, Prefumo F. Accuracy of ultraso-
nography at 11-14 weeks of gestation for
detection of fetal structural anomalies: a
systematic review. Obstet Gynecol. 2013;122:
1160–1167.
54. Spencer K, Nicolaides KH. A first trimester
trisomy 13/trisomy 18 risk algorithm combin-
ing fetal nuchal translucency thickness, mater-
nal serum free beta-hCG and PAPP-A. Prenat
Diagn. 2002;22:877–879.
55. Spencer K, Ong C, Skentou H, et al. Screen-
ing for trisomy 13 by fetal nuchal translucency
and maternal serum free beta-hCG and PAPP-
A at 10–14 weeks of gestation. Prenat Diagn.
2000;20:411–416.
56. Jorgensen PI, Trolle D. Low urinary oestriol
excretion during pregnancy in women giving
birth to infants with Down’s syndrome. Lancet.
1972;2:782–784.
57.  Canick JA, Knight GJ, Palomaki GE, et al. Low
second trimester maternal serum unconjugated
oestriol in pregnancies with Down’s syndrome.
Br J Obstet Gynaecol. 1988;95:330–333.
58. Van Lith JM, Pratt JJ, Beekhuis JR, Mantingh
A. Second-trimester maternal serum immuno-
reactive inhibin as a marker for fetal Down’s
syndrome. Prenat Diagn. 1992;12:801–806.
59. Wallace EM, Grant VE, Swanston IA, Groome
NP. Evaluation of maternal serum dimeric
inhibin A as a first-trimester marker of Down’s
syndrome. Prenat Diagn. 1995;15:359–362.
60. Saller Jr DN, Canick JA, Blitzer MG, et  al.
Second-trimester maternal serum analyte lev-
els associated with fetal trisomy 13. Prenat
Diagn. 1999;19:813–816.
61. Biagiotti R, Cariati E, Brizzi L, et al. Mater-
nal serum screening for trisomy 18 in the
first trimester of pregnancy. Prenat Diagn.
1998;18:907–913.
62. Benacerraf BR, Barss VA, Laboda LA. A
sonographic sign for the detection in the
second trimester of the fetus with Down’s
syndrome. Am J Obstet Gynecol. 1985;151:
1078–1079.
63. Borrell A, Mercade I, Casals E, et al. Combin-
ing fetal nuchal fold thickness with second tri-
mester biochemistry to screen for trisomy 21.
Ultrasound Obstet Gynecol. 2007;30:941–945.
64. Bahado-Singh RO, Oz AU, et al. New Down
syndrome screening algorithm, ultrasono-
graphic biometry and multiple serum mark-
ers combined with maternal age. Am J Obstet
Gynecol. 1998;179:1627–1631.
65. Benn PA, Kaminsky LM, Ying J, et al. Com-
bined second-trimester biochemical and ultra-
sound screening for Down syndrome. Obstet
Gynecol. 2002;100:1168–1176.
66. Miguelez J, Maymon R, Cuckle H, et  al.
Model predicted performance of second tri-
mester Down syndrome screening with ultra-
sound prenasal thickness. J Ultrasound Med.
2010;29:1741–1747.
67. Cuckle H, Maymon R. Role of second tri-
mester ultrasound in screening for Down’s
syndrome. Ultrasound Obstet Gynecol.
2013;41:241–244.
68. Gianferrari EA, Benn PA, Dries L, et al. Absent
or shortened nasal bone length and the detec-
tion of Down syndrome in second-trimester
fetuses. Obstet Gynecol. 2007;109:371–375.
69. Maymon R, Levinsohn-Tavor O, Cuckle
H, et  al. Fetal prenasal thickness combined
with nasal bone length, a new method of
Down’s syndrome screening. Prenat Diagn.
2005;25:906–911.
70. Sonek J, Borenstein M, Downing C, et  al.
Fronto-maxillary facial angles in screening for
trisomy 21 at 14-23 weeks’ gestation. Am J
Obstet Gynecol. 2007;197:160.e1–e5.
71. Molina F, Persico N, Borenstein M, et  al.
Frontomaxillary facial angle in trisomy 21
fetuses at 16-24 weeks of gestation. Ultrasound
Obstet Gynecol. 2008;31:384–387.
72. Sonek J, Molina F, Hiett AK, et al. Prefron-
tal space ratio: comparison between trisomy
21 and euploid fetuses in the second tri-
mester. Ultrasound Obstet Gynecol. 2012;40:
293–296.
73. Yazdi B, Sonek J, Oettling C, et al. The pre-
frontal space ratio in second and third trimes-
ter screening for trisomy 21. Ultrasound Obstet
Gynecol. 2013;41:262–266.
74. Agathokleous M1, Chaveeva P, Poon LC,
et al. Meta-analysis of second-trimester mark-
ers for trisomy 21. Ultrasound Obstet Gynecol.
2013;41:247–261.
75. Aagaard-Tillery KM, Malone FD, Nyberg
DA, et  al. Role of second-trimester genetic
sonography after Down syndrome screening.
Obstet Gynecol. 2009;114:1189–1196.
76. Krantz DA, Hallahan TW, Macri VJ, Macri
JN. Genetic sonography after first-trimester
Down syndrome screening. Ultrasound Obstet
Gynecol. 2007;29:666–670.
77. Wald NJ, Kennard A, Hackshaw A, McGuire
A. Antenatal screening for Down’s syndrome.
Health Technol Assess. 1998;2:1–112.
78. Hutley W, Rudnicka A, Wald NJ. Second-
trimester prenatal screening markers for Down
syndrome in women with insulin-dependent dia-
betes mellitus. Prenat Diagn. 2004;24:804–807.
79. Peled Y, Glilboa Y, Perri T, et al. Strict glycemic
control in the diabetic pregnancy-implications
for second-trimester screening for Down syn-
drome. Prenat Diagn. 2003;23:888–890.
80. Evans MI, Harrison HH, O’Brien JE, et  al.
Correction for insulin-dependent diabetes
in maternal serum α-fetoprotein testing has
outlived its usefulness. Am J Obstet Gynecol.
2002;187:1084–1086.
81. Savvidou MD, Syngelaki A, Muhaisen M,
et  al. First trimester maternal serum free
β-human chorionic gonadotropin and preg-
nancy-associated plasma protein a in preg-
nancies complicated by diabetes mellitus. Br
J Obstet Gynaecol. 2012;119:410–416.
82. Ball S, Wright D, Sodre D. Temporal effect
of Afro-Caribbean race on serum pregnancy-
associated plasma protein-A at 9–13 weeks’
gestation in screening for aneuploidies. Fetal
Diagn Ther. 2012;31:162–169.
83. Madsen H, Ekelund C, Torring N, et  al.
Impact of type 1 diabetes and glycemic control
on fetal aneuploidy biochemical markers. Acta
Obstet Gynecol Scand. 2012;91:57–61.
84. Beneventi F, Simonetta M, Lovati E, et  al.
First trimester pregnancy-associated plasma
protein-A in pregnancies complicated by sub-
sequent gestational diabetes. Prenat Diagn.
2011;31:523–528.
85. Spencer K, Cowans NJ, Spencer CE, Achillea N.
A re-evaluation of the influence of maternal
insulin-dependent diabetes on fetal nuchal
translucency thickness and first-trimester
maternal serum biochemical markers of aneu-
ploidy. Prenat Diagn. 2010;30:937–940.
86. Cheng PJ, Liu CM, Chang SD, et al. Elevated
second-trimester maternal serum hCG in
patients undergoing haemodialysis. Prenat
Diagn. 1999;19:955–958.
87. Cararach V, Casals E, Martinez S, et al. Abnor-
mal renal function as a cause of false-positive
biochemical screening for Down’s syndrome.
Lancet. 1997;350:1295.
88. Karidas CN, Michailidis GD, Spencer K,
Economides DL. Biochemical screening for
down syndrome in pregnancies following
renal transplantation. Prenat Diagn. 2002;22:
226–230.
89. Cuckle H, Maymon R. Down syndrome risk
calculation for a twin fetus taking account of
the nuchal translucency in the co-twin. Prenat
Diagn. 2010;30:827–833.
90. Wright D, Syngelaki A, Staboulidou I, et  al.
Screening for trisomies in dichorionic twins
by measurement of fetal nuchal translucency
thickness according to the mixture model. Pre-
nat Diagn. 2011;31:16–21.
91. Cleary-Goldman J, Rebarber A, Krantz D,
et al. First-trimester screening with nasal bone
in twins. Am J Obstet Gynecol. 2008;199:283.
e1–e3.
92. Maiz N, Staboulidou I, Leal AM, et al. Ductus
venosus Doppler at 11 to 13 weeks of gestation
in the prediction of outcome in twin pregnan-
cies. Obstet Gynecol. 2009;113:860–865.
93. Cuckle H, Moskovitch M, Vaknin Z, et  al.
Nuchal translucency screening in triplets:
Down’s syndrome risk calculation taking
account of between-fetus correlations. Prenat
Diagn. 2012;39:214–219.
94. Chasen ST, Perni SC, Predanic M, et al. Does
a ‘vanishing twin’ affect first trimester bio-
chemistry in Down syndrome risk assessment?
Am J Obstet Gynecol. 2006;195:236–239.
95. Gjerris AC, Loft A, Pinborg A, et al. The effect
of a ‘vanishing twin’ on biochemical and ultra-
sound first trimester screening markers for
Down’s syndrome in pregnancies conceived by
assisted reproductive technology. Hum Reprod.
2009;24:55–62.
96. Gjerris AC, Tabor A, Loft A, et  al. First tri-
mester prenatal screening among women
pregnant after IVF/ICSI. Hum Reprod Update.
2012;18:350–359.
97. Geipel A, Gembruch U, Berg C. Are first-
trimester screening markers altered in assisted
reproductive technologies pregnancies? Curr
Opin Obstet Gynecol. 2011;23:183–189.
98. Lambert-Messerlian G, Dugoff L, Vidaver
J, et  al. First- and second-trimester down
syndrome screening markers in pregnancies
achieved through assisted reproductive tech-
nologies (ART): a faster trial study. Prenatal
Diagn. 2006;26:672–678.

99. Holding S, Cuckle HS. Maternal serum
screening for Downs syndrome taking account
of the result in a previous pregnancy. Prenat
Diagn. 1994;14:321–322.
100. Wald NJ, Huttley WJ, Rudnicka AJ. Prenatal
screening for Down syndrome, the problem
of recurrent false-positives. Prenat Diagn.
2004;24:389–392.
101. Wald NJ, Barnes IM, Birger R, Huttley W.
Effect on Down syndrome screening per-
formance of adjusting for marker levels in a
previous pregnancy. Prenat Diagn. 2006;26:
539–544.
102. Wright D, Syngelaki A, Birdir C, et  al. First-
trimester screening for trisomy 21 with adjustment
for biochemical results of previous pregnancies.
Fetal Diagn Ther. 2011;30:194–202.
103. Benn PA, Gainey A, Ingardia CJ, et al. Second
trimester maternal serum analytes in triploid
pregnancies, correlation with phenotype and
sex chromosome complement. Prenat Diagn.
2001;21:680–686.
104. Spencer K, Liao AW, Skentou H, et  al.
Screening for triploidy by fetal nuchal translu-
cency and maternal serum free beta-hCG and
PAPP-A at 10–14 weeks of gestation. Prenat
Diagn. 2000;20:495–499.
105. Saller Jr DN, Canick JA, Schwartz S, Blitzer
MG. Multiple-marker screening in preg-
nancies with hydropic and nonhydropic
Turner syndrome. Am J Obstet Gynecol.
1992;167:1021–1024.
106. Lambert-Messerlian GM, Saller Jr DN,
Tumber MB, et al. Second-trimester maternal
serum inhibin a levels in fetal trisomy 18 and
Turner syndrome with and without hydrops.
Prenat Diagn. 1998;18:1061–1067.
107. Benn PA, Ying J. Preliminary estimate for
the second-trimester maternal serum screen-
ing detection rate for the 45,X karyotype
using α-fetoprotein, unconjugated estriol and
human chorionic gonadotropin. J Matern
Fetal Neonat Med. 2004;15:160–166.
108. Davis C, Cuckle H, Yaron Y. Screening
for Down syndrome—incidental diagno-
sis of other aneuploidies. Prenat Diagn.
2014;34(11):1044–1048.
109. Spencer K, Tul N, Nicolaides KH. Maternal
serum free beta-hCG and PAPP-A in fetal sex
chromosome defects in the first trimester. Pre-
nat Diagn. 2000;20:390–394.
110. Bradley LA, Palomaki GE, Knight GJ, et  al.
Levels of unconjugated estriol and other
maternal serum markers in pregnancies with
Smith-Lemli-Opitz (RSH) syndrome fetuses.
Am J Med Genet. 1999;82:355–358.
111. Palomaki GE, Bradley LA, Knight GJ, et  al.
Assigning risk for Smith-Lemli-Opitz syn-
drome as part of 2nd trimester screening for
Down’s syndrome. J Med Screen. 2002;9:
43–44.
112. Craig WY, Haddow JE, Palomaki GE, et al.
Identifying Smith-Lemli-Opitz syndrome
in conjunction with prenatal screening for
down syndrome. Prenat Diagn. 2006;26:
842–849.
113. Kashork CD, Sutton VR, Fonda Allen JS,
et  al. Low or absent unconjugated estriol in
pregnancy, an indicator for steroid sulfatase
deficiency detectable by fluorescence in situ
hybridization and biochemical analysis. Prenat
Diagn. 2002;22:1028–1032.
114. Lambert-Messerlian G, Pinar H, Rubin
LP, et  al. Second-trimester maternal serum
markers in twin pregnancies with complete
mole, report of 2 cases. Pediatr Dev Pathol.
2005;8:230–234.
115. Sotiriadis A, Papatheodorou S, Eleftheriades
M, Makrydimas G. Nuchal translucency and
major congenital heart defects in fetuses with
normal karyotype: a meta-analysis. Ultrasound
Obstet Gynecol. 2013;42(4):383–389.
116. Simpson LL, Malone FD, Bianchi DW, et al.
(2007) Nuchal translucency and the risk
of congenital heart disease. Obstet Gynecol.
2007;109:1455–1456.
117. Nicolaides KH. A model for a new pyramid
of prenatal care based on the 11 to 13 weeks’
assessment. Prenat Diagn. 2011;31:3–6.
118. Baschat A, Poon LY, Blitzer M, et al. Impact
of 1st trimester aspirin on population preva-
lence of pre-eclampsia. Ultrasound Obstet
Gynecol. 2009;34:14.
119. Roberge S, Nicolaides KH, Demers H, et al.
Prevention of perinatal death and adverse peri-
natal outcome using: a meta-analysis. Ultra-
sound Obstet Gynecol. 2013;41:491–499.
120. Akolekar R, Syngelaki A, Poon L, et al. Com-
peting risks model in early screening for pre-
eclampsia by biophysical and biochemical
markers. Fetal Diagn Ther. 2013;33(1):8–15.
Erratum in: Fetal Diagn Ther. 2013;34(1):43.
References 175.e3
121. Karagiannis G, Akolekar R, Sarquis R, et  al.
Prediction of small-for-gestation neonates
from biophysical and biochemical markers
at 11-13 weeks. Fetal Diagn Ther. 2011;29:
148–154.
122. Kagan K, Hoopmann M, Abele H, et al. First
trimester combined screening for trisomy
21 with different combinations of placental
growth factor, free β-hCG and PAPP-A. Ultra-
sound Obstet Gynecol. 2012;40:530–535.
123. Rolnik DL, Wright D, Poon LC, et al. Aspi-
rin versus placebo in pregnancies at high
risk for preterm preeclampsia. N Engl J Med.
2017;377(7):613–622.
124. Cuckle H. Strategies for implementing cfDNA
testing. Clin Lab Med. 2016;36(2):213–226.
125. Ginsberg GM, Cuckle H. Cost-utility analysis
of cfDNA screening for Down’s syndrome in
Israel. In: Studd J, Tan SL, FA Chevenak FA,
eds. Current Progress in Obstetrics and Gynecol-
ogy. vol. 4. Mumbai, India: Kothari Medical;
2017.
126. Lawson HW, Frye A, Atrash HK, et al. Abor-
tion mortality, United States, 1972-1987. Am
J Obstet Gynecol. 1994;171:1365–1372.
127. American College of Obstetricians and Gyne-
cologists and the American College of Medi-
cal Genetics. Screening for fetal chromosome
abnormalities. Practice Bulletin No. 77. Obstet
Gynecol. 2007;109:217–227.
128. Driscoll DA, Gross SJ. American college of
medical genetics practice guidelines. First tri-
mester diagnosis and screening for fetal aneu-
ploidy. Genet Med. 2008;10:73–75.
129. Abuhamad A. Technical aspects of nuchal
translucency measurement. Semin Perinatol.
2005;29:376–379.
130. Nix B, Wright D, Baker A. The impact of bias
in MoM values on patient risk and screen-
ing performance for Down syndrome. Prenat
Diagn. 2007;27:840–845.
131. Wright D, Abele H, Baker A, Kagan KO.
Impact of bias in serum free beta-human cho-
rionic gonadotropin and pregnancy-associated
plasma protein-a multiples of the median lev-
els on first-trimester screening for trisomy 21.
Ultrasound Obstet Gynecol. 2011;38:309–313.
132. Wright D, Kagan KO, Molina FS, et  al. A
mixture model of nuchal translucency thick-
ness in screening for chromosomal defects.
Ultrasound Obstet Gynecol. 2008;31:376–383.
19
Ultrasound Screening for Fetal
Abnormalities in the First Trimester
CATERINA M. BILARDO AND FREDRICK USHAKOV
KEY POINTS
• Early diagnosis of structural anomalies is increasingly pos-
sible. About half of the congenital anomalies can be diag-
nosed in the late first trimester.
• Severe and often lethal anomalies can be diagnosed, allowing
parents the options of continuing with the pregnancy or, if
acceptable, termination of pregnancy.
• Women appreciate the opportunity of early reassurance or
early diagnosis, making this scan an essential first step in
screening for congenital anomalies.
Introduction
First trimester ultrasonography (US) was first introduced for accu-
rate dating of pregnancy based on the crown–rump length (CRL)
measurement1 and diagnosis of multiples. However, the rapid
improvement in US imaging in the late 1980s proved that struc-
tural anomalies could already be detected in the first trimester.2 Key
to this development was the introduction of transvaginal US, which
enabled a more detailed visualisation of first trimester fetuses.3-5
Since then numerous reports on early diagnosis of fetal anoma-
lies have followed. The focus was initially on high-risk pregnancies6
but gradually extended towards more unselected populations.7
After the introduction of the most powerful marker for
aneuploidies – the nuchal translucency (NT) measurement8 – US
investigation between 11 and 14 weeks has become the corner-
stone of standard pregnancy care in many countries worldwide,
either as part of screening programs for aneuploidies9,10 or as first
global risk profile assessment in pregnancy.11,12
The Fetal Medical Foundation (FMF) has played a crucial role
in setting standards and providing training and certification for
the performance of first trimester screening, and in 2013, the
International Society of Ultrasound in Obstetrics and Gynecol-
ogy (ISUOG) addressed in a guideline the various aspects of US
investigation in the first trimester of pregnancy, aiming at promot-
ing standardisation and uniformity.13
Overall, in an unselected population and in a routine setting,
about 40% to 50% of the structural anomalies can potentially be
detected in the first trimester, although considerable variations exist
among studies, reflecting operator and population characteristis.7,14
Severe and often lethal anomalies are detected in 100% of cases.7,15
176
A recent systematic review concludes that 30% of structural
anomalies are detectable at first trimester examination, and the
detection can double to 60% when the examination follows a
structured protocol.16
It is clear that the yield of first trimester US goes far beyond
screening for fetal aneuploidy and should therefore be considered
an essential part of routine care for all women, next to noninva-
sive screening for aneuploidies.17 There is little doubt on the role
of first trimester US as first step in ruling out severe congenital
anomalies. What is still missing is a consensus on when this inves-
tigation should be performed (12–14 weeks), on the preferred
approach (transabdominal or transvaginal) and how extended it
should be. It is also important to explain to parents what can be
detected and the limitations of an early US examination. 
Anatomical Survey at 11 to 13+6 weeks
(Fig. 19.1 and Table 19.1)
The mobility of the first trimester fetus can be challenging for the
examiner, but it can also enable a quick visualisation of different
fetal planes within a short time. The use of cine loop is crucial
for this purpose and can expedite first trimester US examination.
Investigation of the fetal head and of the upper thorax, in the
same plane used for the NT measurement, also provides informa-
tion on the nasal bone (NB) and on brain structures, including
the diencephalon, the brainstem and the fourth ventricle with its
choroid plexus (Fig. 19.2). The fourth ventricle appears as a rect-
angular structure called intracranial translucency (IT) delimited
by two echogenic lines.18 Another longitudinal midsagittal view,
including the whole fetal trunk, allows visualisation of the dia-
phragm, of the entire abdominal wall and intraabdominal con-
tents, including bladder filling and size. The fetal spine, although
not yet completely ossified, can also be observed along its whole
extension from the cervical origin to the sacrum. By tilting the
probe on both sides of the fetal body, the extremities are visualised
together with the long bones. A first trimester fetus has commonly
open hands, easily enabling counting of fingers. The legs are often
flexed and the feet close to each other so that in a single sweep,
their positions can be assessed. Cross-sectional planes from cranial
to caudal show in the head the image of the falx (midline) and of
the choroid plexuses, filling at this stage almost entirely the rela-
tively large lateral ventricles. Of note is that the size of the lateral
ventricles (usually 6–8 mm) does not change after 12 to 13 weeks.
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 177

A B C

T
M T C 4v

D E F

G H I
• Fig. 19.1  Anatomical planes used for first trimester fetal investigation. A, Crown–rump length. B, Fetal
profile and nuchal translucency. C, Eyes and lenses. D, Lateral ventricles with choroid plexa (‘butterfly’
sign). E, Thalami (T) and mesencephalon (M) (plane used for head circumference (HC) measurements). F,
Fourth ventricle (4v) with choroid plexus (C). G, Upper lip and palate. H, Diaphragm. I, Stomach.

The typical aspect of the falx and of the two-echogenic structures
in the lateral ventricles has been called the ‘butterfly sign’.19
A cross-sectional view of the thorax at the level of the four-
chamber view allows visualisation of the heart axis and of
the size of atria and ventricles (Fig. 19.3A), whereby use of
colour, or even better directional power Doppler, is a crucial
complement to two-dimensional imaging for rapid visualisa-
tion of cardiac structures. This includes filling of the chambers
(Fig. 19.3B), exclusion of atrioventricular valve regurgitation
(more commonly seen across the tricuspid valve), visualisation
of the crossing of the outflow tracts and confluence of the aortic
arch and ductal arches forming a V (V-sign) pointing towards the
left shoulder of the fetus with (colour) flow in the same direction
(Fig. 19.3C and Video 19.1). More caudally, the fetal stomach
is seen under the heart just above the level of the umbilical cord
insertion. Finally, lower in the pelvis, the bladder is seen, flanked
by the two umbilical arteries. The kidneys can occasionally be
observed as two more echogenic oval structures on both sides
of the spine. This quick and gross anatomical survey enables
exclusions of major and mostly lethal structural anomalies such
as acrania, exencephaly, holoprosencephaly, gross spinal anom-
alies, abdominal wall defects, megacystis and gross skeletal or
limb deformities. Appreciation of the heart axis and of the four-
chamber view and outflow tracts by colour Doppler excludes
gross cardiac anomalies. Although in favourable circumstances,
a transabdominal (TAI) scan performed with high-resolution
US systems gives excellent images, in obese women or in case
of a retroverted uterus, the transvaginal approach can be indi-
cated and improve structure visualisation.20 In obese women, a
transvaginal US can provide even better images than a transab-
dominal midtrimester scan.21-23 Recently, high-frequency linear
array probes have been introduced for use in early fetal anatomi-
cal assessment. In lean women, this probe can provide excellent
images, especially of the fetal heart.24
178 SE C T I O N 6     Prenatal Screening and Diagnosis
J K
M N
Fig. 19.1, cont’d.  J, Abdominal wall with the umbilical cord insertion. K, Bladder and two umbilical arteries.
L, Hand and fingers. M, Lower extremities. N, Kidneys. O, Spine.
The largest study to date, on 44,850 euploid fetuses examined
at the time of the nuchal scan, showed that structural anomalies
were observed in 1.1% of cases.7 Overall 43% of the structural
anomalies were detected at the first trimester scan, and the NT
was enlarged in 30%. The authors state that about one-third of the
structural anomalies are amenable to early diagnosis, and about
40% can potentially be detected in the first trimester. The remain-
ing 30% cannot be detected as they become evident or develop
only at later stages in pregnancy.7 Another study confirmed that
45% of the structural anomalies and 100% of the severe ones are
amenable to early diagnoses. The NT was enlarged in 50% of
cases.15 A recent meta-analysis of 19 studies (115,731 fetuses) on
first trimester US investigation shows an overall DR for structural
anomalies of 46%. In low-risk pregnancies, the DR was 32% and
increased to 60% in high-risk groups and when a protocol was
used.16
Increased Nuchal Translucency (Fig. 19.4) and
Structural Anomalies
In euploid fetuses, excessive nuchal fluid accumulation (large NT,
cystic hygroma) can be regarded as a strong marker for an ever-
growing list of structural and genetic disorders and poor preg-
nancy outcomes.25-28 The chance of a poor outcome depends on
the initial degree of enlargement. The strongest association exists
for cardiac defects, with NT being enlarged in about 40% of the
major cardiac defects.29-31
Of all genetic syndromes, the most commonly associated
with an increased NT is Noonan syndrome, a relatively common
syndrome.32,33 (See more on this in the paragraph Genetic syn­
dromes.)
L
O
An enlarged NT is a common denominator to many develop-
mental disorders, appearing to be a nonspecific US marker shared
by different pathways.34,35
In the attempt to refine risk assessment based on NT screen-
ing, other markers have been investigated and added to the algo-
rithm. The first one was an absent or hypoplastic NB36 followed
by an abnormal ductus venosus (DV) flow (absent or reversed a
wave).37,38 More recently, the appreciation of tricuspid regurgitation
(TR) has been described.39
An absent NB is common in trisomies,36 but some genetic syn-
dromes also share this feature.40 An abnormal DV is especially
associated with cardiac defects, although this association is still
poorly understood.41
Becker and Wegner found in 3094 fetuses investigated at the
11 + 0 to 13 + 6-week scan that an NT of more than 2.5 mm was
observed in 58 of 86 (67.44%) fetuses with a structural anomaly.42
Syngelaki found a highly significant association between NT
above the 95th percentile and acrania (P < .0001), diaphragmatic
hernia (P = .007), exomphalos (P < .001), megacystis (P < .0001),
lethal skeletal dysplasia (P = .0002), bilateral talipes (P = .012) and
body-stalk anomaly (P < .0001) and in cases with multiple defects
(P < .0001).7 A large study from the United States found that an
increased NT gives a threefold increased risk for hydrocephaly,
lung and diaphragm anomalies, bowel obstruction and skeletal
disorders.43
In another study including 6858 fetuses, Becker and colleagues44
found that of all the 220 anomalies (including aneuploidies) pres-
ent in the cohort (prevalence, 2.8%), 111 (1.7%) were observed
in fetuses with a normal NT. Therefore their conclusion was that
although the association between an enlarged NT and structural
fetal anomalies is a given fact, if the aim of the first trimester scan is
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 179

TABLE Suggested Anatomical Assessment at Time of 11 to diagnose as many anomalies as possible, fetuses with a ‘normal’
19.1 to 13+6-Week Scan NT should be thoroughly investigated because about 50% of the
anomalies will be found in this significant group (8.3%).44
Organ or The workup after an enlarged NT includes array comparative
anatomical area Present and/or normal genomic hybridisation (CGH) investigation and repeat scans.
Head Present
Arrays reveal in these fetuses an additional 5% of pathological
Axial copy number variants.45 If the nuchal oedema has completely dis-
Cranial bones appeared in the second trimester and the 20-week scan findings
Midline falx are normal, the chance of an abnormal outcome is extremely low
Choroid plexus–filled ventricles and not dissimilar from the normal population.28 
Midsagittal
Brainstem thicknessa
Fourth ventricle (IT)a
Anomalies of the Central Neural System
Neck Normal appearance The first publication on first trimester US examination of fetal brain
NT thickness if accepted after informed consent appeared in 1989.46 The importance of early diagnosis of central neu-
and trained or certified operator available)a ral system (CNS) anomalies lies in the fact that anomalies are com-
mon, often lethal or associated with severe mental disability or motor
Face Eyes with lensa
Nasal bone
dysfunction. Early detection offers parents the option to terminate
Normal profile and mandible the pregnancy at a stage when termination may be less traumatic.47
Intact lips Brain development and maturation continue throughout pregnancy.
Neurodevelopment starts from the neurulation phase, from around 19
Spine Vertebrae (longitudinal and axial)a days of embryonic life to around day 26. The neural plate becomes
Intact overlying skin the neural tube, and this further develops into prosencephalon (fore-
Chest Symmetrical lung fields brain), mesencephalon (midbrain), rhombencephalon (hindbrain) and
No effusions or masses the spinal cord. At the time of the first trimester scan (11–13 weeks),
rudimentary brain structures are present and can be assessed by US.48
Abdomen Stomach present in left upper quadrant
Bladder
Kidneysa First Trimester Fetal Brain and Spine
Abdominal wall Normal cord insertion Investigation
Extremities Four limbs, each with three segments Cranial bone ossification should be completed by 11 weeks. It is
Hands and feet with normal orientation helpful to look specifically for bone ossification in the axial and
coronal planes. No bony defects (distortion or disruption) of the
Placenta Insertion (with respect to CS scar)
skull should be present. Lateral ventricles are relative large and
Cord Number of vessels filled by the echogenic choroid plexuses in their posterior two
Insertion on the placenta thirds (see Fig. 19.1D). The hemispheres appear symmetrical and
aOptional. separated by a clearly visible interhemispheric fissure and falx (see
CS, caesarean section; IT, intracranial translucency; NT, nuchal translucency. Fig. 19.1D). The thin brain mantle is best appreciated anteriorly,
Modified from Salomon LJ, Alfirevic Z, Bilardo CM, et al. ISUOG practice guidelines: performance lining the large fluid-filled ventricles, an appearance which should
of first-trimester fetal ultrasound scan. Ultrasound Obstet Gynecol. 2013;41(1):102–113. not be mistaken for hydrocephalus. At this early age, some cere-
   bral structures (e.g., corpus callosum, cerebellum) are not yet suf-
ficiently developed to allow accurate assessment.
The midsagittal view of the fetal head, routinely used for the
assessment of NT thickness and NB at 11 to 13 weeks, can also be
used to assess early fetal brain anatomy. The configuration of the
midbrain, brainstem and fourth ventricle changes in case of open
NB spina bifida (OSB). The IT, corresponding to the fourth ventricle,
has been proposed as marker for normal brain development and
intact spine.18,49 In case of OSB, the biparietal diameter is already
affected from the first trimester and is relatively smaller than the
DE head circumference.50,51 Also, cystic abnormalities of the posterior
fossa (Dandy-Walker complex) can be occasionally detected.52
There has been some debate as to which view, midsagittal or axial,
BS is the most informative for early investigation of fetal brain abnormali-
ties.53 The midsagittal (or preferably parasagittal or sagittal-oblique
4V views) is useful for screening purposes, but in case of suspected intra-
cranial findings, the use of parallel axial planes and three-dimensional
+ (3D) neurosonography at referral centres may refine the final diagnosis.
NT
+ Neurosonography can already be performed at 12 to 13
weeks, transabdominally with use of high-frequency transducers
(in lean women), or transvaginally. The use of five axial views can
• Fig. 19.2  Midsagittal plane for nuchal translucency (NT) with nasal bone exclude the most common anomalies amenable to early diagnosis
(NB), diencephalon (DE), brainstem (BS) and fourth ventricle (4V).
180 SE C T I O N 6     Prenatal Screening and Diagnosis

RV LV PA
Ao

A B C

RV
MA PA

D E F

AA

G
• Fig. 19.3  Axial planes used for examination of the heart (A–C). For comparison: hypoplastic left heart
syndrome (HLHS) at 12 weeks (D–G). A, Four-chamber view. B, Ventricular filling by directional power
Doppler. C, V-sign by directional power Doppler. D, HLHS. Mitral atresia (MA) on four-chamber view.
E, HLHS. Right ventricle (RV) filling on four-chamber view. F, HLHS. Big pulmonary artery. G, HLHS.
Reversed flow (red) in aortic arch (AA). Ao, Aorta; LV, left ventricle; PA, pulmonary artery.

(Fig. 19.5). 3D US (box placed around the skull and sweep start-
ing from the ‘butterfly view’) can help systematic assessment in
parallel axial views (plane A), and sagittal reconstructed planes
(plane C) are used for topographic orientation in the axial planes.  or amniotic band syndrome and is associated with aneuploidy in up
Most Common Brain and Neural Tube Anomalies
Acrania. Acrania (Fig. 19.6) (prevalence, ∼3.7 in 10,000 pregnan-
cies), is caused by failure of closure of the rostral part of the neural tube.
In this condition, the cranial bones and scalp skin have not developed,
and the brain tissue is exposed to mechanical and chemical damage.
Eventually, brain tissue ‘dissolves’ in amniotic fluid, producing a ‘milky’
appearance on scan (Fig. 19.6B). Later in pregnancy, acrania evolves
into exencephaly and in the second trimester into anencephaly, in which
hardly no more brain tissue is visible. Acrania can be in 12% of cases
part of chromosomal or genetic condition (Meckel-Gruber syndrome)
to 5% of cases.54 Sonographic features of acrania are present from 9
weeks.55 The usual presentation of acrania at 11 to 13 weeks is an irreg-
ular contour of the head in sagittal plane and absent or severe deficiency
of cranial bones with distortion of the brain structures (Fig. 19.6A
and Video 19.2). To not miss this condition, an early scan should be
performed after 11 weeks by trained sonographers.56
Other rare conditions that can be diagnosed early in gestation are
iniencephaly and craniorachischisis, the latter morphologically similar
to acrania. 
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 181

A B
• Fig. 19.4  Increased nuchal translucency (NT). A, NT 6.0 mm, absent nasal bone. B, Severe hydrops.

View Brain structures to check Pathology seen in this view Example of pathologic conditions
• Falx • Ventriculomegaly (1) 1. 2.
• Hemispheres • Holoprosencephaly (2)
1 Lateral ventricles • Lateral ventricles • Spina bifida
• Midline cysts
• Choroid plexuses • Anterior cephalocele
• Interhemispheric fissure • Choroid plexus cysts
• Skull bones • Anterior cephalocele (3) 3. 4.
• Third ventricle • Spina bifida (4)
2 Third ventricle • Upper thalamus • Holoprosencephaly
• Lateral ventricles • Ventriculomegaly
• Choroid plexuses • Choroid plexus cysts
• Shape of the head 5. 6.
• Size: BPD + HC • Abnormal head shape (5)
Thalamus and • Thalamus • Spina bifida (crash sign) (6)
3
mesencephalon • Basal ganglia • Abnormal head size
• Mesencephalon • Anterior cephalocele
• Aqueduct
7. 8.
• Cerebellar hemispheres • Abnormal cerebellum (7)
4 Cerebellum • Superior vermis • Occipital encephalocele (8)
• Brainstem

• Anterior membranous area
5 Fourth ventricle • Choroid plexus
• Posterior membranous area
• Blake pouch
9. 10.
• Posterior fossa cysts (9)
• Enlarged fourth ventricle (10)
• Spina bifida
• Occipital encephalocelet

• Fig. 19.5  Neurosonography at 11 to 13 weeks: five axial views of the brain. BPD, biparietal diameter;
HC, head circumference.

A B
• Fig. 19.6 Acrania. A, Transabdominal scan at 13 weeks. B, Transvaginal scan at 12 weeks. ‘Milky’
amniotic fluid.
182 SE C T I O N 6     Prenatal Screening and Diagnosis

A B
• Fig. 19.7  Encephalocele at 13 weeks. A, Posterior sagittal view. B, Axial view: herniation of the brainstem
thought occipital bone defect.

TABLE Head and Cranial Signs and Their Sensitivity for

by the neurotoxicity of the amniotic fluid.59 In the first trimes-
19.2 ter, these secondary changes have just started and are therefore
Open Spina Bifida
subtle. Visualisation of the spinal defect and of the myelomenin-
Head or cranial structure Sensitivity (%) gocele (Fig. 19.8) is not always easy, and the defect may remain
IT62 53
undiagnosed.
The IT is the most known simple marker for OSB that can be
CM (nonvisualised;<5th centile)65 50–73 measured in the same plane as the NT.18,49,53,60 It can be identified
brainstem (>95th centile)63,64 97 in 96% of normal fetuses by trained sonographers,61 and a recent
meta-analysis of nine studies indicates that nonvisualisation of the
BSOB (<5th centile)63,64 87 IT has a sensitivity of 53.5% and specificity of 99.7% for OSB.62
BS/BSOB (>95th centile)63,64,66,67 100 To increase sensitivity, other approaches have been proposed,
including measurements of the cisterna magna (CM), the brainstem
Scalloping frontal bones 50–55 and the brainstem occipital bone distance (BSOB) (Fig. 19.9), the
Smaller BPD47-50,71 posterior fossa fluid area and the four-line view62–66 (Fig. 19.10).
Facial angle (<5th centile)72 90 Nonvisualisation of the CM or a CM width below the fifth percentile
are both suggested to have a sensitivity for OSB of 50% to 73%.65 In
BPD/transabdominal diameter <173 77
the same image, it can be appreciated if one of the three posterior brain
IT/CM (R)69 66 spaces is absent62–66 and the BSOB distance can be measured.60–62 In
a retrospective study, a brainstem diameter above the 95th percentile,
Posterior displacement mesencephalon71,75 100
a BSOB distance below the 5th percentile and a brainstem to BSOB
BPD, Biparietal diameter; BS, brainstem; BSOB, brainstem occipital bone distance; CM, cis- ratio above the 95th percentile have a sensitivity for OSB of 96.7%,
terna magna; IT, intracranial translucency; R, ratio. 86.7% and 100%, respectively.65 In the only prospective study, the
   Berlin IT Multicenter Berlin study on 15,526 consecutive patients,
experts were able to diagnose all the 11 OSB cases based on suspicious
findings at the 11- to 13-weeks scan but only by combining all poste-
Encephalocele or Cephalocele. Encephalocele or cephalocele (Fig. rior fossa parameters.66 No single parameter showed a high sensitivity,
19.7) (1 in 5000 live births) is a neural tube defect characterised by and this varied between 18% for IT (present or absent) to 73% when
protrusion of intracranial structures through a defect in the skull. The CM measurements cutoffs were used.64
first trimester detection rate is 80%. It is suggested that the anomaly Two other small prospective studies confirm these results.67,68
is associated with an enlarged rhombencephalic cavity at earlier US The ratio between the IT and the CM (R), a new marker for OSB,
investigation and that absence of one of the three posterior brain showed a sensitivity of 66%.69 Scalloping of the frontal bones and
spaces49 can be helpful to identify fetuses with cephalocele.57 a smaller biparietal diameter (BPD) (50%–55% of cases), both
By visualisation of the fetal skull defect in the axial plain, signs of CSF leakage in OSB leading to ‘dried-up brain’, are also
a differentiation can be made between cranial meningoceles common in first trimester OSB.64,69,70 The abnormal skull shape
(only protrusion of meninges; 37% of cases) and encephaloceles is also reflected in a sharper facial angle that, when corrected for
(protrusion of brain tissue into the cephalocele; 63% of cases) CRL, is about 10 degrees lower than in control participants, and
(Video 19.3).57,58 3D US can be helpful to clearly image the defect.57 it is below the 5th percentile in 90% of cases.71 A ratio between
Additional fetal malformations are seen in 65% of cases. The prog- BPD and transverse abdominal diameter less than 1 can be easily
nosis is variable and depends of associated conditions, localisation, used and detect 69% of OSB cases.72
size and anatomical structures involved. The anomaly can evolve to More recently there is increasing consensus about the fact that
exencephaly. Some defects can lead to intrauterine death.56  markers involving changes in the brain stem and posterior fossa
Spina Bifida (Table 19.2). Open spina bifida (in Europe, ∼1 in configuration seem to be the most predictive of OSB.68,73
2000 pregnancies) is caused by failure of closure of the neural tube Our opinion is that large prospective studies are still manda-
24 to 27 days after conception. Typical associated brain changes tory to define the role of early US in the diagnosis of OSB. A new
are caused by leakage of cerebrospinal fluid (CSF). The developing sign proposed by the University College Hospital group (Fred
spinal cord and exposed nerves are damaged by direct trauma and Ushakov) is the ‘crash sign’, corresponding to the posterior-caudal
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester
A B
• Fig. 19.8  Spina bifida with meningomyelocele. A, Axial view of the spine by transabdominal scan at 11
weeks. B, Coronal view of the spine at 13 weeks (different case).
• Fig. 19.9 Midsagittal plane of the fetal head for brain stem thickness
(yellow arrow), brainstem occipital bone distance (red arrow) and posterior
fossa fluid (cisterna magna) (blue arrow).
1 CM.48,59,73 Direct assessment of the cerebellar vermis is not pos-
2
3 fetal abnormalities.48 Also, in case of suspected vermian anoma-
4
• Fig. 19.10 Midsagittal plane of the fetal head. Four lines: (1) superior
brain stem border, (2) inferior brain stem border, (3) choroid plexus of the
fourth ventricle, and (4) internal border occipital bone.
183
displacement of the mesencephalon that on an axial view ‘crashes’
against the occipital bone in case of OSB (Fig. 19.11), as a highly
sensitive screening parameter.74 It is likely that only by combin-
ing sagittal and axial views of the brain high DRs of OSB can be
achieved. Recently, a new promising marker for the early detection
of spina bifida, the maxillo-occipital line, has been proposed.75 
Midline Defects
Holoprosencephaly. Holoprosencephaly (1:1300 pregnancies)
is characterised by maldevelopment of the prosencephalon into
two hemispheres, resulting in a single ventricle (Fig. 19.12 and
Video 19.4). The anomaly is reported in and is often associated
with severe skull and facial defects and with more than two thirds
of cases associated with chromosomal abnormalities, mainly triso-
mies 13 and 18.75,76
Absence of the butterfly sign17 and the presence of a single
brain cavity anteriorly on the axial plane are specific for alobar
holoprosencephaly with a high sensitivity.8,77
Lobar or semilobar holoprosencephaly, however, is more subtle
to diagnose and usually is not be detected in the first trimester. 
Agenesis of the Corpus Callosum. Agenesis of the corpus cal-
losum (ACC) represents a spectrum of different CNS anomalies
occurring in 0.3% to 0.7% of the population, often as part of a
syndrome.78 The corpus callosum develops relatively late during
fetal life, and the earliest sonographic visualisation is possible from
16 weeks’ gestation onwards and about 1 week earlier in female
fetuses.58,72 Visualisation of the pericallosal artery is possible in
more than 95% of normal fetuses at the 11 to 13 weeks’ scan79 ;
however, we strongly discourage use of Doppler on a developing
fetal brain before 14 weeks’ gestation. 
Dandy-Walker Malformation. In Dandy-Walker malformation
(DWM) (1:30,000 live births), the anomaly may be suggested by
a markedly enlarged intracranial translucency and BSOB, with
absence of the septum separating the fourth ventricle and the
sible because it is only completed at around 18 weeks’ gestation.
DWM can be associated with chromosomal anomalies or other
lies, the final diagnosis should not take place in the first trimester.
The presence of suspicious findings may prompt repetition of the
US scan to after 18 weeks’ gestation. 
Ventriculomegaly. Lateral ventricles measuring more than 10
mm are mainly a second and third trimester diagnosis. Although nor-
mal ranges for the fetal ventricular system in the first trimester are
available, mild ventriculomegaly may still be a normal variant.65,69
184 SE C T I O N 6     Prenatal Screening and Diagnosis

A B
• Fig. 19.11  Spina bifida at 12 weeks: axial view of the brain on the level of Mesencephalon. A, Normal
brain: intact mesencephalon and aqueducts cerebri. B, ‘Crash sign’: the posterior-caudal displacement
of the mesencephalon and its deformation against the occipital bone.

A B
• Fig. 19.12  Holoprosencephaly (alobar): Univentricle in fetuses with trisomy 13 (A) and triploidy (B).

However, lateral ventricle can appear already enlarged in the first tri-
mester in case of chromosomal anomalies,80 especially trisomies 18
and 13, in some genetic syndromes and in aqueduct stenosis (Fig.
19.13). An abducted thumb may suggest X-linked hydrocephalus.81
In conclusion, first trimester diagnosis of conditions such as
acrania, alobar holoprosencephaly and encephalocele is possible,
and these anomalies should actively be excluded at every early
scan. Screening for spina bifida is feasible in specialist centres with
detection rates of 50% to 100% and very low-false positive rates
(FPRs). However, there is controversy as to the best test and how
it will perform in low-risk populations. All other brain anomalies
cannot be diagnosed in early gestation (mild ventriculomegaly,
ACC, migration disorders, tumours, schizencephaly).  high-risk pregnancies, a normal EFEC can provide early reassurance.
Since the widespread use of NT screening and the recognition of
other early markers, paralleled by the improvements in resolution of
US systems, first trimester diagnosis of CHD has been extensively
investigated. Early fetal echocardiography (EFEC) is commonly
offered in high-risk pregnancies or when an increased NT, with or
without additional anomalies, is observed at first trimester screening.86
The importance of an early diagnosis of major CHD is that it allows
additional investigations and, in case of certain diagnosis, for early, less
traumatic and safer termination of pregnancy (TOP), well before the
legal term of 24 weeks (in many countries).48 The challenge is when
there is uncertainty of the diagnosis or clinical significance requiring
assessment at a later gestation, this can increase parental anxiety. In

Facial Anomalies Early Markers for Congenital Heart Disease

Recently, a number of studies have suggested that facial anoma-
lies, such as micrognathia and labiopalatum cleft, can already be
diagnosed in the first trimester.82,83 Besides direct appreciation on
the fetal profile of severe micrognathia and bilateral cleft, the use
of angles can assist in the diagnosis in less obvious cases. Visualisa-
tion of an interrupted maxillary bone, defined as ‘maxillary gap’,
is suggestive of a cleft palate.84 Although useful in specific cases, it
is unlikely that these markers will be used routinely.  mm, 3.4% when between 3.5 and 4.4 mm, 7.5% when between
Congenital Heart Defects
Congenital heart defects (CHDs) are the most common mal-
formations (8–10/1000 live births). About 30% are severe and
responsible for significant mortality and morbidity in the neonatal
period and infancy.85
After the initial suggestion of a strong association between an
increased NT and CHD,29 a recent meta-analysis30 showed an NT
at the 95th percentile or above and at the 99th percentile or above,
a pooled sensitivity and specificity for major CHD of 45.6% and
94.7% and of 21% and 99.2% respectively, with a positive likeli-
hood ratio of 30. The risk for CHD increases with increasing NT
measurement from 1.6% when the NT is between 2.5 and 3.4
4.5 and 5.5 mm, 15% when between 5.5 and 6.4 mm, 19%
when between 6.5 and 8.4 mm and 64% when NT is 8.5 mm or
greater.87,88 All kinds of CHDs can be associated with an increased
NT, without preference for one defect above another.86
An abnormal DV and TR is seen more commonly in fetuses
with CHDs and increases the sensitivity of NT alone as a screen-
ing test for CHD.37–39,89–92
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 185

A B

C D
• Fig. 19.13  Ventriculomegaly at 11 to 13 weeks. A, Axial view of a normal brain on the level of the lateral
ventricles and choroid plexuses. B, Ventriculomegaly: ‘empty’ upper portions of the ventricles. C, Axial
view hypoplastic choroid plexuses. D, Hypoplastic choroid plexuses on three-dimensional rendering.

Abnormal DV flow (absent or reversed A-wave during atrial
contraction) is a sign of cardiac dysfunction in the second and
third trimesters.93 A meta-analysis of seven studies, including 600
chromosomally normal fetuses with NT at the 95th percentile or
greater, found that an abnormal DV flow at 11 to 14 weeks’ gesta-
tion in the presence of a NT of 3.5 mm or greater, was associated
with a threefold increased risk for CHDs, but a normal DV flow
halved the CHD risk.89 Another recent meta-analysis reports that
an abnormal DV A-wave, in association with an increased NT, can
detect 83% of CHDs with an FPR of 20% as opposed to 19%
with a FPR of 4% when the NT is normal.90
The DV can be evaluated as A-wave (positive, absent or
reversed) or pulsatility index (PI). Our group (CB) found an
abnormal ductus venosus PI (DVPI) (≥P95) in two thirds of the
fetuses with an increased NT, normal karyotype and CHD with
a sensitivity and specificity for CHDs of 70% and 62%, respec-
tively.38 There is now consensus that DVPI measurement is supe-
rior to A-wave assessment only as part of screening algorithms.94
The mechanism of the abnormal DV flow in the presence of
CHD is unclear, but the predominance of right-sided obstructive
lesions,44-46 atrioventricular septal defects (AVSD) with atrioven-
tricular (AV) valve regurgitation and hypoplastic left heart syn-
drome (HLHS) suggest that altered both right atrial pressure and
diastolic dysfunction may be involved.35,36
Tricuspid regurgitation is frequently observed in trisomic fetuses
at 11 to 14 weeks and in euploid fetuses with CHDs.36,37,84,85 The
mechanism is not clear but may be related to the reduced diastolic
function and the high afterload at this gestational age.
A recent study of 40,990 fetuses found a NT at the 95th per-
centile or above, TR or reversed A-wave in the DV in 35.3%,
32.9% and 28.2% of the 85 fetuses with major CHD, respec-
tively, and in 4.8%, 1.3% and 2.1% of those without CHD.84,85
Any one of the three markers was found in 57.6% of the fetuses
with CHD (95% confidence interval (CI), 47%–67.6%) and in
8% of those with normal hearts (95% CI, 7.7%–8.2%).84,85
Recently, early measurement of the cardiac axis has been sug-
gested as a very sensitive screening test for CHD. The first tri-
mester normal mean cardiac axis is 44.5 ± 7.4 degrees. In the
CHD group, 74.1%, had an abnormal cardiac axis (110 with left
deviation and 19 with right deviation). Cardiac axis measurement
is suggested to be significantly better than enlarged NT, TR or
reversed a-wave in DV, used alone or in combination, for the in
detection of major CHDs.95
In conclusion, an abnormal DVPIV in the first trimester can
detect about 70% of the major CHDs and is an indication for
referral for specialised EFEC even when the NT is normal. EFEC
is also recommended for TR. The risk for CHD increases with
increasing NT measurement and is further increased in the pres-
ence of an abnormal DV flow or TR but is reduced if these find-
ings are absent. However, the use of Doppler in early pregnancy
should be limited in time and performed only in high-risk cases. 
Accuracy of Congenital Heart Disease Detection
by Early Ultrasound Investigation
A recent review by Khalil and Nicolaides, based on 24 screen-
ing studies for CHD, shows a first trimester DR between 2.3%
and 56%.96 DRs at any stage depend mainly on the experience
of the sonographers and, in nonexpert hands the DR does not
differ greatly if screening is performed at 12 or 18 weeks (11% vs
15%).97
Early DR varies from 16% for coarctation of the aorta (CoA)
and 18% for tetralogy of Fallot (TOF) and transposition of the
great arteries (TGA) to 51% for HLHS.88 In another systematic
186 SE C T I O N 6     Prenatal Screening and Diagnosis
review, Rossi and Prefumo report a 48% DR for CHD with 43%
of the CHDs detected by nontargeted first trimester US examina-
tion, as opposed to 53% when specialised EFEC is carried out.14
When EFEC is performed by experts and in high-risk fetuses, sen-
sitivity and specificity can reach 85% and 99%, respectively,98 and
even higher after 13 to 14 weeks.99  To simplify CHD screening at EFEC, we propose an approach
How to Perform a Complete Early Fetal
Echocardiography
Under normal scanning circumstances, all cardiac structures should
be visible by 13 weeks.100 Initially, the transvaginal approach was
used,101 but with the advent of the NT screening, the transabdominal
approach became the preferred method. Transvaginal echocardiog-
raphy can still be superior in obese patients, but reduced flexibility
in obtaining different scanning planes limits its accuracy.102
The main reason for preferring the transabdominal approach is
that the first trimester fetus often lies in a prone position, probably
because of gravity and shape of the uterine cavity. This position
enables standardisation of cardiac examination.
In axial views of the chest, the cardiac apex points at 1 to 2 or 10
to 11 o’clock positions for cephalic or breech presentations, respec-
tively. Heart and stomach positions are evaluated to determine the
situs. The four-chamber view is used to assess the heart axis, sym-
metry of the ventricles, AV valves and crux (see Fig. 19.3A). Fur-
ther tilting of the transducer cranially demonstrates two parallel
lines, running from the LV to the right, representing the left out-
flow tract and, more superiorly, the three-vessel view. The pulmo-
nary artery (PA) runs straight into the ductus arteriosus (DA) and
meets the left-sided aorta, forming a V sign. Modern US systems
produce reasonable quality image of the four-chamber view in the
majority of cases. The resolution can be improved by the use of
high-frequency probes22 and of proper image magnification (chest
filling at least half of the screen).
The use of colour Doppler is essential in early gestation to over-
come suboptimal visualisation of the great arteries by grey-scale,
with preference for directional power Doppler. However, colour
Doppler should be used if high velocity flow is suspected (like
in TR or aortic stenosis). Mapping of the great arteries reduces
false-negative diagnosis in some CHDs. The first step is to achieve
a good-quality four-chamber view on grey-scale mode and to acti-
vate power Doppler. Ventricular filling is seen as two separate red
(for apical views) stripes equal in size (see Fig. 19.3B and Video
19.1). The LV stripe forms the apex and is slightly longer than the
RV stripe. With good presets, TR is commonly visible as a blue jet
originating in the RV near the interventricular septum. A bit of
TR is a very common in the first trimester and is likely a normal
variant.
By tilting the transducer cranially from the four-chamber view,
the blue stripe of the left ventricular outflow tract (LVOT) is seen.
Sometimes the blood velocity in the LVOT is below the scale, and
it can be better visualised by reducing the pulse repetition frequency
(PRF). The next structure seen by sweeping cranially is the long
blue stripe formed by the right ventricular outflow tract (RVOT). A
normal PA has a very straight course from the RV into the DA. The
vessel appears vertical on the screen. By tilting the transducer farther
cranially, it is possible to see the transverse aorta (aortic arch) on the
right side of the PA (opposite to the heart). The PA–DA and aorta
meet, forming a blue colour V-sign pointing towards the left (see
Fig. 19.3C). When the PA has a straight vertical position, the aorta
may be not visible on colour Doppler because of the relatively slow
blood flow velocity. By gradually reducing the PRF, the V-sign will
appear. Earlier in gestation, at 11 to 12 weeks, the aorta arch is situ-
ated higher than the ductal arch, and in some cases, it is impossible
to get a proper V-sign. However, if starting from the DA, the sweep
is continued caudally, and eventually, the blue stripe of the aorta will
be seen coming from the right.
aimed at detecting the five commonest severe CHD, accounting
for more than 80%: HLHS, AVSD, TGA, TOF and CoA.
The typical sonographic appearance of this severe HLHS (see
Fig. 19.3D) is absence of a normal four-chamber view with sig-
nificant right ventricle–left ventricle (RV–LV) disproportion and
deviation of the heart axis. On colour Doppler, either only the RV
filling (see Fig. 19.3E) is seen or both inflows but with significant
RV–LV disproportion. Sometimes the appearance can be compli-
cated by TR or MR regurgitation(s). The PA is enlarged and straight
(see Fig. 19.3F). There is no V-sign, and retrograde flow in the
transverse aorta is seen just above the ductal arch (see Fig. 19.3G).
The velocity of the retrograde flow in the aorta is generally low, and if
this is not visible, the PRF should be gradually reduced. Some cases
of HLHS are associated with progression of aortic stenosis into atresia
can become apparent only in the second trimesters. Suggestive signs
are RV–LV inflow disproportion and recognition of high velocity
flow at the stenotic AV, which can be easily confused with TR. We
advise a follow-up scan 2 weeks later to confirm or exclude HLHS.
In HLHS, a transvaginal scan gives additional information
about a restricted foramen ovale flow by visualisation of reversed
A-wave in the pulmonary veins.103
Atrioventricular septal defect is commonly associated with tri-
somy 21 (1 in 3) and left atrial isomerism (LAi). The condition varies
in severity, but a complete AVSD is characterised by a common AV
junction with a fused multileaflet AV valve. Large AVSDs are vis-
ible on the four-chamber view (Fig. 19.14 and Video 19.5). Careful
observation of the movements of the common AV valve, especially
during diastole, is a diagnostic hint. Colour Doppler shows a single
ventricular inflow with angled ventricular filling strips instead of
two normal parallel strips. This arrangement is termed the ‘trousers
sign’ (Fig. 19.14A). Insufficiency of the common valve is frequent
in AVSD, and the jet originates from the middle of the heart (Fig.
19.14D).
In cases of AVSD with a normal karyotype, it is important to
exclude LAi. This includes complex types of AVSDs, heart block,
an interrupted inferior vena cava with azygos continuation and
right-sided stomach.104
Transposition of the great arteries is one of the most challeng-
ing diagnoses even at the midtrimester scan with less than 50%
detection rate.105 A great proportion of TGAs have essentially a
normal four-chamber view and normal size of the great arteries.
There is an interfetal variability for the plane where the parallel
course of the great arteries can be visualised (Fig. 19.15A). Detec-
tion at 11 to 13 weeks is quoted to be 18%.95
Grey-scale US is not helpful in the diagnosis of TGA at 11 to 13
weeks because of limited resolution and normal four-chamber view.
There are two different presentations of TGA: (i) parallel vessels seen
at the level of the outflow tracts and (ii) the impression of a ‘single’
vessel caused by the ‘fusion’ of the colour Doppler stripes of the pul-
monary artery-ductal arch and the aorta in a sagittal or oblique view
(Fig. 19.15B). To improve diagnostic ability, we suggest following the
course of the great vessels rather than to look at the three-vessel view.
Tetralogy of Fallot is commonly associated with chromosomal and
genetic conditions and extracardiac anomalies. This CHD has typi-
cally normal four-chamber view and normal filling of the ventricles
by two parallel stripes. The landmark of TOF is visualisation of a large
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 187

C D
• Fig. 19.14  Atrioventricular septal defect (AVSD). A, AVSD (left atrial isomerism) in twin A DCDA at 12
weeks. B, Normal heart in co-twin B. C, AVSD and common AV valve (trisomy 21). D, Common AV valve
regurgitation (trisomy 21 at 13 weeks).

and usually abnormally curved vessel, which arises from the middle
of the heart and forms the aortic arch (Fig. 19.16). The vessel is seen
by grey scale but even better with colour Doppler. Visualisation of
the PA and DA is often difficult because of their hypoplasia and
insufficient resolution. When a ventricular septa defect (VSD) and a
single overriding vessel are seen at 11 to 13 weeks, the diagnostic pos-
sibilities include TOF, pulmonary atresia, double outlet right ventricle
(DORV) and arterial trunk. These are all severe conotruncal CHDs,
and exclusion of 22q.11 microdeletion is recommended.
Coarctation of the aorta is a challenging diagnosis at any stage
of gestation because of high rate of false-negative and false-positive
findings. Suspicion of CoA can arise by disproportion of the ven-
tricles at 11 to 13 weeks. In 40% of cases, there is an associated
VSD, and disproportion may be not evident. We recommend
examination of the diameter of the transverse aorta by grey scale
and of the direction of the flow in the aorta isthmus by colour or
power Doppler. Distinction between CoA and interrupted aortic
arch at 11 to 13 weeks is difficult.
Other serious CHD that can be detected in the first trimester
with an abnormal four-chamber view are Ebstein anomaly and tri-
cuspid atresia. In contrast, diagnosis of DORV, pulmonary atresia,
common arterial trunk and right aortic arch may be more challeng-
ing because it relies on appreciation of abnormal outflow tracts.
The use of four-dimensional spatiotemporal image correlation
(STIC) has been proposed as helpful in EFEC (see Fig. 19.15B
and 19.16A). However, with very active first trimester fetuses,
STIC is only feasible with a shorter acquisition time.106,107 
Conclusions
The diagnosis of major CHDs at 11 to 13 weeks’ gestation is
increasingly feasible. Several early markers give indirect hints and
help identify a group of fetuses at high risk for CHD (increased
NT, abnormal DV flow, presence of TR and abnormal cardiac
axis). Use of a standardised protocol, colour-flow mapping and
training of operators improves early detection of CHD. We expect
188 SE C T I O N 6     Prenatal Screening and Diagnosis

Ao
PA

RV LV

Ao
PA

Ao

B
• Fig. 19.15  Transposition of the great arteries at 13 weeks. A, Parallel arteries on sagittal view. B, Axial
views: four-dimensional echocardiography (spatiotemporal image correlation) combination of diastole and
systole on the same image. Ao, Aorta; LV, left ventricle; PA, pulmonary artery; RV, right ventricle.

that in the future, the 11- to 13-week CHD screening will be
based on direct examination of the cardiac morphology rather
than on examination of markers.
Repetition of the scan at 16 weeks and, in case of uncertainty,
also later in gestation, is mandatory to reduce false-positive and
false-negative diagnosis.  ised by an echogenic aspect of the lungs, currently grouped under
Thorax, Diaphragm, Abdominal Wall and
Bowel
The respiratory system develops from the ventral wall of the fore-
gut starting from the third to fourth weeks (embryonic stage) and
continues developing during the first 2 years of life and beyond. In
the first trimester and up to 17 weeks, the pseudoglandular phase,
one of the four developmental phases, occurs. Unilateral or total
lung agenesis and laryngeal or tracheal occlusion are rare anoma-
lies, potentially amenable to early prenatal diagnosis in view of
their impact on the anatomy of the thorax, but lesions character-
the name congenital pulmonary airways malformation, have never
been reported earlier than at 16 weeks.108
Chronic high airways obstruction syndrome (CHAOS),
because of laryngeal or tracheal occlusion, is usually a lethal
condition (especially if hydrops is present), leading to increased
volume and echogenicity of the lungs, with a visible fluid-filled
trachea and flattening or inversion of the diaphragm. The heart
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 189

Ao

RV
LV

Ao

• Fig. 19.16  Tetralogy of Fallot at 13 weeks: spatiotemporal image correlation combination of diastole and
systole on the same image. Ao, Aorta; LV, left ventricle; RV, right ventricle.

A B
• Fig. 19.17  First trimester echogenic lungs and enlarged nuchal translucency in a case of chronic high
airways obstruction syndrome (CHAOS). A, Sagittal plane. B, Axial plane.

appears small and squeezed by the enlarged hyperechoic lungs
(Fig. 19.17), and the impaired venous return leads sequentially
to ascites, hydrops and heart failure. In less severe forms of occlu-
sions, pulmonary enlargement may resolve later in pregnancy, and
the finding of apparently normal lungs at 11 to 13 weeks does not
exclude CHAOS because the condition can have delayed manifes-
tations (personal experience, FU).
The association between an enlarged NT and diaphragmatic
hernia (CDH) had already been described in 1997.109 Especially
severe CDHs seem to show nuchal oedema, probably owing to
impaired venous return caused by increased intrathoracic pressure.
In left-sided CDH, the stomach can already occasionally be seen
next to the heart, although in milder cases, it may still be situated
intraabdominally and herniate into the thorax only when, later in
pregnancy, the increased intraabdominal pressure pushes it upwards.
In right CDH, the stomach in the first trimester is invariably situ-
ated intraabdominally. In Syngelaki and colleagues’ study,7 three of
eight fetuses with CDH in the cohort (37.5%) had an enlarged NT,
and four of eight were diagnosed at the 11- to 13-week scan. 
Abdominal Wall Defects
Abdominal wall defects can easily be diagnosed in the first trimes-
ter.110 It is important that the diagnosis occurs beyond the phase
of the physiological gut herniation within the umbilical cord. This
is supposed to resolve at about 12 weeks (CRL ≥45 mm).
190 SE C T I O N 6     Prenatal Screening and Diagnosis
C D
• Fig. 19.18 Omphalocele. A, Small with single bowel loop (12 weeks). B, Large with only bowel (12
weeks). C, Large with liver and bowel sagittal (13 weeks). D, Three-dimensional image: large with liver
and bowel (other case).
The prevalence of exomphalos (omphalocele) (Fig. 19.18) at
the first trimester scan is 1 in 419,111 but there is a tendency
for the prevalence to decrease with advancing gestation and
according to the content of the defect. Herniation of the liver
is rare (1 in 3360) and does not resolve (Video 19.6), but for
herniation of bowel only, the prevalence changes from 1 in 98
at a CRL of 45.0 to 54.9 mm to 1 in 798 at a CRL of 55 to
64.9 mm and 1 in 2073 at a CRL of 65.0 to 84.0 mm.112 The
fetal karyotype is frequently abnormal (40.8%) in exomphalos
in both liver- (52%) or bowel-only (55%) cases.110,113 However,
if the karyotype is normal, a bowel-only exomphalos has a 92%
chance of spontaneous resolution by 20 weeks’ gestation and of
an uneventful pregnancy and neonatal outcome. This suggests
that in the large majority of bowel-only exomphalos cases diag-
nosed in the first trimester, this can be regarded as a delayed
resolution of the physiological gut herniation within the umbili-
cal cord. Therefore in case of low risk assessment at first trimester
screening or NIPT, no karyotyping is necessary, and the diagno-
sis of exomphalos should only be reserved for cases persisting in
the second trimester.
In a recent study on 98 cases of exomphalos diagnosed before
14 weeks’ gestation, 46% were associated with other major
structural anomalies: 21.4% had increased NT, and 51% of the
karyotyped fetuses had chromosomal anomalies,82 mainly trisomy
18. In cases of exomphalos associated with other major structural
abnormalities or with increased NT, the incidences of aneuploidy
were 78.9 and 72.2%, respectively.
To improve the prognostic value of first trimester investigation,
Tassin and colleagues investigated whether a standardised ratio
(mean exomphalos diameter/transverse abdominal diameter) and
exomphalos contents is predictive of neonatal morbidity.114 Neo-
natal morbidity was increased when the ratio was greater than 0.8
or when the liver was herniated in the first trimester. The authors
concluded that the outcome of exomphalos (92.6% of liveborn
infants survived the neonatal period, 96% without long-term
sequelae) is relatively good but only when isolated and when the
ratio is below 0.8. Nine percent of the fetuses diagnosed with iso-
lated exomphalos before 14 weeks of gestation had severe malfor-
mations diagnosed later in pregnancy.109
In conclusion, exomphalos diagnosed in the first trimester,
whatever its size or contents, requires additional investigations to
establish the prenatal and postnatal prognosis. When exomphalos
is not associated with aneuploidies or other malformations, its size
could be predictive of prenatal and postnatal outcome.
Gastroschisis (Fig. 19.19 and Video 19.7) is observed less
frequently than exomphalos at the first trimester scan, but its
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester 191

A B
• Fig. 19.19 Gastroschisis. A, Sagittal plane (transabdominal). B, Axial plane (transvaginal).

A B
• Fig. 19.20  Body-stalk anomaly. A, Amnion dividing fetus into intraamniotic and extraamniotic (coelomic)
parts. B, Body distortion on three-dimensional imaging.

prevalence is increasing (from 2.3 to 4.4 in 10.000).115,116 This
abdominal wall defect differs from exomphalos in embryo-
logic development, associated anomalies, risk factors and
outcome. It is characterised by a full-thickness defect of the
anterior abdominal wall, typically on the right side of a nor-
mally inserted umbilical cord, associated with evisceration of
the abdominal contents. Ischaemia of the vascular vitelline ves-
sels is the likely pathological mechanism. Chronic stress and
violence are risk factors for gastroschisis; moreover, mothers
of fetuses with gastroschisis are younger, smokers, more often
users of recreational drugs and exposed to domestic violence
than control participants.117
Although other anomalies involving an ischaemic mechanism
(e.g. lateral limb defect) and cerebral, heart and musculoskeletal
anomalies can be associated with gastroschisis, the defect is usually
isolated.118,119 Rare syndromes have been reported in association
with gastroschisis.120 The most common associated anomalies are
gastrointestinal, especially bowel atresia, occurring in 7% to 28%
of cases.121
Early diagnosis should be made with caution because the phys-
iological gut herniation may mimic the defect or, alternatively, the
defects may be missed because the presence of few bowel loops
herniating through a defect located on the right side of a normally
inserted umbilical cord may not be seen in the midsagittal plane.
The use of transverse planes is therefore indicated.
Management of gastroschisis diagnosed in the first trimester
differs from management of exomphalos because, if the defect is
isolated, karyotyping is not indicated.111
Rare Intraabdominal and Abdominal Wall
Anomalies
Bowel dilation is rarely observed at the first trimester scan, but
its finding (dilated colon) prompts repetition of the scan later in
pregnancy because it may be a feature of anal atresia.122 Intraab-
dominal cysts are occasionally observed and usually disappear later
in pregnancy and have a favourable outcome.123
Body-stalk anomaly is characterised by the presence of a
major abdominal wall defect, severe kyphoscoliosis, probably as
result of a short umbilical cord, whereby half of the fetal body
lies in the amniotic cavity and the other half in the coelomic cav-
ity (Fig. 19.20).124 Owing to its severity, the anomaly is usually
detected at the first trimester scan from as early as 11 weeks.125
Examination of the contents of both the amniotic sac and coelo-
mic cavity and a short umbilical cord help in differentiating this
condition from other abdominal wall defects.
192 SE C T I O N 6     Prenatal Screening and Diagnosis
A B
• Fig. 19.21 Megacystis. A, Moderate enlargement: 10 mm at 12 weeks. B, Massive enlargement: three-
dimensional calculation of the volume by Virtual Organ Computer-aided AnaLysis (VOCAL).
Bladder exstrophy (see also the discussion of urinary tract
anomalies) is a rare (1 in 30,000) defect of closure of the lower
abdominal wall, characterised by protrusion and eversion of the
bladder outside the peritoneal cavity. When no intraabdominal
normally filled bladder is seen at the first trimester scan or the
distance between umbilical cord insertion and the genital tubercle
is shortened, bladder exstrophy should be suspected.126 Alterna-
tively, bladder or cloacal exstrophy can be suspected by the pres-
ence of a large, low abdominal thin-walled cyst.
Another rare anomaly is the omphalocele, bladder or cloacal
exstrophy, imperforate anus, spina bifida complex (OEIS) com-
plex. The prenatal findings are omphalocele, a skin-covered lum-
bosacral neural tube defect, lack of visualisation of the bladder
and limb defects. Anal atresia, bladder exstrophy and abnormal
genitalia are more difficult to visualise in the first trimester. The
OEIS complex is usually identified after 16 weeks even if reports
of earlier diagnoses exist.127
Pentalogy of Cantrell is rare, but owing to its remarkable
aspect of an extracorporeal heart and association with increased
NT and other anomalies, the defect is amenable to first trimester
diagnosis.128  and is detected in 0.06% of first trimester scans (Fig. 19.21 and
Urinary Tract and Kidney Anomalies
Renal development begins from the fourth week of embryonic life129
and is complete by the 12th week of gestation.130 At this time, fetal
kidneys do not function because the placenta works as excretory
organ, and the amniotic fluid is formed by the yolk sac and from
transmembranous exudation from the fetus and the placenta. There-
fore a normal amount of amniotic fluid in early pregnancy does not
exclude a severe genitourinary anomaly with no urine production.
Kidney visualisation may be difficult in the first trimester. At
this stage, fetal renal assessment can include the visualisation of
both kidneys and of a filled bladder.102
Unilateral (URA) and bilateral renal agenesis (BRA) has an inci-
dence of 1 in 1000 and 1 in 4000 live births, respectively. Most cases of
BRA are sporadic, although some familial cases have been reported.131
The prognosis varies widely depending on the sort of agenesis. URA
has a good outcome, with compensatory hypertrophy of the contra-
lateral kidney and normal life expectancy, but BRA has a uniformly
lethal prognosis because of pulmonary hypoplasia secondary to severe
oligohydramnios. Oligohydramnios becomes apparent from the 17th
week.132 In some rare cases, a urachal cyst may produce retrograde
filling of the urinary bladder, masking anuria caused by BRA.
First trimester kidneys can occasionally appear echogenic. This
can be a normal variant, but it can also be a feature of renal dyspla-
sia, chromosomal abnormalities, polycystic disease and other con-
genital syndromes. In case of bilateral large hyperechoic kidneys,
variably associated with an occipital encephalocele and postaxial
polydactyly, Meckel-Gruber syndrome (MKS) should be sus-
pected.133 The earliest prenatal diagnosis of MKS syndrome has
been reported at 10 weeks by embryoscopy134 and between the 11
and 14 weeks of gestation, noninvasively, by US scan. The diagno-
sis of MKS can be easier at this stage because later in pregnancy,
the presence of oligohydramnios may hinder the visualisation of
polydactyly and encephalocele.
The bladder can be recognised as a median hypoechoic structure
in the lower abdomen, surrounded by the umbilical arteries (see
Fig. 19.1K).135 The finding of a single umbilical artery in the first
trimester may be a false-positive diagnosis.136 By 12 to 13 weeks’
gestation, bladder visualisation is successful in 98% of cases. In
case of persisting failure to visualise it, bilateral renal pathology
or bladder exstrophy should be suspected.137 Fetal megacystis is
defined by a bladder longitudinal diameter of 7 mm or greater138
Video 19.8). The underlying cause of this rather rare condition
ranges from lower urinary tract obstructions, to chromosomal
abnormalities and congenital syndromes, to transient enlargement
with spontaneous resolution and a normal urinary system after
birth. From a study of 145 cases of first trimester megacystis, the
outcome was mainly determined by the degree of bladder enlarge-
ment: moderate enlargement, with a longitudinal diameter of 15
mm or less, was associated with chromosomal abnormalities or
spontaneous resolution (2 weeks later) in euploid fetuses. How-
ever, severe distension, with a longitudinal diameter greater than
15 mm, mainly progressed to obstructive uropathy.139 Therefore
in case of a bladder diameter between 7 and 15 mm, karyotyping
and repeating the scan 2 weeks later are recommended. 
Genetic Syndromes
Some typical phenotypic expressions of genetic syndromes may
already be visible at the early US. When parents are known carri-
ers of genetic syndromes with dominant or recessive inheritance
pattern, early US investigation, together with targeted genetic
testing, if available, may help confirming or excluding the occur-
rence of the syndrome in the offspring and reduce the period of
uncertainty.140
 CHAPTER 19  Ultrasound Screening for Fetal Abnormalities in the First Trimester
• Fig. 19.22  Three-dimensional images of a 14-week fetus with Noonan
syndrome diagnosed after a nuchal translucency of 6.0 mm at the first
trimester scan.
Suspicion of a fetal syndrome arises when in a fetus, with a
usually (severely) increased NT, the karyotype and arrays CGH
are normal but there is a delayed resolution of the nuchal fluid
accumulation and additional anomalies are seen at follow-up
scans.25,26 Irrespective of the growing number of reports on associ-
ation between an increased NT and genetic syndromes, their rarity
means that it is impossible to prove whether there is a true associa-
tion or a coincidental finding. The most frequently reported asso-
ciations are with Noonan syndrome (NS) (Fig. 19.22), congenital
adrenal hyperplasia (CAH), fetal akinesia deformation sequence,
Smith-Lemli-Opitz syndrome (SLOS), and spinal muscular atro-
phy (SMA).25,26,33 Thus far, however, a clear association has only
been confirmed for NS and other RASopathies but not for SLOS,
SMA, CAH or 22q11 deletion syndrome.33,141 Genetic workup
for NS should be discussed with the parents in suspected cases.
Genetic testing for NS is complex with multiple genes involved
(the PTPN11 gene accounts for about 50% of cases), so the detec-
tion rate will depend on the panel of genes tested.  Conclusion
Ethical Considerations
Thanks to the continuous improvement in US technology (high-
frequency probes with excellent resolution22 and the widespread
use of US at 11 to 14 weeks’ gestation, early diagnosis of struc-
tural anomalies is increasingly possible. Transvaginal US often
provides even better imaging. Importantly, there is no scientific
evidence that US in the first trimester may be harmful, provided
the thermal indices are kept within acceptable ranges and colour
and spectral Doppler modalities are used under the principle of
‘as low as reasonably achievable’ (ALARA). Exposure of young
fetuses to long-lasting Doppler examination should always be
avoided.
193
The advantages of an early diagnosis are evident because it
allows time for repeating the US, for additional investigations to
be performed and for parents to consider their options, including
TOP. However, it is important to be aware of the fact that certain
findings, such as a thickened NT, bowel herniation or a mega-
cystis, may be transient and not associated with anomalies later
in pregnancy. In addition, as with US at any gestation, there are
findings of unknown significance that require careful counselling
and follow up by experienced personnel. It is therefore mandatory
that early abnormal findings are confirmed by US examinations
carried out by experts. We strongly discourage rushed decisions
to terminate a pregnancy after an early diagnosis of anomalies,
with a few exceptions, such as acrania, anencephaly or body-stalk
anomaly.
Experience, training and adherence to protocols, similar to NT
screening, are key to a good performance of first trimester US. We
recommend that caution should be used in alarming women in
case of uncertain findings or reassuring too early high-risk women
because certain anomalies may only become visible at the midtri-
mester scan or even later. 
Future Directions
The decrease in cost of cell-free DNA will inevitably lead to
the widespread adoption of this screening method as integral
part of prenatal screening from as early as 10 weeks’ gestation.
However, a first trimester scan for the diagnosis of fetal anoma-
lies should remain, together with evaluation of the NT, as the
strongest marker of abnormal development or delayed normal
development.15
Further development in US technique will make new 3D
reconstruction modes and similar technologies, such as virtual
reality, widely available through development of affordable desk-
top function.142 Through this and similar developments, detection
of abnormal embryologic development will become increasingly
possible, in some cases from 9 to 10 weeks’ gestation.143
We expect that in the future, tailored genetic and (precon-
ceptional) risk profile assessment, combined with early US, cell-
free DNA and, when indicated, invasive testing to perform array
CGH or exome sequencing will provide couples with early infor-
mation regarding the health of their offspring but also regarding
the chance of future pregnancy complications.
Because of the multifaceted role of first trimester US preg-
nancy assessment and fetal investigation, it is unthinkable that this
examination may be completely replaced by new emerging fetal
DNA–based techniques. 
The performance of first trimester diagnosis for anomalies is good,
provided experienced sonographers, following a protocol and ide-
ally examine the fetus beyond 12 weeks’ gestation. Training pro-
grams and certification for first trimester US, similar to second
trimester, should become available.
Severe, often lethal anomalies are diagnosed early, offer-
ing parental reproductive choice. In the era of cell-free DNA,
an early fetal anatomical evaluation (including NT assessment)
will remain an essential component of screening for congenital
defects.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Alfirevic Z, Bilardo CM, Salomon LJ, Tabor
A. Women who choose cell-free DNA testing
34. Haak MC, van Vugt JM. Pathophysiology of
increased nuchal translucency: a review of the
should not be denied first-trimester anatomy literature. Hum Reprod Update. 2003;9(2):
1. Robinson HP, Fleming JE. A critical
scan. BJOG. 2017;124(8):1159–1161. 175–184.
evaluation of sonar ‘crown-rump length’
18. Chaoui R, Benoit B, Mitkowska-Wozniak H, 35. Allan LD. The mystery of the nuchal trans-
measurements. Br J Obstet Gynaecol.
et al. Assessment of intracranial translucency lucency. The mannheimer lecture. Cardiol
1975;82(9):702–710.
(IT) in the detection of spina bifida at the Young. 2006;16:11–17.
2. Romero R, Jeanty P, Hobbins JC. Diagnostic
11–13-week scan. Ultrasound Obstet Gynecol. 36. Cicero S, Curcio P, Papageorghiou A, et al.
ultrasound in the first trimester of pregnancy.
2009;34:249–252. Absence of nasal bone in fetuses with trisomy
Clin Obstet Gynecol. 1984;27:286–313.
19. Sepulveda W, Dezerega V, Be C. First-tri- 21 at 11–14 weeks of gestation: an observa-
3. Timor-Tritsch IE, Farine D, Rosen MG.
mester sonographic diagnosis of holopros- tional study. Lancet. 2001;358:1665–1667.
A close look at early embryonic devel-
encephaly: value of the ‘butterfly’ sign. J 37. Matias A, Gomes C, Flack N, et al. Screen-
opment with the high-frequency trans-
Ultrasound Med. 2004;23:761–765. ing for chromosomal abnormalities at 10-14
vaginal transducer. Am J Obstet Gynecol.
20. Ebrashy A, El Kateb A, Momtaz M, et al. 13– weeks: the role of ductus venosus blood flow.
1988;159(3):676–681.
14-week fetal anatomy scan: a 5-year prospec- Ultrasound Obstet Gynecol. 1998;12(6):380–
4. Rottem S, Bronshtein M, Thaler I, Brandes
tive study. Ultrasound Obstet Gynecol. 2010;35: 384.
JM. First trimester transvaginal sono-
292–296. 38. Timmerman E, Clur SA, Pajkrt E, Bilardo
graphic diagnosis of fetal anomalies. Lancet.
21.  Paladini D. Sonography in obese and overweight CM. First trimester measurement of the
1989;1:444–445.
pregnant women: clinical, medicolegal and tech- ductus venosus PIV and the prediction of
5. Achiron R, Tadmor O. Screening for fetal
nical issues. Ultrasound Obstet Gynecol. 2009;33: congenital heart defects. Ultrasound Obstet
anomalies during the first trimester of preg-
720–729. Gynecol. 2010;36(6):668–675.
nancy: transvaginal versus transabdominal
22. Gupta S, Timor-Tritsch IE, Oh C, et al. Early 39. Faiola S, Tsoi E, Huggon IC, et al. Likelihood
sonography. Ultrasound Obstet Gynecol.
second-trimester sonography to improve the ratio for trisomy 21 in fetuses with tricuspid
1991;1:186–191.
fetal anatomic survey in obese patients. J regurgitation at the 11 to 13 + 6-week scan.
6. Yagel S, Achiron R, Ron M, et  al. Trans-
Ultrasound Med. 2014;33(9):1579–1583. Ultrasound Obstet Gynecol. 2005;26:22–27.
vaginal ultrasonography at early preg-
23. Dashe JS, McIntire DD, Twickler DM. 40. Dukhovny S, Shipp T, Benson C, et al. 381:
nancy cannot be used alone for targeted
Effect of maternal obesity on the ultrasound Absent fetal nasal bone: what does it mean
organ ultrasonographic examination in a
detection of anomalous fetuses. Obstet Gyne- for the euploid fetus? Am J Obstet Gynecol.
high-risk population. Am J Obstet Gynecol.
col. 2009;113:1001–1007. 206(1):S177.
1995;172(3):971–975.
24. Persico N, Moratalla J, Lombardi CM, et al. 41. Burger NB, Haak MC, Kok E, de Groot
7. Syngelaki A, Chelemen T, Dagklis T, et  al.
Fetal echocardiography at 11–13 weeks by CJ, et  al. Cardiac defects, nuchal oedema
Challenges in the diagnosis of fetal non-
transabdominal high-frequency ultrasound. and abnormal lymphatic development are
chromosomal abnormalities at 11-13 weeks.
Ultrasound Obstet Gynecol. 2011;37:296– not associated with morphological changes
Prenat Diagn. 2011;31(1):90–102.
301. in the ductus venosus. Early Hum Dev.
8. Nicolaides KH, Azar G, Byrne D, et al. Fetal
25. Souka AP, Nicolaides KH. Diagnosis of fetal 2016;9(101):39–48.
nuchal translucency: ultrasound screening
abnormalities at the 10–14-week scan. Ultra- 42. Becker R, Wegner RD. Detailed screening
for chromosomal defects in first trimester of
sound Obstet Gynecol. 1997;10:429–442. for fetal anomalies and cardiac defects at the
pregnancy. BMJ. 1992;304:867–869.
26. Bilardo CM, Pajkrt E, de Graaf I, et al. Out- 11-13-week scan. Ultrasound Obstet Gynecol.
9. Snijders RJ, Noble P, Sebire N, et  al. UK
come of fetuses with enlarged nuchal trans- 2006;27(6):613–618.
multicentre project on assessment of risk
lucency and normal karyotype. Ultrasound 43. Baer RJ, Norton ME, Shaw GM, et al. Risk
of Trisomy 21 by maternal age and fetal
Obstet Gynecol. 1998;11:401–406. of selected structural abnormalities in infants
nuchal translucency thickness at 10–14
27. Souka AP, Von Kaisenberg CS, Hyett JA, et al. after increased nuchal translucency measure-
weeks of gestation. Fetal medicine founda-
Increased nuchal translucency with normal ment. Am J Obstet Gynecol. 2014;211(6):675.
tion first trimester screening group. Lancet.
karyotype. Am J Obstet Gynecol. 2005;192: 44. Becker R, Schmitz L, Kilavuz S, et al. ‘Nor-
1998;352:343–346.
1005–1021. mal’ nuchal translucency: a justification to
10. Kagan KO, Wright D, Baker A, et al. Screen-
28. Bilardo CM, Müller MA, Pajkrt E, et  al. refrain from detailed scan? Analysis of 6858
ing for trisomy 21 by maternal age, fetal
Increased nuchal translucency thickness cases with special reference to ethical aspects.
nuchal translucency thickness, free beta
and normal karyotype: time for parental Prenat Diagn. 2012;32:550–556.
human chorionic gonadotropin, and preg-
reassurance. Ultrasound Obstet Gynecol. 45. Grande M, Jansen FA, Blumenfeld YJ, et al.
nancy associated plasma protein-A. Ultra-
2007;30(1):11–18. Genomic microarray in fetuses with increased
sound Obstet Gynecol. 2008;31:618–624.
29. Hyett J, Perdu M, Sharland G, et al. Using nuchal translucency and normal karyotype: a
11. Nice guideline CG 62. https://www.nice.org.
fetal nuchal translucency to screen for major systematic review and meta-analysis. Ultra-
uk/guidance/cg129/ifp/chapter/screening-
congenital cardiac defects at 10–14 weeks sound Obstet Gynecol. 2015;46(6):650–658.
and-tests
of gestation: population based cohort study. 46. Kushnir U, Shalev J, Bronstein M, et al. Fetal
12. Nicolaides KH. A model for a new pyramid
BMJ. 1999;318:81–85. intracranial anatomy in the first trimester of
of prenatal care based on the 11 to 13 weeks’
30. Makrydimas G, Sotiriadis A, Huggon IC, pregnancy: transvaginal ultrasonographic eval-
assessment. Prenat Diagn. 2011;31(1):3–6.
et  al. Nuchal translucency and fetal cardiac uation. Neuroradiology. 1989;31(3):222–225.
13. Salomon LJ, Alfirevic Z, Bilardo CM, et al.
defects: a pooled analysis of major fetal echo- 47. Korenromp MJ, Christiaens GCML, van
ISUOG practice guidelines: performance of
cardiography centers. Am J Obstet Gynecol. den Bout J, et  al. Long-term psychological
first-trimester fetal ultrasound scan. Ultra-
2005;192:89–95. consequences of pregnancy termination for
sound Obstet Gynecol. 2013;41:102–113.
31. Sotiriadis S, Papatheodorou M, Elefthe- fetal abnormality: a cross sectional study. Pre-
14.  Rossi AC, Prefumo F. Accuracy of ultrasonog-
riades G, Makrydimas G. Nuchal translu- nat Diagn. 2005;25:253–260.
raphy at 11-14 weeks of gestation for detection
cency and major congenital heart defects in 48. Blaas HG, Eik-Nes SH. Sonoembryology
of fetal structural anomalies: a systematic review.
fetuses with normal karyotype: a meta-anal- and early prenatal diagnosis of neural anoma-
Obstet Gynecol. 2013;122(6):1160–1167.
ysis. Ultrasound Obstet Gynecol. 2013;42(4): lies. Prenat Diagn. 2009;29(4):312–325.
15. Kenkhuis MJA, Bakker M, Bardi F, et  al.
383–389. 49. Chaoui R, Benoit B, Heling KS, et al. Pro-
Effectiveness of 12-13 week scan for early
32. Bakker M, Pajkrt E, Mathijssen IB, Bilardo spective detection of open spina bifida at
diagnosis of fetal congenital anomalies in the
CM. Targeted ultrasound examination 11-13 weeks by assessing intracranial translu-
cell-free DNA era. Ultrasound Obstet Gynecol.
and DNA testing for Noonan syndrome, cency and posterior brain. Ultrasound Obstet
2018;51(4):463–469.
in fetuses with increased nuchal translu- Gynecol. 2011;38(6):722–726.
16. Karim JN, Roberts NW, Salomon LJ,
cency and normal karyotype. Prenat Diagn. 50. Bernard JP, Cuckle HS, Stirnemann JJ,
Papageorghiou AT. Systematic review of
2011;31(9):833–840. et  al. Screening for fetal spina bifida by
first-trimester ultrasound screening for detec-
33. Pergament E, Alamillo C, Sak K, Fiddler ultrasound examination in the first trimes-
tion of fetal structural anomalies and factors
M. Genetic assessment following increased ter of pregnancy using fetal biparietal diam-
that affect screening performance. Ultrasound
nuchal translucency and normal karyotype. eter. Am J Obstet Gynecol. 2012;207(4306):
Obstet Gynecol. 2017;50(4):429–441.
Prenat Diagn. 2011;31(3):307–310. e1–e5.

193.e1
193.e2 References
51. Khalil A, Coates A, Papageorghiou A, et al.
Biparietal diameter at 11–13 weeks’ gestation
in fetuses with open spina bifida. Ultrasound
Obstet Gynecol. 2013;42(4):409–415.
52. Volpe P, Muto B, Passamonti U, et  al.
Abnormal sonographic appearance of poste-
rior brain at 11-14 weeks and fetal outcome.
Prenat Diagn. 2015;35(7):717.
53. Chaoui R, Nicolaides KH. Detecting open
spina bifida at the 11-13-week scan by assess-
ing intracranial translucency and the poste-
rior brain region: mid-sagittal or axial plane?
Ultrasound Obstet Gynecol. 2011;38(6):
609–612.
54. Chen CP. Chromosomal abnormalities
associated with neural tube defects (I):
full aneuploidy. Taiwan J Obstet Gynecol.
2007;46(4):325–335.
55. Becker R, Mende B, Stiemer B, Entezami M.
Sonographic markers of exencephaly at 9 + 3
weeks of gestation. Ultrasound Obstet Gyne-
col. 2000;16(6):582–584.
56. Fleurke-Rozema JH, van Leijden L, van de
Kamp K, et al. Timing of detection of anen-
cephaly in the Netherlands. Prenat Diagn.
2015;35(5):483–485.
57. Tonni G, Grisolia G. Dilated intracranial
translucency and Blake’s pouch cyst: first-
trimester ultrasound markers of occipital
cephalocele diagnosed using novel three-
dimensional reslicing technique. J Clin
Ultrasound. 2014;42(3):157–161.
58. Sepulveda W, Wong AE, Andreeva E, et al.
Sonographic spectrum of first-trimester fetal
cephalocele: review of 35 cases. Ultrasound
Obstet Gynecol. 2015;46(1):29–33.
59. Sival DA, Begeer JH, Staal-Schreinemachers
AL, et  al. Perinatal motor behaviour and
neurological outcome in spina bifida aperta.
Early Hum Dev. 1997;50(1):27–37.
60. Chaoui R, Benoit B, Mitkowska-Wozniak H,
et al. Assessment of intracranial translucency
(IT) in the detection of spina bifida at the
11–13-week scan. Ultrasound Obstet Gynecol.
2009;34(3):249–252.
61. Fong KW, Dengler J, Toi A, et al. Prospec-
tive study of intracranial translucency and
the posterior brain in normal fetuses at the
11- to 13-week scan. J Ultrasound Med.
2014;33(8):1373–1379.
62. Maruotti GM, Saccone G, D’Antonio F,
et  al. Diagnostic accuracy of intracranial
translucency in detecting spina bifida: a
systematic review and meta-analysis. Prenat
Diagn. 2016;36(11):991–996.
63. Karl K, Heling KS, Chaoui R. Fluid area
measurements in the posterior fossa at
11–13 weeks in normal fetuses and fetuses
with open spina bifida. Fetal Diagn Ther.
2015;37(4):289–293.
64. Iuculano A, Zoppi MA, Piras A, et al. Brain
stem/brain stem occipital bone ratio and the
four-line view in nuchal translucency images
of fetuses with open spina bifida. J Matern
Fetal Neonatal Med. 2014:1–4.
65. Garcia-Posada R, Eixarch E, Sanz M, et  al.
Cisterna magna width at 11–13 weeks in the
detection of posterior fossa anomalies. Ultra-
sound Obstet Gynecol. 2013;41(5):515–520.
66. Mangione R, Dhombres F, Lelong N, et al.
Screening for fetal spina bifida at the 11–13-
week scan using three anatomical features of
the posterior brain. Ultrasound Obstet Gyne-
col. 2013;42(4):416–420.
67. Lachmann R, Chaoui R, Moratalla J, et  al.
Posterior brain in fetuses with open spina
bifida at 11 to 13 weeks. Prenat Diagn.
2011;31(1):103–106.
68. Chen FC, Gerhardt J, Entezami M, et  al.
Detection of spina bifida by first trimester
screening—results of the prospective mul-
ticenter Berlin IT study. Ultraschall Med.
2017;38(2):151–157.
69. Kappou D, Papastefanou I, Pilalis A, et  al.
Towards detecting open spina bifida in the
first trimester: the examination of the posterior
brain. Fetal Diagn Ther. 2015;37(4):294–300.
70. Lachmann R, Picciarelli G, Moratalla J, et al.
Frontomaxillary facial angle in fetuses with
spina bifida at 11–13 weeks’ gestation. Ultra-
sound Obstet Gynecol. 2010;36(3):268–271.
71. Orlandi E, Rossi C, Perino A, et  al. Pro-
spective sonographic detection of spina
bifida at 11-14 weeks and systematic lit-
erature review. J Matern Fetal Neonatal Med.
2016;29(14):2363–2367.
72. Loureiro T, Ushakov F, Montenegro N, et al.
Cerebral ventricular system in fetuses with
open spina bifida at 11–13 weeks’ gestation.
Ultrasound Obstet Gynecol. 2012;39(6):620–
624.
73. Simon EG, Arthuis CJ, Haddad G, et  al.
Biparietal/transverse abdominal diameter
ratio ≤1: potential marker for open spina
bifida at 11–13-week scan. Ultrasound Obstet
Gynecol. 2015;45(3):267–272.
74. Ushakov F, Fernandez M. Lesmes Heredia
C, Pandya P. OP06.08: ‘crash sign’: displace-
ment and deformation of mesencephalon
against occipital bone in the diagnosis of
spina bifida at 11–13 weeks. Ultrasound
Obstet Gynecol. 2014;44:80.
75. Ramkrishna J, Araujo Júnior E, Peixoto AB,
Da Silva Costa F, Meagher S. Maxillo-occipital
line: a sonographic marker for screening of
open spina bifida in the first trimester of preg-
nancy. J Matern Fetal Neonatal Med. 2018;13:
1–7. doi:10.1080/14767058.2018.1481384.
76. Blaas HG, Eriksson AG, Salvesen KA, et al.
Brains and faces in holoprosencephaly: pre-
and postnatal description of 30 cases. Ultra-
sound Obstet Gynecol. 2002;19(1):24–38.
77. Kagan KO, Staboulidou I, Syngelaki A,
et  al. The 11–13-week scan: diagnosis and
outcome of holoprosencephaly, exomphalos
and megacystis. Ultrasound Obstet Gynecol.
2010;36(1):10–14.
78. Sepulveda W, Wong AE, Andreeva E, et al.
Biparietal diameter-to-crown-rump length
disproportion in first-trimester fetuses
with holoprosencephaly. J Ultrasound Med.
2014;33(7):1165–1169.
79. Achiron R, Achiron A. Development of the
human fetal corpus callosum: a high-reso-
lution, cross-sectional sonographic study.
Ultrasound Obstet Gynecol. 2001;18(4):343–
347.
80. Diaz-Guerrero L, Giugni-Chalbaud G,
Sosa-Olavarria A. Assessment of pericallosal
arteries by color Doppler ultrasonography at
11–14 weeks: an early marker of fetal corpus
callosum development in normal fetuses and
agenesis in cases with chromosomal anoma-
lies. Fetal Diagn Ther. 2013;34(2):85–89.
81. Loureiro T, Ushakov F, Maiz N, et al. Lat-
eral ventricles in fetuses with aneuploidies at
11–13 weeks’ gestation. Ultrasound Obstet
Gynecol. 2012;40(3):282–287.
82. Senat MV, Bernard JP, Delezoide A, et al. Pre-
natal diagnosis of hydrocephalus-­stenosis of the
aqueduct of Sylvius by ultrasound in the first
trimester of pregnancy. Report of two cases.
Prenat Diagn. 2001;21(13):1129–1132.
83. Hoopmann M, Sonek J, Esser T, et al. Fron-
tal space distance in facial clefts and retrog-
nathia at 11-13 weeks’ gestation. Ultrasound
Obstet Gynecol. 2016;48(2):171–176.
84. Bakker M, Pace M, de Jong-Pleij E, et  al.
Prenasal thickness, prefrontal space ratio
and other facial profile markers in first-
trimester fetuses with aneuploidies, cleft
palate, and micrognathia. Fetal Diagn Ther.
2018;43(3):231–240.
85. Chaoui R, Orosz G, Heling KS, et al. Maxil-
lary gap at 11-13 weeks’ gestation: marker of
cleft lip and palate. Ultrasound Obstet Gyne-
col. 2015;46(6):665–669.
86. Hoffman JI, Kaplan S. The incidence of
congenital heart disease. J Am Coll Cardiol.
2002;39:1890–1900.
87. Clur SA, Bilardo CM. Early detection of fetal
cardiac abnormalities: how effective is it and
how should we manage these patients? Prenat
Diagn. 2014;34(13):1235–1245.
88. Clur SA, Ottenkamp J, Bilardo CM. The
nuchal translucency and the fetal heart: a lit-
erature review. Prenat Diagn. 2009;29:739–
748.
89. Clur SA, Mathijssen IB, Pajkrt E, et  al.
Structural heart defects 391 associated with
an increased nuchal translucency: 9 years
experience in a referral centre. Prenat Diagn.
2008;28:347–354.
90. Maiz N, Plasencia W, Dagklis T, et al. Ductus
venosus Doppler in fetuses with cardiac defects
and increased nuchal translucency. Ultrasound
Obstet Gynecol. 2008;31:256–260.
91. Chelemen T, Syngelaki A, Maiz N, et al. Con-
tribution of ductus venosus doppler in firsttri-
mester screening for major cardiac defects.
Fetal Diagn Ther. 2011;29(2):127–134.
92. Pereira S, Ganapathy R, Syngelaki A, et  al.
Contribution of fetal tricuspid regurgitation
in first trimester screening for cardiac defects.
Obstet Gynecol. 2011;117:1384–1391.
93. Kiserud T, Eik-Nes SH, Hellevik LR, Blaas
H-G. Ductus venosus blood velocity changes
in fetal cardiac disease. J Matern Fetal Invest.
1993;3:15–20.
94. Borrell A, Grande M, Bennasar M, et  al.
First trimester detection of cardiac defects
with the use of ductus venosus blood flow.
Ultrasound Obstet Gynecol. 2013;42:51–94.
95. Sinkovskaya ES, Chaoui R, Karl K, et  al.
Fetal cardiac axis and congenital heart
defects in early gestation. Obstet Gynecol.
2015;125(2):453–460.
96. Khalil A, Nicolaides H. Fetal heart defects:
potential and pitfalls of first-trimester detec-
tion. Semin Fetal Neonatal Med. 2013;18:
251–260.
97. Westin M, Saltvedt S, Bergman, et al. Rou-
tine ultrasound examination at 12 or 18 ges-
tational weeks for prenatal detection of major
congenital heart malformations? A random-
ized controlled trial comprising 36299
fetuses. BJOG. 2006;113:675–682.
98. Rasiah SV, Publicover M, Ewer AK, et al. A
systematic review of the accuracy of first tri-
mester ultrasound examination for detecting
major congenital heart disease. Ultrasound
Obstet Gynecol. 2006;28:110–116.

99. Mogra R, Saaid R, Kesby G, et al. Early fetal
echocardiography: experience of a tertiary
diagnostic service. Aust N Z J Obstet Gynae-
col. 2015;55(6):552–558.
100. Haak MC, Twisk JWR, van Vugt JMG. How
successful is fetal echocardiographic exami-
nation in the first trimester of pregnancy?
Ultrasound Obstet Gynecol. 2002;20:9–13.
101. Carvalho JS, Moscoso G, Ville Y. First-
trimester transabdominal fetal echocardiog-
raphy. Lancet. 1998;351(9108):1023–1027.
Erratum in: Lancet. 1998;352(9124):328.
102. Huggon IC, Ghi T, Cook AC, et  al. Fetal
cardiac abnormalities identified prior to 14
weeks’ gestation. Ultrasound Obstet Gynecol.
2002;20:22–29.
103. Better DJ, Apfel HD, Zidere V, Allan LD.
Pattern of pulmonary venous blood flow in
the hypoplastic left heart syndrome in the
fetus. Heart. 1999;81(6):646–649.
104. Phoon CK, Villegas MD, Ursell PC, Silverman
NH. Left atrial isomerism detected in fetal life.
Am J Cardiol. 1996;77(12):1083–1088.
105. Escobar-Diaz MC, Freud LR, et  al. Prena-
tal diagnosis of transposition of the great
arteries over a 20-year period improved
but imperfect. Ultrasound Obstet Gynecol.
2015;45(6):678–682.
106. Turan S, Turan OM, Desai A, et  al. First-
trimester fetal cardiac examination using spa-
tiotemporal image correlation, tomographic
ultrasound and color Doppler imaging for
the diagnosis of complex congenital heart
disease in high-risk patients. Ultrasound
Obstet Gynecol. 2014;44(5):562–567.
107. Votino C, Cos T, Abu-Rustum R, et  al.
Use of spatiotemporal image correlation
at 11-544 14 weeks’ gestation. Ultrasound
Obstet Gynecol. 2013;42:669–678.
108. Cavoretto P, Molina F, Poggi S, et  al. Pre-
natal diagnosis and outcome of echogenic
fetal lung lesions. Ultrasound Obstet Gynecol.
2008;32:769–783.
109. Sebire NJ, Snijders RJ, Davenport M, et  al.
Fetal nuchal translucency thickness at 10–14
weeks’ gestation and congenital diaphragmatic
hernia. Obstet Gynecol. 1997;90:943–946.
110. Prefumo F, Izzi C. Fetal abdominal wall
defects. Best Pract Res Clin Obstet Gynaecol.
2014;28(3):391–402.
111. Syngelaki A, Guerra L, Ceccacci I, et  al.
Impact of holoprosencephaly, exomphalos,
megacystis and increased nuchal translucency
on first-trimester screening for chromosomal
abnormalities. Ultrasound Obstet Gynecol.
2017;50(1):45–48.
112. Kagan KO, Stamboulidou I, Syngelaki A,
et  al. The 11–13-week scan: diagnosis and
outcome of holoprosencephaly, exomphalos
and megacystis. Ultrasound Obstet Gynecol.
2010;36:10–14.
113. Iacovella C, Contro E, Ghi T, et al. The effect
of the contents of exomphalos and nuchal
translucency at 11–14 weeks on the likeli-
hood of associated chromosomal abnormal-
ity. Prenat Diagn. 2012;32:1066–1070.
114. Tassin M, Descriaud C, Elie C, et  al.
Omphalocele in the first trimester: predic-
tion of perinatal outcome. Prenat Diagn.
2013;33:497–501.
115. Kirby RS, Marshall J, Tanner JP, et  al.
National birth defects prevention network.
Prevalence and correlates of gastroschisis
in 15 states, 1995 to 2005. Obstet Gynecol.
2013;122(2 Pt 1):275–281.
116. Tassin M, Benachi A. Diagnosis of abdomi-
nal wall defects in the first trimester. Curr
Opin Obstet Gynecol. 2014;26(2):104–109.
117. Ortega-Garcia JA, Soldin OP, Sanchez-
Sauco MF, et  al. Violence against women
and gastroschisis: a case-control study. Int J
Environ Res Public Health. 2013;10:5178–
5190.
118. Ruano R, Picone O, Bernardes L, et al. The
association of gastroschisis with other con-
genital anomalies: how important is it? Pre-
nat Diagn. 2011;31:347–350.
119. Kunz LH, Gilbert WM, Towner DR.
Increased incidence of cardiac anomalies in
pregnancies complicated by gastroschisis. Am
J Obstet Gynecol. 2005;193:1248–1252.
120. Mastroiacovo P, Lisi A, Castilla EE, et  al.
Gastroschisis and associated defects: an
international study. Am J Med Genet A.
2007;143:660–671.
121. Kronfli R, Bradnock TJ, Sabharwal A.
Intestinal atresia in association with gas-
troschisis: a 26 year review. J Pediatr Surg.
2010;26:891–894.
122. Lam YH, Shek T, Tang MH. Sonographic
features of anal atresia at 12 weeks. Ultra-
sound Obstet Gynecol. 2002;19(5):523–524.
123. Khalil A, Cooke PC, Mantovani E, et al. Out-
come of first-trimester fetal abdominal cysts:
cohort study and review of the literature. Ultra-
sound Obstet Gynecol. 2014;43(4):413–419.
124. Daskalakis G, Sebire NJ, Jurkovic D,
et  al. Body stalk anomaly at 10–14 weeks
of gestation. Ultrasound Obstet Gynecol.
1997;10:416–418.
125. Panaitescu AM, Ushakov F, Kalaskar A,
Pandya PP. Ultrasound features and man-
agement of body stalk anomaly. Fetal Diagn
Ther. 2016;40(4):285–290.
126. Gearhart JP, Ben-Chaim J, Jeffs RD, et  al.
Criteria for the prenatal diagnosis of clas-
sic bladder exstrophy. Obstet Gynecol.
1995;85:961–964.
127. Wax JR, Pinette MG, Smith R, et al. First-
trimester prenatal sonographic diagnosis of
omphalocele-exstrophy-imperforate anus-
spinal defects complex. J Clin Ultrasound.
2009;37:171–174.
128. Türkçapar AF, Sargın Oruc A, Öksüzoglu
A, Danışman N. Diagnosis of pentalogy of
cantrell in the first trimester using transvagi-
nal sonography and color Doppler. Case Rep
Obstet Gynecol. 2015;2015:179298.
129. Sadler TW, Langman J. Langman’s Medical
Embryology. 12th ed. Philadelphia: Wolters
Kluwer Health/Lippincott Williams &
Wilkins; 2012.
References 193.e3
130. Fong KW, Toi A, Salem S, et  al. Detec-
tion of fetal structural abnormalities with
US during early pregnancy. Radiographics.
2004;21(1):157–174.
131. Roodhooft AM, Birnholz JC, Holmes LB.
Familial nature of congenital absence and
severe dysgenesis of both kidneys. N Engl J
Med. 1984;310(21):1341–1345.
132. Bronshtein M, Amit A, Achiron R, et al. The
early prenatal sonographic diagnosis of renal
agenesis: techniques and possible pitfalls.
Prenat Diagn. 1994;14(4):291–297.
133. Sepulveda W, Sebire NJ, Souka A, et  al.
Diagnosis of the Meckel-Gruber syndrome
at eleven to fourteen weeks’ gestation. Am J
Obstet Gynecol. 1997;176(2):316–319.
134. Dumez Y, Dommergues M, Gluber MC,
et  al. Meckel-Gruber syndrome: prena-
tal diagnosis at 10 menstrual weeks using
embryoscopy. Prenat Diagn. 1994;14:141–
144.
135. McHugo J, Whittle M. Enlarged fetal blad-
ders: aetiology, management and outcome.
Prenat Diagn. 2001;21(11):958–963.
136. Lamberty CO, de Carvalho MH, Miguelez
J, et  al. Ultrasound detection rate of single
umbilical artery in the first trimester of preg-
nancy. Prenat Diagn. 2011;31(9):865–868.
137. Clautice-Engle T, Pretorius DH, Budo-
rick NE. Significance of nonvisualization of
the fetal urinary bladder. J Ultrasound Med.
1991;10(11):615–618.
138. Liao AW, Sebire NJ, Geerts L, et al. Mega-
cystis at 10-14 weeks of gestation: chromo-
somal defects and outcome according to
bladder length. Ultrasound Obstet Gynecol.
2003;21(4):338–341.
139. Sebire NJ, Von Kaisenberg C, Rubio C, et al.
Fetal megacystis at 10-14 weeks of gestation.
Ultrasound Obstet Gynecol. 1996;8(6):387–
390.
140. Conner SN, Longman RE, Cahill AG. The
role of ultrasound in the diagnosis of fetal
genetic syndromes. Best Pract Res Clin Obstet
Gynaecol. 2014;28(3):417–428.
141. Alamillo CM, Fiddler M, Pergament E.
Increased nuchal translucency in the pres-
ence of normal chromosomes: what’s next?
Curr Opin Obstet Gynecol. 2012;24(2):102–
108.
142. Baken L, van Gruting IM, Steegers EA,
et  al. Design and validation of a 3D vir-
tual reality desktop system for sonographic
length and volume measurements in early
pregnancy evaluation. J Clin Ultrasound.
2015;43(3):164–170.
143. Baken L, Exalto N, Benoit B, et  al. Differ-
entiation of early first-trimester cranial neu-
ral tube defects. Ultrasound Obstet Gynecol.
2014;43(6):711–712.
20
Evidence for Routine Ultrasound
Screening for Fetal Abnormalities in the
Second and Third Trimesters
YALDA AFSHAR, RASHMI RAO, ANAHIT MARTIROSIAN AND LAWRENCE D. PLATT
KEY POINTS
• The main objective of routine midtrimester ultrasonography
(US) is to provide accurate diagnostic information for the
delivery of optimised antenatal care, including delivery
planning.
• Routine early US is beneficial because of better estimates of
gestational age.
• Routine US examination leads to earlier detection of clinically
unsuspected fetal malformations and earlier detection of
multiple pregnancies.
• Individuals who routinely perform obstetrics scans should
have specialised training for diagnosis during pregnancy.
• The Eurofetus trial likely approaches the most accurate sensi-
tivity rates for US in the detection of anomalies (∼50%–70%).
• If one screening US examination is performed, the optimal
timing is at 18 to 22 weeks of gestation,1,2 allowing for good
visualisation of anatomy and is early enough to allow comple-
tion of prenatal diagnostic procedures with options for
termination if desired.2
• Routine use of US screening in the third trimester is currently
not supported by available data; however, US that guides
delivery planning is encouraged.
• US-suspected structural malformations as well as hydrops fe-
talis and congenital neuromuscular disorders, among others,
should be considered for delivery at a tertiary care centre with
availability of neonatologists and paediatric subspecialties.
Most congenital anomalies and adverse pregnancy outcomes occur
in pregnancies without known risk factors. That 75% of congeni-
tal malformations are found in low-risk populations and 90% of
infants born with congenital anomalies are born to women with-
out risk factors suggest that routine performance of second and
third trimester ultrasound (US) screening in all pregnancies is
beneficial.3 The prenatal diagnosis of an anomaly will then assist
in the optimisation of antenatal care resulting in the best possible
outcomes for the mother and fetus and optimise delivery plan-
ning, which includes timing and location.1 Although performed
194
routinely in many centres, the extent of benefit of such prena-
tal US screening on pregnancy and neonatal outcomes remains
unproven.
The International Society of Ultrasound in Obstetrics and
Gynecology (ISUOG) has published a practice guideline for the
performance of routine midtrimester fetal US1 and states that rou-
tine US between 18 and 22 weeks aims to provide accurate diag-
nostic information to provide the best outcome for the mother
and the fetus. The recommended components of a standard pre-
natal US examination, are described in Box 20.1. The ISUOG
suggests that midtrimester US between 18 and 22 weeks of gesta-
tion represents a period that allows timely detection of anomalies
and the ability to accurately date a pregnancy (more accurate the
earlier it is done) (Fig. 20.1).
In the United States, the American College of Obstetricians
and Gynecologists (ACOG) supports the use of US to identify a
congenital structural anomaly, to evaluate for an abnormality in
fetal growth or when there is a medical indication. The ACOG
advises against the nonmedical use of prenatal US.4,5 In 2014, the
Eunice Kennedy Shriver National Institute of Child Health and
Human Development (NICHD) hosted a workshop to address
indications, frequency, yield and cost effectiveness for US during
pregnancy. The NICHD published a summary of the consensus
among the NICHD, ACOG, Society for Maternal-Fetal Medi-
cine (SMFM), American Institute of Ultrasound in Medicine
(AIUM), American College of Radiology, Society for Pediatric
Radiology and Society of Radiologists in Ultrasound.3
The workshop consensus agreed upon the following benefits of
US during pregnancy:
1. US provides an accurate determination of gestational age,
fetal number, cardiac activity, placental location and diagnosis
of major fetal anomalies. (Examples of these are seen in Figs.
20.2–20.8.)
2. US is safe for fetuses when used appropriately.
3. US improves the detection of fetal growth abnormalities and
changes in amniotic fluid volume.
4. In the absence of an indicated specific first-trimester examination,
the optimal timing for a single US examination is at 18 to 20
weeks of gestation.
5. The benefits and limitations of US should be discussed with all
patients.
 CHAPTER 20  Evidence for Routine Ultrasound Screening for Fetal Abnormalities in the Second and Third Trimesters
Ultrasonography for the Detection of
Congenital Anomalies
Although it is both recommended and routinely performed, the
sensitivity of routine US screening to detect fetal anomalies in an
unselected population remains controversial.6 Large systematic
reviews report a 16% to 44% detection of anomalies when com-
pleted before 24 weeks of gestation, with higher detection rates of
major and lethal anomalies (see Fig. 20.1 to 20.7).7-9 The overall
detection rate for lethal anomalies is as high as 84%,7 although
the sensitivity of detection varies by anomaly and factors such as
gestational age, maternal body mass index and operator skill and
experience.10-12 The consensus of these reviews agrees that at least
a single US should be routinely offered to all pregnant women
between 18 weeks and 22 weeks of gestation to allow for gesta-
tional dating, evaluation of fetal anatomy, diagnosis of multiple
gestation and chorionicity, abnormal placentation and an assess-
ment of the cervix.
• BOX 20.1  Components of Midtrimester Ultrasound
Examination3,4,17 (see Figs. 20.1 to 20.8)
• Presence or absence of fetal cardiac activity, fetal heart rate and
rhythm
• Fetal number
• Fetal presentation
• Assessment of amniotic fluid volume
• Placental appearance and location (see Figs. 20.3 and 20. 4)
• Umbilical cord vessel number and placental insertion site, if technically
possible
• Fetal biometry (biparietal diameter, head circumference, femoral length,
abdominal circumference) (see Fig. 20.8)
• Fetal anatomic survey (18–20 weeks)
• Includes the following assessments: head (intact cranium, cavum
septi pellucidi, midline falx, thalami, cerebral ventricles, cerebellum,
cisterna magna, choroid plexus), face (orbits, mouth, upper lip
intact), neck (absence of masses), chest and heart (shape and
size of chest and lungs, cardiac activity present, four-chamber
view of heart, aortic and pulmonary outflow tracts, diaphragmatic
hernia), abdomen (stomach in normal position, bowel not dilated,
both kidneys present, cord insertion site, bladder), skeleton (spine,
masses, arms and hands, legs and feet) and genitalia
• Presence or absence of fetal movement
• Evaluation of each fetus of a multiple gestation
A B
• Fig. 20.1  A, Ventriculomegaly diagnosed by 19-week ultrasound. B, Choroid plexus cyst diagnosed by
20-week ultrasound. Caliper demarcates the cyst, and arrow indicates the choroid plexus. C, Anenceph-
aly diagnosed at 15 weeks.
195
In a 2015 Cochrane review of trials of routine US before the
24th week of pregnancy, routine early pregnancy US significantly
increased the detection of fetal abnormalities (relative risk (RR),
3.46; 95% confidence interval (CI), 1.67–7.14).13 However, only
two trials included in this review evaluated the ability to detect
fetal structural anomalies, the Helsinki trial and the Routine Ante-
natal Diagnostic Imaging with Ultrasound (RADIUS) trial. Nota-
bly, a number of factors that affect the detection rate were not
included in determination of the RR. These include gestational
age, type of malformation, number of US examinations per-
formed by the operator, operator experience and the prevalence of
the anomaly in the population. In addition, US imaging technol-
ogy has improved significantly since these trials. Next we review
the trials of routine second trimester US while appreciating that
recent trials are lacking (see Figs. 20.1, 20.2, and 20.5 to 20.7).
Helsinki Trial
The Helsinki trial was performed between 1986 and 1987 and
randomly assigned 4691 women to routine screening US at 16 to
20 weeks and compared outcomes with 4619 control participants
who only underwent clinically indicated US.14 Ninety-five percent
of all pregnant women in Helsinki participated in the study dur-
ing the study period. Seventy-seven percent of women in the con-
trol group underwent US examination during pregnancy. Routine
second trimester US screening increased detection of fetal anoma-
lies. Approximately 50% of serious anomalies were detected; 36%
in the City Hospital and 77% at the University Hospital. There
was a significant reduction in the perinatal mortality rate (4.6 per
1000 vs 9.0 per 1000) in the screened population, most likely
related to the termination of anomalous fetuses resulting in fewer
fetal and neonatal deaths due to congenital anomalies. 
RADIUS Trial
The RADIUS Trial was a multicentre study conducted in the
United States between 1987 and 1991.15,16 The study randomised
15,151 women to either screening US examinations at both 15
to 22 weeks and 31 to 35 weeks or to US for obstetric indica-
tions only. Forty-five percent of the control group had at least one
US examination. Routine second trimester US screening resulted
in an increased detection of anomalies (34.8% vs 11%). Of the
anomalies, half were detected before 24 weeks of gestation in the
screened group. However, despite the US detection of anoma-
lies, there was no improvement in perinatal outcomes with early
C
196 SE C T I O N 6     Prenatal Screening and Diagnosis
• Fig. 20.2 Twenty-three-week ultrasound image demonstrating nasal
bone (left arrow) and corpus callosum with pericallosal artery (right
arrow).
A B
D E
• Fig. 20.3  A–C, Placenta previa with a morbidly adherent placenta. D–F, Vasa previa.
A B
• Fig. 20.4  A, Chorioangioma seen on 36-week ultrasound. B, Colour Doppler demonstrating increased
vascularity of chorioangioma.
detection. There was also no difference in the number of abor-
tions performed for fetal anomalies, and there was no improve-
ment in survival among anomalous fetuses. Antenatal detection of
fetal anomalies did not improve survival over that with postnatal
diagnosis. Again, this trial was conducted at a time with a more
limited US resolution than used today. 
Eurofetus Study
The Eurofetus Study was the largest study of routine US in
unselected pregnancies. This study was conducted between 1990
and 1993.8 Women were routinely scanned between 18 to 22
weeks’ gestation in 61 European obstetric units, and data were
prospectively collected. The sensitivity for the detection of all
anomalies was 56.2%. The detection rate was higher for major
C
F
 CHAPTER 20  Evidence for Routine Ultrasound Screening for Fetal Abnormalities in the Second and Third Trimesters 197

A B
• Fig. 20.5  Colour Doppler demonstrating ventricular septal defect diagnosed on 21-week anatomy ultra-
sound in apical (A) and axial (B) views.

A B
• Fig. 20.6  Fetal pelvic mass in female fetus noted at 29-week (A) and 33-week (B) ultrasound images.

A B C

D E
• Fig. 20.7  Fetal hydrops seen on 23-week ultrasound. A, Scalp oedema. B, Abdominal ascites with skin
oedema. C, Pleural effusion. D, Abdominal ascites. E, Sagittal section of abdominal ascites.
198 SE C T I O N 6     Prenatal Screening and Diagnosis
A B
• Fig. 20.8  Standard fetal biometry. Fetal weight can be estimated by obtaining measurements such as
biparietal diameter (BPD), head circumference (HC), femoral diaphysis length (FL) and abdominal circum-
ference (AC). (Results from prediction models can be compared with fetal 8th percentiles from nomo-
grams.)
(73.7%) versus minor (45.7%) anomalies and higher for anoma-
lies of the central nervous system (CNS) (88.3%) and the urinary
tract (88.5%) than for cardiac abnormalities (20.8%–38.8%).
Overall, 44% of anomalies and 55% of severe anomalies were
detected before 24 weeks. Cardiac and facial defects were diag-
nosed later in pregnancy than abnormalities of the CNS or uri-
nary tract.
Several factors should be considered when reviewing these
trials. Notably, one must consider the prevalence of anomalies,
population demographics and study design. Detection rates for
anomalies are also contingent on follow up. Anomaly prevalence
rates reported in the literature range from 0.3% to 3.2%.17
Studies with low prevalence rates possibly represent incomplete
follow up.
Clinical characteristics of the population studied also affect
the detection rate and the generalizability of the outcomes and
data. For instance, in the RADIUS trial, only 28% of the eligible
women were included in the study after exclusion parameters were
applied. In particular, the study design did not include women
who wished to undergo US and those who might consider termi-
nation in the event of a fetal anomaly. This significantly alters the
study population and decreases the generalizability. The Helsinki
trial was more representative in that it included 95% of pregnant
women in the defined cohort and thus more reliably described the
consequences of detecting fetal anomalies.
The primary study endpoints of the RADIUS trial were the
neonatal consequences of prematurity, not fetal anomalies. As
such, the RADIUS trial was not powered to detect differences
associated with anomalies, including neonatal complications and
financial implications. For example, patients with fetal congenital
heart disease,18 specifically single ventricles19 and truncal abnor-
malities,20 have better outcomes when the diagnosis is made pre-
natally rather than postnatally.
The location of the US examination matters. Both the
RADIUS trial and the Helsinki trial demonstrated that exami-
nations performed in a hospital or tertiary care setting identified
a higher proportion of existing anomalies than those performed
in community centres. This is likely secondary to sonographer or
operator experience and possibly differences in equipment rather
than the location per se. Furthermore, both the RADIUS and
Helsinki trials were performed in the 1980s when US technology
was still new and thus may not be generalizable to current practice
with advanced US technology more readily available throughout
the world.  in decreasing postterm pregnancy.27
C
Additional Uses of Ultrasound Screening
Use of Ultrasound in Screening for Fetal
Aneuploidy
Advances in aneuploidy screening, have decreased the role of sec-
ond trimester US in screening for the common fetal aneuploidies
(trisomies 21, 18 and 13).21,22 Rather than being used as a primary
screening tool, US is now used in conjunction with combined
screening at 11 to 14 weeks or second trimester serum screening
to modify the risk (see Chapter 16). The identification or absence
of US ‘soft markers’ of aneuploidy such as echogenic bowel, short
long bones, dilated renal pelvis or an echogenic intracardiac foci
should not be interpreted independently.3 Although US is limited
in its ability to identify aneuploidy, it does help identify other
anomalies that may be associated with abnormal serum markers
(e.g. neural tube defects) and copy number variants.
Increasingly, women may choose to have analysis of cell-free DNA
(cfDNA) (see Chapter 21) after a high risk result from aneuploidy
screening, or some may choose this option as their primary screen-
ing test.23-26 In these cases, the positive and negative predictive value
of cfDNA is such that the presence or absence of soft markers is not
relevant. Confirmation of a positive cfDNA result requires an invasive
diagnostic test for confirmation.23 In woman who have had invasive
genetic testing, the presence of US markers is not relevant. 
Better Estimate of Gestational Age
The determination of the expected date of delivery (EDD) is abso-
lutely essential to obstetric management. A body of evidence has
accumulated demonstrating that routine US examination results
in a more accurate assessment of the EDD than last menstrual
period dating even with regular and certain menstrual dates (see
Fig. 20.8).3-5 The benefits of accurate estimation of gestational
age include a reduction of planned caesarean delivery before 39
weeks, a decrease in intervention for postterm pregnancy and a
reduction in diagnosis of fetal growth restriction. In a Cochrane
review of 11 trials, the routine use of early US and the subsequent
adjustment of the EDD led to a reduction in induction of labour
for postterm pregnancy (RR, 0.59; 95% CI, 0.42–0.83).9 A ran-
domised trial looking specifically at the timing of the examination
demonstrated that first trimester US examination in a low-risk
population was more effective than second trimester examination
 
 CHAPTER 20  Evidence for Routine Ultrasound Screening for Fetal Abnormalities in the Second and Third Trimesters
Second Trimester Cervical Length
The role of routine cervical length measurement in the second tri-
mester remains controversial. A 2013 Cochrane review did not find
sufficient evidence to recommend routine cervical length screening
of all pregnant women.28 Based on a meta-analysis of progester-
one treatment of women with asymptomatic midtrimester cervical
shortening, cervical length screening for all singleton pregnancies
in the midtrimester coupled with treatment of women found to
have a short cervix was felt appropriate.29 In women undergo-
ing obstetrical US examination, ACOG has recommended that
the cervix be examined when clinically appropriate and techni-
cally feasible.2 The Society of Obstetricians and Gynaecologists of
Canada (SOGC) previously concluded that routine transvagi-
nal cervical length assessment was not indicated in women at
low risk.30 In 2016, the SMFM recommended routine cervical
length screening in singleton women with a prior preterm birth;
however, there were no recommendations for routine screening.31
Review of the current literature supports no consensus on routine
cervical length screening; however, given the availability for treat-
ment with vaginal progesterone, we believe it may be a reasonable
approach at every detailed anatomical evaluation between 18 and
22 weeks of gestation.  high-risk factors (see Figs. 20.3 and 20.4).33 However, some
Multiple Gestations
Routine US screening provides for the early identification of mul-
tiple gestations. In the 2015 Cochrane review of trials of routine
US before the 24th week of pregnancy, a multiple gestation was
diagnosed earlier in routinely scanned pregnancies and US in
early pregnancy significantly reduced the failure to detect mul-
tiple pregnancy by 24 weeks of gestation (RR, 0.07; 95% CI,
0.03–0.17).9 In the RADIUS trial, women who did not have a
routine second trimester US examination had 38% of twin preg-
nancies unrecognised until after 26 weeks of gestation, and 13%
of twins were not diagnosed until delivery; no twin pregnancies
were missed on US examination.16 Again, this trial was conducted
in the 1980s. Similar results were found in the Helsinki trial.14 All
twin pregnancies were detected before 21 weeks of gestation in the
screened group versus 76.3% in the control group. The perinatal
mortality rate of twins in the screened group was 27.8 per 1000
compared with 65.8 per 1000 in the control group, which did not
reach statistical significance. Other retrospective series have sug-
gested improved neonatal outcomes with early diagnosis of twin
pregnancy.32 Ideally, the diagnosis of multiple pregnancy should
be made in the first trimester at a time when chorionicity can be
accurately determined.  images and their interpretation falls on the physician. In other
Routine Third Trimester Ultrasound
An early US determination of gestational age provides a baseline
against which later examinations can be compared to evaluate
subsequent fetal growth. In a 2015 review of trials of routine US
versus selective US, routine use of early US did not result in a
significant reduction in the diagnosis of small for gestational age
(SGA) fetuses (RR, 1.05; 95% CI, 0.81–1.35).9 Furthermore, the
value of later gestation universal growth USs (see Fig. 20.8) to
identify fetal growth restriction remains unclear even though early
identification of growth-restricted fetuses allows for closer surveil-
lance and earlier intervention. A 2015 Cochrane review of con-
trolled trials of routine late pregnancy US found that routine US
did not improve detection of neonates with birth weight less than
199
the 10th percentile (RR, 0.98; 95%, CI 0.74–1.28) or lead to a
decrease in perinatal mortality (RR, 1.13; 95% CI, 0.58–2.19).33
Subsequently, the Pregnancy Outcome Prediction study, a pro-
spective cohort study addressed this in a population of unselected
nulliparous women with singleton gestations who underwent early
US for pregnancy dating.34 Women who agreed to participate (n =
4512) underwent additional US examinations at about 20, 28 and
36 weeks of gestation. Half of the nonparticipating women eventually
underwent clinically indicated third trimester US examinations. Uni-
versal US in the third trimester tripled the detection of infants born
SGA compared with clinically indicated sonography (57% vs 20%);
however, universal US overdiagnosed SGA more often than clinically
indicated US (positive predictive value, 35% vs 50%). Among fetuses
with an estimated fetal weight (EFW) less than the 10th percentile,
only those with an abdominal circumference growth velocity at the
lowest decile were at increased risk for neonatal morbidity.
Although US examination late in pregnancy can diagnose clin-
ically important placental abnormalities, fetal malpresentation,
disorders of amniotic fluid, and excessive fetal growth, a 2015
Cochrane review found no evidence that routine late pregnancy
US screening results in better maternal, fetal or neonatal outcomes
than performance of US when indicated by clinical findings or
anomalies, such as certain skeletal dysplasias, can often only be
detected in the third trimester.34,35 
Routine Use of Doppler Velocimetry
Studies have evaluated the use of Doppler US as a screening tool
in low-risk women.3 These have found neither maternal nor fetal
benefit, which is in contrast to the proven benefit in high-risk,
growth-restricted pregnancies. In a 2015 Cochrane review of
14,185 women, routine Doppler US in low-risk or unselected
populations did not result in increased antenatal, obstetric and
neonatal interventions, and no overall differences were detected
for clinical outcomes.36 
Who Should Perform the Scan?
There is significant disparity in the skill and experience of opera-
tors performing US examinations which differs according to local
rules and regulations as well as location of the practice. In some
countries, including the United States, sonographers often per-
form the US examination, and physicians review the images. In
the United States, the registered diagnostic medical sonographer
does not practice independently, and the responsibility for the
countries, physicians do the complete examination. In certain
locations, physicians only perform targeted US examinations.
Whatever the practice pattern, the operator that is performing the
US should be skilled and trained to perform these examinations.
Both the AIUM and ISUOG provide guidelines and qualifica-
tions for US imaging which describe the minimal standards set
forth by the societies.4,37 
Equipment
Obstetric USs should be performed using the transabdominal or
transvaginal approach (or both) in real time. The choice of trans-
ducer frequency is a delicate balance between the resolution of
the US and the penetration. Generally, a 3- to 5-MHz transab-
dominal transducer allows enough penetration while providing
200 SE C T I O N 6     Prenatal Screening and Diagnosis
adequate resolution for most patients. In obese patients, a lower
frequency transducer may be needed to provide increased pen-
etration of adiposity. For more detailed scans in high-risk cases,
state-of-the-art equipment includes pulse and continuous-wave
Doppler and three-dimensional imaging capabilities, which have
demonstrated advantages in evaluating many fetal malformations.
Images obtained should be archived digitally, and the machine
should undergo appropriate preventive maintenance on a regular
basis and be upgraded as appropriate. Adequate attention must be
placed on maintaining and cleaning the transducer, and infection
control protocols must be maintained in both transabdominal and
transvaginal US.38,39  evaluation of the benefit of US screening may vary by location.
Documentation and Communication of
Results
Adequate documentation of the images and results is essential to
appropriate care of the patient, regardless of where the US is per-
formed. There should exist a permanent record of the US examina-
tion and its interpretation by the physician. It is the opinion of these
authors that US should be used to supplement a trained physicians’
history and physical examination in formulating a differential diag-
nosis and direct patient contact optimises the diagnostic yield of
a physical examination, including the US. As such, telemedicine
should not be a replacement for direct contact with the patient.
Quality assurance programs have been set forth by AIUM,
among others, to streamline and standardise both documenta-
tion and communication.37 Images, both normal and abnormal,
should always be recorded and archived in a retrievable format,
preferably digitally. Retention of the US images and the thorough
report should be consistent both with clinical needs and with rel-
evant legal and local health care facility requirements.
Communication between the interpreting physician and refer-
ring provider should be clear, timely and fully respect patient con-
fidentiality. Final US reports should be available within 24 hours
of completion of the examination. This finalised report must
include (at least): the patient’s name and other identifying infor-
mation (e.g. date of birth), the facility’s identifying information,
the physician identifying information, the date and time of the
US, interpretation, differential diagnosis and recommendations
for follow-up. Limitations in image quality and interpretation, as
well as a review of any prior or relevant imaging studies should be
included in this communication.
Following a standardised detailed protocol for imaging and
reporting will minimise both human error and near-misses. Many
newer machines have built-in protocols meant to reduce misses
that guide the examiner through the scan. Ideally, images are
stored and audited digitally to allow ongoing review between stud-
ies and for retrieval.
Quality assurance programs are essential to maintain excellence
in all practice settings. Each US unit should have in place a review
process that allows for the rapid review of archived images for the
identification of adverse outcomes and missed diagnoses as well
as the ability to perform random checks to ensure a standardised,
thorough and patient-centred approach of the imaging by all
trained providers.  was comparable in the two groups. Routine US was associated
Resource Utilisation
The effect of routine screening on the health care system has yet to be
determined. Some reports have analysed the cost-to-benefit ratio of
screening low-risk women with obstetric US. The value that each indi-
vidual patient places on childbirth or on the avoidance of a child with
an unsuspected anomaly needs to be considered in any cost-versus-
benefit equation. There is presently not a standardised way to incor-
porate this patient-centred metric. Furthermore, the World Health
Organization (WHO) Study Group has recognised that worldwide,
‘much of the US currently performed is carried out by individuals
with in fact little or no formal training’. To address this, organisa-
tions such as the ISUOG and AIUM have provided minimum stan-
dardised practice guidelines1 and suggest that consideration should be
given to local circumstances and resources further confirming that the
As discussed, the RADIUS trial concluded that routine US
was not cost effective and that offering the procedure routinely
would produce a significant health care system burden. Although
the overall perinatal mortality rate was not improved with rou-
tine screening, there was a higher rate of detection of anomalies.
The study was not powered to analyse what affect this detection
rate had on secondary outcomes. The cost analysis also did not
account for the societal costs of the US examinations performed
on patients who did not qualify for randomisation. Investigators
not involved in the RADIUS study have performed a cost-to-
benefit analysis of routine second trimester US using data from
the RADIUS trial, taking into consideration all of the findings
of the trial.40 Costs were estimated for each type of fetal anomaly
and projected for the follow-up each would need in neonatal life.
Savings from diagnosis of postterm pregnancy were also included
and concluded that routine US screening was associated with sig-
nificant savings but only if and when the US was performed in
tertiary care centres.
The Helsinki trial addressed the use of antenatal care services
in its trial of routine US screening.41 US screening reduced the
need for specialist services with no differences in hospital admis-
sions. A cost-effectiveness analysis on the same data set suggested
that second trimester screening with US is cost effective, at least
in a society with a publicly financed health system42 and an abso-
lute cost per US of $86 (of note, dollars as of 1995). When
the costs of additional screening-induced examinations and pro-
cedures were added, the cost per US rose to $102. Cost sav-
ings were calculated based upon decreased utilisation of health
services and visits and amounted to $182 per US, yielding a
net saving of $80 per patient in the screened group. The gross
cost of avoiding one perinatal death was $21,938, as based upon
the decreased perinatal mortality rate reported in that trial. This
evaluation assumes the availability and acceptability of termina-
tion of pregnancy.
A randomised trial from a centre in South Africa looked at
the costs of routine US screening and perinatal outcomes.43
The study group consisted of patients without risk factors for
congenital anomalies referred for US at 18 to 24 weeks of ges-
tation; the control group had routine care only. Both groups
could be referred for additional scans as indicated. Women in
the control group were more likely to be diagnosed with post-
term pregnancies and undergo amniocenteses for confirmation
of lung maturity, yet the incidence of adverse perinatal outcomes
with increased costs that were not accounted for by improved
outcomes. A systematic review of studies examining cost effec-
tiveness in screening US concluded that the available data were
of poor quality and that data could not be summarised to draw
useful conclusions.44 
 CHAPTER 20  Evidence for Routine Ultrasound Screening for Fetal Abnormalities in the Second and Third Trimesters
Safety and Ethical Considerations
The responsibility for the safe and ethical use of US is shared
among stakeholders, including the manufacturer, who should
ensure accuracy. As such, the ISUOG mandates that US be per-
formed by health professionals trained in its clinical use and bioef-
fects while disapproving of the use of US for the sole purpose
of souvenir images. Although no reports of fetal harm have been
reported, US is still considered energy, and there is a theoretical
potential of a biological effect.45-47 Prenatal US appears safe for
clinical practice and relies on the ALARA principle, that is, as low
as reasonably achievable.
Concomitantly, patients’ autonomy in decision making
about their medical choices should be considered. They
should be informed that although US has demonstrated ben-
efits in prenatal screening, there are inconsistent data regard-
ing the benefits of universal screening and cost effectiveness,
while the demonstration of improved perinatal outcome and
delivery planning has been demonstrated. Shared decision
making with the patient regarding the benefits and limitations
of routine US respects their autonomy by providing them
with the evidence-based information they need to make these
choices.6,48  Self-assessment questions available at ExpertConsult.com.
201
Conclusions
• The main objective of routine midtrimester US is to provide
accurate diagnostic information for optimising antenatal care
and delivery planning.
• Routine early US is beneficial because of better estimates of
gestational age, diagnosing multiple pregnancies and deter-
mining chorionicity
• Routine US examination can lead to earlier detection of clinically
unsuspected fetal malformations and earlier detection of multiple
pregnancies. The Eurofetus trial likely approaches the most accurate
sensitivity rates for US in the detection of anomalies (∼50%–70%).
• Individuals who routinely perform obstetric scans should have
specialised training for diagnosis during pregnancy.
• If one screening US examination is performed, the optimal tim-
ing is at 18 to 22 weeks of gestation,1,2 allowing for good visuali-
sation of anatomy early enough to allow completion of prenatal
diagnostic procedures with options for termination if desired.2
• Routine use of US screening examination in the third trimester
is not supported by available data.
Access the complete reference list online at ExpertConsult.com.
References 23. Committee Opinion No. 640. Cell-free DNA screening for fetal aneu-
ploidy. Obstet Gynecol. 2015;126(3):e31–e37.
24. Bianchi DW, Parker RL, Wentworth J, CARE study group, et al. DNA
1. Salomon LJ, Alfirevic Z, Berghella V, et al. ISUOG Clinical Standards
sequencing versus standard prenatal aneuploidy screening. N Engl J Med.
Committee. Practice guidelines for performance of the routine mid-
2014;370(9):799–808.
trimester fetal ultrasound scan. Ultrasound Obstet Gynecol. 2011;37(1):
25. Bianchi DW, Platt LD, Goldberg JD, et al. Maternal blood is source to
116–126.
accurately diagnose fetal aneuploidy (MELISSA) study group. Genome-
2.  American College of Obstetricians and Gynecologists. ACOG Prac-
wide fetal aneuploidy detection by maternal plasma DNA sequencing.
tice Bulletin No. 101: ultrasonography in pregnancy. Obstet Gynecol.
Obstet Gynecol. 2012;119(5):890–901.
2009;113(2 Pt 1):451–461.
26. Norton ME, Jacobsson B, Swamy GK, et al. Cell-free DNA analysis for
3. Reddy UM, Abuhamad AZ, Levine D, Saade GR. Fetal imaging work-
noninvasive examination of trisomy. N Engl J Med. 2015;372(17):1589–
shop invited participants. Fetal imaging: executive summary of a joint
1597.
Eunice Kennedy Shriver National Institute of Child Health and Human
27. Bennett KA, Crane JM, O’shea P, et al. First trimester ultrasound screen-
Development, Society for Maternal-Fetal Medicine, American Institute
ing is effective in reducing postterm labor induction rates: a randomized
of Ultrasound in Medicine, American College of Obstetricians and Gyne-
controlled trial. Am J Obstet Gynecol. 2004;190(4):1077–1081.
cologists, American College of Radiology, Society for Pediatric Radiol-
28. Berghella V, Baxter JK, Hendrix NW. Cervical assessment by ultra-
ogy, and Society of Radiologists in Ultrasound Fetal Imaging Workshop.
sound for preventing preterm delivery. Cochrane Database Syst Rev.
Obstet Gynecol. 2014;123(5):1070–1082.
2013;(1):CD007235.
4. Abuhamad AZ. ACOG committee on practice bulletins-obstetrics.
29. Romero R, Nicolaides K, Conde-Agudelo A, et al. Vaginal progesterone
ACOG practice bulletin, clinical management guidelines for obstetrician-
in women with an asymptomatic sonographic short cervix in the midtri-
gynecologists number 98, October 2008 (replaces practice bulletin num-
mester decreases preterm delivery and neonatal morbidity: a systematic
ber 58, December 2004). Ultrasonography in pregnancy. Obstet Gynecol.
review and metaanalysis of individual patient data. Am J Obstet Gynecol.
2008;112(4):951–961.
2012;206(2):124.e1–19.
5. ACOG Committee on Ethics. ACOG committee opinion. Number 297,
30. Lim K, Butt K, Crane JM. SOGC clinical practice guideline. Ultrasono-
august 2004. Nonmedical use of obstetric ultrasonography. Obstet Gyne-
graphic cervical length assessment in predicting preterm birth in single-
col. 2004;104(2):423–424.
ton pregnancies. J Obstet Gynaecol Can. 2011;33(5):486–499.
6. Levi S. Ultrasound in prenatal diagnosis: polemics around routine ultra-
31. Society for Maternal-Fetal Medicine (SMFM), McIntosh J, Feltovich H,
sound screening for second trimester fetal malformations. Prenat Diagn.
Berghella V, Manuck T. The role of routine cervical length screening in
2002;22(4):285–295.
selected high- and low-risk women for preterm birth prevention. Am J
7. Antenatal care, ed. Routine care for the healthy pregnant woman. London:
Obstet Gynecol. 2016;215(3):B2–B7.
RCOG Press; March 2008. https://www.ncbi.nlm.nih.gov/pubmed/
32. Hughey MJ, Olive DL. Routine ultrasound scanning for the detection
21370514. National Institute for Health and Clinical Excellence:
and management of twin pregnancies. J Reprod Med. 1985;30(5):427–
­Guidance.
430.
8. Grandjean H, Larroque D, Levi S. The performance of routine ultraso-
33. Bricker L, Medley N, Pratt JJ. Routine ultrasound in late pregnancy
nographic screening of pregnancies in the Eurofetus study. Am J Obstet
(after 24 weeks’ gestation). Cochrane Database Syst Rev. 2015;(6):
Gynecol. 1999;181(2):446–454.
CD001451.
9. Whitworth M, Bricker L, Neilson JP, Dowswell T. Ultrasound for fetal
34. Sovio U, White IR, Dacey A, et  al. Screening for fetal growth restric-
assessment in early pregnancy. Cochrane Database Syst Rev. 2010;(4):
tion with universal third trimester ultrasonography in nulliparous women
CD007058.
in the pregnancy outcome prediction (POP) study: a prospective cohort
10. Abuhamad A, Minton KK, Benson CB, et  al. Obstetric and gynecologic
study. Lancet. 2015;386(10008):2089–2097.
ultrasound curriculum and competency assessment in residency training pro-
35. Renna MD, Pisani P, Conversano F, et  al. Sonographic markers for
grams: consensus report. Ultrasound Obstet Gynecol. 2018;51(1):150–155.
early diagnosis of fetal malformations. World J Radiol. 2013;5(10):356–
11. Best KE, Tennant PW, Bell R, Rankin J. Impact of maternal body
371.
mass index on the antenatal detection of congenital anomalies. BJOG.
36. Alfirevic Z, Stampalija T, Medley N. Fetal and umbilical Doppler ultra-
2012;119(12):1503–1511.
sound in normal pregnancy. Cochrane Database Syst Rev. 2015;(4):
12. Pasko DN, Wood SL, Jenkins SM, et al. Completion and sensitivity of
CD001450.
the second-trimester fetal anatomic survey in obese gravidas. J Ultrasound
37. Reddy UM, Abuhamad AZ, Levine D, Saade GR. Fetal imaging work-
Med. 2016;35(11):2449–2457.
shop invited participants. Fetal imaging: executive summary of a joint
13. Whitworth M, Bricker L, Mullan C. Ultrasound for fetal assessment in
Eunice Kennedy Shriver National Institute of Child Health and Human
early pregnancy. Cochrane Database Syst Rev. 2015;(7):CD007058.
Development, Society for Maternal-Fetal Medicine, American Institute
14. Saari-Kemppainen A, Karjalainen O, Ylöstalo P, Heinonen OP. Ultra-
of Ultrasound in Medicine, American College of Obstetricians and Gyne-
sound screening and perinatal mortality: controlled trial of systematic
cologists, American College of Radiology, Society for Pediatric Radiology,
one-stage screening in pregnancy. The Helsinki ultrasound trial. Lancet.
and Society of Radiologists in Ultrasound Fetal Imaging Workshop. Am J
1990;336(8712):387–391.
Obstet Gynecol. 2014;210(5):387–397.
15. Crane JP, LeFevre ML, Winborn RC, et al. A randomized trial of prenatal
38. American Institute of Ultrasound in Medicine (AIUM). AIUM Prac-
ultrasonographic screening: impact on the detection, management, and
tice Parameter for Documentation of an Ultrasound Examination. 2015.
outcome of anomalous fetuses. The RADIUS study group. Am J Obstet
https://www.aium.org/resources/guidelines/documentation.pdf.
Gynecol. 1994;171(2):392–399.
39. Benacerraf BR, Minton KK, Benson CB, et  al. Proceedings: beyond
16. Ewigman BG, Crane JP, Frigoletto FD, et  al. Effect of prenatal ultra-
ultrasound first forum on improving the quality of ultrasound imaging in
sound screening on perinatal outcome. RADIUS study group. N Engl J
obstetrics and gynecology. J Ultrasound Med. 2018;37(1):7–18.
Med. 1993;329(12):821–827.
40. Vintzileos AM, Ananth CV, Smulian JC, et al. Routine second-trimester
17. American Institute of Ultrasound in Medicine. AIUM practice guideline
ultrasonography in the United States: a cost-benefit analysis. Am J Obstet
for the performance of obstetric ultrasound examinations. J Ultrasound
Gynecol. 2000;182(3):655–660.
Med. 2013;32(6):1083–1101.
41. Saari-Kemppainen A. Use of antenatal care services in a controlled
18. Friedberg MK, Silverman NH, Moon-Grady AJ, et al. Prenatal detection
ultrasound screening trial. Acta Obstet Gynecol Scand. 1995;74(1):12–
of congenital heart disease. J Pediatr. 2009;155(1):26–31.31 e1.
14.
19. Tworetzky W, McElhinney DB, Reddy VM, et al. Improved surgical out-
42. Leivo T, Tuominen R, Saari-Kemppainen A, et al. Cost-effectiveness of
come after fetal diagnosis of hypoplastic left heart syndrome. Circulation.
one-stage ultrasound screening in pregnancy: a report from the Helsinki
2001;103(9):1269–1273.
Ultrasound Trial. Ultrasound Obstet Gynecol. 1996;7(5):309–314.
20. Bonnet D, Coltri A, Butera G, et al. Detection of transposition of the
43. Geerts LT, Brand EJ, Theron GB. Routine obstetric ultrasound exami-
great arteries in fetuses reduces neonatal morbidity and mortality. Circu-
nations in South Africa: cost and effect on perinatal outcome—a pro-
lation. 1999;99(7):916–918.
spective randomised controlled trial. Br J Obstet Gynaecol. 1996;103(6):
21. Evans MI, Wapner RJ, Berkowitz RL. Noninvasive prenatal screen-
501–507.
ing or advanced diagnostic testing: caveat emptor. Am J Obstet Gynecol.
44. Roberts T, Henderson J, Mugford M, et al. Antenatal ultrasound screen-
2016;215(3):298–305.
ing for fetal abnormalities: a systematic review of studies of cost and cost
22. Rink BD, Norton ME. Screening for fetal aneuploidy. Semin Perinatol.
effectiveness. BJOG. 2002;109(1):44–56.
2016;40(1):35–43.

201.e1
201.e2 R E F E REN C ES
45. Bly S, Van den Hof MC. Diagnostic imaging committee, Society of
Obstetricians and Gynaecologists of Canada. Obstetric ultrasound bio-
logical effects and safety. J Obstet Gynaecol Can. 2005;27(6):572–580.
46. Marinac-Dabic D, Krulewitch CJ, Moore Jr RM. The safety of prenatal ultra-
sound exposure in human studies. Epidemiology. 2002;13(suppl 3):S19–S22.
47. Miller MW, Brayman AA, Abramowicz JS. Obstetric ultrasonography: a
biophysical consideration of patient safety—the ‘rules’ have changed. Am
J Obstet Gynecol. 1998;179(1):241–254.
48. Chervenak FA, McCullough LB. Ethical dimensions of ultrasound
screening for fetal anomalies. Ann N Y Acad Sci. 1998;847:185–190.
21
Noninvasive Screening for Cytogenetic
Disorders (Fetal Aneuploidy Including
Microdeletions)
KAREN WOU AND RONALD J. WAPNER
KEY POINTS
• The origins of intact fetal circulating trophoblast cells and
nucleated red blood cells in maternal plasma are reviewed,
and their pitfalls and promises for noninvasive prenatal test-
ing are discussed.
• The discovery of cell-free fetal DNA (cffDNA) in maternal
circulation and how it has allowed the development of an ex-
cellent noninvasive screening test for common aneuploidies
and sex chromosomal abnormalities in both the high-risk and
general populations is discussed.
• The clinical performance of cffDNA is reviewed, including sen-
sitivity, specificity, positive predictive value and false-positive
and false-negative rates.
• The different techniques using cffDNA to detect fetal aneuploi-
dies and factors affecting the fetal DNA fraction are discussed.
• The challenges of using cffDNA testing to identify fetal copy
number variants are presented, including the depth of se-
quencing. Clinical use of cffDNA to identify subchromosomal
abnormalities is currently not recommended by major societies.
Introduction
Prenatal screening to identify pregnancies at risk for fetal cytogenetic
abnormalities has been practiced for almost half a century, although
the approach has constantly evolved with the goal of increasing the
detection of fetal aneuploidies while simultaneously decreasing unnec-
essary false positives.1 Fifty years ago, advanced maternal age was the
sole risk factor used in population-based screening, and identification
of pregnancies with Down syndrome was the primary goal. At that
time, the detection rate was approximately 30% with about 5% of the
population at risk because of advanced maternal age. By the 1990s,
second trimester multiple serum markers (human chorionic gonado-
trophin (hCG), alpha-fetoprotein (AFP), unconjugated estriol (uE3))
were added to modify the maternal age risk, which improved detec-
tion of trisomies 21, 18 and 13 to approximately 80% for the same
5% screen positive rate. Subsequently, the addition of nuchal trans-
lucency (NT) measurement to first trimester serum biochemical ana-
lytes (pregnancy-associated placental protein A (PAPP-A) and hCG)
brought these detection rates up to 92% to 95%.2 (These screening
methods are reviewed extensively in Chapter 19.)
202
Despite the excellent screening performance of indirect bio-
markers, acquisition of fetal tissue or DNA would allow complete
genetic analysis of a fetus. Presently, this is only possible using
invasive diagnostic procedures such as chorionic villus sampling or
amniocentesis. Despite multiple attempts over the past 20 years to
retrieve and isolate fetal cells from the maternal circulation or cer-
vix, this has not proven clinically viable.3 A major paradigm shift
has recently occurred in which the analysis of fetal cell-free DNA
(cfDNA) circulating in the maternal circulation has been dem-
onstrated to detect more than 99% of Down syndrome pregnan-
cies with less than a 0.1% false-positive rate (FPR). This screening
method has been rapidly introduced into clinical care and is pres-
ently recommended by major societies worldwide for use in preg-
nancies at high risk for common whole-chromosome aneuploidies
(trisomies 21, 18 and 13). This includes populations such as
women of advanced maternal age, those with positive biochemi-
cal and NT screening, those with a previous offspring having a
common trisomy, a known parental balanced Robertsonian trans-
location increasing the risk for an unbalanced offspring involving
chromosomes 21 or 13 or fetuses with ultrasonographic findings
indicative of an increased risk for aneuploidy (Table 21.1).4-7 Its
use in lower risk populations is now being considered because data
are progressively showing that its technical performance is equally
reliable. Further work in the development of noninvasive prena-
tal diagnostic testing using cfDNA is ongoing with the ultimate
goal of detecting other cytogenetic abnormalities similar to those
attainable with direct fetal testing. 
Origins of Fetal Cells and Cell-Free DNA in
the Maternal Circulation
Fetal Cells in the Maternal Circulation
The passage of fetal material, including intact cells, across the so-
called ‘placental’ barrier into the maternal circulation has become a
focus of intense research over the past 20 years8-11 because retrieval
of a small number of such cells would provide a complete copy of
the fetal genome amenable to analysis. This has the potential to be
a true diagnostic test using improved molecular technologies such
as chromosomal microarray and sequencing.
The first report of fetal cells in the maternal circulation
occurred in 1893 by a German pathologist Georg Schmorl
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions) 203

when he described the presence of trophoblast cells in mater- chromosome material.13 It was subsequently recognised using
nal lungs in patients dying from eclampsia.12 Not until 1969 fluorescence in situ hybridisation (FISH) and polymerase chain
was the presence of fetal cells in the circulation of pregnant reaction (PCR) that fetomaternal cellular trafficking occurs in
women with normal pregnancies confirmed with the identi- all gestations.14-16
fication in maternal blood of fetal lymphocytes containing Y Multiple studies have shown the paucity of fetal cells in mater-
nal blood. Their prevalence is estimated at 1 fetal to 104 to 108
maternal mononuclear cells.17,18 To quantify the prevalence
TABLE Recommendations for Noninvasive Prenatal
of fetal cells, Emad and colleagues used automated scanning of
21.1 Screening from Major Societies4-7 microscopic slides to determine the concentration of fetal cells in
Major Society Recommendations maternal blood.19 With this approach, the number of fetal cells
identified per 1 millilitre of maternal blood ranged from 3 to 6
ACOG/SMFM NIPS is currently only recommended as a screen-
(2015) ing option for women at increased risk for fetal in normal pregnancies and up to 13 to 21 cells in pregnancies
aneuploidy. affected with Down syndrome. Because of the rarity of these cells,
most research on noninvasive prenatal diagnosis using fetal cells
SOGC (2013) NIPS should be an option available to women at has focused on cell separation techniques to both enrich the popu-
increased risk as an alternative to amniocentesis. lation of fetal cells and to deplete contaminating maternal cells
ISPD (2015) NIPS may be considered in women classified as high with the goal of finding a sufficient population of the ‘ideal’ fetal
risk based on serum and ultrasound screen- target cells for testing.20,21
ing, contingently to women considered high or Types of fetal cells in the maternal circulation. Four types
intermediate risk after conventional screening or of circulating fetal cells have been described: trophoblasts, fetal
all pregnant women. nucleated red blood cells (fNRBCs), leukocytes and undifferenti-
RCOG (2014) No specific recommendations (ROCG Scientific ated stem cells and progenitors.22 However, the latter two types
Impact Paper No. 15) persist in the maternal circulation up to 27 years postpartum,23
making them unusable for prenatal diagnosis of a current preg-
ACMG (2016) Inform all pregnant women that NIPS is the most
nancy. Prenatal diagnostic research has thus focused on tropho-
sensitive screening option for traditionally screened
aneuploidies. (ACMG Policy Statement)
blasts and fNRBCs which are cleared from the maternal blood
rapidly after delivery. 
ESHG/ASHG No specific recommendations (ASHG/ESHG Joint Isolation of fetal cells collected from the maternal circulation.
(2015) Statement) By far the biggest challenge with using these rare and fragile cells
ACMG, American College of Medical Genetics and Genomics; ACOG, American Congress of
for noninvasive testing remains their isolation from contaminat-
Obstetricians and Gynecologists; ASHG, American Society of Human Genetics; ESHG, European ing maternal cells without damage. The primary approaches have
Society of Human Genetics; ISPD, International Society of Prenatal Diagnosis; NIPS, noninvasive used gradient separation to increase the relative concentration of
prenatal screening; RCOG, Royal College of Obstetricians and Gynaecologists; SMFM, Society the cells of interest followed by their isolation through unique cell
of Maternal-Fetal Medicine; SOGC, Society of Obstetricians and Gynecologists of Canada surface or cytoplasmic markers (Fig. 21.1).
  
Ficoll-hypaque separation of MNC from
peripheral blood

Magnetic positive selection

• Fig. 21.1  A, Ficoll-Hypaque separation of mononuclear cells (MNC) from peripheral blood. The separa-
tion of mononuclear cells from other cells (e.g., red blood cells) in the maternal circulation is demonstrated.
A Ficoll-Hypaque gradient is used, separating the cells based on density. The mononuclear cell layer will
have an increased concentration of fetal cells. B, Magnetic positive selection. Further separation in which
the cells are stained with antibodies attached to ferrous beads known to be fetal cell specific. Magnetic
cell separation then performed to select the labelled cells. (Courtesy of Ronald J Wapner.)
204 SE C T I O N 6     Prenatal Screening and Diagnosis
Trophoblast cells. Trophoblasts were the first fetal cell type to
be detected in the maternal circulation24 but present challenges
to their use for noninvasive prenatal diagnosis. First, few highly
specific antibodies are available for isolation and enrichment. Sec-
ond, these multinucleated cells are not easily amenable to stan-
dard cytogenetic techniques, such as FISH, but require molecular
techniques such as chromosomal microarray. Third, because they
are placental in origin, there is a 1% incidence of confined placen-
tal mosaicism similar to that seen with chorionic villus sampling.
Despite these difficulties, contemporary studies continue to show
their potential value.
Paterlini-Brechot’s group reported the genetic diagnosis of 63
fetuses at risk for either cystic fibrosis (CF) or spinal muscular
atrophy (SMA) using circulating trophoblast cells.25 Recovery of
trophoblast cells was performed by the initial isolation of epithe-
lial tumour/trophoblastic cells (ISET) by size using a calibrated
filter. Cells were subsequently laser dissected off the filter and gen-
otyped using maternal and paternal short tandem repeats (STRs)
to confirm those that were trophoblast. Of the cells greater than
15 μM in size selected by filtering, half were confirmed as fetal.
These cells then underwent specific diagnostic testing. Approx-
imately, 1.5 cells per millilitre of maternal blood were present,
and approximately 5 to 10 cells were analysed per case. All fetuses
affected by CF or SMA were correctly diagnosed and confirmed
by chorionic villus sampling (CVS).
Further evidence of the potential value of trophoblast recov-
ery was presented by Hatt and colleagues using specific markers
for cells most likely belonging to the endovascular subgroup of
extravillous trophoblast (EVTs).26 Expression of the endothelial/
vascular marker of these originally ectodermal cells is the result
of adaptation to their vascular environment. Fetal cells were iso-
lated from maternal blood (gestational age, 11–13 weeks) using
magnetic cell sorting enrichment (with a novel antibody combi-
nation for the endothelial/vascular markers CD105 and CD141).
Trophoblast cells were demarcated using a cocktail of cytokeratin
antibodies. In 85% of the samples, it was possible to obtain X and
Y signals by FISH for gender determination with a 91% specific-
ity. Concordance to fetal sex of 100% was possible if three or more
fetal cells could be found in a sample.26
Recently, Beaudet and his group developed methods for the
detection of chromosomal and subchromosomal abnormalities
in fetal trophoblast cells in maternal circulation using array com-
parative genomic hybridisation (CGH), next-generation sequenc-
ing (NGS), or both.27,28 They first separated nucleated cells from
maternal blood collected at 10 to 16 weeks’ gestation by density
fractionation and then immunostained them to identify cytokera-
tin-positive and CD45-negative trophoblasts. Array CGH, NGS,
or both was used for analysis after whole-genome amplification
and genotyping and identified normal fetuses and fetuses affected
with Klinefelter syndrome; trisomies 21, 18 and 13; and chromo-
some 15 deletion syndrome.27,28
The presence of trophoblast cells in cervical mucus has also
been reported, first by Shettles in 1971 using quinacrine mustard
fluorescent staining for identification.29,30 The cells were readily
identified by their morphology and unique immunohistochemis-
try using specific antibodies.31-33 However, since then, identifica-
tion of fetal cells in cervical mucus has been inconsistent with
detection rates between 50% and 60%.34,35
More recently, Bolnick and colleagues published results on 56
women between 5 and 20 weeks of gestation36 in whom cervi-
cal specimens were collected using a cytobrush and human leu-
kocyte antigen G trophoblast-specific antibodies to identify cells.
The average number of cells recovered per patient was 746 ±
59 across the gestational ages. There was minimal maternal cell
contamination, and 95% to 100% of the cells isolated expressed
confirmatory fetal-specific parameters. Nonetheless, to date, tech-
nical challenges have prevented this technology from developing
into a clinical tool. Although some groups are still exploring this
approach, none has consistently demonstrated success. 
Fetal nucleated red blood cells. Fetal nucleated red blood
cells have been detected in the maternal circulation and have a
relative short half-life, making them specific to the current preg-
nancy.37 This has led to their evaluation as a target for prenatal
diagnosis.38,39 The advantages of this cell are that they can be iden-
tified through a marker profile which is characteristic for erythroid
precursor cells and which varies from other blood cell subpopu-
lations. For example, fNRBCs express the transferrin receptor
(CD71) on their cell surfaces and do not express CD45 as do
maternal leukocytes.
Stringent isolation criteria are required to isolate and identify
the fetal cells which are present at a concentration of 1 in 105 to
107 maternal nucleated cells. After initial staining with cell-specific
markers, enrichment of fetal cells most often uses immunomag-
netic or flow cytometric cell separation techniques, either alone or
in combination. Other approaches attempted include separation
by centrifugation using a Ficoll gradient, filtration on a chip, lat-
eral displacement and magnetophoresis, lectin-binding, dielectro-
phoresis, micromanipulation, laser microdissection and pressure
catapulting.40 This relatively large assortment of approaches shows
the lack of consensus on the single best approach for isolation.
Confirmation of the fetal origin of the cells is required after
enrichment because maternal immature nucleated RBCs are pres-
ent and can express markers similar to those on fetal cells such as
the transferrin receptors.9,14 Immune labelling with embryonic or
fetal globin antibodies followed by selected cell dissection or cap-
ture is presently the most frequently used approach.
Initially, cytogenetic techniques such as FISH were used for
cytogenetic evaluation but proved difficult because of the cellular
disruption that occurred during separation and isolation. More
recently, molecular approaches which only require a small amount
of fetal DNA have proven more efficient.
The National Institute of Child Health and Human Develop-
ment Fetal Cell Isolation Study (NIFTY) is the largest to date
to evaluate fNRBC-based techniques for noninvasive prenatal
diagnosis.41 Results showed that the sensitivity and specificity
of the cell-based methods were not satisfactory for aneuploidy
detection. In the NIFTY trial, the authors used various poten-
tially unique NRBC cell surface identifiers and both magnetic-
activated cell sorting (MACS) and fluorescent-activated (FACS)
approaches.41,42 The results of this trial, which included 2744
samples, showed an aneuploidy detection rate by FISH of triso-
mies 13, 18 and 21 and the sex chromosomes of 74.4% with an
FPR of 0.6% to 4.1%. This study demonstrated the limitations
of NRBC identification, separation and analysis using approaches
available at the time. Isolation of fetal nucleated cells for prena-
tal diagnosis continues to be a costly, labour-intensive and time-
consuming process and will remain so until reliable automated
approaches become more readily available. 
Future Directions
In the past decade, various groups have developed new antibodies
to fNRBCs and have used advanced single-cell analytic techniques
to continue to explore the possibility of retrieving and analysing
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)
fetal cells. One example is the life science biotechnology com-
pany RareCyte, which has developed an innovative method using
sequential density fractionation to recover intact fetal cells.43 A
two-step centrifugation method takes advantage of the nucleated
cells’ density to concentrate and separate cells of interest away
from plasma and maternal RBCs. Multiplex imaging using pro-
prietary software then detects and ranks potential cells of interest
by analysing individual cell’s morphology and biomarker expres-
sion profile. After discovery and characterisation, cells of interest
are individually retrieved for additional downstream analysis.
KellBenX focuses on a proprietary monoclonal antibody (4 B9)
found uniquely on fNRBCs and their precursors and not on sur-
face antigens of maternal RBCs.44 When the antibody is bound
to an epitope expressed by fNRBCs, fetal cells can be isolated and
can undergo full analysis by sequencing, PCR, FISH, microarray
or immunohistochemistry.
Although these advancements are intriguing, they have not yet
proven useful for clinical analysis because the complexity of the
approaches and the need to validate that the retrieved cells are
exclusively fetal make it difficult to scale for routine screening.  Gestational age (wk)
Cell-Free Fetal DNA in the Maternal Circulation
Circulating cell-free nucleic acids in plasma and serum have
been described in other fields of medicine, such as oncology and
trauma, as novel biomarkers for various diseases. Mandel and
Metais were the first to report the presence of extracellular nucleic
acids in the circulation in 1948.45 In 1997, real-time PCR target-
ing SRY, a single-copy Y-chromosome-specific sequence, was used
to demonstrate and quantify the amount of fetal DNA in preg-
nant women carrying male fetuses. This discovery of cell-free fetal
DNA (cffDNA) in the maternal plasma opened a new perspective
in prenatal diagnosis.46
The main advantage of cffDNA is that it is up to 1000-fold
more prevalent than the DNA obtained from the limited number
of circulating fetal cells. It also diminishes rapidly after birth with
a half-life estimated to be 16.3 minutes. cffDNA levels are unde-
tectable by 2 hours postpartum, making it specific to the current
pregnancy.47
Initially, cffDNA fragments were examined by conventional
or real-time quantitative PCR to identify fetal DNA sequences
that were completely absent from the maternal genome (e.g. the
Y chromosome from a male fetus or the fetal RhD gene absent
from Rhesus-negative pregnant women).48,49 Since then, the clini-
cal utility and success of cffDNA analysis has continued to expand
with the detection of fetal aneuploidies, sex chromosomal abnor-
malities and more recently microdeletions and microduplications.
Origin of cell-free fetal DNA in the maternal circulation. Many
cells go through a life cycle, which ends in programmed cell death
called ‘apoptosis’. During this process, DNA gets cleaved into
short fragments of 150 to 200 base pairs that are released into
the blood as ‘cell-free DNA’ (cfDNA). During pregnancy, DNA
fragments arise from cytotrophoblast cells of the placenta through
a constant turnover of villous trophoblasts and are released into
the maternal circulation, resulting in maternal plasma containing
a mixture of both maternal and cffDNA. The sum of the fetal
DNA fragments in the maternal circulation represents the entire
fetal genome.
The term cell-free ‘fetal’ DNA is misleading when applied to
that found in the maternal circulation because the DNA ema-
nates predominately from the placenta. It is therefore not surpris-
ing that cffDNA is elevated in a number of conditions associated
205
40
30
Fetal clDNA (%)
20
10
0
10 15 20 25 30 35 40
• Fig. 21.2  Gestational age and maternal weight effects on fetal cell-free
DNA (cfDNA) levels in maternal plasma. Relationship between percentage
of fetal cfDNA and gestational age. The cfDNA percentage for 22,384 preg-
nancies is plotted with respect to gestational age (weeks). At 10 weeks,
0 days to 10 weeks, 6 days of gestation, the median cfDNA percentage
was 10.2%. Between 10 and 21 weeks of gestation (black open circles),
cfDNA increased at 0.10% per week (P  < .0001; blue dashed line within
black open circles), and 2% of the pregnancies during this period were
below 4% cfDNA. Starting at 21 weeks’ gestation, the cfDNA percent-
age increased at a rate of 1% per week (P  < 0.0001) a 10-fold increase
in the amount of cfDNA percentage per week compared with the weeks
between 10 and 21 weeks’ gestation. (Adapted from Wang E, Batey A,
Struble C, et al. Gestational age and maternal weight effects on fetal cell-
free DNA in maternal plasma. Prenat Diagn. 2013;33(7):662–666.)
with placental pathology such as preeclampsia, preterm labour,
placenta previa, hyperemesis gravidarum and fetal intrauterine
growth restriction.47 
Amount of fetal cell-free DNA in the maternal plasma. The
amount of cffDNA in the maternal plasma represents approxi-
mately 3% to 10% of the total and occasionally may reach 30%,
depending on multiple factors.50-53 cffDNA can be reliably
detected after 10 weeks of gestation; however, reports have shown
detection as early as 5 to 7 weeks.54
Fetal fraction. The fetal fraction is the ratio of the fetal cfDNA
to the total circulating cfDNA and has a bell-shaped distribution
with an average peak of 10% to 20% (Fig. 21.2). The ff remains
relatively stable between 10 and 21 weeks of gestation but then
increases toward term.
The most commonly used approach to quantifying fetal frac-
tion differentiates maternal from fetal DNA in male pregnancies
using genes on the Y chromosome such as SRY, DYS14 and DAZ.46
The fetal fraction can also be measured in female pregnancies
using molecular counting, amplification of differentially methyl-
ated sequences or detection of unique fetal polymorphisms.55,56
Estimates based on the use of epigenetic or genotype differences
between maternal and fetal DNA fragments may be more accurate
than measurements of Y-specific sites.57
The fetal fraction can vary from woman to woman and from
one pregnancy to another and changes throughout gestation sec-
ondary to many exogenous factors, including gestational age,
maternal weight and fetal aneuploidies. 
206 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE 72
21.2 Main Companies Offering Noninvasive Prenatal Screening
Sequenom, MaterniT21+ Illumina, Verifi Ariosa, Harmony Natera, Panorama Labcorp, InformaSeq
Trisomies 21, 18, 13 21, 18, 13 21, 18, 13 21, 18, 13 21, 18, 13
Sex chromosomes XX, YY, XXX, XYY, XXY XX, YY, XXX, XYY, XXY XX, YY, XXX, XYY, XXY, XX, YY, XXX, XYY, XXY XX, YY, XXX, XYY, XXY
XXYY
Monosomy X X — X X
Other tests Microdeletions (7); — — Microdeletions (7); —
trisomies 16, 22 triploidy
Method MPS MPS Targeted microarray SNP MPS
Sensitivity for:
Trisomy 21 >99.1% >99% >99% >99% >99.1%
Trisomy 18 >99/9% 97.4% 97.4% 96.4% 98.3%
Trisomy 13 91.7% 87.5% 93.8% >99% 98.2%
Gestation 10 wk 10 wk 10 wk 9 wk 10 wk
Twin gestation Yes Yes Yes No Yes

MPS, Massively parallel sequencing; SNP, single nucleotide polymorphism

Gestational age. The rate of placental cell apoptosis and the
fetal DNA concentration increase as gestation advances50,58,59
(see Fig. 21.2). At 10 weeks’ gestation, the median fetal fraction is
approximately 10% and rises by 0.1% per week between 10 and
20 weeks of gestation followed by a 10-fold increase to 1% per
week from 21 weeks to term.59 Approximately 2% of pregnancies
less than 21 weeks’ gestation have a fetal fraction below 4%, which
is considered too low to be used for aneuploid screening.  the total cfDNA in the maternal plasma, requiring that labora-
Maternal weight. There is a significant negative correlation
between fetal fraction and maternal weight59 from a mean of
about 12% for a maternal weight of 60 kg to 6% for a weight of
120 kg.60 Accordingly, the proportion of pregnancies with a fetal
fraction less than 4% increases with increasing maternal weight.59
For example, 20% of women over 250 lb will have a value less
than 4% as will 50% at more than 350 lb.61 Obesity may be asso-
ciated with a lower fetal fraction caused by a dilutional effect from
an increased maternal circulatory volume59-61 but is more likely
related to an increase in maternal cfDNA from a higher adipocyte
turnover rate.62 
Fetal aneuploidies. Specific fetal chromosome abnormalities
may alter the fetal fraction.63-65 Specifically, in fetuses with Down
syndrome, the DNA fragments result in a higher proportion of
fetal DNA than in disomic fetuses.63-66 This observation has also
been observed for other fetal aneuploidies such as trisomy 13 and
sex chromosomal abnormalities such as 47,XXY but perhaps less
significantly. On the other hand, other studies have shown fetal
fraction to be lower with cases of trisomy 18 and monosomy X,
possibly related to a smaller placental size.65,67  sequenced by the addition of fluorescently labelled nucleic acids.
Twin gestations. The median fetal fraction per fetus in twin
pregnancies is lower than for singleton gestations.62 For instance,
Srinivasan and colleagues found the overall fetal fraction per fetus
to be reduced by up to 50% in both mono- and dizygotic twins,68
resulting in a clinically significant higher proportion of twin gesta-
tions with a fetal fraction too low for accurate assessment.  sequence all informative chromosome regions and then count the
Other factors. As illustrated by their correlation with three-
dimensional ultrasound measurements, serum concentrations of
β-hCG and PAPP-A are an indirect measure of placental mass69
so that the fetal fraction increases in a linear fashion with these
analytes independent of maternal weight and other maternal char-
acteristics. Fetal fraction is not affected by other prenatal analyte
results, maternal age, fetal sex, previous blood donations, race,
smoking or ultrasound measurements such as NT or crown–rump
length.60–71 
Analysis of cell-free DNA for the prediction of fetal cytoge-
netic abnormalities. Fetal DNA represents only about 10% of
tory methods to identify any small increase or decrease associated
with a fetal chromosomal aneuploidy must be robust. Today, more
than 10 companies are offering this noninvasive prenatal screen-
ing (NIPS) test worldwide, and seven of them distribute from the
United States.72 Each company is using one of three different test-
ing approaches and offering different testing options (Table 21.2),
which are described next. 
Techniques Using Cell-Free DNA to Detect
Common Fetal Aneuploidies
Massively Parallel Sequencing and
Next-Generation Sequencing
Shotgun or Random Massively Parallel Sequencing. The tech­
nology underlying massively parallel sequencing (MPS) is
illustrated in Fig. 21.3. In summary, fragments of both maternal
and fetal DNA are clonally amplified and then each clone is
One million to 43 billion reads of 40 to 500 base pairs (bp) each
can be obtained per run.73,74 Using bioinformatics, the reads
are aligned to a reference copy of the human genome and the
chromosomal source of the fragment identified.
The basic goal of shotgun MPS for aneuploidy detection is to
DNA fragments assigned to a single-locus.75,76 In this method,
maternal and fetal fragments of 150 to 200 bp in size are sequenced
simultaneously and approximately the first 36 bp are aligned and
mapped to their chromosome of origin. Because the entire human
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions) 207

DNA fragments in
maternal plasma

Chr1
36 bp Bioinformatics Chr7
alignment ChrX
AAGCT... Chr13
Sequence and CTAGT... Chr1
align TAGGC... Chr21
GCATG... Chr18
ChrY and so on...
nth sequence

Sequence
counting

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

• Fig. 21.3  Mass parallel (shotgun) sequencing analysis of fetal DNA. The process of mass parallel next-
generation sequencing and sequence counting to the analysis of maternal cell-free DNA for fetal aneu-
ploidy. Cell-free DNA fragments between 120 and 200 kb are sequenced. Bioinformation analysis assigns
sequences to their genomic origins, and the number of sequences per genomic assignment is counted.
chr, Chromosome. (Adapted from Zhong, X, Holzgreve, W. Circulating cell-free DNA in women’s medicine.
Glob Libr Womens Med 2009; DOI 10.3843/GLOWM.10270.)

genome sequence is known, MPS maps between 12 and 25 mil-
lion fragments per sample to discrete loci on each chromosome.
The number of fragment reads from each specific chromosome are
then quantified and compared with values from a normal refer-
ence chromosome.
A relative excess or deficiency in the number of counts on the
chromosome of interest compared with a reference chromosome
will determine the risk for aneuploidy (trisomy or monosomy) for
that specific chromosome. This difference is usually expressed as a
Z-score.75-78 A large number of reads is needed because the differ-
ence between aneuploidy and euploidy is small (∼1.05 in 1 for a
trisomy) because on average only 10% of the DNA fragments will
come from an aneuploid fetus and 90% from the euploid mother.
The difference is even smaller with lower fetal fractions.  aneuploidy analyses SNPs and determines the relative quantitative
Targeted or Directed Massively Parallel
Sequencing
Massively parallel sequencing is highly accurate but relatively inef-
ficient if screening only for the common aneuploidies because the
majority of the fragment reads come from chromosomes that are
never analysed. Targeted MPS improves efficiency by including a
presequencing step that selectively amplifies only fragments from
the chromosomes of interest, thereby creating mapped reads that
are specific to these chromosomes alone. A small number of single
nucleotide polymorphisms (SNPs) are also analysed, allowing cal-
culation of the fetal fraction. This chromosome-selective sequenc-
ing is referred to as digital analysis of selected regions (DANSR)
and is a more efficient use of sequencing to simultaneously quan-
tify hundreds of loci on selected chromosomes.52,79
After the fragments are counted, a ‘fetal-fraction optimised risk
for trisomy evaluation’ (FORTE) algorithm is used to calculate
the risk for aneuploidy.52,79 In addition to the actual fragment
counts, the FORTE algorithm includes the a priori maternal
age-related risks and the fetal fraction to provide an estimate of
patient-specific risk for trisomy. Using this approach, fewer than
400 loci per chromosome are sufficient to enable aneuploidy dis-
crimination.79 Using the DANSR assay and FORTE algorithm,
targeted MPS has a sensitivity approaching 100% for detection
of trisomy 21 and a sensitivity of 98% for detection of trisomy
18.80 The main advantage is a substantial reduction in the overall
sequence data and thus a decrease in costs.55 The increased depth
of sequencing of DANSR may also result in better discrimination
between euploid and aneuploid cases. 
Single Nucleotide Polymorphism
The third method for noninvasive prenatal testing (NIPT) for fetal
contributions of maternal and fetal DNA in the plasma (Fig. 21.4).
SNPs are highly polymorphic DNA sequence differences in which
only a single nucleotide varies among the population and can be
easily determined.81 The fetus will have SNPs from its father at
multiple alleles that differ from those transmitted from its mother.53
The SNP approach selectively amplifies and sequences nearly
20,000 polymorphic loci on chromosomes of interest (13, 18, 21,
X and Y), mathematically subtracts out the contribution of the
maternal SNPs and then uses a proprietary algorithm for analy-
sis, referred to as the next-generation aneuploidy test using SNPs
(NATUS) algorithm.53,82 This technology does not compare the
sequencing results from specific chromosomes with control chro-
mosomes but instead uses complex computer bioinformatics and
a Bayesian-based maximum likelihood statistical method to deter-
mine the chromosomal count of the fetal chromosomes interro-
gated in each sample in comparison with the maternal genotype.
The distribution of fetal SNPs, compared with maternal, will
determine the likelihood that the fetus is monosomic, disomic or
trisomic. The SNP-based method also requires a minimum fetal
fraction of 3% to 4% to avoid test failure, and its performance is
comparable with the NGS counting methods.
208 SE C T I O N 6     Prenatal Screening and Diagnosis

Data from Human

Genotypic data Multiple hypotheses for Genome Project
from mother each chromosome (HapMap)

x x x x x x x x

y y y y y y y y
z z z z z z z z

Subhypotheses with
different crossover points

Sequencing
measurements
from maternal plasma

Compare

B
• Fig. 21.4  Single nucleotide polymorphism (SNP)-based analysis for aneuploidy detection from cell-free
DNA. A, The maternal genotype is evaluated, and SNPs from the chromosomes of interest are deter-
mined. Using this data and data from the human genome project on the frequency and location of cross-
over events, a subhypothesis of the fetal genotype is determined for monosomy, disomy and trisomy.
B, Maximum likelihood estimation determines correct hypothesis. The fetal hypothesis is then compared
with the sequencing results from the maternal plasma cell-free DNA. Then using the maximum likelihood
estimation of each hypothesis, the most likely correct hypothesis is selected to determine the fetal geno-
type. The subpicture to the side is an illustration of the readout in which the red upper line is the frequency
of aa alleles (a being the more frequent allele) and the blue line being the bb allele (b being the less frequent
allele SNP). The green frequency is of the other allele (ab). Note that y-axis or the relative allele frequency
of genotype at each allele in the mixture of the determined of the green frequency. (Reprinted with permis-
sion from Natera, Inc. Copyright, 2017 Natera, Inc.)

The potential advantages of a SNP-based approach are that
additional information is provided concerning the parent of ori-
gin of aneuploidy, recombination and inheritance of mutations.
It also allows the detection of triploidy, which NGS cannot.83
SNP-based methods can also identify regions of homozygosity in
a fetus, indicating consanguinity or uniparental disomy. In certain
cases, a sample from the father can be helpful and improve test
performance. There is a no-call rate of 5.4%, comparable with
other approaches to NIPS.82 On the other hand, the disadvan-
tages include the inadvertent detection of nonpaternity as well
as the need for enrichment of fetal DNA, deeper sequencing or
higher levels of high-fidelity amplification given that SNPs only
account for 1.6% of the human genome.84  reduced cost because of individual hybridisation instead of sample
Microarray-Based Technology
More recently, an approach has been developed using a micro-
array-based technology to quantify the relative contributions
from specific chromosomes, hence not requiring NGS and thus
potentially reducing the cost and turnaround time.85 In this
approach, DNA fragments from the chromosomes of interest are
selected using DANSR and hybridised to and analysed using a
specially designed array allowing millions of genomic locations
to be studied simultaneously with each sample. Juneau and col-
leagues showed that microarray-based NIPS in combination with
DANSR assays and the FORTE algorithm for targeted analysis
resulted in high sensitivity (>99.9%, 97.5% and 93.8% for tri-
somy 21, 18 and 13, respectively) and high specificity (>99.9%
for each of the common trisomies) for fetal aneuploidies, screen-
ing comparable to NGS.85,86 The advantages of microarray-based
NIPS are: decreased variability that will allow testing samples with
lower fetal fraction to be analysed, improved turnaround time and
multiplexing.85 
Methylation-Based Technology
Another approach to cfDNA analysis focuses on epigenetic mark-
ers to differentiate fetal DNA from maternal DNA.87 In 2005,
Chim and colleagues showed that the maspin gene, among
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)
others, is hypomethylated in placental cells but hypermethylated
in maternal blood cells.88 A large number of fetomaternal meth-
ylation differences have subsequently been detected, primarily
on chromosome 21.89,90 This differential pattern of DNA meth-
ylation offers a basis for differential analysis of fetal and mater-
nal fragments for NIPS.91-93 However, this approach is limited
by the relatively small number of regions that are differentially
methylated and have a restriction site suitable for testing with a
methylation-sensitive restriction enzyme.
The modern use of immunoprecipitation with antibodies spe-
cific to 5-methylcytidine has allowed enrichment of hypermeth-
ylated DNA sequences specific to chromosome 21 in placental
cells.94 In 2009, Papageorgiou and colleagues identified 2000 dif-
ferentially methylated regions on chromosomes 21, 18, 13, X and
Y using this method, the majority in CG-poor nongenic regions.94
Immunoprecipitation allowed the selective enrichment of cffDNA
to undergo real-time PCR to measure differences in the amount of
chromosome 21-derived DNA from the fetus allowing detection
of trisomy 21.90,95
Recent studies continue to define new fetal specific epigenetic
markers. Hatt and colleagues presented a comprehensive microar-
ray-based analysis of the methylation status of more than 450,000
CpG sites (regions of DNA where a cytosine nucleotide is followed
by a guanine nucleotide in the linear sequence of bases) in mater-
nal blood and CVS samples.96 They defined a list of markers on
chromosomes 21, 18, 13 and other autosomes that contain restric-
tion sites for one of 16 different methylation-sensitive restriction
enzymes and could potentially be used for NIPS. However, this
can only be applied to CpG islands and promoter regions, which
cover only a small fraction of the genome. This approach is prom-
ising but still has to undergo further clinical trials. If successful,
such methodology could reduce the cost of screening dramatically
compared to sequencing-based methods.
Methods for Detection of Subchromosomal
Abnormalities (Microdeletions and
Microduplications)
Even before clinical validation, a number of companies offering
NIPS have launched and have been offering the option to include
a set of five to seven microdeletions: 22 q deletion syndrome
(DiGeorge), 5 p (cri-du-chat syndrome), 15 q (Prader–Willi/
Angelman syndromes), 1 p36 deletion syndrome, 4 p (Wolf-
Hirschhorn syndrome), 8 q (Langer–Giedion syndrome) and 11 q
(Jacobsen syndrome). The frequencies of these microdeletion syn-
dromes are not affected by maternal age and often have no asso-
ciated fetal anomalies on ultrasound. Although their individual
prevalence is overall low ranging from 1 in 4000 (22 q 11.2) to 1
in 50,000 (cri-du-chat), the combined prevalence of all microdele-
tion and microduplication syndromes is significant.
Microdeletions and microduplications are now identifi-
able by NIPT, but with present technologies, there are still
limitations.97,98 Companies quote detection rates ranging
from 60% to greater than 99%,98 but there is no doubt that
the specificity and positive predictive value (PVV) are signifi-
cantly lower than NIPS for common aneuploidies because of
a lower prevalence in addition to a higher FPR for genome-
wide deletion detection.99 Data are still lacking to determine
accurate sensitivity and specificity and more important, the
positive predictive value (PPV) and negative predictive value
(NPV). At present, because of the limited clinical testing to
209
evaluate noninvasive detection of deletions, no major national
obstetric or genetic organisation recommends screening for
microdeletions.100
Depth of sequencing to detect microdeletions and duplica-
tions. The statistical power of MPS largely depends on the read
depth and the size of the fetal copy-number variants (CNVs) anal-
ysed. To identify increasingly small alterations, deeper sequencing
is required to assure sufficient fragments are analysed from the areas
of interest. In one approach, sequencing is performed at the level
of 1 billion tags as opposed to 10 to 20 million tags with current
MPS for fetal whole-chromosome aneuploidy screening.101 Only
at this level can fragments from smaller regions of the genome be
differentiated. However, although theoretically possible, attaining
noninvasive detection of CNVs with the resolution of a chromo-
somal microarray on amniocytes or villi (CNVs <100 kb) remains
unproven. Srinivasan and colleagues showed that deep sequenc-
ing, using 1 billion reads, could potentially detect fetal CNVs as
small as 300 kb by partitioning the genome into 1-Mb bins, but
these studies have small numbers and are underpowered to pro-
vide any proof of clinical validation.101
Detection of subchromosomal abnormalities of 3 Mb or greater
is feasible with NGS.102,103 Zhao and colleagues have described
a novel approach using low-coverage whole-genome sequencing
to detect genome-wide CNVs followed by a statistical method
to search for consistently increased or decreased regions.104 Their
algorithm achieved a sensitivity of 94.4% and a specificity of
99.4% in 18 cases in which the CNVs ranged from 3 to 40 Mb.
The limiting factors in their experience were fetal fraction, CNV
size, coverage and biological and technical variability of the region
of interest. Lo and colleagues described a calling pipeline based
on a segmentation algorithm to detect selected subchromosomal
abnormalities.105 They sequenced 565 samples, 31 with CNV.
Using the same read depth as their standard pipeline for testing
aneuploidy, they detected 83% of samples with CNVs of 6 Mb
or greater, 20% of samples with CNV of less than 6 Mb and had
2 false-positives cases of 534 normal samples. The authors found
a sensitivity of 83% (95% confidence interval (CI), 61%–94%),
specificity of 99.6% (95% CI, 98.6%–99.9%) and a PPV of 55%
for a frequency of 0.6%. They concluded that the test sensitiv-
ity depends on fetal fraction, read depth and CNV size.105 Thus
NIPS for subchromosomal abnormalities is not yet ready for clini-
cal implementation. 
Single nucleotide polymorphism-based noninvasive prenatal
screening to detect microdeletions and duplications. The previ-
ously described SNP-based method is expandable to include addi-
tional imbalances, including microdeletions and duplications,
by identifying sufficient informative SNPs within the region of
interest. The theoretical advantage of this approach is again that
the maternal genotype is ‘subtracted out’, allowing smaller fetal
genomic abnormalities to be detected. Although encouraging,
the approach is limited by the need for SNP probes through-
out the genome because clinically relevant microdeletions can
occur on any chromosome. At present, applicability is restricted
to identification of a limited number of microdeletions and not
validated yet for smaller and less frequent microdeletions and
microduplications.98,106,107
A proof of principle study demonstrated the feasibility of this
approach.98 The samples underwent targeted multiplex PCR and
were then sequenced. The analysis was done with the NATUS
algorithm with a set of primers to amplify more than 4000 SNPs
in the regions of interest to target these microdeletion regions; one
example is a total of 672 SNPs targeting a 2.91 Mb in the 22q11.2
210 SE C T I O N 6     Prenatal Screening and Diagnosis
TABLE 109,110
21.3 Updated Meta-Analyses of cffDNA for Fetal Aneuploidies
Detection False-Positive
Rate (%) Rate (%) Sensitivity (%)
Trisomy 21 99.2 0.09 99.3
Trisomy 18 96.3 0.13 97.4
Trisomy 13 91.0 0.13 97.4
Monosomy X 90.3 0.23 — —
Other sex chro- 93.0 0.14 — —
mosomes
Trisomy 21 in 93.7 0.23 — —
TWINS
cffDNA, Cell-free fetal DNA.
region. The NATUS algorithm would then predict the copy num-
ber for the fetus for target regions based on the allele distribution
pattern. Wapner and colleagues demonstrated that the SNP-based
NIPS for screening a set of 5 microdeletions on 469 samples was
highly accurate with detection rates of 97.8% for a 22q11.2 dele-
tion and 100% for Prader-Willi, Angelman, 1p36 deletion and
cri-du-chat syndromes.98 FPRs were less than 1%, and no false
positives occurred.
More recently, Gross and colleagues published their experience
using SNP-based NIPS for 22q11.2 deletion syndrome.108 The
method is exactly as described earlier with 672 SNPs targeting
this specific region. Ninety-five of 21,948 samples were reported
as high risk for fetal 22q11.2 deletion; 61 cases had subsequent
diagnostic testing. The true positive rate was 18.0%, and the FPR
82.0% giving an overall PPV of 18.0% and a lower PPV of 4.9%
for cases with no ultrasound abnormalities. The sensitivity in this
study was not available since all negative cases were not followed.  because a reliable result cannot be assured.51-52,112 As explained
Clinical Performances of Noninvasive
Screening Using Cell-Free Fetal DNA
Initial studies of the performance of cfDNA screening were per-
formed on women undergoing diagnostic testing for either advanced
maternal age or a positive sequential or combined screening test thus
having an exceptionally high risk for the common fetal aneuploidies.
These studies demonstrated sensitivities for trisomy 21 of between
97% and 99% and for trisomies 18 and 13 of approximately 85%.
The FPR was approximately 1 to 3 per 1000. For sex chromosome
detection, the sensitivity was approximately 90%. Two recent meta-
analyses of up to 41 studies have recently evaluated this performance
and demonstrated even higher sensitivities and reduced FPRs in both
low- and high-risk populations109,110 (Table 21.3).
To evaluate the performance of NIPS in the general risk popu-
lation, the Noninvasive Examination of Trisomy (NEXT) study
completed in 2015 evaluated almost 16,000 sequential cases
regardless of maternal age or a priori risk.111 The mean maternal
age was 31 years, and the overall trisomy 21 risk was 1 in 417. All
cases of trisomy 21 were detected as were 90% (9 of 10 cases) of
trisomy 18 and 100% (2 2 cases) of trisomy 13. The screen-pos-
itive rate for each of the common aneuploidies was 3 in 10,000.
Positive Predictive Value (Low-/ False-Negative Rate
Specificity (%) High-Risk Population) (%) (Low/High Risk Pop)
99.9 82/91 1 in 5570 / 1 in 1054
99.9 37/ 84 1 in 7194 / 1 in 930
>99.9 49/87 1 in 8506 / 1 in 4265
— --
— --
— --
In a subgroup analysis of women younger than 35 years of age,
the detection rate for trisomy 21 was 100%, and for the lowest
risk group of woman having negative biochemical and NT screen-
ing and an a priori risk of less than 1 in 270, the performance
was equally good. All patients in this study also had conventional
first trimester screening performed. In the overall group and the
subgroups, cfDNA performed better than NT and biochemistry
having both a higher sensitivity and a significantly lower FPR.111
Importance of Fetal Fraction
The ability to accurately screen for cytogenetic abnormalities
depends on the relative proportion of fetal to maternal cfDNA
because lower fetal fractions require deeper sequencing to assure
adequate fetal fragment representation.112 If the fetal fraction
is below 3% to 4%, many laboratories will not perform NIPS
earlier, one of the more common causes of low fetal fraction is
maternal obesity, most likely caused by an excess of DNA frag-
ments from the mother, although this mechanism is still poorly
understood.59-60,112,113
Specific fetal aneuploidies are associated with smaller placen-
tas and hence lower levels of fetal cfDNA.65,67 One example is
trisomy 18, known to be associated with a smaller placental vol-
ume by three-dimensional ultrasound imaging114 and thus having
a lower fetal fraction of cfDNA. Rava and colleagues showed the
mean fetal fraction for trisomy 18 and trisomy 13 to be 29.7%
and 28.3% lower than in euploid pregnancies.67 Several series
have shown that failed cffDNA screens increase aneuploidy risk
with an odds ratio of 2.5 to 6.2.111,115 Because of this, when a low
fetal fraction (<4%) is identified, a repeat sample can be obtained,
and if the fetal fraction is sufficiently increased, the results of the
NIPT should be valid. If the fetal fraction remains low, diagnostic
testing by CVS or amniocentesis should be offered because the
risk for aneuploidy may be 2% or greater.116 
False-Negative Noninvasive Prenatal Testing
Results
As with any screening test, there will be false-negative and false-
positive results. False-negative results are inherent to techniques
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)
based on a statistical assessment of the number of cfDNA frag-
ments originating from euploid and aneuploid cells because to
maintain reasonable screen-positive rates, cutoff values are neces-
sary to discriminate between normal and abnormal results. As a
consequence, NIPS is considered a screening rather than a diag-
nostic test.117
False-negative results will be more frequent if the fetal frac-
tion is low, resulting in a smaller number of trisomic fetal frag-
ments, for example, when NIPT is done too early in gestation
(<10 weeks),112,118 in obese women59,61,118 and in cases of sub-
optimal and prolonged storage of blood samples before pro-
cessing.119,120 To address this, most laboratories now measure
the fetal fraction as part of their routine analysis, and when it
is less than 4%, a result will not be reported. Other laborato-
ries address this by reporting a lower reliability of a negative
result.
False-negative cases can also result from placental mosaicism
in which despite fetal aneuploidy, a proportion of the placental
cells are euploid. In approximately 0.8% to 1% of pregnancies
sampled by CVS, confined placental mosaicism (CPM) exists
in which the karyotype of the cytotrophoblast (the source of
cfDNA) differs from that of the fetus.121,122 In a retrospective
study based on 52,673 pregnancies, placental mosaicism was pre-
dicted to be the likely cause of a false-negative NIPS result in 1 of
136 of trisomy 13, 1 of 64 of trisomy 18 and 1 of 135 of trisomy
21 cases.123
In some false-negative cases, a cause of the discrepant NIPS
results cannot be identified. Hochstenbach and colleagues
described two such cases involving trisomies 13 and 18.124 The
authors also reviewed previously published cases of false-negative
NIPS with molecular/cytogenetic follow up showing that CPM
may be the most frequent explanation (Table 21.4).125-131  Choice of the Confirmatory Diagnostic
False-Positive Results
In a cohort of 52,673 patients, Grati and colleagues showed
using mosaic results found on CVS that CPM could results in
a false-positive NIPS rate for the common trisomies of 0.033%
(1 in 3006) and a FP rate of 0.08% (1 in 1243) if monosomy
X is included.123 In NIPS series, the pooled FP rates among all
positives were calculated to be 5.6% for trisomy 21, 40.5% for
trisomy 18, 55.6% for trisomy 13 and 62.1% for monosomy X
(Table 21.5).132-134
Many biologic conditions can result in false-positive NIPS
results. As with false-negative results, CPM may be causative;
however, in these situations, the fetal karyotype will be normal,
but there will be aneuploid cells isolated to the placenta. CPM
has been calculated to result in an FPR in 5.0% of trisomy 21
cases. Trisomy 18 appears to have a higher FPR of 12.6%.123 An
aneuploid vanishing twin continues to undergo apoptosis and is
a common cause of a false-positive NIPS because approximately
5% to 36% of initially twin gestations will have a vanishing
twin. Futch and colleagues have recently shown that in clinical
use, approximately 15 % of discordant results had a vanishing
twin.135
Maternal mosaicism will also result in FPRs and appears to
occur most commonly when a sex chromosome abnormality
is suggested by NIPS. Wang and colleagues have reported that
8.6% of suspected sex chromosome abnormalities were second-
ary to maternal mosaicism.136 This was almost equally distributed
between X chromosome gains (9.5%) and losses (8.1%). Because
211
of this possibility, practitioners may consider a maternal karyotype
when a sex chromosome aneuploidy is suggested by NIPS and the
fetus in euploid.
A rare cause of false-positive NIPT results is maternal malig-
nancy. Bianchi and colleagues demonstrated that of 3757 positive
NIPT cases, 10 were associated with maternal cancer in which
the abnormal cfDNA came from an aneuploid tumour line.137 In
most of these cases, aneuploidy was suspected for multiple chro-
mosomes, including double trisomies reported by NIPS. Until
recently, laboratories were reticent to report aneuploidy results for
chromosomes other than 21, 18 and 13, but because of the asso-
ciation with malignancy, this has now become common practice.
The management of such cases and the need for maternal imaging
remain uncertain. 
Importance of Positive Predictive Value
As with any screening test, the significance of a positive result
depends on the disease frequency. Even with superior sensitivity
and specificity, when screening for rare disorders, the likelihood
of an affected pregnancy may be low. For example, in a popula-
tion of women older than 35 years old in whom the frequency
of trisomy 21 is approximately 1 in 100, even with a 99.3%
sensitivity and a 1 per 1000 FPR, about 9 of every 10 positive
test results will have Down syndrome. Alternatively, in a popu-
lation in which the disease frequency is 500, the PPV is 67%.
In a low-risk group with negative conventional screening, only
half of the positive NIPS results will be affected. For this rea-
son, all positive NIPS results must be confirmed by a diagnostic
procedure. 
Procedure
As addressed earlier, CPM can result in false-positive NIPS
results when the fetus is euploid.138,139 This raises the issue of
whether CVS sampling of the placenta or amniocentesis should
be used to confirm the result. Amniocentesis has the benefit of
providing fetal rather than placental tissue and should not be
altered by placental mosaicism. In a large series, the likelihood
of finding mosaicism with CVS and needing a follow-up amnio-
centesis after a high-risk NIPS is 2%, 4%, 22% and 59% for
trisomies, 21, 18, 13 and monosomy X, respectively.140 In these
cases of mosaicism, 44%, 14%, 4% and 26% of the abnormal
results were confirmed on amniocentesis to be of fetal origin.140
Therefore in certain fetal aneuploidies such as trisomy 13 and
monosomy X, amniocentesis may be a better choice given the
higher likelihood of CPM.
However, many woman having NIPS at 9 to 10 weeks pre-
fer not to wait more than 1 month until amniocentesis can be
safely performed. In these cases, CVS can be performed, but there
are caveats. Firstly, if the CVS is mosaic, performing a follow-up
amniocentesis is absolutely necessary to confirm fetal aneuploidy.
Second, because the majority of confined placental mosaic results
come from aneuploid cytotrophoblast cells, performing analysis
of all villus tissue sources (cytotrophoblast and mesenchymal core)
by both FISH or direct preparation and culture is recommended.
If all sources confirm nonmosaic aneuploidy, there is minimal risk
for a false-positive result. Ultrasound scanning after a positive
NIPS result can also be beneficial in deciding on the preferred
diagnostic test. An abnormal scan result, including an elevated
212 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE 124-131
21.4 Summary of Published False-Negative Cases with Cytogenetic Follow-Up

Trisomy Indication for ff Result of

[Reference] NIPT MA (yr) GA (wk) (%) NIPT Karyotype Explanation for FN NIPT
13 [124] 1/190 risk for 35 13+5 8.8 <1/10,000 47,XY,+13 in Nonmosaic 47,XY, +13 karyotype;
T18 for T13, amniocytes sampling of placenta
T18, T21 (9 biopsies) gave no evidence
for the presence of euploid cells
13 [125] Ultrasound 34 14 6 Z 0.08 for 46,XX/47,XX,+13 in Amniocentesis showed10% mosa-
abnormalities chr 13 amniocytes icism for a cell line with +13
18 [124] Maternal age 40 11 10.7 <1/10,000 47,XX,+18 in Nonmosaic 47,XX, +18 karyotype;
for T13, amniocytes sampling of placenta (10 biop-
T18, T21 sies) showed that a maximum of
20%–30% euploid cells may have
been present in the cytotrophoblast
18 [125] Maternal age 39 12 23 Z 0.22 for 47,XY,+21/48,XY,+18,+21 CVS showed 45% mosaicism for a
chr 18 in CVS cell line with both +18 and +21
18 [126] 1/70 risk for T21 43 13 7.4 High risk for 48,XXX,+18 in Estimated 20%–30% of placental
by combined XXY amniocytes cells studied by FISH were +18,
FTS with QF-PCR showing variable
levels of +18 cells across the
placenta
18 [127] 1/313 risk for 22 17 11.6 Z 0.035 for 47,XX,+18 in Placental biopsies showed on average
T18 by serum T18 amniocytes 50% +21, 35% +18, and15%
screen Z 4.4 for T21 normal cells but at least one
region had 61% +21 but only 22%
+18
18 [128] 1/45 risk for T21 24 18 N/A T -0.52 for 47,XX,+18 in ∼30% of placental cells studied by
by combined T18 amniocytes FISH were +18, and ∼67% were
test T -4.05 for 45,X; SNP-array was indicative of
X chr 50% cells with +18
18 [129] 1/360 risk for 29 19 5.3 Aneuploidy 47,Xy,+18 in POC Six placental biopsies showed
T21 by com- not 20%–40% cells with +18
bined test detected
18 [129] 1/45 risk for T21 24 20 9.5 Indicative of 47,XX,+18 in In placental tissue, 30% of the cells
by combined 45,X amniocytes showed +18, and 60% showed
test 45,X
21 [125] Maternal age 44 11 17 Z 2.03 for 46,XX[20]/47,XY,+21[2] CVS showed 9% mosaicism for a cell
chr 21 in CVS line with +21
21 [125] Maternal age 41 12 9 Z -1.25 for 46,XY/47/XY,+21 50% mosaicism by CVS; possibly
chr 21 in CVS this was lower in the placenta as
a whole
21 [130] 1/370 risk for 32 18 15.6 Z 2.043 for 46,XX,der(21;21) Placental biopsies had17%, 21%,
T21 by serum chr 21 (q10;q10),+21 in 23% and 53% cells
screen fetal blood with +21
21 [130] Two spontane- 35 18 19.7 Z 1.33 for 47,XY,+21 in Placental biopsies had 2%, 51% and
ous abortions chr 21 amniocytes 76% cells with +21
21 [131] CAVCD 32 20 N/A Negative for 47,XX,+21 in postnatal No study of placenta or umbilical
T21 blood cord; no explanation provided

CAVCD, Complete atrioventricular canal defect; chr, chromosome; CVS, chorionic villus sampling; ff, fetal DNA fraction; FISH, fluorescence in situ hybridisation; FN, false negative; FTS, first trimester
screen; GA, gestational age; MA, maternal age; N/A: not available; NIPT, noninvasive prenatal testing; POC, products of conception; QF-PCR, quantitative fluorescence polymerase chain reaction; T, t score;
T13, trisomy 13; T18, trisomy 18; T21, trisomy 21; Z, z score.
  
 CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions) 213

TABLE Reported False-Positive Rates Among All Conclusion

21.5 Positive Results124-126
Prenatal screening through noninvasive methods is not a new con-
Choy Meck Wang cept but one that has evolved tremendously since its introduction
et al132 et al133 et al134 Total (%) half a century ago. The ultimate goal is to develop a noninvasive test
that allows the complete genetic analysis of the fetus comparable to
Trisomy 21 3/55 1/30 3/41 5.6
that offered with current invasive diagnostic procedures such as CVS
Trisomy 28 6/12 2/5 9/25 40.5 or amniocentesis. Initial research focused on intact fetal cells circu-
Trisomy 13 3/7 3/4 9/16 55.6
lating in the maternal plasma, but this has presented many techni-
cal challenges in terms of retrieval and isolation of these rare cells.
Sex chromosome 2/6 6/7 10/16 62.1 On the other hand, cffDNA has quickly become the cell of choice
abnormality for noninvasive testing for common aneuploidies, sex chromosomal
abnormalities and a small number of microdeletion and microdu-
   plication syndromes. At this stage, cffDNA remains a screening test
given the clinically significant false-positive and false-negative rates.
However, with rapid advances in laboratory methods, a noninvasive
NT, would suggest that CVS is the preferred approach. Patients diagnostic test will eventually be available for clinical use.
with normal scans who defer testing until the second trimes-
ter should be made aware that a normal scan does not preclude Access the complete reference list online at ExpertConsult.com.
an aneuploid fetus and that delaying testing may lead to a later Self-assessment questions available at ExpertConsult.com.
and more difficult pregnancy termination if an affected fetus is
confirmed. 
References application for detection of fetal cells in mater-
nal blood. Prenat Diagn. 2014;34(6):538–546.
38. Bianchi DW, Mahr A, Zickwolf GK, et  al.
Detection of fetal cells with 47,XY,+21 karyo-
20. Ganshirt-Ahlert D, Borjesson-Stoll R, Bur- type in maternal peripheral blood. Hum
1. Cuckle H, Maymon R. Development of pre-
schyk M, et al. Detection of fetal trisomies 21 Genet. 1992;90:368–370.
natal screening—a historical overview. Semin
and 18 from maternal blood using triple gra- 39. Parano E, Falcidia E, Grillo A, et  al. Fetal
Perinatol. 2016;40:12–22.
dient and magnetic cell sorting. Am J Reprod nucleated red blood cell counts in peripheral
2. Malone FD, Canick JA, Ball RH, et al. First-
Immunol. 1993;30:194–201. blood of mothers bearing Down syndrome
trimester or second-trimester screening, or
21. Troeger C, Holzgreve W, Hahn S. A compari- fetus. Neuropediatrics. 2001;32:147–149.
both, for Down’s syndrome. N Engl J Med.
son of different density gradients and antibod- 40. Choolani M, Mahyuddin AP, Hahn S. The
2005;353:2001–2011.
ies for enrichment of fetal erythroblasts by promise of fetal cells in maternal blood. Best
3. Holzgreve W1, Hahn S. Fetal cells in cervical
MACS. Prenat Diagn. 1999;19:521–526. Pract Res Clin Obstet Gynaecol. 2012;26:655–
mucus and maternal blood. Baillieres Best Pract
22. Choolani M, Mahyuddin AP, Hahn S. The 667.
Res Clin Obstet Gynaecol. 2000;14:709–722.
promise of fetal cells in maternal blood. Best Pract 41. Bianchi DW, Simpson JL, Jackson LG,
4. Committee Opinion No. 640. cell-free DNA
Res Clin Obstet Gynaecol. 2012;26:655–667. et  al. Fetal gender and aneuploidy detection
screening for fetal aneuploidy. Obstet Gynecol.
23. Bianchi DW, Zickwolf GK, Weil GJ, et  al. using fetal cells in maternal blood: analysis
2015;126:31–37.
Male fetal progenitor cells persist in maternal of NIFTY I data. National institute of child
5. Gregg AR, Gross SJ, Best RG, et  al. ACMG
blood for as long as 27 years postpartum. Proc health and development fetal cell isolation
statement on noninvasive prenatal screening for
Natl Acad Sci U S A. 1996;93:705–708. study. Prenat Diagn. 2002;22:609–615.
fetal aneuploidy. Genet Med. 2013;15:395–398.
24. Snyder MW, Simmons LE, Kitzman JO, 42. Wang JY, Zhen DK, Falco VM, et  al. 2000.
6. Soothill PW, Lo YMD. Non-invasive prenatal
et al. Noninvasive fetal genome sequencing: a Fetal nucleated erythrocyte recovery: fluo-
testing for chromosomal abnormality using
primer. Prenat Diagn. 2013;33:547–554. rescence activated cell sorting–based positive
maternal plasma DNA (Scientific Impact Paper
25. Mouawia H, Saker A, Jais JP, et al. Circulating selection using anti-gamma globin versus
No. 15). Guidelines from the Royal College of Obste-
trophoblastic cells provide genetic diagnosis in magnetic activated cell sorting using anti-
tricians & Gynaecologists. 2014. https://www.
63 fetuses at risk for cystic fibrosis or spinal CD45 depletion and anti-gamma globin posi-
rcog.org.uk/en/guidelines-research-services/
muscular atrophy. Reprod Biomed Online. tive selection. Cytometry. 2000;39:224–230.
guidelines/sip15/.
2012;25:508–520. 43. RareCyte. http://www.rarecyte.com.
7. Langlois S, Brock JA, Genetics Committee, et al.
26. Hatt L, Brinch M, Singh R, et  al. A new 44. KellBenX. http://www.kellbenx.com.
Current status in non-invasive prenatal detection
marker set that identifies fetal cells in maternal 45. Mandel P, Metais P. Les acides nucleiques du
of Down syndrome, trisomy 18, and trisomy 13
circulation with high specificity. Prenat Diagn. plasma sanguin chez l’homme. C R Acad Sci
using cell-free DNA in maternal plasma. J Obstet
2014;34:1066–1072. Paris. 1948;142:241–243.
Gynaecol Can. 2013;35:177–183.
27. Bi W, Breman A, Shaw CA, et al. Detection of 46. Lo YM, Corbetta N, Chamberlain PF, et  al.
8. Holzgreve W, Garritsen HS, Ganshirt-Ahlert
≥1 Mb microdeletions and microduplications Presence of fetal DNA in maternal plasma and
D. Fetal cells in the maternal circulation. J
in a single cell using custom oligonucleotide serum. Lancet. 1997;350:485–487.
Reprod Med. 1992;37:410–418.
arrays. Prenat Diagn. 2012;32:10–20. 47. Lo YM, Zhang J, Leung TN, et  al. Rapid
9. Ganshirt-Ahlert D, Burschyk M, Garritsen
28. Breman AM, Chow JC, U’Ren L, et al. Evi- clearance of fetal DNA from maternal plasma.
HS, et al. Magnetic cell sorting and the trans-
dence for feasibility of fetal trophoblastic Am J Hum Genet. 1999;64:218–224.
ferrin receptor as potential means of prenatal
cell-based noninvasive prenatal testing. Prenat 48. Zhu YJ, Zheng YR, Li L, et  al. Diagnostic
diagnosis from maternal blood. Am J Obstet
Diagn. 2016;36:1009–1019. accuracy of non-invasive fetal RhD genotyp-
Gynecol. 1992;166:1350–1355.
29. Shettles LB. Use of the Y chromosome ing using cell-free fetal DNA: a meta analy-
10. Simpson JL, Elias S. Isolating fetal cells from
in prenatal sex determination. Nature. sis. J Matern Fetal Neonatal Med. 2014;27:
maternal blood. Advances in prenatal diag-
1971;230:52–53. 1839–1844.
nosis through molecular technology. JAMA.
30. Shettles LB. Cells in cervical mucus reveal 49. Bianchi DW, Parsa S, Bhatt S, et al. Fetal sex
1993;270:2357–2361.
sex of infant. Am J Obstet Gynecol. 1978;130: chromosome testing by maternal plasma DNA
11. Lo YM, Lo ES, Watson N, et  al. Two-way cell
248–249. sequencing: clinical laboratory experience and
traffic between mother and fetus: biologic and
31. Rodeck C, Tutschek B, Sherlock J, Kingdom biology. Obstet Gynecol. 2015;125:375–382.
clinical implications. Blood. 1996;88:4390–4395.
J. Methods for the trans cervical collection of 50. Lo YM, Tein MS, Lau TK, et al. Quantitative
12. Schmorl G. Pathologisch-Anatomische Unter-
fetal cells during the first trimester of preg- analysis of fetal DNA in maternal plasma and
suchungen über Puerperal-Eklampsie. Leipzig:
nancy. Prenat Diagn. 1995;15:933–942. serum: implications for noninvasive prenatal
FCW Vogel; 1893.
32. Sargent IL, Johansen M, Chau S, Redman diagnosis. Am J Hum Genet. 1998;62:768–775.
13. Walknowska J, Conte FA, Grumbach MM.
CWG. Clinical experience: isolating tropho- 51. Palomaki GE, Kloza EM, Lambert-Messerlian
Practical and theoretical implications of
blasts from maternal blood. Ann N Y Acad Sci. GM, et  al. DNA sequencing of maternal
fetal-maternal lymphocyte transfer. Lancet.
1994;731:154–161. plasma to detect Down syndrome: an inter-
1969;1:1119–1122.
33. van Wijk IJ, van Vugt JM, Mulders MA, et al. national clinical validation study. Genet Med.
14. Bianchi DW, Flint AF, Pizzimenti MF, et al.
Enrichment of fetal trophoblast cells from the 2011;13:913–920.
Isolation of fetal DNA from nucleated eryth-
maternal peripheral blood followed by detec- 52. Sparks AB, Struble CA, Wang ET, et al. Non-
rocytes in maternal blood. Proc Natl Acad Sci
tion of fetal deoxyribonucleic acid with a invasive prenatal detection and selective analy-
U S A. 1990;87:3279–3283.
nested X/Y polymerase chain reaction. Am J sis of cell-free DNA obtained from maternal
15. Bianchi DW, Mahr A, Zickwolf GK, et  al.
Obstet Gynecol. 1996;174:871–878. blood: evaluation for trisomy 21 and trisomy
Detection of fetal cells with 47,XY,+21 karyo-
34. Fang CN, Kan YY, Hsiao CC. Detection of 18. Am J Obstet Gynecol. 2012;206:311–319.
type in maternal peripheral blood. Hum
fetal cells from transcervical mucus plug before 53. Zimmermann B, Hill M, Gemelos G, et  al.
Genet. 1992;90:368–370.
first-trimester termination of pregnancy by Noninvasive prenatal aneuploidy testing of
16. Ganshirt-Ahlert D, Borjesson-Stoll R, Bur-
cytokeratin-7 immunohistochemistry. J Obstet chromosomes 13, 18, 21, X, and Y, using tar-
schyk M, et al. Detection of fetal trisomies 21
Gynaecol Res. 2005;31:500–507. geted sequencing of polymorphic loci. Prenat
and 18 from maternal blood using triple gra-
35. Katz-Jaffe MG, Mantzaris D, Cram DS. DNA Diagn. 2012;32:1233–1241.
dient and magnetic cell sorting. Am J Reprod
identification of fetal cells isolated from cervi- 54. Wright CF, Burton H. The use of cell-free
Immunol. 1993;30:194–201.
cal mucus: potential for early non-invasive pre- fetal nucleic acids in maternal blood for
17. Price JO, Elias S, Wachtel SS, et  al. Prenatal
natal diagnosis. BJOG. 2005;112:595–600. non-invasive prenatal diagnosis. Hum Reprod
diagnosis with fetal cells isolated from mater-
36. Bolnick JM, Kilburn BA, Bajpayee S, et  al. Update. 2009;15:139–151.
nal blood by multi parameter flow cytometry.
Trophoblast retrieval and isolation from the 55. Norton ME, Brar H, Weiss J, et  al. Non-
Am J Obstet Gynecol. 1991;165:1731–1737.
cervix (TRIC) for noninvasive prenatal screen- Invasive Chromosomal Evaluation (NICE)
18. Hamada H, Arinami T, Kubo T, et  al. Fetal
ing at 5 to 20 weeks of gestation. Fertil Steril. Study: results of a multicenter prospective
nucleated cells in maternal peripheral blood:
2014;102:135–142.e6. cohort study for detection of fetal trisomy 21
frequency and relationship to gestational age.
37. Bianchi DW, Flint AF, Pizzimenti MF, et al. and trisomy 18. Am J Obstet Gynecol. 2012;
Hum Genet. 1993;91:427–432.
Isolation of fetal DNA from nucleated eryth- 207:137.e1-8.
19. Emad A, Bouchard EF, Lamoureux J, et  al.
rocytes in maternal blood. Proc Natl Acad Sci 56. Nygren AO, Dean J, Jensen TJ, et  al.
Validation of automatic scanning of micro-
U S A. 1990;87:3279–3283, 1990. Quantification of fetal DNA by use of
scope slides in recovering rare cellular events:

213.e1
213.e2 References
methylation-based DNA discrimination. Clin
Chem. 2010;56:1627–1635.
57. Lun FM, Chiu RW, Allen Chan KC, et  al.
Microfluidics digital PCR reveals a higher
than expected fraction of fetal DNA in mater-
nal plasma. Clin Chem. 2008;54:1664–1672.
58. Kolialexi A, Tsangaris GT, Antsaklis A, et al.
Apoptosis in maternal peripheral blood during
pregnancy. Fetal Diagn Ther. 2001;16:32–37.
59. Wang E, Batey A, Struble C, et al. Gestational
age and maternal weight effects on fetal cell-
free DNA in maternal plasma. Prenat Diagn.
2013;33:662–666.
60. Ashoor G, Poon L, Syngelaki A, et al. Fetal frac-
tion in maternal plasma cell-free DNA at 11–13
weeks gestation: effect of maternal and fetal fac-
tors. Fetal Diagn Ther. 2012;31:237–243.
61. Ashoor G, Syngelaki A, Poon LC, et al. Fetal
fraction in maternal plasma cell-free DNA at
11-13 weeks’ gestation: relation to maternal
and fetal characteristics. Ultrasound Obstet
Gynecol. 2013;41:26–32.
62. Bevilacqua E, Gil MM, Nicolaides KH, et al.
Performance of screening for aneuploidies by
cell-free DNA analysis of maternal blood in
twin pregnancies. Ultrasound Obstet Gynecol.
2015;45:61–66.
63. Lo YM, Lau TK, Zhang J, et al. Increased fetal
DNA concentrations in the plasma of preg-
nant women carrying fetuses with trisomy 21.
Clin Chem. 1999;45:1747–1751.
64. Zhong XY, Bürk MR, Troeger C, et al. Fetal
DNA in maternal plasma is elevated in preg-
nancies with aneuploid fetuses. Prenat Diagn.
2000;20:795–798.
65. Wataganara T, LeShane ES, Farina A, et  al.
Maternal serum cell-free fetal DNA levels are
increased in cases of trisomy 13 but not tri-
somy 18. Hum Genet. 2003;112:204–208.
66. Wright A, Zhou Y, Weier JF, et al. Trisomy 21
is associated with variable defects in cytotro-
phoblast differentiation along the invasive path-
way. Am J Med Genet. 2004;130A:354–364.
67. Rava RP, Srinivasan A, Sehnert AJ, Bianchi
DW. Circulating fetal cell-free DNA fractions
differ in autosomal aneuploidies and mono-
somy X. Clin Chem. 2014;60:243–250.
68. Srinivasan A, Bianchi DW, Liao W, et  al.
Maternal plasma DNA sequencing: effects of
multiple gestation on aneuploidy detection
and the relative cell-free fetal DNA (cff DNA)
per fetus. Am J Obstet Gynecol. 2013;208:S31.
69. Metzenbauer M, Hafner E, Hoefinger D, et al.
Three-dimensional ultrasound measurement
of the placental volume in early pregnancy:
method and correlation with biochemical pla-
centa parameters. Placenta. 2001;22:602–605.
70. Zhong XY, Hahn S, Kiefer V, Holzgreve W.
Is the quantity of circulatory cell-free DNA in
human plasma and serum samples associated
with gender, age and frequency of blood dona-
tions? Ann Hematol. 2007;86:139–143.
71. Brar H, Wang E, Struble C, et  al. The fetal
fraction of cell-free DNA in maternal plasma
is not affected by a priori risk of fetal tri-
somy. J Matern Fetal Neonatal Med. 2013;26:
143–145.
72. Allyse M, Minear MA, Berson E, et al. Non-
invasive prenatal testing: a review of interna-
tional implementation and challenges. Int J
Womens Health. 2015;7:113–126.
73. Staden R. A strategy of DNA sequencing
employing computer programs. Nucleic Acids
Res. 1979;6:2601–2610.
74. Anderson S. Shotgun DNA sequencing using
cloned DNase I-generated fragments. Nucleic
Acids Res. 1981;9:3015–3327.
75. Fan HC, Blumenfeld YJ, Chitkara U,
et  al. Noninvasive diagnosis of fetal aneu-
ploidy by shotgun sequencing DNA from
maternal blood. Proc Natl Acad Sci U S A.
2008;105:16266–16271.
76. Chiu RW, Chan KC, Gao Y, et  al. Non-
invasive prenatal diagnosis of fetal chro-
mosomal aneuploidy by massively parallel
genomic sequencing of DNA in maternal
plasma. Proc Natl Acad Sci U S A. 2008;105:
20458–20463.
77. Sehnert AJ, Rhees B, Comstock D, et al. Opti-
mal detection of fetal chromosomal abnormal-
ities by massively parallel DNA sequencing: of
cell free fetal DNA from maternal blood. Clin
Chem. 2011;57:1042–1049.
78. Dan S, Wang W, Ren J, et al. Clinical appli-
cation of massively parallel sequencing based
prenatal noninvasive fetal trisomy test for
trisomies 21 and 18 in 11,105 pregnan-
cies with mixed risk factors. Prenat Diagn.
2012;32:1225–1232.
79. Sparks AB, Wang ET, Struble CA, et al. Selec-
tive analysis of cell free DNA in maternal
blood for evaluation of fetal trisomy. Prenat
Diagn. 2012;32:3–9.
80. Ashoor G, Syngelaki A, Wagner M, et  al.
Chromosome-selective sequencing of mater-
nal plasma cell-free DNA for first-trimester
detection of trisomy 21 and trisomy 18. Am J
Obstet Gynecol. 2012;206:322.e1–5.
81. Ding C, Chiu RW, Lau TK, et al. MS analysis
of single-nucleotide differences in circulat-
ing nucleic acids: application to noninvasive
prenatal diagnosis. Proc Natl Acad Sci U S A.
2004;101(29):10762–10767.
82. Nicolaides KH, Syngelaki A, Gil M, et  al.
Validation of targeted sequencing of single-
nucleotide polymorphisms for non-invasive
prenatal detection of aneuploidy of chromo-
somes 13, 18, 21, X and Y. Prenat Diagn.
2013;33:575–579.
83. Nicolaides KH, Syngelaki A, Del Mar Gil M,
et al. Prenatal detection of fetal triploidy from
cell-free DNA testing in maternal blood. Fetal
Diagn Ther. 2014;35(3):212–217.
84. Liao GJ, Chan KC, Jiang P, et al. Noninvasive
prenatal diagnosis of fetal trisomy 21 by allelic
ratio analysis using targeted massively parallel
sequencing of maternal plasma DNA. PLoS
One. 2012;7:e38154.
85. Juneau K, Bogard PE, Huang S, et al. Micro-
array-based cell-free DNA analysis improves
noninvasive prenatal testing. Fetal Diagn Ther.
2014;36:282–286.
86. Stokowski R, Wang E, White K, et al. Clinical
performance of non-invasive prenatal testing
(NIPT) using targeted cell-free DNA analysis
in maternal plasma with microarrays or next
generation sequencing (NGS) is consistent
across multiple controlled clinical studies. Pre-
nat Diagn. 2015;35:1243–1246.
87. Poon LLM, Leung TN, Lau TK, et  al. Dif-
ferential DNA methylation between fetus and
mother as a strategy for detecting fetal DNA in
maternal plasma. Clin Chem. 2002;48:35–41.
88. Chim SSC, Tong YK, Chiu RWK, et  al.
Detection of the placental epigenetic signature
of the maspin gene in maternal plasma. Proc
Natl Acad Sci U S A. 2005;102:14753–14758.
89. Chim SS, Jin S, Lee TY, et al. Systematic search
for placental DNA-methylation markers on
chromosome 21: toward a maternal plasma-
based epigenetic test for fetal trisomy 21. Clin
Chem. 2008;54:500–511.
90. Chiu RW, Chim SS, Wong IH, et al. Hyper-
methylation of RASSF1A in human and
rhesus placentas. Am J Pathol. 2007;170:
941–950.
91. Tong YK, Ding C, Chiu RW, et  al. Nonin-
vasive prenatal detection of fetal trisomy 18
by epigenetic allelic ratio analysis in maternal
plasma: theoretical and empirical consider-
ations. Clin Chem. 2006;52:2194–2202.
92. Tong YK, Jin S, Chiu RW, et al. Noninvasive
prenatal detection of trisomy 21 by an epigen-
etic-genetic chromosome-dosage approach.
Clin Chem. 2010;56:90–98.
93. Old RW, Crea F, Puszyk W, Hulten MA. Can-
didate epigenetic biomarkers for non-invasive
prenatal diagnosis of Down syndrome. Reprod
Biomed Online. 2007;15:227–235.
94.  Papageorgiou EA, Fiegler H, Rakyan V, et al.
Sites of differential DNA methylation between
placenta and peripheral blood: molecular mark-
ers for noninvasive prenatal diagnosis of aneu-
ploidies. Am J Pathol. 2009;174:1609–1618.
95. Tsaliki E, Papageorgiou EA, Spyrou C, et  al.
MeDIP real-time qPCR of maternal periph-
eral blood reliably identifies trisomy 21. Prenat
Diagn. 2012;32:996–1001.
96. Hatt L, Aagaard MM, Graakjaer J, et  al.
Microarray-based analysis of methylation sta-
tus of CpGs in placental DNA and maternal
blood DNA—potential new epigenetic bio-
markers for cell free fetal DNA-based diagno-
sis. PLoS One. 2015;10:e0128918.
97. Benn P, Cuckle H. Theoretical performance of
non-invasive prenatal testing for chromosome
imbalances using counting of cell-free DNA
fragments in maternal plasma. Prenat Diagn.
2014;34:778–783.
98. Wapner RJ, Babiarz JE, Levy B, et al. Expand-
ing the scope of non–invasive prenatal testing:
detection of fetal micro deletion syndromes.
Am J Obstet Gynecol. 2015; 212:332.e1–e9.
99. Helgeson J, Wardrop J, Boomer T, et  al.
Clinical outcome of subchromosomal events
detected by whole-genome noninvasive prena-
tal testing. Prenat Diagn. 2015;35:999–1004.
100. Dondorp W, de Wert G, Bombard Y, et  al.
Non-invasive prenatal testing for aneuploidy
and beyond: challenges of responsible inno-
vation in prenatal screening. Summary and
recommendations. Eur J Hum Genet. 2015.
https://doi.org/10.1038/ejhg.2015.56.
101. Srinivasan A, Bianchi DW, Huang H, et  al.
Noninvasive detection of fetal subchro-
mosome abnormalities via deep sequenc-
ing of maternal plasma. Am J Hum Genet.
2013;92:167–176.
102. Peters D, Chu T, Yatsenko SA, et al. Nonin-
vasive prenatal diagnosis of a fetal microde-
letion syndrome. N Engl J Med. 2011;365:
1847–1848.
103. Jensen TJ, Dzakula Z, Deciu C, et al. Detec-
tion of micro deletion 22 q11.2 in a fetus
by next-generation sequencing of maternal
plasma. Clin Chem. 2012;58:1148–1151.
104. Zhao C, Tynan J, Ehrich M, et al. Detection of
fetal subchromosomal abnormalities by sequenc-
ing circulating cell-free DNA from maternal
plasma. Clin Chem. 2015;61:608–616.
105. Lo KK, Karampetsou E, Boustred C, et  al.
Limited clinical utility of non-invasive prena-
tal testing for subchromosomal abnormalities.
Am J Hum Genet. 2016;98:34–44.

106. Yatsenko SA, Peters DG, Saller DN, et  al.
Maternal cell-free DNA-based screening
for fetal microdeletion and the importance
of careful diagnostic follow-up. Genet Med.
2015;17:836–838.
107. Rabinowitz M, Savage M, Pettersen B, et  al.
Noninvasive cell-free DNA-based prena-
tal detection of micro deletions using single
nucleotide polymorphism-targeted sequenc-
ing. Obstet Gynecol. 2014;123(suppl 1):167S.
108. Gross SJ, Stosic M, McDonald-McGinn DM,
et al. Clinical experience with single-nucleotide
polymorphism-based non-invasive prenatal
screening for 22q11.2 deletion syndrome. Ultra-
sound Obstet Gynecol. 2016;47(2):177–183.
109. 
MM1 Gil, Quezada MS, Revello R, et  al.
Analysis of cell-free DNA in maternal blood
in screening for fetal aneuploidies: updated
meta-analysis. Ultrasound Obstet Gynecol.
2015;45:249–266.
110. Taylor-Phillips S, Freeman K, Geppert J,
et  al. Accuracy of non-invasive prenatal
testing using cell-free DNA for detection
of Down, Edwards and Patau syndromes: a
systematic review and meta-analysis. BMJ
­ of CVS mosaicism and non-mosaic discrep-
Open. 2016;6:e010002.
111. Norton ME, Jacobsson B, Swamy GK,
et  al. Cell-free DNA analysis for noninva-
sive examination of trisomy. N Engl J Med.
2015;372:1589–1597.
112. Canick JA, Palomaki GE, Kloza EM, et  al.
The impact of maternal plasma DNA fetal
fraction on next generation sequencing tests
for common fetal aneuploidies. Prenat Diagn.
2013;33:667–674.
113. Haghiac M, Vora NL, Basu S, et al. Increased
death of adipose cells, a path to release
cell-free DNA into systemic circulation
of obese women. Obesity (Silver Spring).
2012;20:2213–2219.
114. Wegrzyn P, Faro C, Falcon O, et  al. Placen-
tal volume measured by three-dimensional
ultrasound at 11 to 13 + 6 weeks of gestation:
relation to chromosomal defects. Ultrasound
Obstet Gynecol. 2005;26:28–32.
115. Pergament E, Cuckle H, Zimmermann B,
et  al. Single-nucleotide polymorphism-based
noninvasive prenatal screening in a high-
risk and low-risk cohort. Obstet Gynecol.
2014;124:210–218.
116. Norton ME, Jelliffe-Pawlowski LL, Currier
RJ. Chromosome abnormalities detected
by current prenatal screening and non-
invasive prenatal testing. Obstet Gynecol.
2014;124(5):979–986.
117. Benn P, Borell A, Chiu R, et  al. Position
statement from the aneuploidy screening
committee on behalf of the board of the inter-
national society for prenatal diagnosis. Prenat
Diagn. 2013;33:622–629.
118. Bianchi DW, Wilkins-Haug L. Integration of
noninvasive DNA testing for aneuploidy into
prenatal care: what has happened since the rub-
ber met the road? Clin Chem. 2014;60:78–87.
119. Fernando MR, Chen K, Norton S, et  al. A
new methodology to preserve the original pro-
portion and integrity of cell-free fetal DNA in
maternal plasma during sample processing and
storage. Prenat Diagn. 2010;30:418–424.
120. Barrett AN, Zimmermann BG, Wang D, et al.
Implementing prenatal diagnosis based on
cell-free fetal DNA: accurate identification of
factors affecting fetal DNA yield. PLoS One.
2011;6:e25202.
121. Ledbetter DH, Zachary JM, Simpson JL,
et  al. Cytogenetic results from the U.S. col-
laborative study on CVS. Prenat Diagn.
1992;12:317–345.
122. JM1 Hahnemann, Vejerslev LO. Accuracy
of cytogenetic findings on chorionic villus
sampling (CVS)—diagnostic consequences
ancy in centres contributing to EUCROMIC
1986-1992. Prenat Diagn. 1997;17:801–820.
123. Grati FR, Malvestiti F, Ferreira JC, et al. Feto-
placental mosaicism: potential implications
for false-positive and false-negative nonin-
vasive prenatal screening results. Genet Med.
2014;16:620–624.
124. Hochstenbach R, Page-Christiaens GC, van
Oppen AC, et  al. Unexplained false negative
results in noninvasive prenatal testing: two
cases involving trisomies 13 and 18. Case Rep
Genet. 2015;2015:926545.
125. Canick JA, Palomaki GE, Kloza EM, et  al.
The impact of maternal plasma DNA fetal
fraction on next generation sequencing tests
for common fetal aneuploidies. Prenat Diagn.
2013;33(7):667–674.
126. Gao Y, Stejskal D, Jiang F, Wang W. False-
negative trisomy 18 non-invasive prenatal test
result due to 48,XXX,+18 placental mosaicism.
Ultrasound Obstet Gynecol. 2014;43(4):477–
478.
127. Mao J, Wang T, Wang BJ, et al. Confined pla-
cental origin of the circulating cell free fetal
DNA revealed by a discordant non-invasive
prenatal test result in a trisomy 18 pregnancy.
Clin Chim Acta. 2014;433:190–193.
128. Pan Q, Sun B, Huang X, et al. A prenatal case
with discrepant findings between non-invasive
prenatal testing and fetal genetictestings. Mol
Cytogenet. 2014;16(7):48.
REFERENCES 213.e3
129. Zhang H, Gao Y, Jiang F, et al. Non-invasive
prenatal testing for trisomies 21, 18 and 13:
clinical experience from 146,958 pregnancies.
Ultrasound Obstet Gynecol. 2015;45:530–538.
130. Wang Y, Zhu J, Chen Y, et  al. Two cases of
placental T21 mosaicism: challenging the
detection limits of non-invasive prenatal test-
ing. Prenat Diagn. 2013;33:1207–1210.
131. Smith M, Lewis KM, Holmes A, Visootsak
J. A case of false negative NIPT for Down
syndrome—lessons learned. Case Rep Genet.
2014;2014:823504.
132. Choy KW, Kwok KY, Lau ET, et al. Discor-
dant karyotype results among non-invasive
prenatal screening positive cases. In: The Shift-
ing Landscape of Genetic Testing: Approaches
and Success Stories, Platform Session, Abstract
#19, American Society of Human Genetics
2013 Annual Meeting, Boston, MA. 2013.
133. Meck JM, Kramer Dugan E, et al. Noninva-
sive prenatal screening for aneuploidy: positive
predictive values based on cytogenetic find-
ings. Am J Obstet Gynecol. 2015;213:214.e1–
e5.
134. Wang JC, Sahoo T, Schonberg S, et al. Discor-
dant noninvasive prenatal testing and cytoge-
netic results: a study of 109 consecutive cases.
Genet Med. 2015;17:234–236.
135. Futch T, Spinosa J, Bhatt S, et  al. Initial
clinical laboratory experience in noninvasive
prenatal testing for fetal aneuploidy from
maternal plasma DNA samples. Prenat Diagn.
2013;33:569–574.
136. Wang Y, Chen Y, Tian F, et  al. Maternal
mosaicism is a significant contributor to dis-
cordant sex chromosomal aneuploidies associ-
ated with noninvasive prenatal testing. Clin
Chem. 2014;60:251–259.
137. Bianchi DW, Chudova D, Sehnert AJ, et  al.
Noninvasive prenatal testing and incidental
detection of occult maternal malignancies.
JAMA. 2015;314:162–169.
138. Grati FR, Malvestiti F, Branca L, et  al.
Chromosomal mosaicism in the fetoplacen-
tal unit. Best Pract Res Clin Obstet Gynaecol.
2017;42:39–52.
139. Grati FR. Implications of fetoplacental mosa-
icism on cell-free DNA testing: a review of a
common biological phenomenon. Ultrasound
Obstet Gynecol. 2016;48:415–423.
140. Grati FR, Bajaj K, Malvestiti F, et  al. The
type of feto-placental aneuploidy detected
by cfDNA testing may influence the choice
of confirmatory diagnostic procedure. Prenat
Diagn. 2015;35:994–998.
22
Noninvasive Prenatal Diagnosis for
Single-Gene Disorders
MELISSA HILL AND LYN S. CHITTY
KEY POINTS
• Noninvasive prenatal diagnosis (NIPD) based on analysis of
cell-free DNA in maternal plasma for fetal sex determination is
now an established clinical service in many countries.
• NIPD for a small number of single-gene disorders, including
achondroplasia, thanatophoric dysplasia, Apert and Crouzon
syndromes, congenital adrenal hyperplasia and cystic fibrosis,
is now offered in accredited clinical practice in the United
Kingdom.
• NIPD for monogenic disorders offers definitive diagnosis
and, unlike noninvasive prenatal testing or screening (NIPT
or NIPS) for aneuploidy, does not require an invasive test for
confirmation of diagnosis.
• The potential to test for a wide range of other conditions has
been demonstrated, and new technologies and approaches
are allowing the development of NIPD for conditions with a
more complex genetic basis.
• Potential service users and health professionals value the
opportunity to have a test with no risk for miscarriage that is
available early in pregnancy and easy to access.
• Uptake of NIPD is likely to be high, and many women unlikely
to request invasive testing would have NIPD if available to
inform decisions about termination or to prepare for the birth
of an affected child.
• NIPD care pathways using straightforward approaches for
the exclusion of paternal or de novo mutant alleles are less
expensive than invasive testing. However, more complex
approaches and likely increased uptake will increase costs
above those for invasive testing.
Introduction
The identification of cell-free fetal DNA (cffDNA) circulating in
maternal plasma, which is present from early in pregnancy, has
paved the way for the development of noninvasive prenatal diag-
nosis (NIPD), avoiding the small miscarriage risk associated with
invasive prenatal diagnosis. The cffDNA is rapidly cleared from
the maternal circulation after delivery,1 and because the whole
fetal genome is represented,2 it offers scope for comprehensive
prenatal diagnosis. NIPD for monogenic disorders is diagnos-
tic, and results do not require confirmation on chorionic villi or
214
amniocytes obtained by invasive testing as diagnosis is undertaken
in pregnancies known to be at increased risk because of family
history or sonographic findings and is targeted at a specific gene
or panel of genes. This contrasts with noninvasive prenatal testing
or screening (NIPT or NIPS) for aneuploidy, which is considered
a very sensitive screening test requiring an invasive test to confirm
a positive result (Table 22.1). This is because cffDNA emanates
from the placenta,3 and testing maternal plasma can detect con-
fined placental mosaicism, and because the majority of cell-free
DNA (cfDNA) in maternal plasma comes from the mother her-
self, NIPT can reveal unexpected maternal chromosomal rear-
rangements. Although maternal mosaicism can complicate NIPD
for monogenic disorders, this can be taken into account by testing
maternal genomic DNA (gDNA) in parallel with the cfDNA, but
as far as we are aware, placental mosaicism for cell lines containing
mutations found in single-gene disorders has not been reported.
Differences between NIPD and NIPT also apply to the tim-
ing of testing, largely because any cffDNA-based test is affected
by the fetal fraction. The fetal fraction increases as pregnancy
progresses, usually reaching a level that can be used for testing in
straightforward applications such as the detection or exclusion of
an allele not present in the maternal genome, for example, fetal
sex determination, by 7 weeks’ gestation.4 However, for more
complex applications that require accurate quantification (e.g.,
aneuploidy screening), accurate testing usually requires higher
levels of cffDNA, which are attained around 10 to 12 weeks in
most pregnancies.5 It is therefore critical that the gestational age is
determined using ultrasonography before embarking on cfDNA
testing. Furthermore, ultrasonography should be used to look for
evidence of multiple pregnancies, which may complicate results.
If an empty sac or ‘vanishing twin’ is identified, this may lead
to false-positive results4 because the placenta continues to shed
cffDNA in the absence of a fetal pole.3 NIPD is possible in mul-
tiple pregnancies, but in the absence of any abnormal ultrasound
findings, it is not possible to tell which fetuses are affected, and
invasive testing will be needed for definitive diagnosis. However,
in dizygotic twins, it may be possible to analyse single nucleotide
polymorphisms (SNPs) to measure variations in the fetal fraction
between genomic regions to determine zygosity and analyse the
fetal fragments for each fetus.6
It is not possible to separate maternal and fetal cfDNA and,
as described earlier, any test based on analysing cfDNA in mater-
nal plasma will analyse both the maternal and fetal fractions,
the maternal fraction being the most abundant. Consequently,
 CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders
TABLE Comparison of the Features of Noninvasive Prenatal Diagnosis for Monogenic Disorders and Noninvasive
22.1 Prenatal Testing (or Screening) for Aneuploidy
NIPD for Single-Gene Disorders
Type of test Diagnostic
Confirmation by invasive testing Not required
Approach Analysis targets possible changes to one or more
specific genes
Placental mosaicism Placental mosaicism for cell lines containing
mutations for single gene disorders not reported;
does not detect chromosomal mosaicism
Maternal mosaicism Controlled for by concurrently testing maternal
genomic DNA
Maternal chromosomal Not detected
rearrangements
Maternal tumour cell lines Not detectable
CPM, Confined placental mosaicism; NIPD, noninvasive prenatal diagnosis; NIPT, noninvasive prenatal testing.
the early clinical applications of NIPD focussed on the detection
of paternal alleles or those arising de novo that were therefore not
present in the mother (e.g., fetal sex determination,7 fetal RHD
typing in RhD-negative mothers,8 paternally inherited single-
gene disorders9, or single-gene disorders arising de novo, such as
achondroplasia10). NIPD can also be used in pregnancies at risk
for autosomal recessive conditions if the parents are heterozygous
for different mutations. In these cases, termed ‘paternal exclusion
testing’, if the paternal allele is identified in maternal plasma, an
invasive test is still required for definitive prenatal diagnosis to
determine inheritance of the maternal allele and see whether the
fetus is affected or not.11
Many different laboratory approaches have been reported for
NIPD, largely in research settings (Table 22.2). New technolo-
gies, such as massively parallel sequencing (MPS), that allow accu-
rate quantification of specific sequences has meant that tests are
now being developed that take into account the presence of the
maternal cfDNA, thus allowing diagnosis of autosomal recessive
X-linked conditions.
Confirming the presence of cffDNA is critical to delivering
a reliable NIPD result. In situations in which testing requires
determination of the presence or absence of an allele not pres-
ent in the mother, detection allows definitive diagnosis, but if
absent, although it may reflect a true negative. it may also result
from absence or very low levels of cffDNA with consequent fail-
ure to amplify the target sequence. Inclusion of a fetal specific
marker in an NIPD assay is required to confirm the presence of
cffDNA and allows a negative result to be definitive. In male-
bearing pregnancies, Y-chromosome sequences can be used,
but there is no straightforward approach for female-bearing
pregnancies. Paternally inherited SNPs, short tandem repeats
or small insertion or deletions (indel markers) that are either
absent or different in the mother57,58 could be used but add to
the cost of the test as maternal and paternal DNA require analy-
sis, and even a large panel of SNPs may not always be informa-
tive. Alternative approaches to confirm the presence of cffDNA
take advantage of epigenetic differences between the fetus and
215
NIPT for Aneuploidy
Screening
Required
Analysis across all chromosomes or selected chromosomes
False positives can occur because of detection of
chromosomal cell lines confined to the placenta (CPM)
False positives can occur because of maternal mosaicism
for chromosomal cell lines
Discordant results may result from detection of maternal
chromosomal rearrangements, particularly if using the
whole-genome sequencing approach
May be detected if using the whole-genome sequencing approach
the mother which arise as a result of the differential expression of
maternally and paternally inherited alleles. The Ras-association
domain family member 1, transcript variant A (RASSF1A)
which is hypomethylated in the mother and hypermethylated
in the fetus, is the allele most frequently used,59 but the assay is
complex and not ideally suited for use in a busy service labora-
tory. Developing robust assays to measure fetal fraction may be
particularly important in women with high body mass indexes
because cffDNA levels tend to be lower in obese women, probably
because of increase shedding of maternal cfDNA from adipose
tissue.60 In addition, strategies that optimise sample collection
and processing (e.g., separation of the plasma within a few hours
of blood draw or the use of cell stabilising tubes) are impor-
tant to optimise cffDNA levels and minimise inconclusive or
failed cases.61
Although the commercial sector has driven the development
of NIPT for aneuploidy, which is now available worldwide,62
development of NIPD for monogenic disorders has been led by
the academic sector, presumably because of the rarity of most
disorders together with the cost and time required to develop
individual tests do not make NIPD an attractive commercial
prospect. In this chapter, we discuss the development of a clini-
cal service focused on NIPD for monogenic disorders, which
includes NIPD for fetal sex determination to inform the need for
definitive diagnosis in pregnancies at risk for sex-linked disorders.
Implementation of any new test into clinical practice requires
robust validation, both in the laboratory but also in terms of clin-
ical validity and economic viability, and must meet service users’
needs. Development of an NIPD clinical service in the United
Kingdom has been based on the UK Genetic Testing Network
(UKGTN) standards, as required by the UK National Health
Service (NHS), to commission and thus fully fund any molecu-
lar genetic test for a monogenic disorder (Table 22.3). Here we
discuss current applications of NIPD (in the United Kingdom
and elsewhere) and provide an overview of clinical utility, eco-
nomic and social issues and stakeholder viewpoints, all of which
are required for UKGTN approval. 
216 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Studies Reporting Noninvasive Prenatal Diagnosis for Single-Gene Disorders Published Between January
22.2 2000 and August 2016
Condition Method Total Samples Results Reference
AUTOSOMAL DOMINANT CONDITIONS
Achondroplasia Restriction digest 1 1 affected Saito et al.12 (2000)
Restriction digest 1 1 affected Li et al.13 (2004)
MALDI-TOF MS 2 2 affected Li et al.14 (2007)
PCR-RED 6 4 affected Chitty et al.10 (2011)
2 unaffected
QF-PCR 2 1 affected Lim et al.15 (2011)
1 unaffected
NGS PCR-RED: 14/14 affected Chitty et al.16 (2015)
MPS: 8/9 affected
Apert syndrome Allele-specific real-time PCR 1 1 affected Au et al.17 (2011)
PCR-RED 2 1 affected Raymond et al.18 (2010)
1 unaffected
Crouzon syndrome PCR-RED 1 1 unaffected; recurrence excluded Raymond et al.18 (2010)
Huntington disease QF-PCR 1 1 unaffected Gonzalez-Gonzalez et al.19 (2003)
QF-PCR 4 2/3 affected Bustamante-Aragones et al.20
1/1 unaffected (2008)
QF-PCR 1 1 unaffected Gonzalez-Gonzalez et al.21 (2008)
Myotonic dystrophy Nested PCR 1 1 affected Amicucci et al.22 (2000)
Thanatophoric dysplasia PCR-RED 4 3 affected Chitty et al.23 (2013)
types 1 and 2 1 recurrence excluded at 12 wk
MPS PCR-RED: 9/11 affected Chitty et al.16 (2015)
MPS: 9/9 affected
Torsion dystonia RT-PCR 2 2 affected Meaney and Norbury24 (2009)
AUTOSOMAL RECESSIVE: PARENTS CARRYING DIFFERENT MUTATIONS
Congenital adrenal Fluorescent SNPs 1 1 unaffected Chiu et al.25 (2002)
hyperplasia
Craniosynostosis COLD-PCR 1 1 affected Galbiati et al.26 (2014)
Cystic fibrosis PCR-RFLP 1 1 affected Gonzalez-Gonzalez et al.27 (2002)
SnaPshot 3 2 affected Bustamante-Aragones et al.20,28
1 unaffected (2008)
NGS panel with 10 4 4 correctly classified: 2 inherited Hill et al.11 (2015)
common mutations paternal mutations
Haemoglobin E Nested PCR and 5 3 affected Fucharoen et al.29 (2003)
restriction digestion 2 unaffected
Seminested and nested 39 beta(E) All correctly classified Tungwiwat et al.30 (2007)
real-time PCR for three 12 beta(17) 26 affected/carrier beta(E)
different mutations 9 beta(41/42) 6 affected/carrier beta(17)
5 affected/carrier beta(41/42)
HB Lepore Allele-specific PCR 1 1 unaffected Lazaros et al.31 (2006)
Leber congenital amaurosis Denaturing HPLC 1 1 affected Bustamante-Aragones et al.32
(2008)
Propionic acidaemia SnaPshot; melt curve analysis. 1 1 affected (positive using both methods) Bustamante-Aragones et al.33
(2008)
α-Thalassaemia Real-time nested PCR 13 8 carriers, 1 HbH, 2 HbBarts, Tungwiwat et al.34 (2006)
2 no mutation
QF-PCR 30 10/30 paternal allele Ho et al.35 (2010)
correctly excluded

TABLE Studies Reporting Noninvasive Prenatal Diagnosis for Single-Gene Disorders Published Between January
22.2 2000 and August 2016—cont’d
Condition Method
β-Thalassaemia COLD-PCR
AS-PCR for SNPs
APEX
Genome-wide MPS and
SPRT analysis
Fetal DNA enrichment
and real-time PCR
AUTOSOMAL RECESSIVE: PARENTS CARRYING THE SAME MUTATIONS
Congenital adrenal Targeted MPS and
hyperplasia haplotype analysis
Targeted MPS and haplo-
type analysis – hidden
Markov model
Cystic fibrosis Single-cell short tandem
repeat genotyping
Maple syrup urine disease Targeted MPS and haplo-
type analysis
Methylmalonic Relative mutation dosage
academia with digital droplet PCR
and parental SNP analysis
Sickle cell disorder Relative mutation dosage
using digital RT-PCR
PAP
Spinal muscular atrophy Single-cell short tandem
repeat genotyping
Targeted MPS and haplo-
type analysis
α-Thalassaemia Real-time quantitative PCR
Allele-specific real-time PCR
β-Thalassaemia Relative mutation dosage
using digital PCR
Targeted MPS and relative
haplotype dosage
(PAP)
Targeted MPS and
haplotype analysis
High-resolution melting
analysis
CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders 217
Total Samples Results Reference
35 10/21 affected Cd39 Galbiati et al.36 (2011)
11/21 unaffected
12/14 affected IVSI-110
2/14 unaffected
2 1 affected Papasavva et al.37 (2006)
1 unaffected
7 3 inherited paternal mutations Papasavva et al.38 (2008)
3 unaffected
1 incorrect
1 1 carrier Lo et al.2 (2010)
10 10 paternal mutations Ramezanzadeh et al.39 (2016)
correctly identified
14 14 affected New et al.40 (2014)
1 1 unaffected Ma et al.41 (2014)
32 32 correctly classified (7 affected) Mouawia et al.42 (2012)
1 1 correctly classified You et al.43 (2014)
1 1 correctly classified Gu et al.44 (2014)
65 52 correctly classified Barrett et al.45 (2012)
7 incorrectly classified
5 unclassified
1 1 negative for linked paternal Phylipsen et al.46 (2012)
SNP allele
31 31 correctly classified (7 affected) Mouawia et al.42 (2012)
5 5 correctly classified Chen et al.47 (2016)
158 61/62 affected Sirichotiyakul et al.47 (2012)
Sensitivity: 98.4%
False-positive rate: 20.8%
67 33/67 correctly classified unaffected Yan et al.49 (2011)
10 5 correctly classified Lun et al.50 (2008)
1 incorrect
4 unclassified
2 2 correctly classified as carriers Lam et al.51 (2012)
13 Paternal SNP allele detected in Phylipsen et al.46 (2012)
maternal plasma in 13 cases
10 NIPD possible in 8/10 cases Papasavva et al.52 (2013)
50 25/27 correctly classified as affected Zafari et al.53 (2016)
19/23 correctly classified as unaffected
Continued
218 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Studies Reporting Noninvasive Prenatal Diagnosis for Single-Gene Disorders Published Between January
22.2 2000 and August 2016—cont’d
Condition Method Total Samples Results Reference
X-LINKED
Haemophilia A and B Relative mutation dosage 7 3/3 affected haemophilia A Tsui et al.54 (2011)
using digital PCR 4/4 affected haemophilia B
Retinitis pigmentosa Sequencing 1 1 mutation on paternal Bustamante-Aragones et al.55
(X-linked) allele detected (2006)
DMD and Becker Targeted MPS and haplo- 9 2/2 affected DMD Parks et al.56 (2016)
muscular dystrophy type analysis 7/7 unaffected

APEX, Arrayed primer extension; AS-PCR, allele specific-polymerase chain reaction; COLD-PCR, cold-polymerase chain reaction;
DMD, Duchenne muscular dystrophy; HbBarts, haemoglobin Barts; HbH, haemoglobin H; HPLC, high-performance liquid chromatography;
MALDI-TOF MS, matrix-assisted laser desorption ionisation time of flight mass spectrometry; MPS, massively parallel sequencing;
NGS, next-generation sequencing; PAP, pyrophosphorolysis-activated polymerisation; PCR, polymerase chain reaction; PCR-RED, polymerase chain reaction-restriction enzyme digest;
PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; QF-PCR, quantitative fluorescence-polymerase chain reaction; SnaPshot, single base primer extension; SNP, single
nucleotide polymorphism; SPRT, sequential probability ratio test.
Adapted from Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn 33:555–562, 2013.
  

TABLE UK Genetic Testing Network Considerations
22.3
Required for Implementation of New Molecular
Genetic Tests Into UK National Health Service
Clinical Practice
• Seriousness of the condition
• Prevalence of the condition
• The context in which the test is to be used: population groups
• Complexity of the test
• Clinical sensitivity, specificity and predictive value
• Utility of the test: benefit to patient management
• Test purpose: diagnosis, treatment, prognosis, management and so on
• Availability of alternative diagnostic procedures
• Ethic al legal and social considerations
• Economics of the test
Adapted from NHS. UK Genetic Testing Network. http://www.ukgtn.nhs.uk.
  
Fetal Sex Determination
Fetal sex determination was one of the first applications of cfDNA
to be used in clinical practice with a variety of methods being
applied across centres in Europe from the late 1990s.7 This test,
now well-established in several countries,4,7,63–65 can be per-
formed reliably from 7 weeks’ gestation to guide the management
of pregnancies at risk for X-linked conditions, congenital adrenal
hyperplasia (CAH) and in cases in which there is genital ambi-
guity.4,65 In pregnancies at risk for X-linked conditions, such as
Duchenne muscular dystrophy (DMD) or adrenoleukodystrophy,
identification of a male fetus using NIPD indicates the need for
an invasive test to determine inheritance of the maternal mutant
allele and subsequent definitive diagnosis, but this is not required
if the fetus is found to be female. In pregnancies at risk for CAH,
early maternal treatment with dexamethasone, although contro-
versial, can reduce the degree of virilisation of external genitalia
in affected female fetuses.66 Because this is a recessive condition,
males can have CAH, but they are not at risk for genital virilisa-
tion. Early determination of fetal sex can be used to guide mater-
nal dexamethasone therapy and direct the need for invasive testing
for definitive diagnosis, which, as will be discussed later, is now
possible in many families using NIPD for CAH. NIPD for fetal
sex determination is also helpful for the clarification of fetal ultra-
sound findings such as genital ambiguity or to provide additional
information for diagnosing genetic conditions such as campo-
melic dysplasia or Smith-Lemni-Opitz syndrome in which genital
ambiguity or sex reversal is a feature of the condition.67
Noninvasive prenatal diagnosis for fetal sex determination is
relatively straightforward as it is performed by identifying the pres-
ence or absence of Y-chromosome sequences in maternal plasma.
Detection of the Y-chromosome sequence indicates that the fetus is
male. If the Y-chromosome sequence is not detected, it is assumed
that the fetus is female. A systematic review and meta-analysis that
included 57 studies with 3524 male- and 3017 female-bearing
pregnancies tested over a wide range of gestations and with a variety
of laboratory methodologies, largely performed on a research basis,
indicated that NIPD for fetal sex determination is reliable after 7
weeks in pregnancy.65 A large national audit of NIPD for sex deter-
mination performed in UK public-sector laboratories using the
most common technique, real-time quantitative polymerase chain
reaction (RT-qPCR) of Y-chromosome targets (the single copy SRY
gene or the multicopy DYS14 sequence located within the TSPY
gene) showed a sensitivity of 99.5% (95% confidence interval,
98.2–99.9%)4. A failure rate of individual tests of approximately
4% to 5% has been seen in many studies,65 a rate which some
suggest may be reduced by using digital PCR68 or by using assays
that target both the SRY and DYS14 genes.69 However, these latter
studies only report small numbers, and further evaluation of these
methods is required. Test failures and false-negative results caused
by technical issues are likely to occur with any method and, as
discussed earlier, the concurrent use of a universal cffDNA marker
to confirm the presence of cffDNA will minimise false-negative
results by identifying pregnancies in which very low fetal fraction
results in failure to amplify the Y-chromosome sequences rather
than absence of Y-markers because the fetus is female.
Because NIPD for fetal sex determination has been in clini-
cal practice for several years, it has been possible to demonstrate
clinical utility. In the UK audit, the rates of invasive testing in
women at risk for very severe X-linked conditions (excluding hae-
mophilia) were only 43%, and 38% in those at risk for CAH.4
However, the same audit failed to demonstrate clinical utility in
 CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders
Mother
Unaltered
Parental DNA
Fetal DNA or
Affected
Volumes of
altered and > =
unaltered genes
in maternal plasma
• Fig. 22.1  Relative mutation dosage (RMD) for the diagnosis of single-gene disorders.
pregnancies at risk for haemophilia, in which invasive testing was
infrequent (16.9% compared with 43%) for other severe X-linked
disorders.70 This is largely because in the majority of these preg-
nancies, knowledge of fetal sex is required to direct management
of labour rather than inform parental decisions regarding inva-
sive testing for definitive diagnosis and possible termination of
pregnancy. Sex determination in these pregnancies can readily
be performed using routine midtrimester ultrasonography; thus
clinical utility could not be demonstrated as an alternative, more
cost-effective approach is available. Clinical utility has been fur-
ther demonstrated in a French multicentre study of fetal sex deter-
mination in 258 pregnancies at risk for CAH (134 male and 124
female fetuses), which showed that prenatal maternal steroid treat-
ment had been avoided in 68% of male-bearing pregnancies.64
Health economic aspects must be considered when implement-
ing new tests, particularly in publicly funded health care systems. A
detailed health economic analysis in the United Kingdom demon-
strated that NIPD for sex determination was cost effective for severe
X-linked conditions such as DMD because the costs of NIPD are
offset by the smaller proportion of women who require invasive
testing, reducing costs by an average of £87 (-£303 to £131) per
pregnancy.71 Cost savings were slightly greater, a reduction of £193
(-£301 to -£84), in pregnancies at risk for CAH because of the
additional savings related to maternal dexamethasone treatment.
It must be noted that these costs relate to the United Kingdom,
where the cost of labour is high and laboratory consumables may
be lower than in other countries. Thus the labour costs related to
invasive testing are relatively high. In health economies with a dif-
ferent balance of labour: consumable costs, benefits and costs may
be different.71 UK service users welcome the availability of the
NIPD and report practical benefits from safe early testing as well as
psychological benefits, such as a feeling of having control over the
pregnancy and peace of mind.72 Health professionals also welcome
the advent of safer and earlier fetal sex determination with NIPD.67
The UKGTN gave approval for NIPD for fetal sex determina-
tion in the UK NHS, but their recommendation, and thus sub-
sequent commissioning and public-sector funding, was limited
to NIPD for serious X-linked disorders (excluding haemophilia)
and CAH. Since gaining UKGTN approval, there has been a sig-
nificant increase in the use of NIPD for fetal sex determination,
with it now being one of the most frequently performed prenatal
molecular tests in pregnancies at risk for monogenic disorders.73
However, there remains significant discordance in practice in
pregnancies at risk for haemophilia with some services routinely
219
Father
Altered Unaltered Altered
or or
Carrier Carrier Unafferected
and not a carrier
= <
offering NIPD to all carriers of haemophilia or to all carriers of
severe haemophilia but others primarily offering NIPD as a first
step to invasive testing.70 Development of definitive NIPD for hae-
mophilia may resolve this inequality of access as this would enable
safe optimisation of pregnancy management for all pregnancies. 
Noninvasive Prenatal Diagnosis for
Monogenic Disorders
Noninvasive prenatal diagnosis for a small number of monogenic dis-
orders is available clinically in some countries, and the potential to test
for a wide range of other conditions has been clearly demonstrated in
many proof-of-principle studies on a research basis (see Table 22.2).
At the time of writing, so far as can be established, the only clinically
accredited laboratories offering a definitive NIPD service not requir-
ing confirmation by invasive testing are in the United Kingdom
(http://www.ukgtn.nhs.uk). Others have reported their experience,
but as these tests have largely been undertaken in a research setting
they have recommend invasive testing for confirmation of results
before changing pregnancy management.74 The first NIPD tests for
monogenic disorders developed were for autosomal dominant condi-
tions, which are paternally inherited or arise as a result of a de novo
mutation and, as such, the mutant allele would not be present in the
mother. These tests are possible using straightforward molecular tech-
niques, similar to fetal sex determination because in these cases any
mutation detected in maternal blood must derive from the fetus (see
Table 22.2). NIPD has also been used to direct the need for definitive
invasive testing in autosomal recessive conditions when the parents
carry different mutations (see Table 22.2). In these paternal exclusion
assays, if the paternal mutation is present, an invasive test is required
to determine if the fetus also has the maternal mutation.
Noninvasive prenatal diagnosis for autosomal recessive condi-
tions, in which parents carry the same mutation, or for X-linked
conditions is more complex because both the maternal and fetal
contribution to cfDNA must be taken into account. Technologi-
cal developments, such as digital PCR and MPS, that allow single-
molecule counting and thereby permit sensitive quantification of
alleles have facilitated development of NIPD for a wider range of con-
ditions. Using these techniques and a statistical analytical approach
called relative mutation dosage (RMD) analysis,50 the fetal genetic
status can be determined by evaluating the ratio of a specific mutant
to wild-type (WT) alleles present in maternal plasma (Fig. 22.1).
The main challenge for RMD is the need to accurately estimate
220 SE C T I O N 6     Prenatal Screening and Diagnosis

Type I Prenat Diagn 2013;33:416–423, C from Chitty LS, Griffin DR, Meaney C, et al.
New aids for the non-invasive prenatal diagnosis of achondroplasia: dysmor-
Normal cfDNA Test cfDNA phic features, charts of fetal size and molecular confirmation using cell-free
fetal DNA in maternal plasma. Ultrasound Obstet Gynecol 2011;37:283–289.)

BsiHKAL

BsiHKAL
Uncut

Uncut
Dralll

Dralll
Afel

Afel
the fetal fraction, which is relatively straightforward in male fetuses
using Y-chromosome sequences. However, without a universal fetal
marker, quantification of fetal fraction when the fetus is female is
127 bp problematic and requires the detection of paternally inherited SNPs.
Furthermore, when using sequencing approaches, allele drop-out
means it is likely that such SNPs will need to be closely associated
with the gene being tested for greatest accuracy. This can be com-
1 2 3 4 5 6 7 8 9 10 plicated and may require the development of specific fetal fraction
A quantification assays for individual conditions.
Type II Here we describe how we established a clinical NIPD service in
Normal Test our public-sector regional genetics laboratory in the United King-
Normal Test
cfDNA cfDNA cfDNA cfDNA
dom, how we have moved to using MPS to deliver more compre-
hensive NIPD for paternal exclusion assays and are now developing
Uncut

Uncut

Uncut

Uncut

NIPD for recessive conditions in which parents carry the same

Bbsl

Bbsl

Bbsl

Bbsl

mutation. We discuss the economic aspects and the psychosocial

B 1 2 3
Primer set 1
ones. The work done was largely funded by the National Institute
for Health Research (NIHR) through RAPID a 5-year programme
grant which aimed to develop the standards required to implement
NIPD into NHS practice for fetal sex determination and selected
4 5 6 7 8 9 10 monogenic disorders (http://www.rapid.nhs.uk).
Primer set 2

Noninvasive Prenatal Diagnosis for Autosomal

Dominant Conditions and Exclusion of Paternal
Uncut PCR product

400 µL 800 µL or De Novo Mutations

Positive control
Normal control

plasma plasma
50bp ladder

The relatively simple technique of PCR restriction enzyme digest

(PCR-RED) can be applied to determine presence or absence of some
10 µL
20 µL

10 µL

20 µL

NTC
5 µL

5 µL

mutations. This method ideally requires knowledge of the mutation

150 bp
before testing and is limited to those in which a restriction enzyme
exists which can recognise the mutant or WT allele. This method has
C 100 bp been applied by many researchers (see Table 22.2), and it was our ini-
• Fig. 22.2  Polymerase chain reaction restriction enzyme digest of cell- tial approach to NIPD which we used for the diagnosis FGFR3-related
free DNA (cfDNA) from women bearing pregnancies with thanatophoric skeletal dysplasias.10,23 Achondroplasia was one of the early condi-
dysplasia type I (FGFR3 c. 742C>T) (A) and type II (FGFR3 c. 1948A>G) tions diagnosed using NIPD (see Table 22.2) and is an ideal starting
(B) and achondroplasia caused by FGFR3 c.1138G>A (C)ty et al. 201110). point for development and implementation of NIPD into the routine
A, Restriction digest of PCR products for two thanatophoric dysplasia (TD) diagnostic laboratory repertoire as 98% of cases are caused by a single
mutations. (A) The c.742C>T type I TD mutation was detected using PCR mutation (FGFR3 c. 1138G>A), most cases arise sporadically (result-
followed by digestion with AfeI, BsiHKAI and DraIII. cffDNA from a woman ing from a de novo paternal mutation) and presentation is usually late
carrying an unaffected fetus is digested with AfeI (lane 3) but remains uncut in pregnancy, when levels of cffDNA are higher, after detection of short
using BsiHKAI (lane 4) and DraIII (lane 5); conversely, in an affected fetus,
limbs on ultrasonography.10 The same technique was also applied for
the AfeI site is destroyed, leaving some of the cffDNA undigested (lane 8),
and a BbsI site (lane 9) and a DraIII site (lane 10) are created.
the development of NIPD for thanatophoric dysplasia, also caused
by mutations in the FGFR3 gene.23 However, in this condition, there
B, The c.1948A>G mutation was detected using digestion with the BbsI
enzyme and two different primer sets. In the presence of an unaffected
are about 12 causative mutations, some of which are not amenable to
fetus, all cffDNA is digested by BbsI (lanes 3 and 8), whereas with an the PCR-RED approach. Furthermore, because this is a lethal domi-
affected fetus, the BbsI restriction site is destroyed, leaving some of the nant condition, there is no prior knowledge of the causative mutation
cffDNA uncut (lanes 5 and 10). when offering diagnosis in cases detected by fetal ultrasound arising
C, Polymerase chain reaction (PCR) showing amplification of the 132 base de novo. The results delivered by PCR-RED require subjective inter-
pair (bp) region of exon 8 containing the mutation causative for achondropla- pretation of an electrophoresis image (Fig. 22.2), limiting utility of
sia using 5, 10 or 20 μL of deoxyribonucleic acid (DNA) extracted from 400 the approach, but still yielding high sensitivity and specificity.16 In a
μL or 800 μL of plasma, as well as on genomic DNA from a normal and a study of 75 cases at risk for achondroplasia or thanatophoric dysplasia,
positive control. In the unaffected DNA sample, restriction digest of the PCR there were five (7%) inconclusive and one false-negative (c.742C>T)
product with BsrG1 does not cut the DNA, giving rise to a single 132 bp frag- result, the latter caused by low fetal fraction despite being tested after
ment, whereas if the mutation is present a BsrG1 restriction site is created, 20 weeks’ gestation. For thanatophoric dysplasia, there was a further
and digestion produces fragments of 132, 112 and 20 bp, which results in
three cases with other thanatophoric dysplasia-causing mutations not
two bands, rather than the single band seen in the control sample. In all six
dilutions of the maternal plasma sample the faint second band can be seen,
covered by the PCR-RED assays, further demonstrating the limited
indicating the presence of the mutation in the maternal plasma sample. utility of tests based on individual mutation detection.16 Digital PCR
(A and  B from  Chitty LS, Khalil A, Barrett AN, et al. Safe, accurate, prena-
is an alternative approach to PCR-RED which does not rely on sub-
tal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA. jective interpretation because it gives a digital readout and is more

TABLE Estimated Target Molecules for Wild-Type (WT)
22.4 and Mutant Alleles for Fraser Syndrome and
ARPKD Cases
Sample WT Targets
Fraser Maternal gDNA 1363
syndromea
Paternal gDNA 4328
First pregnancy 120
Second pregnancy (1) 90
Second pregnancy (2) 206
ARPKD Maternal gDNA 2878
Paternal gDNA 2698
First pregnancy 935
Second pregnancy 361
aTwo samples were tested for the second Fraser syndrome pregnancy because the first
sample was such an early gestation.
Adapted from Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive
prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn
33:555–562, 2013.
ARPKD, autosomal recessive polycystic kidney disease.
sensitive, detecting mutant alleles in maternal plasma that were not
detected by PCR-RED.9 Examples are given in Table 22.4 and Figure
22.3, which show results in pregnancies with Fraser syndrome and
autosomal recessive polycystic kidney disease (ARPFD).
Both PCR-RED and digital PCR have a number of limitations,
including low throughput, the need to know (or be able to easily sur-
mise) the mutation in question and a separate assay being required
to confirm the presence of cffDNA in the absence of fetal-specific
mutation detection. MPS has become increasingly feasible over recent
years as sequencing costs and processing time reduce, along with
increased output. It is more sensitive, the presence of cffDNA can be
confirmed in the same assay as mutation detection, and because it is
scalable in terms of sample numbers and regions of interest that can
be analysed simultaneously, it is more appropriate for use in a busy
service laboratory. Other technologies may offer more potential in the
future, but at the moment, MPS offers the best flexibility as illustrated
by the paternal mutation exclusion assays now in routine clinical prac-
tice in the United Kingdom for a number of diseases using the highly
targeted approach of sequencing of specific PCR products (amplicon
sequencing).11,16 Primers have been designed to create an FGFR3-
skeletal dysplasia panel to screen pregnancies with sonographic find-
ings suggestive of achondroplasia or thanatophoric dysplasia and
to exclude a recurrence for couples with a previously affected preg-
nancy.16 As part of the validation process before clinical implementa-
tion, 47 samples were tested using the MPS panel, 27 having also been
tested by PCR-RED. MPS was 96.2% sensitive (81%–99.3%) and
100% specific (85%–100%), with no inconclusive results, including
one positive which was inconclusive by PCR-RED. The MPS panel
also detected thanatophoric dysplasia mutations in three cases where
no PCR-RED assay was available. There was one false-negative result
caused by a rare FGFR3 mutation not covered by the panel, but assay
redesign is straightforward, allowing additional mutations to be read-
ily incorporated as required.16 Similar assays have been developed for
Apert syndrome9 and to cover a panel of the most common cystic
fibrosis (CF) mutations for paternal exclusion in families in which
parents carry different mutations.11
CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders 221
Extension of the sequencing approach to include families at
risk for a wider range of much rarer conditions has recently been
reported, including tuberous sclerosis, neurofibromatosis, Rhab-
doid tumour predisposition, early infantile epileptic encephalopa-
Mutant Targets thy, osteogenesis imperfecta and Fraser syndrome.75 Development
of these assays includes sequencing of parental gDNA and, because
1.5
MPS is a very sensitive technology, in the course of working up
4116 tests for these families low level mosaicism has been identified in
17
three mothers and two fathers. This not only increases recurrence
risks but, in the case of maternal mosaicism, also renders NIPD
0 impractical because of the background of maternal mutation.
1 Overall, more than 30% of all prenatal molecular genetic tests
for monogenic disorders performed in our public-sector Regional
0 Genetics Laboratory in North East Thames in 2015 were delivered
0 via NIPD. Considering all results, 4.3% were inconclusive, one
(0.4%) was false negative and others (confirmed after delivery)
86 included 31% mutation positive, and in three families (1.2%),
0 NIPD was not offered because of low-level maternal mosaicism.
The relatively high throughput and use of MPS has allowed sam-
ple multiplexing to reduce costs and optimise turnaround times
to within 5 days. However, because the sensitivity of sequencing
can identify unexpected mosaicism in parents, analysis of mater-
nal gDNA should be done in parallel with cfDNA testing to avoid
false-positive results caused by low-level maternal mosaicism. 
Noninvasive Prenatal Diagnosis for Autosomal
Recessive and Sex-Linked Disorders
The paternal exclusion approach to NIPD for recessive conditions
in which parents carry different mutations has been described
by a number of groups for conditions, including CAH, CF and
β-thalassaemia (see Table 22.2), but an invasive test is still required
if the paternal mutation is present to determine inheritance of the
maternal mutation. Approaches that enable very sensitive estima-
tion of mutation load are required if the paternal allele has been
inherited, when parents carry the same mutation and for diagnosis
in X-linked conditions because the high background level of the
maternal mutation needs to be taken into account. Here RMD
can be applied to determine the significance of any allelic imbal-
ance caused by the fetal contribution to cfDNA. Calculation of
fetal fraction is required because fetal mutation load will vary with
fetal fraction, and thus an additional assay targeting a fetal specific
marker is required. Digital PCR and MPS are both very sensitive
techniques which allow for single-molecule counting and have
been used with RMD for NIPD in these circumstances. Digital
PCR has been used for NIPD for β-thalassaemia, haemophilia
and sickle cell disorder (see Table 22.2), but it has the same draw-
backs discussed earlier – the mutation must be known, a separate
assay is required for the fetal-specific marker and multiplexing is
not possible. Thus MPS will offer a more practical approach, par-
ticularly as some of the more commonly requested prenatal tests
for monogenic disorders are for conditions such as β-thalassaemia
and CF in which there are multiple causative mutations.
Some conditions and mutations require more complex tech-
nical approaches. Pathogenic genes with known pseudogenes or
regions of high homology are challenging to assess using cfDNA
because the methods such as long-range PCR which can be used
on gDNA to specifically amplify the gene of interest cannot be
applied to the short fragments of cfDNA. As discussed earlier,
for recessive or X-linked disorders, RMD needs to be performed
to determine the fetal contribution. Although digital PCR has
shown some application to definitive diagnosis of recessive dis-
ease, standard PCR amplicon methods do not resolve down to the
222 SE C T I O N 6     Prenatal Screening and Diagnosis

Panel01 - Maternal gDNA-c.10261C Panel02 - Maternal gDNA-c.10261C Panel01 - Maternal gDNA-c.10261T Panel02 - Maternal gDNA-c.10261T
(wild type) (wild type) (mutant) (mutant)

Panel03 - Paternal gDNA-c.10261C Panel04 - Paternal gDNA-c.10261C Panel03 - Paternal gDNA-c.10261T Panel04 - Paternal gDNA-c.10261T
(wild type) (wild type) (mutant) (mutant)

Panel05 - 1st Pregnancy cfDNA- Panel06 - 1st Pregnancy cfDNA- Panel05 - 1st Pregnancy cfDNA- Panel06 - 1st Pregnancy cfDNA-
c.10261C (wild type) c.10261C (wild type) c.10261T (mutant) c.10261T (mutant)

Panel07 - 2nd Pregnancy cfDNA- Panel08 - 2nd Pregnancy cfDNA- Panel07 - 2nd Pregnancy cfDNA- Panel08 - 2nd Pregnancy cfDNA-
c.10261C (wild type) c.10261C (wild type) c.10261T (mutant) c.10261T (mutant)

Panel09 -Unaffected cfDNA-c.10261C Panel10 -Unaffected cfDNA-c.10261C Panel09 -Unaffected cfDNA-c.10261T Panel10 -Unaffected ffDNA-c.10261T
(wild type) (wild type) (mutant) (mutant)

Panel11 -NTC -c.10261C (wild type) Panel12 -NTC -c.10261C (wild type) Panel11 -NTC -c.10261T (mutant) Panel12 -NTC -c.10261T (mutant)

A
Panel01 - Maternal gDNA-c.9374C Panel02 - Maternal gDNA-c.9374C Panel01 - Maternal gDNA-c.9374T Panel02 - Maternal gDNA-c.9374T
(wild type) (wild type) (mutant) (mutant)

Panel03 - Paternal gDNA-c.9374C Panel04 - Paternal gDNA-c.9374C Panel03 - Paternal gDNA-c.9374T Panel04 - Paternal gDNA-c.9374T
(wild type) (wild type) (mutant) (mutant)

Panel05 - 1st Pregnancy cfDNA- Panel06 - 1st Pregnancy cfDNA- Panel05 - 1st Pregnancy cfDNA- Panel06 - 1st Pregnancy cfDNA-
c.9374C (wild type) c.9374C (wild type) c.9374T (mutant) c.9374T (mutant)

Panel07 - 2nd Pregnancy cfDNA- Panel08 - 2nd Pregnancy cfDNA- Panel07 - 2nd Pregnancy cfDNA- Panel08 - 2nd Pregnancy cfDNA-
c.9374C (wild type) c.9374C (wild type) c.9374T (mutant) c.9374T (mutant)

Panel09 -Unaffected Control cfDNA- Panel10 -Unaffected Control cfDNA- Panel09 -Unaffected Control cfDNA- Panel10 -Unaffected Control cfDNA-
c.9374C (wild type) c.9374C (wild type) c.9374T (mutant) c.9374T (mutant)

Panel11 -NTC -c.9374C (wild type) Panel12 -NTC -c.9374C (wild type) Panel11 -NTC -c.9374T (mutant) Panel12 -NTC -c.9374T (mutant)

B
• Fig. 22.3  Heat map images showing digital polymerase chain reaction for Fraser syndrome and auto-
somal recessive polycystic kidneydisease (ARPKD). Samples were run in duplicate, with wild-type (WT)
alleles labelled red and mutant alleles blue. A, For a family with pregnancies at risk for Fraser syndrome
(FRAS1 c. 10261C>T), whereas maternal genomic DNA (gDNA) contained only the WT sequence, the
paternal had approximately equal numbers of WT and mutant, clearly indicating that he was a carrier of
the recessive condition. In the first pregnancy, the fetus had inherited the paternal mutant allele (17 mutant
allele counts and 120 WT) ,and the second pregnancy had no paternal mutant allele (see Table 22.4) and
thus could not be affected. B, ARPKD samples with WT signal present in all samples but the nonmaternal
causative mutation (c.9374C>CT on PKHD1) only found in the affected pregnancy, not in the maternal or
paternal gDNA or the cfDNA from the second (unaffected) pregnancy, indicating that this mutation arose
de novo. ARPKD, autosomal recessive polycystic kidney disease (With permission from Lench N, Barrett
A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single gene disor-
ders: challenges and progress made. Prenat Diagn 33:555–562, 2013.)
 CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders 223

single molecule level, and PCR bias can be introduced. In addi- Step 1. Step 2.
tion, the mutation must be known and be a single base change or
a small indel. RMD is not a useful tool for pathogenic large dele-
tions or rearrangements. However, the haplotyping method for
NIPD initially described by the Hong Kong group,2,51 who linked
SNPs to the mutation to determine phase and deliver NIPD for
β-thalassaemia, has potential for broad application, although it
requires sequencing of parental and affected proband DNA. This
approach has been reported for CAH,40 maple syrup urine dis- Maternal Paternal Proband Proband
ease43 and DMD56 and uses the affected proband and parental
samples to construct mutant and normal haplotypes by linking Step 3. Step 4.
SNPs to the mutation in question (Fig. 22.4). This has the benefit
of not needing to sequence the specific mutation and can therefore
be applied in cases of pseudogenes, deletion or conversions and
means that a generic assay can be applied to multiple families with
different mutations in the same gene.
In our laboratory, we have designed, validated, gained UKGTN
approvals and implemented definitive MPS haplotyping assays for
CF (including cases in which parents carry the same mutation)
and for CAH. The workflow for these assays varies.75 For CAH,
the first stage is to determine fetal sex. If the fetus is male, no fur-
ther investigation is required unless the family require definitive
diagnosis regardless of fetal sex. For both conditions, if the paternal
allele is not inherited, then further investigation is not required.
To establish inheritance of the maternal allele, relative haplotype
dosage analysis (RHDO) analysis is required. This is based on the
principles of RMD described earlier, except that instead of using
the single mutation point for RMD, an accumulation of counts
from multiple SNPs linked to the maternal mutation are used for
a robust assessment of inheritance of maternal alleles. To date, we
have found this a very accurate means of prenatal diagnosis, but it
can only be applied when both parents and an offspring (affected
Maternal mutant allele Paternal wild-type allele
or unaffected) are available to determine phase. 

Ethical and Social Issues

The clinical benefits of introducing NIPD for fetal sex determina-
tion and direct diagnosis of single-gene disorders are compelling
because these tests are safe, available early in pregnancy and easy
to access. It is, however, important that implementation addresses
stakeholder views and ethical concerns. Several studies have been
undertaken to assess stakeholder attitudes to NIPD, with view-
points sought from parents who have had NIPD for fetal sex
determination72 or NIPD for skeletal dysplasias,76 as well as with
carriers of single-gene disorders77-79 and health professionals.70,80
Attitudes to NIPD are generally positive, and stakeholders high-
light the many practical and psychological benefits NIPD affords
through opportunities for safe and early testing.76,77,79 One key
benefit is that decision making may be emotionally easier because
parents do not have to consider the risk for miscarriage.77,79,80
Notably, easier decision making was highlighted by parents with a
low risk for recurrence after a pregnancy with a skeletal dysplasia
because NIPD in subsequent pregnancies allows early reassurance
without putting the pregnancy at risk.76
The most common concerns held by stakeholders were the
potential for NIPD to be viewed as routine or expected, which
may undermine informed choice and increased pressure to have
prenatal testing as NIPD because the test is safe and simple to
perform. For most, the benefits of NIPD were thought to bal-
ance out these concerns, which stakeholders thought could be
addressed with formal regulation of services and appropriate pre-
test counselling in which the consent process is highlighted and
Maternal wild-type allele SNP linked maternal mutant allele
Paternal mutant allele SNP linked paternal mutant allele
• Fig. 22.4 Haplotyping approach for noninvasive prenatal diagnosis of
recessive conditions. Step 1: DNA from couple at risk for affected preg-
nancy and their affected proband is obtained. Step 2: DNA enriched for
heterozygous single nucleotide polymorphisms (SNPs), and these SNPs
are linked to affected allele. Step 3: In subsequent at-risk pregnancy, cell-
free DNA (cfDNA) is extracted from maternal plasma and cfDNA enriched
for SNPs linked to affected allele. Step 4: To determine if paternal mutant
allele inherited by fetus, the presence or absence of SNPs linked to pater-
nal mutation (blue stars) is established. Inheritance of maternal mutant
allele is established if there is an overrepresentation of SNPs linked to
maternal mutation (red stars).
discussion includes the benefits and limitations of NIPD, alterna-
tives, encouragement to reflect on the reasons for choosing NIPD
and the impact of results.76-78 For service delivery, women and
health professionals highlighted the need for NIPD to be provided
through specialised genetics services so that counselling would
be undertaken by health professionals with specialist knowledge
of the condition and trained in counselling for prenatal testing.
Implementation must be accompanied, but a health educational
224 SE C T I O N 6     Prenatal Screening and Diagnosis
programme to ensure that those offering NIPD highlight all
aspects of testing to encourage informed parental choice.72,76,77
In a number of studies, carriers of recessive conditions, includ-
ing those who had previously declined invasive prenatal diagnosis,
said they would choose to have NIPD in subsequent pregnan-
cies, not necessarily to inform decisions regarding termination
of pregnancy but because they would value the information to
prepare for the birth of an affected child.77,79 There were ethical
concerns over the appropriateness of directing resources to test-
ing that would not change pregnancy management, particularly
in state-funded health systems.  changing prenatal care. Fetal sex determination is well established
Economic Issues
Comparison of total costs of prenatal diagnosis in the United
Kingdom using NIPD or invasive testing for a representative set
of single-gene disorders has shown that for autosomal dominant
conditions with straightforward molecular techniques, NIPD
was cheaper than invasive testing (£314 less), but the more com-
plex and, as a result, more expensive NIPD approaches needed
for autosomal recessive and X-linked conditions increased costs
above those of invasive testing (£141–£1090 more).81 However, as
discussed earlier, research with stakeholders strongly suggests that
uptake of NIPD is likely to be high, and many of the couples that
would not consider invasive testing because of the risk for miscar-
riage will want NIPD. This increased uptake is expected to result
in the overall costs of the NIPD care pathways being consider-
ably more expensive than current invasive testing pathways.11,81
However, as sequencing costs continue to fall, the extent of these
discrepancies will be reduced. The cost of delivering a NIPD ser-
vice can be reduced by multiplexing testing for conditions, such
as CF or sickle cell disorder, or testing for several different con-
ditions or multiple causative mutations in a single assay. As we
have moved most of our NIPD for monogenic disorders to MPS
platforms, we have seen a reduction in cost but also a reduction
in turnaround times because the volume of testing done is such
that we have several sequencing runs a week. This then raises the
question as to how many laboratories should develop these ser-
vices. Furthermore, for the very rare single-gene disorders, there is
a question as to the cost and benefits of developing NIPD when
tests are performed so infrequently, an issue that raises potential
ethical concerns regarding restricting access to safer testing. This
is particularly the case for bespoke testing because costs are high
because of the cost of consumables and staff time involved in
working up a test.75 
Conclusions
Noninvasive prenatal diagnosis based on cfDNA is dramatically
as a clinical service in many countries and enables accurate deter-
mination of fetal sex from 7 to 9 weeks’ gestation. NIPD is now
available for some single-gene disorders which arise de novo, such
as achondroplasia, and new technologies such as digital PCR and
MPS are allowing NIPD to be offered through some accredited
public-sector laboratories for a range of recessive conditions,
including CF and CAH. The availability of NIPD for monogenic
disorders still seems largely limited to laboratories in the United
Kingdom, and we have a long way to go to offer equity of access
to families at risk for monogenic disorders worldwide. There is
significant scope for the future of NIPD for single-gene disor-
ders as proof-of-principle studies have shown that is possible to
map the entire fetal genome using cffDNA. Although currently
restricted by the cost of the large amount of sequencing involved,
this technology will ultimately allow testing for mutations when
there is a known family history as well as de novo mutations. How-
ever, any advance must be accompanied by rigorous and large-
scale evaluations before clinical implementation, and continued
research considering ethical issues, stakeholder views and imple-
mentation strategies is essential to ensure cfDNA testing is being
offered appropriately. Finally, after tests are introduced into clini-
cal practice, ongoing audit and monitoring of both test accuracy
and service delivery through recognised quality assurance schemes
is important.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References and thanatophoric dysplasia: next-generation
sequencing allows for a safer, more accurate,
32. Bustamante-Aragones A, Vallespin E,
Rodriguez de Alba M, et al. Early non-invasive
and comprehensive approach. Prenat Diagn. prenatal detection of a fetal CRB1 mutation
1. Lo YM, Zhang J, Leung TN, et  al. Rapid
2015;35:656–662. causing Leber congenital amaurosis. Mol Vis.
clearance of fetal DNA from maternal plasma.
17. Au PK, Kwok YK, Leung KY, Tang LY, Tang 2008;14:1388–1394.
Am J Hum Genet. 1999;64:218–224.
MH, Lau ET. Detection of the S252W muta- 33. Bustamante-Aragones A, Pérez-Cerdá C,
2. Lo Y, Chan K, Sun H, et al. Maternal plasma
tion in fibroblast growth factor receptor 2 Pérez B, et al. Prenatal diagnosis in maternal
DNA sequencing reveals the genome-wide
(FGFR2) in fetal DNA from maternal plasma plasma of a fetal mutation causing propionic
genetic and mutational profile of the fetus.
in a pregnancy affected by Apert syndrome. acidemia. Mol Genet Metab. 2008;95:101–
Sci Transl Med. 2010;2:61ra91.
Prenat Diagn. 2011;31:218–220. 103.
3. Alberry M, Maddocks D, Jones M, et  al.
18. Raymond FL, Whittaker J, Jenkins L, et  al. 34. Tungwiwat W, Fucharoen S, Fucharoen G,
Free fetal DNA in maternal plasma in anem-
Molecular prenatal diagnosis: the impact et al. Development and application of a real-
bryonic pregnancies: confirmation that the
of modern technologies. Prenat Diagn. time quantitative PCR for prenatal detection
origin is the trophoblast. Prenat Diagn.
2010;30:674–681. of fetal alpha(0)-thalassemia from maternal
2007;27:415–418.
19. González-González MC, Trujillo MJ, Rodrí- plasma. Ann N Y Acad Sci. 2006;1075:103–
4. Hill M, Finning K, Martin P, et  al. Non-
guez de Alba M, et  al. Huntington disease- 107.
invasive prenatal determination of fetal sex:
unaffected fetus diagnosed from maternal 35. Ho SS, Chong SS, Koay ES, et al. Noninva-
translating research into clinical practice. Clin
plasma using QF-PCR. Prenat Diagn. sive prenatal exclusion of haemoglobin Bart’s
Genet. 2011;80:68–75.
2003;23:232–234. using foetal DNA from maternal plasma. Pre-
5. Norton ME, Brar H, Weiss J, et  al. Non-
20. Bustamante-Aragones A, Trujillo-Tiebas MJ, nat Diagn. 2010;30(1):65–73.
Invasive Chromosomal Evaluation (NICE)
Gallego-Merlo J, et  al. Prenatal diagnosis 36. Galbiati S, Brisci A, Lalatta F, et  al. Full
study: results of a multicenter prospective
of Huntington disease in maternal plasma: COLD-PCR protocol for non-invasive pre-
cohort study for detection of fetal trisomy 21
direct and indirect study. Eur J Neurol. natal diagnosis of genetic diseases. Clin Chem.
and trisomy 18. Am J Obstet Gynecol. 2012;207:
2008;15:1338–1344. 2011;57:136–138.
137.e1–e8.
21. González-González MC, Garcia-Hoyos M, 37. Papasavva T, Kalakoutis G, Kalikas I, et  al.
6. Leung TY, Qu JZ, Liao GJ, et al. Noninva-
Trujillo-Tiebas MJ, et  al. Improvement in Non-invasive prenatal diagnostic assay for the
sive twin zygosity assessment and aneuploidy
strategies for the non-invasive prenatal diag- detection of beta-thalassemia. Ann N Y Acad
detection by maternal plasma DNA sequenc-
nosis of Huntington disease. J Assist Reprod Sci. 2006;1075:148–153.
ing. Prenat Diagn. 2013;33:675–681.
Genet. 2008;25:477–481. 38. Papasavva T, Kalikas I, Kyrri A, Kleanthous
7. Hyett JA, Gardener G, Stojilkovic-Mikic T,
22. Amicucci P, Gennarelli M, Novelli G, Dal- M. Arrayed primer extension for the non-
et al. Reduction in diagnostic and therapeutic
lapiccola B. Prenatal diagnosis of myotonic dys- invasive prenatal diagnosis of beta-thalassemia
interventions by non-invasive determination
trophy using fetal DNA obtained from maternal based on detection of single nucleotide poly-
of fetal sex in early pregnancy. Prenat Diagn.
plasma. Clin Chem. 2000;46:301–302. morphisms. Ann N Y Acad Sci. 2008;1137:
2005;25:1111–1116.
23. Chitty LS, Khalil A, Barrett AN, et al. Safe, 302–308.
8. Finning K, Martin P, Daniels G. A clinical
accurate, prenatal diagnosis of thanatophoric 39. Ramezanzadeh M, Salehi M, Farajzadegan Z,
service in the UK to predict fetal Rh (Rhe-
dysplasia using ultrasound and free fetal Kamali S, Salehi R. Detection of paternally
sus) D blood group using free fetal DNA
DNA. Prenat Diagn. 2013;33:416–423. inherited fetal point mutations for beta-
in maternal plasma. Ann N Y Acad Sci.
24. Meaney C, Norbury G. Non-invasive pre- thalassemia in maternal plasma using simple
2004;1022:119–123.
natal diagnosis of early onset primary dys- fetal DNA enrichment protocol with or with-
9. Lench N, Barrett A, Fielding S, et  al. The
tonia I in maternal plasma. Prenat Diagn. out whole genome amplification: an accuracy
clinical implementation of non-invasive
2009;29:1218–1221. assessment. J Matern Fetal Neonatal Med.
prenatal diagnosis for single gene disorders:
25. Chiu RW, Lau TK, Cheung PT, et al. Non- 2016;29:2645–2649.
challenges and progress made. Prenat Diagn.
invasive prenatal exclusion of congenital adre- 40. New MI, Tong YK, Yuen T, et al. Noninva-
2013;33:555–562.
nal hyperplasia by maternal plasma analysis: sive prenatal diagnosis of congenital adre-
10. Chitty LS, Griffin DR, Meaney C, et al. New
a feasibility study. Clin Chem. 2002;48:778– nal hyperplasia using cell-free fetal DNA in
aids for the non-invasive prenatal diagnosis of
780. maternal plasma. J Clin Endocrinol Metab.
achondroplasia: dysmorphic features, charts
26. Galbiati S, Stenirri S, Sbaiz L, et al. Identifi- 2014;99:E1022–E1030.
of fetal size and molecular confirmation using
cation of an 18 bp deletion in the TWIST1 41. Ma D, Ge H, Li X, et  al. Haplotype-based
cell-free fetal DNA in maternal plasma. Ultra-
gene by CO-amplification at lower denatur- approach for noninvasive prenatal diag-
sound Obstet Gynecol. 2011;37:283–289.
ation temperature-PCR (COLD-PCR) for nosis of congenital adrenal hyperplasia by
11. Hill M, Twiss P, Verhoef TI, et  al. Non-
non-invasive prenatal diagnosis of craniosyn- maternal plasma DNA sequencing. Gene.
invasive prenatal diagnosis for cystic fibrosis:
ostosis: first case report. Clin Chem Lab Med. 2014;544:252–258.
detection of paternal mutations, exploration
2014;52:505–509. 42. Mouawia H, Saker A, Jais JP, et al. Circulat-
of patient preferences and cost analysis. Prenat
27. González-González MC, García-Hoyos M, ing trophoblastic cells provide genetic diag-
Diagn. 2015;35:950–958.
Trujillo MJ, et al. Prenatal detection of a cystic nosis in 63 fetuses at risk for cystic fibrosis
12. Saito H, Sekizawa A, Morimoto T, et  al.
fibrosis mutation in fetal DNA from maternal or spinal muscular atrophy. Reprod Biomed
Prenatal DNA diagnosis of a single-gene
plasma. Prenat Diagn. 2002;22:946–948. Online. 2012;25:508–520.
disorder from maternal plasma. Lancet.
28. Bustamante-Aragones A, Gallego-Merlo J, 43. You Y, Sun Y, Li X, et al. Integration of tar-
2000;356:1170.
Trujillo-Tiebas MJ, et al. New strategy for the geted sequencing and NIPT into clinical
13. Li Y, Holzgreve W, Page-Christiaens GC,
prenatal detection/exclusion of paternal cystic practice in a Chinese family with maple syrup
et  al. Improved prenatal detection of a fetal
fibrosis mutations in maternal plasma. J Cyst urine disease. Genet Med. 2014;16:594–600.
point mutation for achondroplasia by the
Fibros. 2008;7:505–510. 44. Gu W, Koh W, Blumenfeld YJ, et  al. Non-
use of size-fractionated circulatory DNA in
29. Fucharoen G, Tungwiwat W, Ratanasiri T, invasive prenatal diagnosis in a fetus at risk
maternal plasma - case report. Prenat Diagn.
Sanchaisuriya K, Fucharoen S. Prenatal detec- for methylmalonic acidemia. Genet Med.
2004;24:896–898.
tion of fetal hemoglobin E gene from maternal 2014;16:564–567.
14. Li Y, Page-Christiaens GC, Gille JJ, et  al.
plasma. Prenat Diagn. 2003;23(5):393–396. 45. Barrett AN, McDonnell TCR, Allen Chan
Non-invasive prenatal detection of achon-
30. Tungwiwat W, Fucharoen G, Fucharoen S, KC, Chitty LS. Digital PCR analysis of mater-
droplasia in size-fractionated cell-free DNA
et  al. Application of maternal plasma DNA nal plasma for non-invasive detection of sickle
by MALDI-TOF MS assay. Prenat Diagn.
analysis for non-invasive prenatal diagno- cell anemia. Clin Chem. 2012;58:1026–1032.
2007;27:11–17.
sis of Hb E-beta-thalassemia. Transl Res. 46. Phylipsen M, Yamsri S, Treffers EE, et  al.
15. Lim JH, Kim MJ, Kim SY, et al. Non-invasive
2007;150:319–325. Non-invasive prenatal diagnosis of beta-
prenatal detection of achondroplasia using
31. Lazaros L, Hatzi E, Bouba I, et  al. Non- thalassemia and sickle-cell disease using
circulating fetal DNA in maternal plasma.
invasive prenatal detection of paternal origin pyrophosphorolysis-activated polymerization
J Assist Reprod Genet. 2011;28:167–172.
Hb lepore in a male fetus at the 7th week of and melting curve analysis. Prenat Diagn.
16. Chitty LS, Mason S, Barrett AN, et al. Non-
gestation. Fet Diagn Ther. 2006;21:506–509. 2012;32:578–587.
invasive prenatal diagnosis of achondroplasia

224.e1
224.e2 References
47. Chen M, Lu S, Lai Z, et al. Targeted sequenc-
ing of maternal plasma for haplotype-based
noninvasive prenatal testing of spinal muscu-
lar atrophy. Ultrasound Obstet Gynecol. 2017;
49:799–802.
48. Sirichotiyakul S, Charoenkwan P,
Sanguansermsri T. Prenatal diagnosis of
homozygous alpha-thalassemia-1 by cell-free
fetal DNA in maternal plasma. Prenat Diagn.
2012;32(1):45–49.
49. Yan TZ, Mo QH, Cai R, et al. Reliable detec-
tion of paternal SNPs within deletion break-
points for non-invasive prenatal exclusion
of homozygous α-thalassemia in maternal
plasma. PLoS One. 2011;6(9):e24779.
50. Lun FM, Tsui NB, Chan KC, et al. Noninva-
sive prenatal diagnosis of monogenic diseases
by digital size selection and relative mutation
dosage on DNA in maternal plasma. Proc Natl
Acad Sci U S A. 2008;105:19920–19925.
51. Lam KW, Jiang P, Liao GJ, et  al. Noninva-
sive prenatal diagnosis of monogenic diseases
by targeted massively parallel sequencing of
maternal plasma: application to beta-thalas-
semia. Clin Chem. 2012;58:1467–1475.
52. Papasavva T, van Ijcken WF, Kockx CE,
et al. Next generation sequencing of SNPs for
non-invasive prenatal diagnosis: challenges
and feasibility as illustrated by an applica-
tion to β-thalassaemia. Eur J Hum Genet.
2013;21(12):1403–1410.
53. Zafari M, Gill P, Kowsaryan M, Alipour A,
Banihashemi A. High-resolution melting
analysis for noninvasive prenatal diagnosis
of IVS-II-I (G-A) fetal DNA in minor beta-
thalassemia mothers. J Matern Fetal Neonatal
Med. 2016;29:3323–3328.
54. Tsui NB, Kadir RA, Chan KC, et  al. Non-
invasive prenatal diagnosis of hemophilia by
microfluidics digital PCR analysis of maternal
plasma DNA. Blood. 2011;117:3684–3691.
55. Bustamante-Aragones A, Garcia-Hoyos M,
Rodriguez De Alba M, et  al. Detection of a
paternally inherited fetal mutation in mater-
nal plasma by the use of automated sequenc-
ing. Ann N Y Acad Sci. 2006;1075:108–117.
56. Parks M, Court S, Cleary S, et al. Non-invasive
prenatal diagnosis of Duchenne and Becker
muscular dystrophies by relative haplotype
dosage. Prenat Diagn. 2016;36:312–320.
57. Pertl B, Sekizawa A, Samura O, et al. Detec-
tion of male and female fetal DNA in maternal
plasma by multiplex fluorescent polymerase
chain reaction amplification of short tandem
repeats. Hum Genet. 2000;106:45–49.
58. Page-Christiaens GC, Bossers B, VAN DER
Schoot CE, DE Haas M. Use of bi-allelic
insertion/deletion polymorphisms as a posi-
tive control for fetal genotyping in maternal
blood: first clinical experience. Ann N Y Acad
Sci. 2006;1075:123–129.
59. White HE, Dent CL, Hall VJ, Crolla JA,
Chitty LS. Evaluation of a novel assay for
detection of the fetal marker RASSF1A:
facilitating improved diagnostic reliability of
non-invasive prenatal diagnosis. PLoS One.
2012;7:e45073.
60. Wang E, Batey A, Struble C, et al. Gestational
age and maternal weight effects on fetal cell-
free DNA in maternal plasma. Prenat Diagn.
2013;33:662–666.
61. Barrett AN, Zimmermann BG, Wang D,
Holloway A, Chitty LS. Implementing pre-
natal diagnosis based on cell-free fetal DNA:
accurate identification of factors affecting
fetal DNA yield. PLoS One. 2011;6:e25202.
62. Minear MA, Lewis C, Pradhan S,
Chandrasekharan S. Global perspectives on
clinical adoption of NIPT. Prenat Diagn.
2015;35:959–967.
63. Bustamante-Aragones A, Rodriguez de Alba
M, Gonzalez-Gonzalez C, et  al. Foetal sex
determination in maternal blood from the
seventh week of gestation and its role in diag-
nosing haemophilia in the foetuses of female
carriers. Haemophilia. 2008;14:593–598.
64. Tardy-Guidollet V, Menassa R, Costa
JM, et  al. New management strategy of
pregnancies at risk of congenital adrenal
hyperplasia using fetal sex determination
in maternal serum: French cohort of 258
cases (2002-2011). J Clin Endocrinol Metab.
2014;99:1180–1188.
65. Devaney SA, Palomaki GE, Scott JA, Bian-
chi DW. Noninvasive fetal sex determination
using cell-free fetal DNA: a systematic review
and meta-analysis. JAMA. 2011;306:627–636.
66. Heland S, Hewitt JK, McGillivray G, Walker
SP. Preventing female virilisation in congeni-
tal adrenal hyperplasia: the controversial role
of antenatal dexamethasone. Aust N Z J Obstet
Gynaecol. 2016;56:225–232.
67. Everett TR, Chitty LS. Cell-free fetal DNA:
the new tool in fetal medicine. Ultrasound
Obstet Gynecol. 2015;45:499–507.
68. Sillence KA, Roberts LA, Hollands HJ, et al.
Fetal sex and RHD genotyping with digital
PCR demonstrates greater sensitivity than
real-time PCR. Clin Chem. 2015;61:1399–
1407.
69. Jacob RR, Saxena R, Verma IC. Noninvasive
diagnosis of fetal gender: utility of combining
DYS14 and SRY. Genet Test Mol Biomarkers.
2015;19:505–511.
70. Hill M, Compton C, Lewis C, Skirton H,
Chitty LS. Determination of foetal sex in
pregnancies at risk of haemophilia: a qualita-
tive study exploring the clinical practices and
attitudes of health professionals in the United
Kingdom. Haemophilia. 2011;18:575–583.
71. Hill M, Taffinder S, Chitty LS, Morris S.
Incremental cost of non-invasive prenatal
diagnosis versus invasive prenatal diagno-
sis of fetal sex in England. Prenat Diagn.
2011;31:267–273.
72. Lewis C, Hill M, Skirton H, Chitty LS.
Non-invasive prenatal diagnosis for fetal sex
determination: benefits and disadvantages
from the service users’ perspective. Eur J Hum
Genet. 2012;20:1127–1133.
73. Association of Clinical Genetics Science.
Association of Clinical Genetics Science Activity
Audit 2014 -15; 2016. Available from: http:
//www.acgs.uk.com/media/982691/acgsaudit
14_15_reviewedv2a.pdf.
74. Bustamante-Aragones A, Rodriguez de Alba
M, Perlado S, et  al. Non-invasive prenatal
diagnosis of single-gene disorders from mater-
nal blood. Gene. 2012;504:144–149.
75. Jenkins LA, Deans ZC, Lewis C, Allen S.
Delivering an accredited non-invasive pre-
natal diagnosis service for monogenic disor-
ders and recommendations for best practice.
Prenat Diagn. 2018;38(1):44–51.
76. Lewis C, Hill M, Chitty LS. Non-invasive
prenatal diagnosis for single gene disor-
ders: experience of patients. Clin Genet.
2014;85:336–342.
77. Hill M, Compton C, Karunaratna M, Lewis
C, Chitty L. Client views and attitudes to
non-invasive prenatal diagnosis for sickle
cell disease, thalassaemia and cystic fibrosis.
J Genet Couns. 2014;23:1012–1021.
78. Skirton H, Goldsmith L, Chitty LS. An
easy test but a hard decision: ethical issues
concerning non-invasive prenatal testing for
autosomal recessive disorders. Eur J Hum
Genet. 2015;23:1004–1009.
79. Hill M, Suri R, Nash E, Morris S, Chitty LS.
Preferences for prenatal tests for cystic fibro-
sis: a discrete choice experiment to compare
the views of adult patients, carriers of cystic
fibrosis and health professionals. J Clin Med.
2014;3:176–190.
80. Hill M, Karunaratna M, Lewis C, Forya
F, Chitty L. Views and preferences for the
implementation of non-invasive prenatal
diagnosis for single gene disorders from health
professionals in the United Kingdom. Am J
Med Genet A. 2013;161A:1612–1618.
81. Verhoef TI, Hill M, Drury S, et  al. Non-
invasive prenatal diagnosis (NIPD) for single
gene disorders: cost analysis of NIPD and
invasive testing pathways. Prenat Diagn.
2016;36:636–642.
23
Invasive Diagnostic Procedures
ANTHONY O. ODIBO AND GANESH ACHARYA
KEY POINTS
• Amniocentesis is used from 15 weeks of gestation onwards
for prenatal diagnosis of chromosomal abnormalities,
single-gene disorders, fetal lung maturity, fetal infections and
inflammation.
• Chorionic villus sampling (CVS) is used from 10 weeks of ges-
tation onwards for prenatal diagnosis of single-gene defects
and chromosomal abnormalities.
• Early amniocentesis (<15 weeks) and early CVS (<10 weeks)
have been proscribed because of increased risk for fetal loss
rate and structural abnormalities
• The procedure-related fetal loss rates associated with
amniocentesis and first trimester CVS performed by trained
specialists at appropriate gestations appear to be low (∼0.1%
and 0.2%, respectively).
• In equally experienced hands, both the transcervical and the
transabdominal routes for CVS have a similar fetal loss rate.
• The indications for diagnostic fetal blood sampling and other
invasive procedures are now limited because of the availabil-
ity of less invasive or noninvasive methods.
Introduction
Chorionic villus sampling (CVS), amniocentesis and to a lesser
extent fetal blood sampling are the most common invasive diag-
nostic procedures performed prenatally. Amniocentesis was first
introduced in the early 1880s as a therapeutic procedure to treat
polyhydramnios.1,2 The technique evolved over the years to be
used for the assessment of fetal well-being, including monitoring
of Rh-alloimmunised pregnancies.3-6 The use of amniocentesis for
exclusively genetic indications began in the mid-1950s from early
work on fetal sex determination by X-chromatin analysis of amni-
otic fluid cells (AFCs), to several reports of successful diagnosis of a
wide variety of chromosomal and metabolic disorders over the next
few years.7-16a CVS was introduced16b and has been used solely for
genetic diagnosis and remains the invasive procedure of choice in
the first trimester. Fetal blood sampling is used less frequently for
genetic diagnosis (generally after 18 weeks), but its main role is in
confirming the diagnosis fetal anaemia.
This chapter addresses current technique and the safety of genetic
amniocentesis, CVS and fetal blood sampling. Indications and meth-
ods of prenatal diagnosis are considered in detail throughout this
text.17-22 In addition, a brief consideration of the impact of noninvasive
prenatal testing on the role of these diagnostic procedures is included.
Whenever possible, preconception counselling to discuss genetic
risks and available antenatal testing options before pregnancy should
be made available to every couple.17,21,23 When an indication for
an invasive diagnostic test has been identified, the couple must be
informed about the risks associated with such procedures, the accu-
racy and limitations of prenatal diagnosis, the time required before
results become available, technical problems potentially necessitat-
ing a repeat procedure, and the rare possibility of an inability to
make a diagnosis. 
Amniocentesis
Amniocentesis should be performed only by a trained obstetri-
cian who has acquired adequate skill and experience in this pro-
cedure, has the availability of high-quality ultrasonography and
has access to a laboratory with experience in performing prenatal
diagnostic tests.20,24,25 The recommendation that the procedure
should be performed by an obstetrician is not because of techni-
cal difficulty but because the operator must always be prepared to
deal with the potential complications of the procedure. Accord-
ing to the American College of Obstetricians and Gynecologists
(ACOG), if a serious abnormality is detected and the couple
elects to terminate the pregnancy, the obstetrician must either
perform the abortion or refer the family to a provider who will
act on their request.26
Amniocentesis can be performed from about the 15th week
of gestation, when the ratio of viable to nonviable cells is great-
est.27 Early amniocentesis (EA) performed before 14 weeks’ gesta-
tion, and transvaginal amniocentesis is only of historical interest
because of the technical difficulty as well as associated risks of
infection and spontaneous abortion.28
Technique of Amniocentesis
After a thorough ultrasonographic examination, a needle inser-
tion site is selected. Under continuous real-time ultrasound guid-
ance, the needle is inserted to the optimal pocket of amniotic fluid
(AF) while avoiding the fetus. Avoiding needle insertion through
the placenta is desirable but not mandatory. Although Tabor
and coworkers29 reported that transplacental needle insertion
increased the risk for the procedure, this has not been confirmed
by others.30,31
Local anaesthetic is typically not needed.32-35 It is uncertain if
the site of needle placement affects the level of pain.36 Counselling
before amniocentesis should emphasise that the actual pain and
anxiety experienced during the procedure are significantly lower
than expected.37,38
225
226 SE C T I O N 6     Prenatal Screening and Diagnosis
Ultrasound
transducer
Amniotic fluid
Fetus
• Fig. 23.1  Schematic illustration of amniocentesis being performed under
direct, continuous ultrasound scanning (sector transducer, 3.5 MHz).
The maternal skin is cleansed with an iodine- or alcohol-based
solution; sterile drapes are then placed around the needle inser-
tion site to help maintain an aseptic field. A disposable 22-gauge
spinal needle with stylet is most frequently used. During the
entire procedure, the needle tip should be continuously visualised
using two-dimensional real-time ultrasound monitoring. In view
of the extensive anterior placenta, this procedure was performed
transplacentally. Use of four-dimensional ultrasound guidance
has been suggested, but there are no objective data to indicate
improved outcomes39 (Fig. 23.1 and Video 23.1).
After confirming that the needle is in its proper location,
the stylet is removed, and a 10- or 20-cc syringe is attached to
the hub of the needle. The initial 1- to 2-mL sample is usually
discarded. Ten to 20 mL of AF is usually aspirated into ster-
ile disposable plastic syringes, although as little as 3 to 5 mL
of AF has been shown to suffice for reliable prenatal cytoge-
netic results.40,41 Maternal cell contamination appears to occur
more frequently in genetic amniocentesis samples that are
obtained by physicians who perform fewer than 50 genetic
amniocentesis annually.42 Investigators have described using a
vacuum container aspiration technique for amniocentesis,43 but
there appears to be little, if any, advantage over using a syringe
technique.
Amniocentesis had been reported to be unsuccessful with rates
as high as 5.9% to 10.6%,44,45 especially in the era when con-
current ultrasound guidance was not the norm. Because real-time
ultrasound guidance has become routine, failure to obtain AF
occurs far less.46,47 However, this is much more problematic with
EA. When performed at 15 to 16 weeks of gestation by experi-
enced practitioners, failure to obtain AF should occur in fewer
than 1% of cases.24,48-50
Training in performing amniocentesis has traditionally been by
trainees observing experienced operators followed by the trainees
performing the procedure under direct supervision of the mentors.
Modern high-fidelity simulator-based models might be useful for
teaching amniocentesis because trainees’ performance has been
shown to improve with experience on the simulator.51-53 
Amniocentesis in Twins and Higher-Order
Multiple Pregnancies
Amniocentesis can be performed successfully in most twin preg-
nancies perhaps with no increased risk compared with singleton
gestations undergoing amniocentesis,54 although the risk is difficult
to quantify accurately because of the lack of randomised studies.55
Separate amniocentesis of each sac is used in most centres in the
United States to assess individual fetuses irrespective of the chori-
onicity. Each amniotic sac may be identified if the clinician injects
a dye (indigo carmine) immediately after aspiration of the first AF
sample but before withdrawal of the needle. After completion of
the first amniocentesis, a second amniocentesis is performed in
the ultrasonographically located area of the other fetus. Aspiration
of clear AF indicates that the second sac was successfully entered;
aspiration of blue-tinged AF indicates that the original sac was
reentered.56-60 However, this is not necessary to perform routinely
because visualisation of the membranes separating the sacs is gener-
ally possible. Methylene blue dye is proscribed because it has been
associated with high risk for small intestine atresia and fetal death.61
Single-needle insertion under ultrasound guidance to sample
both sacs in twins has been reported.62,63 The main concern is that
the single-puncture technique could lead to cross-contamination
between sacs, resulting in diagnostic inaccuracy. The technique
described by Jeanty and colleagues64 uses a single myometrial
needle puncture into the first amniotic sac and then through the
membranous septum into the second sac. This technique has been
validated by Sebire and coworkers, with no increase in cell contam-
ination between twin fetuses or increased risk for pregnancy loss.65
Using the above techniques, experienced investigators have
been successful in obtaining information regarding both fetuses in
more than 90% to 95%.66-83 The reported loss rates after amnio-
centesis in twins varies from 0.6% to 2.7%.77-88
Amniocentesis has been performed in several triplet pregnancies,
with successful aspiration of fluid from all gestational sacs.82,83,86
Still, data are insufficient to make any statement regarding risks of
amniocentesis in triplet gestations. 
Maternal Risks of Amniocentesis
Life-threatening maternal risks are extremely rare. Amnionitis
occurs in approximately 1 per 1000 women who undergo amnio-
centesis.89-91 This may lead to fetal loss but is seldom life threaten-
ing to the mother.
Minor maternal problems, however, are not rare.44 Approxi-
mately 2% to 3% of women experience transient vaginal spotting
or leakage of AF after amniocentesis. Although almost always lim-
ited in amount and duration, AF leakage could persist and lead to
oligohydramnios and pregnancy loss. Oligohydramnios is a well-
known cause of fetal deformation and pulmonary hypoplasia.92
Uterine contractions or cramping immediately after amniocen-
tesis are not rare. Again, expectant management and reassurance
are generally all that is required. 
Fetal Risks of Amniocentesis
Potential fetal risks include spontaneous abortion, injuries caused by
needle puncture, placental separation, chorioamnionitis, premature

labour and injury caused by the withdrawal of AF (e.g., amniotic
bands). Rare but reported direct needle injuries include ileocuta-
neous fistula, peritoneoparietal fistula, gangrene of an arm, ocular
trauma, ileal atresia, porencephalic cysts, patellar disruption, brain
injuries, peripheral nerve injury and umbilical cord haematoma.93-106
Some of these problems are more logically attributed to amniocen-
tesis than others, and all except a few of these case reports are from
the era before concurrent use of real-time ultrasound guidance.  of 2256 pairs were recruited. There were no significant differences
HIV Transmission After Amniocentesis
Amniocentesis has been associated with an increase in the rate
of vertical transmission of human immunodeficiency virus (HIV)
type 1.107-109 However, with the advent of retroviral chemopro-
phylaxis, the risk for transmission as a result of amniocentesis has
been markedly reduced. Bucceri and coworkers108 reported nine
HIV-infected women who underwent amniocentesis between 16
and 20 weeks of gestation. Six of these women were on chemo-
prophylaxis, and none of 10 infants born to these women were
infected. The International Perinatal HIV Group110 reported
that five of nine HIV-infected women not on chemoprophylaxis
undergoing amniocentesis delivered infected infants, but none
of five infants born to women taking zidovudine were infected.
Recently, a 4-year report from an Italian registry including selected
HIV-infected pregnant women receiving combined antiretroviral
prophylaxis found no cases of vertical transmission after amnio-
centesis or CVS.111 It appears from this limited evidence that
HIV-infected women electing to have amniocentesis may benefit
from chemoprophylaxis.  Development-sponsored multicentre first and second trimes-
Pregnancy Losses After Amniocentesis
Although most spontaneous abortions occur during the first tri-
mester, these may also occur during the second trimester. More-
over, older women are relatively more likely to have a spontaneous
abortion than are younger women.112 Age-related phenomena
could possibly influence frequencies of premature delivery and
other adverse pregnancy outcomes. Thus the only reports to which
any real weight can be attached are those in which participants
undergoing amniocentesis are matched with control participants
not undergoing the procedure, after which the excess fetal loss in
the subject group may thus be assessed. Four major national col-
laborative studies (US, UK, Canadian and Danish studies) of the
risks of amniocentesis have been published.44,45,88,113,114
The UK study reported a higher loss rate, but the participants
were significantly older than the control participants, and age
alone might account for some of the increased fetal losses and
antepartum haemorrhage.88 Indeed, in comparison with the US
and Canadian studies, the British study showed an apparent defi-
cit of fetal loss among the control participants rather than excess
among the participants. Longitudinal studies in the United King-
dom and North America of ultrasonographically monitored preg-
nancies revealed surprisingly few losses in pregnancies that were
viable at 8 to 16 weeks.113,115,116
Tabor and colleagues published results of the only randomised
controlled study in 1986 of amniocentesis performed on 4606
women aged 25 to 34 years who were at low risk for fetal genetic
abnormalities.50 Amniocentesis was performed under real-time
ultrasound guidance with an 18-gauge needle. Thus this was the
first collaborative study of amniocentesis safety that routinely
required ultrasound. The spontaneous abortion rate after 16 weeks
was 1.7% in patients who had undergone amniocentesis compared
CHAPTER 23  Invasive Diagnostic Procedures 227
with 0.7% in control participants (P < .01), with a 2.6-fold rela-
tive risk for spontaneously aborting if the placenta was traversed.
Tongsong and coworkers117 reported a large-scale cohort study
from Thailand in which singleton pregnant women between
15 and 24 weeks of gestation undergoing amniocentesis were
matched prospectively to control participants on a one-to-one
basis for maternal age, parity and socioeconomic status. A total
in fetal loss rates, premature deliveries or placental abruptions
between the two groups (P > 0.5).
Papantoniou and colleagues118 reported a retrospective analysis
of 1006 women undergoing amniocentesis with singleton
pregnancies. Control participants consisted of 4024 women
undergoing amniocentesis and who had no risk factors. In both
groups, amniocentesis was performed between 16 and 18 weeks
of gestation. When cases and control participants were strati-
fied according to maternal age, a statistically significant differ-
ence in the fetal loss rate was observed between women aged 20
to 34 years (2.54%) and women older than 40 years (5.1%).
Women with a history of vaginal bleeding during the current
pregnancy also had a higher fetal loss rate (6.5%) compared
with control participants (2.8%). Women with a history of
previous spontaneous abortions or terminations had a fetal
loss rate of 8% compared with a 2.8% loss rate among control
participants.
In 2006, Eddleman and coworkers reported the procedure-
related fetal loss rate after midtrimester amniocentesis using the
database from the National Institute of Child Health and Human
ter evaluation of risk (FASTER) trial designed to compare first
trimester Down syndrome screening to second trimester screen-
ing.119 Among a total of 35,003 patients who were enrolled in
the FASTER trial, 3096 underwent midtrimester amniocentesis
(study group), and 31,907 did not (control group). The rate of
fetal loss before 24 weeks’ gestation was compared between the two
groups, and multiple logistic regression analysis was used to adjust
for potential confounders. The spontaneous fetal loss rates were
1.0% in the amniocentesis group and 0.94% in the no amniocen-
tesis group. The difference between these groups was not signifi-
cant (P = .74; 95% confidence interval (CI) –0.26% to 0.49%).
Several studies have subsequently reported lower loss rates, thereby
validating the findings of the FASTER trial group.120-124
Overall, we can conclude that the conventionally stated preg-
nancy loss rate of 0.5% is no longer appropriate in experienced
hands. Surely, it is illogical to counsel the same 0.5% risk offered
a quarter century ago when ultrasound was not available. In sup-
port, Armstrong and coworkers125 followed the outcome after
28,613 procedures performed by obstetricians throughout the
United States, mostly for advanced maternal age. The total loss
rate (combined background plus procedure related) was only 1 in
362. In comparing 11,746 women undergoing genetic midtrimes-
ter amniocentesis and 39,811 women who did not have invasive
procedures over a 16-year period, Odibo and coworkers126 con-
cluded that the fetal loss rate attributable to amniocentesis was
0.13%, or 1 in 769.
Recently, Akolekar et al performed a systematic review of stud-
ies published after 2000 that had included at least 1000 proce-
dures and had a control group and reported a pooled risk for fetal
loss of 0.11% (95% CI, –0.04% to 0.26%) after amniocentesis
before 24 weeks.127 When limited to studies published over the
past 10 years, the procedure-related loss rate after amniocentesis is
0.16% (95% CI, –0.57% to 0.51%). See Table 23.1. 
228 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE
23.1 Summary of Contemporary Amniocentesis Studies Between 2006 and 2016
Procedure-Related Loss Rate
Study (Year) Amniocentesis Loss Rate (% (95% CI)) Control Group Loss Rate (% (95% CI)) (% (95% CI))
Eddleman et al.119 (2006) 1.00 (0.68–1.42) 0.94 (0.84–1.05) 0.06 (–0.30–0.42)
Caughey et al.120 (2006) 0.83 (0.73–0.94) — —
Towner et al.121 (2007) 0.46 (0.36–0.58) 0.53 (0.52–0.65) −0.07(–0.22–0.09)
Odibo et al.126 (2008) 0.97 (0.80–1.16) 0.85 (0.76–0.94) 0.12 (–0.07–0.31)
Tabor et al.122 (2009) 1.39 (1.27–1.52) 0.90 (0.88–0.92) 0.49 (0.39–0.60)
Pitukkijronnakorn et al.123 (2011) 0.37 (0.18–0.66) 0.20 (0.04–0.59) 0.17 (–0.18–0.51)
Corrado et al.124 (2012) 1.00 (0.68–1.43) 0.82 (0.22–2.09) 0.18 (–0.77–1.13)
Pooled loss rate 0.86 (0.67–1.10) 0.70 (0.54–1.04) 0.16 (-0.57–0.51)

CI, Confidence interval.

Modified from Akolekar R, Beta J, Picciarelli G, et al. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet
Gynecol 45(1):16–26, 2015.
  

Early Amniocentesis allowed routine obstetric management.157,158 Because these are

Early amniocentesis was explored to obviate the inconvenience for
patients of having to be rescheduled if they presented for CVS but
were determined to be beyond 14 weeks of gestation but earlier
than 15 weeks of gestation.
The technique for EA is essentially the same as for traditional
amniocentesis, except that a smaller volume of AF is with-
drawn. One limitation of EA was a higher prevalence of tent-
ing of the membrane and dry tap. The earlier in gestation one
attempts amniocentesis, the more problematic membrane tent-
ing becomes, given incomplete fusion of the chorion and the
amnion.128 Tenting of the membranes is seen in about 10% of
EA procedures.
The use of EA gained popularity in the late 1980s, but several
studies over the next decade highlighted the complications associ-
ated with the procedure.129-150
Based on the data showing that EA results in significantly
higher rates of pregnancy loss and complications than performing
traditional amniocentesis, the ACOG has recommended that EA
(<14 weeks’ gestation) should not be performed.151  Chorionic villus sampling is typically performed between 10 and
Third Trimester Amniocentesis
Amniocentesis in the third trimester of pregnancy has been mostly
performed to document fetal lung maturity. It can also be indi-
cated for fetal anomalies detected after the typical second trimester
screening window, and these tend to pose little or no risk for fetal
loss. The technique is similar to that used for diagnostic amnio-
centesis in the second-trimester. The challenge is with finding an
adequate pocket of AF that is free from umbilical cord or fetal
parts to tap.152-154 Reports of neonatal complications even in the
presence of documented positive test results for fetal lung matu-
rity has limited the role of amniocentesis for this indication.155,156
Third trimester amniocentesis has also been reported in small
series to be useful for the diagnosis of inherited bleeding disor-
ders before delivery. Bleeding disorders such as moderate to severe
haemophilia A and B and type 3 von Willebrand disease can confer
an increased risk for bleeding during delivery. For affected cases,
restrictive birth plans are implemented, and unaffected cases are
still based on small case series, larger studies are needed to confirm
the safety and reliability of this approach. 
Chorionic Villus Sampling
Chorionic villus sampling was formally introduced in the 1980s
and has become established as the prenatal diagnostic procedure
in the first trimester after an early feasibility report in 1968 by
Mohr.16b The indications for CVS are similar to those discussed
for amniocentesis. CVS also offers the advantage of early and
rapid diagnosis for pregnancies at high-risk (e.g., 25%–50%
risk) for certain genetic disorders. In these scenarios, rapid
DNA testing can be performed on uncultured DNA cells and
the couple offered the option of early pregnancy termination if
affected.
Technique
14 weeks’ gestation. However, CVS can be performed at much
later gestational ages if amniocentesis or other testing is not pos-
sible (e.g., with oligohydramnios). The drawback with late CVS
is a higher rate of culture failure of the villi. Reports of possible
association between CVS performed earlier than 10 weeks and
fetal limb constriction and other anomalies led to proscription of
the procedure before this gestational age.159
Chorionic villus sampling can be performed through trans-
abdominal and transcervical routes (Figs. 23.2 and 23.3 and
Video 23.2). There is no evidence that one route is safer or more
reliable than the other.160 Operator preference and position of
the placenta are the most influential factors regarding the route
chosen for CVS. In a high anterior or fundal location of the
placenta, a transabdominal route is preferred; in a posterior loca-
tion, a transcervical route is optimal.161,162
In the transabdominal technique, the ideal site exposing the
longest axis of the placenta is identified under ultrasound guid-
ance. The skin is disinfected with iodine- or alcohol-based solu-
tion, and ideally, a local anaesthetic is given (when a needle larger

A B
• Fig. 23.2  Chorionic villus sampling being performed. A, Transcervical route. B, Transabdominal route.
FR 27Hz
R1
M5
Z 1.4
2D
33%
C 52
P Low
Res
x 5
• Fig. 23.3  Sonographically guided transcervical chorionic villus sampling.
The catheter with an intact guidewire can be seen as an echogenic line
within the posteriorly located placenta.
than 20 gauge is used). We prefer the double-needle technique.
This involves using an 18-gauge needle as a trocar through
which a smaller gauge needle (20 or 21 gauge) is inserted into
the placenta. A 20-cc syringe containing Roswell Park collec-
tion medium mixed with a small concentration of heparin is
attached to the end of the needle, and a negative pressure is
created. The needle is moved up and down through the pla-
centa several times while maintaining the negative pressure. On
removal, the sample is emptied unto a petri dish and exam-
ined for the presence of a sufficient amount of chorionic villi.
With the double-needle technique, multiple passes at the pla-
centa can be made without reinsertion through the uterine wall.
Some operators, however, report good results using a single-
needle technique.
For the transcervical route, the patient is placed in a lithotomy
position. Then a sterile speculum is introduced to expose and
cleanse the cervix with iodine solution. In our centre, we generally
do not use a tenaculum to steady the cervix, but in rare situa-
tions, this may be needed. Under ultrasound guidance, a 16-gauge
catheter with a malleable guidewire is inserted in the region of
the trophoblast. The guidewire is then removed, a 20-cc syringe
containing heparinised medium is attached to the end of the cath-
eter and a negative pressure created. The catheter is withdrawn
slowly and the sample transferred to a petri dish and examined
for adequacy of villi concentration. Transcervical CVS can also
be performed using especially designed small biopsy forceps.163
This technique may be associated with less pain and less failure to
obtain adequate sample.164  their experience with similar loss rates.181-188 The technique
CHAPTER 23  Invasive Diagnostic Procedures 229
Complications of Chorionic Villus Sampling
Overall, CVS is considered safe, but as with most invasive proce-
dures, there is a risk for potential complications. Vaginal bleeding
is rare with transabdominal CVS but may occur in 7% to 10% of
cases of transcervical CVS. Other complications of CVS include
chorioamnionitis (incidence <1 per 1000 cases), acute rupture of
membranes, oligohydramnios (0.3%), preterm rupture of mem-
branes and preterm labour.165 Previous suggestions associating
CVS with hypertensive disorders of pregnancy have not been con-
firmed by more recent studies.166-170
In experienced hands, the procedure-related loss rate after CVS
is low. It was reported to be 0.22% (95% CI, –0.71% to 1.16%)
by a recent systematic review and similar to that for amniocente-
sis.127,145 The Canadian Collaborative experience showed no sig-
nificant difference in loss rates: 7.6% in the amniocentesis group
compared with 7% in the CVS group.171 Similarly, the US collab-
orative group reported no significant difference in loss rates between
amniocentesis and CVS.172 In contrast to these studies, the Medi-
cal Research Council (MRC) working party on the evaluation of
CVS reported a 4.6% higher loss rate after CVS compared with
amniocentesis.173 The most commonly reported rates of loss from
the time of CVS up to 28 weeks’ gestation is between 2% and 3%,
but a recent report suggests that the rate may be much lower.160,174
It is also important to consider the higher background pregnancy
loss rate in the first trimester that may not be related to the proce-
dure. Selected studies on loss rates from CVS are summarised on
Table 23.2.174-176 The overall loss rate from the systematic review
by Akolekar and associates is 0.22 (95% CI, –0.71 to 1.16). These
studies have the common limitation that they are not randomised.
The CVS loss rate does not appear to be significantly affected
by the route of the procedure. Table 23.3 summarises four studies
comparing transabdominal to transcervical CVS.177-179 When all
studies are pooled, the loss rate is not significantly different irre-
spective of route.179 
Safety of Chorionic Villus Sampling in Multiple
Pregnancies
Chorionic villus sampling can be performed successfully in mul-
tiple gestations by experienced operators. Wapner and associates
reported a 6-year experience with the successful performance of
CVS on 81 set of twins with an overall pregnancy loss rate of
3.2% before 28 weeks’ gestation.180 Other groups have reported
230 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE
23.2 Summary of Contemporary Chorionic Villus Sampling (CVS) Studies Between 2005 and 2016
Study (Year) CVS Loss Rate (% (95% CI)) Control Group Loss Rate (% (95% CI)) Procedure-Related Loss Rate (% (95% CI))
Lau et al.175 (2005) 1.85 (1.20–2.71) 1.16 (0.62–1.97) 0.69 (–0.29–1.67)
Odibo et al.174 (2008) 2.68 (2.26–3.16) 3.35 (2.86–3.90) −0.67(–1.35–0.01)
Akolekar et al.176 (2011) 1.84 (1.34–2.46) 1.14 (1.03–1.27) 0.69 (0.24–1.15)
Pooled loss rate 2.18 (1.61–2.82) 1.79 (0.61–3.58) 0.22 (–0.71–1.16)

CI, Confidence interval.

Modified from Akolekar R, Beta J, Picciarelli G, et al. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet
Gynecol 45(1):16–26, 2015.
  

TABLE Fetal Loss Rates (<28 Weeks’ Gestation) From the fetus with a resultant abnormality confined to the placenta.
23.3
Trials Comparing Route of Chorionic Villus There is potential for this to occur because only few of the cells
constituting the inner cell mass in early embryonic period eventu-
Sampling
ally become part of the fetus. The rest develop into extraembryonic
TC-CVS TA-CVS tissues with potential for trisomies confined to these tissues. The
Loss Loss Relative Risk mosaicism tends to be confined within the trophoblast because of
Study (Year) Rate (%) Rate (%) (95% CI) two mechanisms, postzygotic nondisjunction within the placenta
or trisomic rescue in the fetus.192
Bovicelli et al.177 (1986) 3.3 3.3 1.0 (0.15–6.87) Confined placental mosaicism occurs in 1.3% of CVS proce-
Brambati et al.178 (1991) 7.9 7.4 1.07 (0.72–1.58) dures.193 Although follow-up procedures such as amniocentesis
or fetal blood sampling may be needed to confirm the diagnosis,
Smidt-Jensen et al.179 8.2 3.0 2.72 (1.82–4.07)
(1992)
CPM may also be a marker for a pregnancy that needs closer fol-
low-up for risk for intrauterine growth restriction, perinatal death
Pooled loss rate 7.9 4.6 1.72 (0.79–3.58) or uniparental disomy. 
CI, Confidence interval; TA-CVS, transabdominal chorionic villus sampling; TC-CVS, transcer-
vical chorionic villus sampling. Fetal Limb-Reduction Abnormalities and
   Chorionic Villus Sampling
The possibility of an association between CVS and limb-reduction
is similar to that used for singletons, and both transabdominal defects has been the subject of many reports. Following the first
and transcervical approaches are safe. Sometimes a combination report by Firth and colleagues of four infants with oromandibular-
of both approaches may be used depending on the location of limb hypogenesis and one with terminal transverse limb reduc-
the placentas. With monochorionic twins, many operators would tion defect after CVS,194 similar case series were reported by other
sample only one fetus, and although a heterokaryotypic genotype groups. These reports also indicated that the complication appears
is possible, this is so rare and does not warrant routine sampling of confined to CVS procedures performed before 70 days of gesta-
both. With dichorionic twins, both fetuses must be sampled, and tion.195,196 However, a large World Health Organisation regis-
the increased risks quoted earlier may apply more to this type of try of more than 200,000 CVS procedures found no significant
placentation. A recent systematic review of loss rates from CVS in association between the procedure and limb-reduction defects.197
multiple pregnancies found no randomised trial to evaluate and Given the controversy regarding this association, it is prudent to
from a summary of pooled studies, a loss rate of 2.75% (95% inform women requesting CVS of these reports and that if such a
CI, 1.28–4.75) before 20 weeks’ gestation and 3.44% (95% CI, risk exists, it is less than 1 in 3000 procedures and not reported for
1.67–5.81) before 28 weeks’ gestation.189  procedures performed after 70 days’ gestation.198 

Laboratory Aspects of Chorionic Villus Sampling Fetal Blood Sampling

Chorionic villi have three major components: syncytiotrophoblasts,
cytotrophoblasts and an inner mesodermal layer that contains fetal
capillaries. These components arise from multiple sources with the
potential to yield confounding results. These issues were problem-
atic in the early period of the CVS technique.190 The US collab-
orative study reported only a 1.1% incidence of needing another
confirmatory test with the most common indications being labo-
ratory failure, maternal cell contamination and confined placental
mosaicism (CPM).191 This has become a rare occurrence because
of improved laboratory techniques. CPM occurs when there is a
discrepancy in the cytogenetic material between the placenta and
Fetal blood sampling is most often used for rapid fetal karyotyp-
ing, evaluation of fetal haematologic disorders, identification of
fetal infection (by culture or molecular typing), drug therapy and
the diagnosis and treatment of fetal anaemia by transfusion.
When fetal blood is withdrawn from the umbilical cord, the
procedure may be referred to in several ways, as fetal blood sam-
pling, percutaneous umbilical blood sampling (PUBS), funicente-
sis or cordocentesis.
Percutaneous umbilical blood sampling for fetal blood chro-
mosome analysis has been used to help clarify purported chromo-
somal mosaicism detected in cultured AF cells, chorionic villi or
 CHAPTER 23  Invasive Diagnostic Procedures 231

both.199 Rapid assessment of fetal chromosome complement has

been accomplished by “direct” cytogenetic analysis of nonculti-
vated nucleated blood cells.200 In addition, some fetal abnormali-
ties do not become apparent until later in pregnancy, and in such
instances, rapid results may prove useful for decision making with
regard to obstetric management and mode of delivery.201,202

Indications for Fetal Blood Sampling

Fetal blood sampling was once used for the prenatal evaluation
of many fetal haematologic and biochemical abnormalities.203-205
Fetal haematocrit can be directly measured to assess fetal hae-
molysis resulting from Rh or other antigen incompatibility and
alloimmunisation states.206-208 Fetal haemoglobin can be directly
evaluated to diagnose sickle cell disease, α- or β-thalassemias or
other haemoglobinopathies.209 However, these disorders can now
also be correctly identified by using DNA analysis of chorionic
villi or AF cells. Fetal blood sampling can also be used to assess • Fig. 23.4  Fetal blood sampling at the umbilical cord insertion site.
platelet quantity and quality of function.210,211
Fetal blood has also been used for the diagnosis of various
coagulation factor abnormalities in fetuses, such as haemophilia
A, haemophilia B and von Willebrand disease.205,212 In addition
to haematologic studies, fetal blood samples have been used to
diagnose autosomal recessive or X-linked immunologic deficien-
cies, including severe combined immunodeficiency (SCID), Ché-
diak-Higashi syndrome, Wiskott-Aldrich syndrome and chronic
granulomatous disease.213-216
Recovery of fetal blood permits assessment of viral, bacterial
and parasitic infections of the fetus. Serum fetal blood titres per-
mit quantification of antibody titres.217,218 In addition to anti-
body titres, PUBS can be used for direct analysis of viral, bacterial
and parasitic infections by culture of or molecular amplification of
vector-specific DNA sequences in fetal blood.218-223 

Technique of Fetal Blood Sampling

Fetal blood sampling is typically performed under continuous
real-time ultrasound guidance from 18 weeks of gestation onward.
However, successful procedures have been reported as early as
12 weeks of gestation.224-226 Maternal sedation is usually unneces-
sary, but when a prolonged procedure is anticipated (e.g., with
fetal blood transfusion), injection of 1% lidocaine to the local site
may be of benefit, and occasionally, fetal neuromuscular block-
ade with pancuronium or atracurium can be considered to abolish
fetal movements temporarily.
A sterile field is established by cleansing the skin with an iodine-
based solution or alcohol and sterile drapes are applied. Most
commonly, two-dimensional ultrasonographic needle guidance is
used. Although some have suggested using four-dimensional nee-
dle guidance, there is no evidence that this newer technology is an
improvement over two-dimensional visualisation.39,227
There are several potential sampling sites. Because of its fixed
position, the umbilical cord insertion site to the placenta is usu-
ally the site of choice whenever it is clearly visible and accessible.
Other possibilities are a free loop of the umbilical cord or the fetal
intrahepatic umbilical vein (Figs. 23.4 and 23.5).203,225,226 Many
practitioners prefer using a 22-gauge, acute-angle echo-enhanced
needle, but others have advocated even smaller gauged needles (e.g.,
25 gauge).228 The amount of blood aspirated for diagnosis depends
on the indication for diagnosis by PUBS but rarely exceeds 5 cc.
On completion of the fetal blood sampling procedure, the
needle is withdrawn, and an ultrasound examination is performed
• Fig. 23.5  Fetal blood sampling via the intrahepatic vein.
to evaluate fetal condition. All women at risk for Rh isoimmunisa-
tion should receive 300 mg of Rh immunoglobulin (RhIG) after
the procedure. 
Safety of Fetal Blood Sampling
Maternal complications from PUBS are rare but include amnionitis
and transplacental haemorrhage.212,229 Data from large perinatal
centres estimate the fetal risks of death in utero or subsequent spon-
taneous abortion to be 3% or less after PUBS.225-234 Studies directly
comparing loss rates in control and treated groups have been pub-
lished, but none of these are randomised. The only case control
study is by Tongsong and associates,209 who followed 1281 women
undergoing freehand cordocentesis between 16 and 24 weeks’ gesta-
tion. Women with no overt fetal anomalies (and thus not requiring
a procedure) served as control participants. Indications for PUBS
were increased risk for thalassemia (61%), rapid karyotyping (21%)
or both (8.7%). Loss rates were 3.2% (PUBS group) versus 1.8%
(control participants) with no differences in obstetric complications.
232 SE C T I O N 6     Prenatal Screening and Diagnosis
These studies have a common confounding that baseline loss
rates for patients undergoing PUBS or fetal blood sampling vary
greatly with the indication of the procedure.235 Loss rates are far
greater for fetuses with ultrasound-detected anomalies than for
fetuses evaluated for haemolytic diseases secondary to maternal
blood group sensitisation for late booking or for clarification of
mosaicism at amniocentesis. Overall, procedure-related loss rates
of 1% to 1.5 %, should be assumed.
The relationship between fetomaternal transfusion and preg-
nancy outcome was studied.236 Despite a positive correlation
between fetomaternal haemorrhage and bleeding time, there was
no association between the degree of fetomaternal transfusion and
pregnancy outcome. Others also showed that PUBS is frequently
associated with fetomaternal haemorrhage, which in turn was cor-
related with anterior position of the placenta, duration of the pro-
cedure and number of needle insertions.237,238
Finally, in a retrospective analysis of 59 PUBS procedures per-
formed in 30 multiple pregnancies (29 twins and 1 triplet) at a
gestational age of 19.5 ± 1.6 weeks, Tongprasert and colleagues239
reported a 98.3% sampling success rate. In cases of continuing
pregnancy, the total fetal loss rate was 10.5%; however, there were
no fetal losses within 2 weeks of the procedure. There is therefore
insufficient data to give a reliable procedure-related loss rate for
PUBS in twins.  reasons, but the few low- or mixed-risk studies published suggest
Rh-Alloimmunisation After Invasive Diagnostic
Procedures
There is an increased risk for fetomaternal transfusion after amnio-
centesis, CVS and fetal blood sampling, which might have an
immunising effect. However, the magnitude of this putative risk
has not been determined. Nonetheless, Rh sensitisation after sec-
ond trimester amniocentesis has clearly been observed.240-242 Thus
there is a rationale for administering RhIG to Rh-negative women
after genetic amniocentesis to prevent sensitisation. Indeed, Khalil
and coworkers243 reported only one sensitisation among 300
(0.3%) at-risk women who received 300 mg of RhIG after amnio-
centesis. By contrast, among 615 Rh-negative women at risk for
sensitisation who did not receive RhIG after amniocentesis, Gol-
bus and coworkers241 reported that 12 (2.1%) became sensitised.
Similarly, an increase in alpha-fetoprotein and fetal red blood
cells in maternal circulation has been demonstrated after CVS and
fetal blood sampling.244,245 Therefore virtually all operators advo-
cate routine use of RhIG after invasive procedures. However, the
dose to be administered remains controversial. The ACOG246 cur-
rently recommends a 300-mg dose of RhIG after second trimester
amniocentesis.  able to busy clinicians to adequately review the available options
Effect of Noninvasive Prenatal Testing on
Diagnostic Procedures
The risk for pregnancy loss after prenatal diagnostic testing resulted
in interest in isolating fetal DNA from maternal serum. After the
discovery by Lo and coworkers that pregnant women have cell-
free DNA (cfDNA) in their plasma,247 several studies have been
published confirming this finding and also that the proportion of
cell-free fetal fraction can vary from 3% to 20% of maternal cir-
culation.248,249 The initial challenges posed by the relatively small
concentration of cfDNA in maternal plasma has been circum-
vented because of advances in molecular biology and sequencing
technology.
Rapid advances in the field of noninvasive prenatal testing
(NIPT) have occurred, resulting in the publication of results from
several clinical trials.250-254 There are significant differences in the
approach used in these trials that are important considerations
when evaluating the potential impact on the prenatal diagnostic
procedures. The study designs vary from retrospective cohorts
to case-control to prospective approaches. In addition, the algo-
rithms for generating results vary from z-score based systems to
others incorporating both maternal and gestational ages.255 The
majority of these studies are, however, from high-risk populations.
The advent of cell-free fetal DNA (cffDNA) in maternal blood
has allowed near-diagnostic ability for fetal Down syndrome. It
is, however, important to highlight some of the current limita-
tions of NIPT. As mentioned earlier, the majority of published
studies enrolled women at high risk for aneuploid for pragmatic
that NIPT may be equally effective in these populations.252,256,257
As has been emphasised by many professional bodies, these and
other limitations of NIPT necessitate labelling it a good screen-
ing test and not a diagnostic test.258-260 It is therefore important
to emphasise that the current data and reports on the efficiency
of cffDNA require a confirmatory cytogenetic testing for posi-
tive NIPT results because false-positive cases are beginning to be
reported in the literature.261 Furthermore, with the availability
of newer cytogenetic techniques that can detect subtle chromo-
somal abnormalities, indications for CVS and amniocentesis are
expanding. There is, however, clear evidence that the introduction
of NIPT has resulted in a substantial decline in the demand for
invasive procedures.262,263 The complexity of choices available to
women currently underlies the necessity for adequate counselling
before diagnostic prenatal testing.264 
Conclusion
Several options for prenatal diagnostic procedures are now avail-
able to women. The increased availability of screening options has
also created a complex environment for women to make informed
choices. The situation is compounded by insufficient time avail-
and their associated complications. This calls for judicious use of
genetic counsellors to bridge the existing gap and provide better
guidance for these anxious women.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 24. Gerbie AB, Elias S. Technique for midtri-
mester amniocentesis for prenatal diagnosis.
44. NICHD National Registry for Amniocente-
sis Study Group. Midtrimester amniocente-
Semin Perinatol. 1980;4:159. sis for prenatal diagnosis: safety and accuracy.
1. Lambl D. Ein seltener fall von hydramnios.
25. Gerbie AB, Elias S. Amniocentesis for ante- JAMA. 1976;236:1471.
Zentralbl Gynaekol. 1881;5:329.
natal diagnosis of genetic defects. Clin Obstet 45.  Simpson NE, Dallaire L, Miller JR, et al. Pre-
2. Schatz F. Eine besondere art von ein seitiger
Gynecol. 1980;7:5. natal diagnosis of genetic disease in Canada:
polyhdramnic mit anderseitiger oligohydram-
26. American College of Obstetricians and Gyne- report of a collaborative study. Can Med Assoc
nie bei zwillingen. Arch Gynecol. 1882;19:392.
cologists. The limits of conscientious refusal in J. 1976;15:739.
3. Menees TD, Miller JD, Holly LE. Amniog-
reproductive medicine. ACOG Committee 46.  Finberg HJ, Frigoletto FD. Sonographic dem-
raphy: preliminary report. Am J Roentgenol.
Opinion No 385. Obstet Gynecol. 2007;110(5): onstration of uterine contraction during amnio-
1930;24:363.
1203–1208. centesis. Am J Obstet Gynecol. 1981;139:740.
4. Aburel ME. Le declenchement du travail
27. Emery AEH. Antenatal diagnosis of genetic 47. Platt LD, Devore GR, Gim OV, et al. Failed
par injections intraamniotique de serum sale
disease. Mod Trends Hum Genet. 1970;1:267. amniocentesis: the role of membrane tenting.
hypertonique. Gynecol Obstet. 1937;36:398.
28.  Scrimegeour JB. Amniocentesis: technique Am J Obstet Gynecol. 1982;144:479.
5. Gadow EC. Reaching the fetal environment:
and complications. In: Emery AEH, ed. Ante- 48. Golbus MS, Loughman WD, Epstein CJ,
a tribute to Dr. Hermógenes Alvarez. Prenat
natal Diagnosis of Genetic Disease. Baltimore, et  al. Prenatal diagnosis in 3000 amniocen-
Diagn. 1998;18:870.
MD: Williams and Wilkins; 1973:11. teses. N Engl J Med. 1979;300:157.
6. Bevis DCA. The antenatal prediction of
29. Tabor A, Philip J, Bang J, et al. Needle size 49. Romero R, Jeanty P, Reece EA, et al. Sono-
haemolytic disease of the newborn. Lancet.
and risk of miscarriage after amniocentesis. graphically monitored amniocentesis to
1952;1:395.
Lancet. 1988;1:183. decrease intraoperative complications. Obstet
7. Fuchs F, Riis P. Antenatal sex determination.
30.  Tharmaratnam S, Sadek S, Steele EK, et al. Trans- Gynecol. 1985;65:426.
Nature. 1956;117:330.
placental early amniocentesis and pregnancy 50. Tabor A, Philip J, Madsen MI, et  al. Ran-
8. Shettles LB. Nuclear morphology of cells in
outcome. Br J Obstet Gynaecol. 1998;105:228. domized controlled trial of genetic amnio-
human amniotic fluid in relation to sex of
31. Bombard AT, Powers JF, Carter SM, et  al. centesis in 4606 low-risk women. Lancet.
infant. Am J Obstet Gynecol. 1956;71:834.
Procedure related fetal losses in transplacen- 1986;1:1287.
9. Makowski EL, Prem K, Kaiser IH. Detec-
tal versus non-transplacental genetic amnio- 51. Pittini R, Oepkes D, Macrury K, et al. Teach-
tion of sex of fetuses by the incidence of sex
centesis. Am J Obstet Gynecol. 1995;172:868. ing invasive perinatal procedures: assessment
chromatin in nuclei of cells in amniotic fluid.
32. Gordon M, Ventura-Braswell A, Higby K, of a high fidelity simulator-based curriculum.
Science. 1956;123:542.
Ward J. Does local anesthesia decrease pain Ultrasound Obstet Gynecol. 2002;19:478.
10. Steele MW, Breg Jr WR. Chromosome anal-
perception in women undergoing amniocen- 52. Forest C, Comas O, Vaysiere C, et al. Ultra-
ysis of human amniotic fluid cells. Lancet.
tesis? Am J Obstet Gynecol. 2007;196:55. sound and needle insertion simulators built
1966;1:383.
33. Van Schoubroeck D, Verhaeghe J. Does local on real patient-based data. In: Westwood JD,
11. Jacobson CB, Barter RH. Intrauterine diag-
anesthesia at mid-trimester amniocentesis Hoffman HM, eds. Medicine Meets Virtual
nosis and management of genetic defects. Am
decrease pain experience? A randomized trial Reality 14: Accelerating Change in Healthcare.
J Obstet Gynecol. 1967;99:795.
in 220 patients. Ultrasound Obstet Gynecol. IOS Press; 2007:136.
12. Valenti C, Schutta EJ, Kehaty T. Prena-
2000;16:536. 53. Zubair I, Marcotte MP, Weinstein L, Brost
tal diagnosis of Down’s syndrome. Lancet.
34.  Pongrojpaw D, Somprasit C, Chanthasenanont BC. A novel amniocentesis model for learn-
1968;2:220.
A. The efficacy of lidocaine-prilocaine cream to ing stereotactic skills. Am J Obstet Gynecol.
13.  Nadler HL. Antenatal detection of hereditary
reduce pain in genetic amniocentesis. J Med 2006;194:846.
disorders. Pediatrics. 1968;42:912.
Assoc Thai. 2007;10:1992. 54. Anderson RL, Goldberg JD. Prenatal diag-
14. Milunsky A. The Prenatal Diagnosis of Hered-
35. Wax JR, Pinette MG, Carpenter M, et  al. nosis in multiple gestations: 20 years’ expe-
itary Disorders. Springfield, IL: Charles C.
Reducing pain with genetic amniocentesis— rience with amniocentesis. Prenat Diagn.
Thomas; 1973.
a randomized trial of subfreezing versus room 1991;11:263.
15. Nadler HL, Gerbie AB. Role of amniocen-
temperature needles. J Matern Fetal Neonatal 55. Agrawal K, Alfirevic Z. Pregnancy loss
tesis in the intrauterine detection of genetic
Med. 2005;18:221. after chorionic villus sampling and genetic
disorders. N Engl J Med. 1970;282:596.
36. Lekskul N, Tannirandorn Y. The loca- amniocentesis in twin pregnancies: a sys-
16a. Littlefield JW. The pregnancy at risk for a
tion of needle insertion effect on maternal tematic review. Ultrasound Obstet Gynecol.
genetic disorder. N Engl J Med. 1970;282:627.
pain in amniocentesis. J Med Assoc Thai. 2012;40(2):128–134.
16b. Mohr J. Foetal genetic diagnosis: development of
2006;89(suppl 4):137. 56. Henrion R, Papa F, Rouvillois JL, et  al.
techniques for early sampling of foetal cells. Acta
37. Ferber A, Onyeije CI, Zelop CM, et  al. L’amniocentese precoce en cas de grossesse
Pathol Microbiol Scand. 1968;73(1):73–77.
Maternal pain and anxiety in genetic amnio- gemellaire. Nouv Press Med. 1978;7:4119.
17.  Elias S, Annas GJ. Reproductive Genetics and the
centesis: expectation versus reality. Ultra- 57. Benacerraf BR, Frigoletto FD. Amniocen-
Law. Chicago: Year Book; 1987.
sound Obstet Gynecol. 2002;19:13. tesis under continuous ultrasound guid-
18.  Simpson JL, Elias S. Prenatal diagnosis of
38. Karasahin E, Gungor S, Goktolga U, et  al. ance: a series of 232 cases. Obstet Gynecol.
genetic disorders. In: Creasy RK, Resnik R, eds.
Anticipated and perceived pain from midtri- 1983;62:760.
Maternal-fetal Medicine: Principles and Practice.
mester amniocentesis. Int J Gynecol Obstet. 58.  Williamson RA, Vamer MW, Grant SS.
2nd ed. Philadelphia: WB Saunders; 1989:78.
2008;101:290. Reduction in amniocentesis risks using a real-
19.  Farrell PM, Elias S. Prenatal diagnosis and
39. Dolkart L, Harter M, Snyder M. Four- time needle guide procedure. Obstet Gynecol.
neonatal screening. In: Gilbert-Barness E, ed.
dimensional ultrasonographic guidance for 1985;65:751.
Potter’s Pathology of the Fetus, Infant and Child.
invasive obstetric procedures. J Ultrasound 59.  Cruikshank DP, Vamer MW, Cruikshank JE,
2nd ed. Philadelphia: Elsevier; 2007:611.
Med. 2005;24:1261. et al. Midtrimester amniocentesis: an analysis
20.  Simpson JL, Elias S. Genetics in Obstetrics and
40. Sikkema-Raddatz B, van Echten J, van der of 923 cases with neonatal follow-up. Am J
Gynecology. 3rd ed. Philadelphia: WB Saun-
Vlag J, et  al. Minimal volume of amniotic Obstet Gynecol. 1983;146:204.
ders; 2003.
fluid for reliable prenatal cytogenetic diagno- 60. Elias S, Gerbie A, Simpson JL. Genetic
21.  Shulman LP, Elias S. Techniques for prena-
sis. Prenat Diagn. 2002;22:164. amniocentesis in twin gestations. Am J Obstet
tal diagnosis. In: Rimoin DL, Connor JM,
41.  Guven M, Ceylaner G, Coskun A. Volume of Gynecol. 1980;138:169.
Pyeritz RE, Korf BR, eds. Emory and Rimoin’s
sampled amniotic fluid and prenatal cytogenetic 61. Kidd SA, Lancaster PAL, Anderson JC,
Principles and Practice of Medical Genetics. 5th
diagnosis. Int J Gynecol Obstet. 2006;95:157. et  al. Fetal death after exposure to methy-
ed. Philadelphia: Elsevier; 2007:679.
42. Welch RA, Salem-Elgharib S, Wiktor A, lene blue dye during mid-trimester amnio-
22. Shulman LP, Simpson JL, Elias S. Invasive
et al. Operator experience and sample quality centesis in twin pregnancy. Prenat Diagn.
prenatal genetic techniques. In: Sciarra JJ,
in genetic amniocentesis. Am J Obstet Gyne- 1996;16:39.
ed. Gynecology and Obstetrics. vol. 3. Phila-
col. 2006;194:189. 62. Van Vugt JM, Nieuwint A, van Geijn HP.
delphia: JB Lippincott; 1992:1.
43.  Borrell A, Borobio V, Hernandez S, et  al. Single-needle insertion: n alternative tech-
23. Elias S. Prenatal diagnosis of genetic disor-
Vacuum container aspiration as a new tech- nique for early second trimester genetic
ders. In: Givens JR, ed. Endocrinology of Preg-
nique for genetic amniocentesis. Prenat Diagn. twin amniocentesis. Fetal Diagn Ther.
nancy. Chicago: Year Book; 1980:327.
2008;28:962. 1995;10:178.

232.e1
232.e2 References
63. Buscaglia M, Ghisoni L, Bellotti M, et  al.
Genetic amniocentesis in biamniotic twin
pregnancies by a single insertion of the nee-
dle. Prenat Diagn. 1995;15:17.
64. Jeanty P, Shah D, Roussis P. Single-needle
insertion in twin gestations. J Ultrasound
Med. 1990;9:5111.
65. Sebire NJ, Noble PL, Odibo A, et al. Single
uterine entry for genetic amniocentesis in
twin pregnancies. Ultrasound Obstet Gynecol.
1996;7:26–31.
66. Henry G, Robinson A. Genetic amniocen-
tesis in twin pregnancies. Am J Hum Genet.
1975;30:695.
67. Wolfe DA, Scheible FW, Young FE, et  al.
Genetic amniocentesis in multiple preg-
nancy. J Clin Ultrasound. 1979;7:208.
68. Jassani MN, Merkatz IR, Brennan IN, et  al.
Twin pregnancy with discordance for Down’s
syndrome. Obstet Gynecol. 1980;55(suppl):455.
69. Bovicelli L, Michelacci L, Rizzo N, et  al.
Genetic amniocentesis in twin pregnancy.
Prenat Diagn. 1983;3:83.
70. Goldstein AI, Stills SM. Mid-trimester
amniocentesis in twin pregnancies. Am J
Ostet Gynecol. 1983;62:659.
71. Palle C, Anderson JW, Tobar A, et al. Increased
risk of abortion after genetic amniocentesis in
twin pregnancies. Prenat Diagn. 1983;3:83.
72. Taylor MB, Anderson RL, Golbus MS. One
hundred twin pregnancies in a prenatal diagno-
sis program. Am J Med Genet. 1984;148:585.
73. Filkins K, Russo J. Genetic amniocentesis in
multiple gestations. Prenat Diagn. 1984;4:
223.
74. Librach CL, Doran TA, Benzie RJ, et  al.
Genetic amniocentesis in seventy twin pre­
gnancies. Am J Obstet Gynecol. 1984;148:
585.
75. Pijpers L, Jahoda MG, Vosters RP, et  al.
Genetic amniocentesis in twin pregnancies.
Br J Obstet Gynaecol. 1988;95:323.
76. Simpson JL. Procedures for prenatal diagno-
sis of genetic disorders. Genetics in obstetrics
and gynecology. In: Golbus SG, Simpson
JL, eds. 2nd ed. Philadelphia: WB Saunders;
1992.
77. Dacus JV, Wilroy RS, Summitt RL, et  al.
Genetic amniocentesis: a twelve years’ expe-
rience. Am J Med Genet. 1985;20:443.
78. Yukobowich E, Anteby EY, Cohen SM, et al.
Risk of fetal loss in twin pregnancies under-
going second trimester amniocentesis (1).
Obstet Gynecol. 2001;98:231.
79. Millaire M, Bujold E, Morency AM,
Gauthier RJ. Mid-trimester genetic amnio-
centesis in twin pregnancy and the risk of fetal
loss. J Obstet Gynaecol Can. 2006;28:512.
80. Ghidini A, Lynch L, Hicks C, et al. The risk
of second-trimester amniocentesis in twin
gestations: a case-control study. Am J Obstet
Gynecol. 1993;169:1013.
81. Weisz B, Rodeck CH. Invasive diagnos-
tic procedures in twin pregnancies. Prenat
Diagn. 2005;25:751.
82. Rochon M, Eddleman K, Stone J. Invasive
procedures in multifetal pregnancies. Clin
Perinatol. 2005;32:355.
83. Appelman Z, Furman B. Invasive genetic
diagnosis in multiple pregnancies. Obstet
Gynecol Clin North Am. 2005;32:97.
84. Bhide A, Thilaganathan B. What prenatal
diagnosis should be offered in multiple preg-
nancy? Best Pract Res Clin Obstet Gynaecol.
2004;18:531.
85.  Toth-Pal E, Ban Z, Papp C, et  al. Genetic
amniocentesis in twin pregnancy—experience
of eleven years. Orv Hetil. 2004;145:1127.
86. Toth-Pal E, Papp C, Beke A, et  al. Genetic
amniocentesis in multiple pregnancy. Fetal
Diagn Ther. 2004;19:138.
87. Cahill AG, Macones GA, Stamilio DM,
et al. Pregnancy loss rate after mid-trimester
amniocentesis in twin pregnancies. Am J
Obstet Gynecol. 2009;200:257.e1–e6.
88. Working Party on Amniocentesis. An assess-
ment of hazards of amniocentesis. Br J Obstet
Gynaecol. 1978;85(suppl 2):1.
89. Ayadi S, Carbillon L, Varlet C, et  al. Fatal
sepsis due to Escherichia coli after second-
trimester amniocentesis. Fetal Diagn Ther.
1998;13:98.
90. Elchalal U, Shachar IB, Peleg D, Schenker
JG. Maternal mortality following diagnostic
2nd-trimester amniocentesis. Fetal Diagn
Ther. 2004;19:195.
91. Thorp JA, Helfgott AW, King EA, et  al.
Maternal death after second-trimester genetic
amniocentesis. Obstet Gynecol. 2005;105(5,
Part 2):1213.
92. Thomas IT, Smith DW. Oligohydramnios,
cause of the nonrenal features of the Potter’s
syndrome, including pulmonary hypoplasia.
J Pediatr. 1974;84:811.
93. Lamb MP. Gangrene of a fetal limb due to
amniocentesis. Br J Obstet Gynaecol. 1975;
82:829.
94. Karp LE, Hayden PW. Fetal puncture during
mid-trimester amniocentesis. Obstet Gynecol.
1977;49:115.
95. Rickwood AMK. A case of ileal atresia and
ileocutaneous fistula caused by amniocente-
sis. J Pediatr. 1977;1:720.
96. Eply SL, Hanson JW, Cruikshank DP. Fetal
injury with midtrimester diagnostic amnio-
centesis. Obstet Gynecol. 1979;53:77.
97. Swift PFG, Driscoll IB, Vovles KDJ. Neona-
tal small bowel obstruction associated with
amniocentesis. BMJ. 1979;1:720.
98. Youroukos S, Papadelis F, Matsaniotis N.
Porencephalic cysts after amniocentesis. Arch
Dis Child. 1980;55:814.
99.  Merin S, Byth Y. Uniocular congenital blind-
ness as a complication of midtrimester amnio-
centesis. Am J Ophthalmol. 1980;80:299.
100. Isenberg SJ, Heckenlively JR. Traumatized
eye with retinal damage from amniocentesis. J
Pediatr Ophthalmol Strabismus. 1988;25:196.
101. Adrnoni MM, Ben Ezra D. Ocular trauma
following amniocentesis as the cause of leu-
kocoria. J Pediatr Opthalmol Strabismus.
1988;25(4):196–197.
102. Gounot E, Cuzin B, Louis D, et  al. Fistule
peritoneo-parietale au decours d’une amnio-
centese precoce: a propos d’un cas. Revue de
la literature. Chir Pediatr. 1989;30:52.
103. Mancini J, Lethel V, Hugonenq C, et  al.
Brain injuries in early foetal life: conse-
quences for brain development. Dev Med
Child Neurol. 2001;43:52.
104. Fines B, Ben-Ami TE, Yousefzadeh DK. Trau-
matic prenatal sigmoid perforation due to
amniocentesis. Pediatr Radiol. 2001;31:440.
105. DeLong GR. Mid-gestation right basal gan-
glia lesion: clinical observations in two chil-
dren. Neurology. 2002;59:54.
106. Vilar Coromina N, Vicente Villa A, Puigar-
nau Vallhonrat R, et  al. Skin dimpling: a
complication of amniocentesis. An Pediatr
(Barc). 2007;66:4.
107. Mandelbrot L, Mayaux MJ, Bongain A, et al.
Obstetric factors and mother-to-child trans-
mission of human immunodeficiency virus
type 1: the French perinatal cohorts. SERO-
GEST French Pediatric HIV Infection Study
Group. Am J Obstet Gynecol. 1996;175:661.
108. Bucceri AM, Somigliana E, Vignali M. Early
invasive diagnostic techniques during preg-
nancy in HIV-infected women. Acta Obstet
Gynecol Scand. 2001;80:82.
109. de Decker HP. Mother-to-fetus HIV transmis-
sion during amniocentesis—ethical concerns.
S Afr Med J. 2002;92:124.
110. International Perinatal HIV Group. The mode
of delivery and the risk of vertical transmission
of human immunodeficiency virus type 1.
N Engl J Med. 1999;179:590.
111. Floridia M, Masuelli G, Meloni A, et al. Ital-
ian Group on Surveillance on Antiretroviral
Treatment in Pregnancy. Amniocentesis and
chorionic villus sampling in HIV-infected
pregnant women: a multicentre case series.
BJOG. 2016. https://doi.org/10.1111/1471-
0528.14183. [Epub ahead of print].
112. Stein Z, Kline J, Susser E, et al. Maternal age
and spontaneous abortion. In: Porter IH,
Hook EB, eds. Human Embryonic and Fetal
Death. New York: Academic Press; 1980:107.
113. Simpson JL. Incidence and timing of preg-
nancy losses: relevance to evaluating safety
of early prenatal diagnosis. Am J Med Genet.
1990;35:165.
114. Medical Research Council. Diagnosis of
Genetic Disease by Amniocentesis During the
Second Trimester of Pregnancy. Ottawa: Medical
Research Council; 1977.
115. Christiaens GCML, Stoutenbeek PH. Spon-
taneous abortion in proven intact pregnan-
cies. Lancet. 1984;2:572.
116. Wilson RD, Kendrick V, Wittman BK, et al.
Risks of spontaneous abortion in ultraso-
nographically normal pregnancies. Lancet.
1984;2:290.
117. Tongsong T, Wanapirak C, Sirivatanapa
P, et  al. Amniocentesis-related fetal loss: a
cohort study. Obstet Gynecol. 1998;92:64.
118. Papantoniou NE, Daskalakis GJ, Tziotis JG,
et  al. Risk factors predisposing to fetal loss
following a second trimester amniocentesis.
Br J Obstet Gynaecol. 2001;108:1053.
119. Eddleman KA, Malone FD, Sullivan L,
et  al. Pregnancy loss rates after midtrimes-
ter amniocentesis by the First And Second
Trimester Evaluation of Risk (FASTER)
Trial Research Consortium. Obstet Gynecol.
2006;109(2, Part 1):1067.
120. Caughey AB, Hopkins LM, Norton ME.
Chorionic villus sampling compared with
amniocentesis and the difference in the rate of
pregnancy loss. Obstet Gynecol. 2006;108:612–
616.
121. Towner D, Currier RJ, Lorey FW, et  al.
Miscarriage risk from amniocentesis per-
formed for abnormal maternal serum scr­
eening. Am J Obstet Gynecol. 2007;196:
608.e1–e5.
122. Tabor A, Vestergaard CH, Lidegaard O. Fetal
loss rate after chorionic villus sampling and
amniocentesis: an 11-year national registry
study. Ultrasound Obstet Gynecol. 2009;34:
19–24.
123. Pitukkijronnakorn S, Promsonthi P, Pan-
burana P, et  al. Fetal loss associated with
second-trimester amniocentesis. Arch Gyne-
col Obstet. 2011;284:793–797.

124. Corrado F, Cannata ML, La Galia T, et  al.
Pregnancy outcome following mid-trimester
amniocentesis. J Obstet Gynaecol. 2012;32:117–
119.
125. Armstrong J, Cohen AW, Bombard AT, et al.
Comparison of amniocentesis-related loss
rates between obstetrician-gynecologists and
perinatologists. Obstet Gynecol. 2002;99:65S.
126. Odibo AO, Gray DL, Dicke JM, et al. Revis-
iting the fetal loss rate after second-trimester
genetic amniocentesis: a single center’s 16-year
experience. Obstet Gynecol. 2008;111:589–
595.
127. Akolekar R, Beta J, Picciarelli G, et  al.
Procedure-related risk of miscarriage follow-
ing amniocentesis and chorionic villus sam-
pling: a systematic review and meta-analysis.
Ultrasound Obstet Gynecol. 2015;45(1):16–
26.
128. Henry GP, Miller WA. Early amniocentesis.
J Reprod Med. 1992;37:396.
129. Hanson FW, Zorn EM, Tennant FR.
Amniocentesis before 15 weeks’ gestation:
outcome, risks, and technical problems. Am J
Obstet Gynecol. 1987;156:1524.
130. Evans MI, Grogan A, Koppitch III MS, et al.
Genetic diagnosis in the first trimester: the
norm for the 1990s. Am J Obstet Gynecol.
1989;160:1332.
131. Elejalde BR, de Elejalde MM, Acuna JM.
Prospective study of amniocentesis per-
formed between weeks 9 and 16 of gestation:
its feasibility, risks, complications and use in
early genetic prenatal diagnosis. Am J Med
Genet. 1990;35:188.
132. Bombard T, Rigdon DT. Prospective pilot
evaluation of early (11–14 weeks’ gesta-
tion) amniocentesis in 75 patients. Mil Med.
1992;157:339.
133. Calhoun BC, Brehm W, Bombard AT. Early
genetic amniocentesis and its relationship to
respiratory difficulties in paediatric patients:
a report of findings in patients and matched
controls 3–5 years post-procedure. Prenat
Diagn. 1994;14:209.
134. Stripparo L, Buscaglia M, Longatti L.
Genetic amniocentesis: 505 cases performed
before the sixteenth week of gestation. Prenat
Diagn. 1990;10:359.
135. Penso CA, Sandstrom MM, Garber MF,
et al. Early amniocentesis: report of 407 cases
with neonatal follow-up. Obstet Gynecol.
1990;76:1032.
136. Hackett GA, Smith ill, Rebello CTH, et al.
Early amniocentesis at 11–14 weeks gesta-
tion for the diagnosis of fetal chromosomal
abnormality: a clinical evaluation. Prenat
Diagn. 1991;11:311.
137. Bombard AT, Carter SM, Nitowsky HM.
Early amniocentesis versus chorionic vil-
lus sampling for fetal karyotyping. Lancet.
1994;344:826.
138. Brumfield CG, Lin S, Conner W, et al. Preg-
nancy outcome following genetic amniocen-
tesis at 11–14 versus 16–19 weeks’ gestation.
Obstet Gynecol. 1996;88:114.
139. Wilson RD, Johnson J, Dansereau J. Preg-
nancy outcome following genetic amniocen-
tesis at 11–14 versus 16–19 weeks’ gestation.
Obstet Gynecol. 1996;88:638.
140. Shulman LP, Elias S, Phillips OP, et  al.
Amniocentesis performed at 14 weeks ges-
tation or earlier: comparison with first-
trimester chorionic villus sampling. Obstet
Gynecol. 1994;83:543.
141. Bravo RR, Shulman LP, Phillips OP, et  al.
Transplacental needle passage in early amnio-
centesis and pregnancy loss. Obstet Gynecol.
1995;86:437.
142. Shulman LP, Elias S, Phillips OP, et al. Early
twin amniocentesis prior to 14 weeks gesta-
tion. Prenat Diagn. 1992;12:625.
143. Diaz Vega M, De La Cueva P, Leal C, et al.
Early amniocentesis at 10–12 weeks’ gesta-
tion. Prenat Diagn. 1996;16:307.
144. Rao N, Pettenati M, Barry M, et  al. Early
amniocentesis: a cytogenetic evaluation of
1010 consecutive cases. Am J Hum Genet.
1990;47:A283.
145. Nicolaides KH, Brizot ML, Patel F, et  al.
Comparison of chorionic villus sampling and
amniocentesis for fetal karyotyping at 10–13
weeks gestation. Lancet. 1994;344:435.
146. Vandenbussche FPHA, Kanhai HHH, Kei-
rse MJNC. Safety of early amniocentesis.
Lancet. 1994;344:1032.
147. Canadian Early and Mid-Trimester Amnio-
centesis Trial Group. Randomized trial
to assess safety and fetal outcome of early
and midtrimester amniocentesis. Lancet.
1998;351:242.
148. Johnson JM, Wilson RD, Singer J, et  al.
Technical factors in early amniocentesis pre-
dict adverse outcome. Results of the Cana-
dian Early (EA) versus Mid-trimester (MA)
Amniocentesis Trial. Prenat Diagn. 1999;19:
732.
149. Sundberg K, Bang J, Smidt-Jensen S, et  al.
Randomised study of risk of fetal loss related
to early amniocentesis versus chorionic villus
sampling. Lancet. 1997;350:697.
150. Philip J, for the NICHD EATA Study Group.
Greater risk associated with early amniocen-
tesis compared to chorionic villus sampling:
an international randomized trial. Am J Obstet
Gynecol. 2002;187:39A.
151. ACOG. Invasive prenatal testing for aneu-
ploidy. ACOG Practice Bulletin No 88.
Obstet Gynecol. 2007;110:1459.
152. Gluck L, Kulovich MV, Borer Jr RC, Keidel
WN. The interpretation and significance of
the lecithin-sphingomyelin ratio in amniotic
fluid. Am J Obstet Gynecol. 1974;120:142–
155.
153. Field NT, Gilbert WM. Current status of
amniotic fluid tests of fetal maturity. Clin
Obstet Gynecol. 1997;40:366–386.
154. McElrath TF, Colon I, Hecht J, et al. Neona-
tal respiratory distress syndrome as a function
of gestational age and an assay for surfactant-
to-albumin ratio. Obstet Gynecol. 2004;103:
463–468.
155. Spong CY, Mercer BM, D’alton M, et  al.
Timing of indicated late-preterm and early-
term birth. Obstet Gynecol. 2011;118:323–
333.
156. Varner S, Sherman C, Lewis D, et al. Amnio-
centesis for fetal lung maturity: will it become
obsolete? Rev Obstet Gynecol. 2013;6:126–
134.
157. Cutler J, Chappell LC, Kyle P, Madan B.
Third trimester amniocentesis for diagnosis
of inherited bleeding disorders prior to deliv-
ery. Haemophilia. 2013;19:904–907.
158. Simcox L, Tower C1, Byrd L, et  al. Third
trimester amniocentesis for inherited bleed-
ing disorders can be used to inform delivery
management for at risk male fetuses. Arch
Dis Child Fetal Neonatal Ed. 2014;99(suppl
1):A87.
References 232.e3
159. Christiaens GC1, Van Baarlen J, Huber
J, et  al. Fetal limb constriction: a pos-
sible complication of CVS. Prenat Diagn.
1989;9(1):67–71.
160. Jackson LG, Zachary JM, Fowler SE, et al. A
randomized comparison of transcervical and
transabdominal chorionic-villus sampling.
The U.S. National Institute of Child Health
and Human Development Chorionic-Villus
Sampling and Amniocentesis Study Group.
N Engl J Med. 1992;327(9):594–598.
161. Silver R, MacGregor S, Sholl J. Initiating a
chorionic villus sampling program. Relying
on placental location as the primary deter-
minant of the sampling route. J Reprod Med.
1990;35:964–968.
162. Brambati B, Oldrini A, Lanzani A. Transab-
dominal and trans-cervical chorionic villus
sampling: efficiency and risk evaluation of
2,411 cases. Am J Med Genet. 1990;35:160–
164.
163. Fortuny A, Borrell A, Soler A, et  al. Cho-
rionic villus sampling by biopsy forceps.
Results of 1580 procedures from a single
centre. Prenat Diagn. 1995;15(6):541–550.
164. Young C, von Dadelszen P, Alfirevic Z.
Instruments for chorionic villus sampling for
prenatal diagnosis. Cochrane Database Syst
Rev. 2013;(1):CD000114.
165. Weiner S, Kurjak A. Interventional ultrasound.
Parthenon: New York; 1999.
166. Adusumalli J, Han CS, Beckham S, et  al.
Chorionic villus sampling and risk for hyper-
tensive disorders of pregnancy. Am J Obstet
Gynecol. 2007;196(6):591.e1–e7.
167. Daskalakis G, Papapanagiotou A, Antonako-
poulos N, et al. Invasive diagnostic procedures
and risk of hypertensive disorders in pregn­
ancy. Int J Gynaecol Obstet. 2014;125(2):146–
149.
168. Basaran A, Basaran M, Topatan B. Chorionic
villus sampling and the risk of preeclampsia:
a systematic review and meta-analysis. Arch
Gynecol Obstet. 2011;283:1175–1181.
169. Khalil A, Akolekar R, Pandya P, et al. Cho-
rionic villus sampling at 11 to 13 weeks of
gestation and hypertensive disorders in preg-
nancy. Obstet Gynecol. 2010;116:374–380.
170. Odibo AO, Singla A, Gray DL, et al. Is cho-
rionic villus sampling associated with hyper-
tensive disorders of pregnancy? Prenat Diagn.
2010;30:9–13.
171. 
Multicentre randomised clinical trial of
chorion villus sampling and amniocentesis.
First report. Canadian Collaborative CVS-
Amniocentesis Clinical Trial Group. Lancet.
1989;7:1–6.
172. Rhoads G, Jackson I, Schlesselman S. The
safety and efficacy of chorionic villus sampling
for early prenatal diagnosis of cytogenetic
abnormalities. N Engl J Med. 1989;320:609–
619.
173. Medical Research Council European trial of
chorion villus sampling. MRC working party
on the evaluation pf chorion villus sampling.
Lancet. 1991;337:1491–1499.
174. Odibo AO, Dicke JM, Gray DL, et al. Evalu-
ating the rate and risk factors for fetal loss after
chorionic villus sampling. Obstet Gynecol. 2008;
112:813–819.
175. Lau KT, Leung YT, Fung YT, et  al. Out-
come of 1,355 consecutive transabdominal
chorionic villus samplings in 1,351 patients.
Chin Med J (Engl). 2005;118:1675–1681.
232.e4 References
176. Akolekar R, Bower S, Flack N, et al. Predic-
tion of miscarriage and stillbirth at 11–13
weeks and the contribution of chorionic vil-
lus sampling. Prenat Diagn. 2011;31:38–45.
177. Bovicelli L, Rizzo N, Montacuti V, Morandi
R. Transabdominal versus transcervical routes
for chorionic villus sampling. Lancet. 1986;
2(8501):290.
178. Brambati B, Terzian E, Tognoni G. Random-
ized clinical trial of transabdominal versus
transcervical chorionic villus sampling meth-
ods. Prenat Diagn. 1991;11(5):285–293.
179. Smidt-Jensen S, Permin M, Philip J, et  al.
Randomised comparison of amniocentesis and
transabdominal and transcervical chorionic vil-
lus sampling. Lancet. 1992;340(8830):1237–
1244.
180. Wapner RJ, Johnson A, Davis G, et al. Pre-
natal diagnosis in twin gestations: a compari-
son between second-trimester amniocentesis
and first-trimester chorionic villus sampling.
Obstet Gynecol. 1993;82:49–56.
181. Simonazzi G, Curti A, Farina A, et  al.
Amniocentesis and chorionic villus sampling
in twin gestations: which is the best sampling
technique? Am J Obstet Gynecol. 2010;202:
365.e1–e5.
182. Casals G, Borrell A, Martinez JM, et al. Tran-
scervical chorionic villus sampling in multi-
ple pregnancies using a biopsy forceps. Prenat
Diagn. 2002;22:260–265.
183. Antsaklis A, Souka AP, Daskalakis G, et al.
Second-trimester amniocentesis vs. chorionic
villus sampling for prenatal diagnosis in mul-
tiple gestations. Ultrasound Obstet Gynecol.
2002;20:476–481.
184. De Catte L, Liebears I, Foulon W. Out-
come of twin gestations after first trimester
chorionic villus sampling. Obstet Gynecol.
2000;96:714–720.
185. Aytoz A, De Catte L, Camus M, et  al.
Obstetric outcome after prenatal diagno-
sis in pregnancies obtained after intracy-
toplasmic sperm injection. Hum Reprod.
1998;13:2958–2961.
186. De Catte L, Liebaers I, Foulon W, et al. First
trimester chorionic villus sampling in twin
gestations. Am J Perinatol. 1996;13:413–
417.
187. Pergament E, Schulman JD, Copeland K,
et  al. The risk and efficacy of chorionic vil-
lus sampling in multiple gestations. Prenat
Diagn. 1992;12:377–384.
188. Antsaklis A, Gougoulakis A, Mesogitis S,
et  al. Invasive techniques for fetal diagnosis
in multiple pregnancy. Int J Gynaecol Obstet.
1991;34:309–314.
189. Agarwal K, Alfirevic Z. Pregnancy loss after
chorionic villus sampling and genetic amnio-
centesis in twin pregnancies: a systematic
review. Ultrasound Obstet Gynecol. 2012;40:
128–134.
190. Wapner RJ. Invasive prenatal diagnostic tech-
niques. Semin Perinatol. 2005;29(6):401–
404.
191. Ledbetter DH, Martin AO, Verlinsky Y, et al.
Cytogenetic results of chorionic villus sam-
pling: high success rate and diagnostic accu-
racy in the United States collaborative study.
Am J Obstet Gynecol. 1990;162:495–501.
192. Wolstenholme J. Confined placental mosa-
icism for trisomies 2, 3, 7, 8, 9, 16, and 22:
their incidence, likely origins, and mecha-
nisms for cell lineage compartmentalization.
Prenat Diagn. 1996;16:511–524.
193. Goldberg JD, Wohlferd MM. Incidence
and outcome of chromosomal mosaicism
found at the time of chorionic villus sam-
pling. Am J Obstet Gynecol. 1997;176(6):
1349–1352.
194. Firth H, Boyd P, Chamberlain P. Severe limb
abnormalities after chorion villus sampling at
56–66 days gestation. Lancet. 1991;337:726.
195. Mastroiacovo P, Botto L, Cavalcanti D.
Limb anomalies following chorionic villus
sampling: a registry based case control study.
Am J Med Genet. 1992;44:856–863.
196. Brambati B, Simoni G, Traui M. Genetic
diagnosis by chorionic villus sampling before
8 gestational weeks: efficiency, reliability, and
risks on 317 completed pregnancies. Prenat
Diagn. 1992;12:784–799.
197. Froster U, Jackson L. Limb defects and
chorionic villus sampling: results from an
international registry, 1992–1994. Lancet.
1996;347:489.
198. Olney R, Khoury M, Alo C. Increased risk
for transverse digital deficiency after chori-
onic villus sampling: results of the United
States Multistate Case-Control Study, 1988–
1992. Teratology. 1995;1:20–29.
199. Gosden C, Nicolaides KH, Rodeck CH.
Fetal blood sampling in investigation of
chromosome mosaicism in amniotic fluid
culture. Lancet. 1988;2:613.
200. Tipton RE, Therapel AT, Chang HT, et al.
Rapid chromosome analysis using sponta-
neously dividing cells from umbilical cord
blood (fetal and neonatal). Am J Obstet Gyne-
col. 1990;161:1546.
201. Liou JD, Chen CP, Breg WR, et  al. Fetal
blood sampling and cytogenetic abnormali-
ties. Prenat Diagn. 1993;13:1.
202. Porreco RP, Harshbarger B, McGavran L.
Rapid cytogenetic assessment of fetal blood
samples. Obstet Gynecol. 1993;82:242.
203. Ryan G, Rodeck CH. Fetal blood sampling.
In: Simpson JL, Elias S, eds. Essentials of Pre-
natal Diagnosis. New York: Churchill Living-
stone; 1993:63.
204. Daffos F. Fetal blood sampling. Annu Rev
Med. 1989;40:319.
205. Forestier F, Cox WL, Daffos F, et  al. The
assessment of fetal blood samples. Am J Obstet
Gynecol. 1988;158:1184.
206. Nicolaides KH, Clewel WH, Rodeck CH.
Measurement of human fetoplacental blood
volume in erythroblastosis fetalis. Am J Obstet
Gynecol. 1987;157:50.
207. Pardi G, Marconi M, Cetin I, et al. Fetal blood
sampling during pregnancy: risks and diag-
nostic advantages. J Perinat Med. 1994;22:
513.
208. Bahado-Singh RO, Morotti R, Pirhonen J,
et  al. Invasive techniques for prenatal diag-
nosis: current concepts. J Assoc Acad Minority
Phys. 1995;6:28.
209. Tongsong T, Wanapirak C, Pkunavikatikul
C, et al. Fetal loss rate associated with cordo-
centesis at midgestation. Am J Obstet Gynecol.
2001;184:719.
210. Donnenfeld AE, Wiseman B, Lavi E, et  al.
Prenatal diagnosis of severe combined immu-
nodeficiency. J Pediatr. 1990;10:29.
211. Udom-Rice I, Bussel JB. Fetal and neonatal
thrombocytopenia. Blood Rev. 1995;9:57.
212. Bussel JB, Berkowitz RL, Mcfarland JG,
et  al. Antenatal treatment of neonatal allo-
immune thrombocytopenia. N Engl J Med.
1988;319:1374.
213. Weiner CP. Cordocentesis for diagnostic
indications: two years experience. Obstet
Gynecol. 1987;70:664.
214. Durandy A, Dumez Y, Guy-Grand D, et al.
Prenatal diagnosis of severe combined immu-
nodeficiency. J Pediatr. 1982;101:995.
215. Holmberg L, Gustavii B, Jonsson A. A pre-
natal study of fetal platelet count and size
with application to fetus at risk for Wiskott-
Aldrich syndrome. J Pediatr. 1983;102:773.
216. Diukman R, Tanigawa S, Cowan MJ, et al.
Prenatal diagnosis of Chediak-Higashi syn-
drome. Prenat Diagn. 1992;12:1877.
217. Rodeck CH, Nicolini U. Fetal blood sam-
pling. Eur J Obstet Gynecol Reprod Biol.
1988;28:85.
218. Daffos F, Forestier F, Capella-Pavlovsky M,
et al. Prenatal management of 746 pregnan-
cies at risk for congenital toxoplasmosis. N
Engl J Med. 1988;318:271.
219. Peters MT, Nicolaides KH. Cordocentesis
for the diagnosis and treatment of human
fetal parvovirus infection. Obstet Gynecol.
1990;75:501.
220. Hsieh PI, Ko TM, Chang FM, et  al. Percu-
taneous ultrasound-guided fetal blood sam-
pling: experience in the first 100 cases. Taiwan
I Hsueh Hui Tsa Chi. 1989;88:137.
221. Viscarello RR, Cullen MT, DeGennaro NJ,
et al. Fetal blood sampling in human immu-
nodeficiency virus seropositive women. Am J
Obstet Gynecol. 1992;167:1075.
222. Newton ER. Diagnosis of perinatal TORCH
infections. Clin Obstet Gynecol. 1999;42:59.
223. Azam AZ, Vial Y, Fawer CL, et  al. Prena-
tal diagnosis of congenital cytomegalovirus
infection. Obstet Gynecol. 2001;97:443.
224. Orlandi F, Damiani G, Jakil C, et  al. The
risks of early cordocentesis (12–21 weeks):
analysis of 500 procedures. Prenat Diagn.
1990;10:425.
225. Chinaiya A, Venkat A, Dawn C, et al. Intra-
hepatic vein fetal blood sampling: current
role in prenatal diagnosis. J Obstet Gynaecol
Res. 1998;24:239.
226. Nicolini U, Nicolaides KH, Fisk NM, et al.
Fetal blood sampling from the intrahepatic
vein: analysis of safety and clinical experi-
ence with 214 procedures. Obstet Gynecol.
1990;76:47.
227. Kim SR, Won HS, Lee PR, Kim A. Four-
dimensional ultrasound guidance of prenatal
invasive procedures. Ultrasound Obstet Gyne-
col. 2005;26:663.
228. Kawakami Y, Matsuda H, Shibasaki T, et al.
Safer cordocentesis by new 25-gauge needles.
Fetal Diagn Ther. 2008;24:211.
229. Nicolini U, Kochenour NK, Greco P, et al.
Consequences of fetomaternal hemor-
rhage after intrauterine transfusion. BMJ.
1988;297:1379.
230. Ghidini A, Sepulveda W, Lockwood CJ,
et al. Complications of fetal blood sampling.
Am J Obstet Gynecol. 1993;168:1339.
231. Wilson RD, Farquarhson DF, Wittman BK,
et  al. Cordocentesis: overall pregnancy loss
rate as important as procedure loss rate. Fetal
Diagn Ther. 1994;9:142.
232. Buscaglia M, Ghisoni L, Bellotti M, et al. Percu-
taneous umbilical blood sampling: indication,
changes, and procedure loss rates in nine years’
experience. Fetal Diagn Ther. 1996;11:106.
233. Weiner CP, Okamura K. Diagnostic fetal
blood sampling-technique related losses.
Fetal Diagn Ther. 1996;11:169.

234. Liao C, Wei J, Li Q, et al. Efficacy and safety
of cordocentesis for prenatal diagnosis. Int J
Gynecol Obstet. 2006;93:13.
235. Antsaklis A, Daskalakis G, Papantoniou N,
et al. Fetal blood sampling—indication-related
losses. Prenat Diagn. 1998;18:934.
236. Sikovanyecz J, Horvath E, Sallay E, et  al.
Fetomaternal transfusion and pregnancy out-
come after cordocentesis. Fetal Diagn Ther.
2001;16:83.
237. Van Selm M, Kanhai HHH, Van Loon J.
Detection of fetomaternal hemorrhage asso-
ciated with cordocentesis using serum alpha-
fetoprotein and the Kleihauer technique.
Prenat Diagn. 1995;15:313.
238. Rujiwetpongstorn J, Tongsong T, Wanapirak
C, et al. Fetomaternal hemorrhage after cor-
docentesis at Maharaj Nakorn Chiang Mai
Hospital. J Med Assoc Thai. 2005;88:145.
239. Tongprasert F, Tongsong T, Wanapirak C,
et  al. Cordocentesis in multifetal pregnan-
cies. Prenat Diagn. 2007;27:1100.
240. Hill LM, Platt LD, Collage B. Rh-sensitization
after genetic amniocentesis. Obstet Gynecol.
1980;56:459.
241. Golbus MS, Stephens JD, Can HM, et  al.
Rh-isoimmunization following genetic
amniocentesis. Prenat Diagn. 1982;2:149.
242. Wyskowski DK, Flynt JW, Goldberg MF,
et  al. Rh hemolytic disease: epidemiologic
surveillance in the United States, 1968 to
1975. JAMA. 1979;242:1376.
243. Khalil MA, Tabsh MA, Lobherz TB, et  al.
Risks of prophylactic anti-D immunoglobu-
lin after second-trimester amniocentesis. Am
J Obstet Gynecol. 1984;149:225.
244. Katiyar R1, Kriplani A, Agarwal N, et  al.
Detection of fetomaternal hemorrhage fol-
lowing chorionic villus sampling by Kleihauer
Betke test and rise in maternal serum alpha feto
protein. Prenat Diagn. 2007;27(2):139–142.
245. Meleti D, Caetano AC, Boute T, et  al.
Assessment of fetomaternal hemorrhage by
Kleihauer-Betke test, flow cytometry and
α-fetoprotein after invasive obstetric pro-
cedures. Clin Exp Obstet Gynecol. 2012;
39(3):303–306.
246. ACOG. Prevention of RhD Isoimmuni-
zation. ACOG Practice Bulletin No 4.
Washington, DC: American College of
Obstetricians and Gynecologists.
247. Lo YMD, Corbetta N, Chamberlain PF,
et  al. Presence of fetal DNA in maternal
plasma and serum. Lancet. 1997;350:485–
487.
248. Lo YMD, Tein MS, Lau TK, et  al. Quan-
titative analysis of fetal DNA in maternal
plasma and serum: implications for nonin-
vasive prenatal diagnosis. Am J Hum Genet.
1998;62:768–775.
249. Lun FM, Chiu RW, Allen Chan KC, et al.
Microfluidics digital PCR reveals a higher
than expected fraction of fetal DNA in
maternal plasma. Clin Chem. 2008;54:1664–
1672.
250. Nicolaides KH, Syngelaki A, Ashoor G, et al.
Noninvasive prenatal testing for fetal triso-
mies in a routinely screened first-trimester
population. Am J Obstet Gynecol. 2012;207:
374.e1–e6.
251. Norton ME, Brar H, Weiss J, et  al. Non-
Invasive Chromosomal Evaluation (NICE)
Study: results of a multicenter prospective
cohort study for detection of fetal trisomy
21 and trisomy 18. Am J Obstet Gynecol.
2012;207(2):137.e1–e8.
252. Norton ME, Jacobsson B, Swamy GK,
et  al. Cell-free DNA analysis for noninva-
sive examination of trisomy. N Engl J Med.
2015;372(17):1589–1597.
253. Bianchi DW, Platt LD, Goldberg JD, et al.
Genome-wide fetal aneuploidy detection by
maternal plasma DNA sequencing. Obstet
Gynecol. 2012;119:890–901.
254. Palomaki GE, Kloza EM, Lambert-Messer-
lian GM, et al. DNA sequencing of maternal
plasma to detect Down syndrome: an inter-
national clinical validation study. Genet Med.
2011;13:913–920.
255. Benn P, Cuckle H, Pergament E. Non-
invasive prenatal testing for aneuploidy: cur-
rent status and future prospects. Ultrasound
Obstet Gynecol. 2013;42:15–33.
References 232.e5
256. Dan S, Wang W, Ren J, et al. Clinical appli-
cation of massively parallel sequencing-based
prenatal noninvasive fetal trisomy test for
trisomies 21 and 18 in 11,105 pregnan-
cies with mixed risk factors. Prenat Diagn.
2012;32:1225–1232.
257. Gil MM, Quezada MS, Bregant B, et  al.
Implementation of maternal blood cell-free
DNA testing in early screening for aneuploi-
dies. Ultrasound Obstet Gynecol. 2013;42:34–
40.
258. 
American College of Obstetricians and
Gynecologists. Noninvasive prenatal testing
for fetal aneuploidy. Committee Opinion
No. 545. Obstet Gynecol. 2012;120:1532–
1534.
259. Benn P, Borell A, Chiu R, et  al. Position
Statement from the Aneuploidy Screen-
ing Committee on behalf of the Board
of the International Society for Prenatal
Diagnosis. Prenat Diagn. 2013. https://doi.
org/10.1002/pd4139. [Epub ahead of print].
260. Langlois S, Brock J-A. Current status in
non-invasive prenatal detection of Down
syndrome, trisomy 18, and trisomy 13 using
cell-free DNA in maternal plasma. J Obstet
Gynaecol Can. 2013;35:177–181.
261. Mennuti MT, Cherry AM, Morrissette
JJ, et  al. Is it time to sound an alarm about
false-positive cell-free DNA testing for fetal
aneuploidy? Am J Obstet Gynecol. 2013;
209:415–419.
262. Larion S, Warsof SL, Romary L, et al. Asso-
ciation of combined first-trimester screen and
noninvasive prenatal testing on diagnostic pro-
cedures. Obstet Gynecol. 2014;123(6):1303–
1310.
263. Chetty S, Garabedian MJ, Norton ME.
Uptake of noninvasive prenatal testing (NIPT)
in women following positive aneuploidy
screening. Prenat Diagn. 2013;33(6):542–
546.
264. Chard RL, Norton ME. Genetic counseling
for patients considering screening and diag-
nosis for chromosomal abnormalities. Clin
Lab Med. 2016;36(2):227–236.
24
Prenatal Diagnosis of Chromosome
Abnormalities
COLLEEN G. BILANCIA, MYTHILY GANAPATHI AND BRYNN LEVY
KEY POINTS
• Chorionic villi, amniotic fluid and fetal blood are the speci-
men types currently used for prenatal diagnostic testing for
chromosome abnormalities.
• The spectrum of chromosomal alterations seen during prena-
tal testing include autosomal or sex chromosome aneuploidy,
balanced or unbalanced structural rearrangements, triploidy,
supernumerary marker chromosomes, submicroscopic dele-
tions and duplications, mosaicism and uniparental disomy.
• Methods for detecting chromosomal abnormalities in prena-
tal specimens include karyotype of G-banded chromosomes,
fluorescence in situ hybridisation, chromosomal microarray,
quantitative fluorescence polymerase chain reaction, multi-
plex ligation-dependent probe amplification and next-
generation sequencing (NGS).
• Chromosomal single nucleotide polymorphism microarrays
are invaluable for diagnosing clinically relevant microdele-
tions and microduplications. Unlike whole-chromosome
aneuploidy from nondisjunction, the risk for submicroscopic
copy number variants is not dependent on maternal age.
With these advances in our knowledge and testing capabili-
ties, all pregnant women should be offered prenatal diagnos-
tic testing for chromosome abnormalities.
• Noninvasive prenatal screening using maternal peripheral
blood is currently used to assess the risk for common triso-
mies, sex chromosome aneuploidies and select microdeletion
syndromes. Follow-up with confirmatory diagnostic testing is
recommended for all screen positive pregnancies.
• Challenges in the diagnosis of chromosome abnormalities
include interpreting mosaicism and copy number variants
with incomplete penetrance or variable expressivity, as well
as the risk in de novo balanced rearrangements.
• Future prospects for prenatal detection of chromosome
abnormalities are currently centred on NGS to improve the
capabilities of noninvasive prenatal screening.
Introduction
Prenatal diagnosis of chromosome abnormalities has been in prac-
tice since the 1960s and continues to be a crucial part of pre-
natal care.1–4 Initially, testing was performed during the second
trimester by amniocentesis. Concerns over safety of the proce-
dure led to studies to assess increased risks to the pregnancy as a
result of amniocentesis. These studies showed the increased rate
for miscarriage or spontaneous abortion from amniocentesis to
be approximately 1 in 200 or 0.5%.5–7 This risk was lower than
the risk for a woman older than the age of 35 years at the time of
delivery having a fetus with a chromosomal abnormality by karyo-
type. Therefore by the 1970s the standard of care was to offer these
women amniocentesis. Advances in prenatal testing continued
through the 1980s with the advent of chorionic villus sampling
(CVS), a procedure that could be performed in the first trimester,
offering families prenatal diagnosis earlier in the pregnancy.8–11
Cohort studies have shown that gestational timing is important to
reduce the risk for limb defects from CVS, which occur when the
procedure is done at less than 8 weeks. The standard of care is to
perform CVS between 10 and 12 weeks’ gestation. CVS may carry
a slightly higher risk for miscarriage compared with amniocente-
sis,12,13 but the risk from either procedure is significantly lower
than the original studies suggested. Recently, a large meta-analysis
found the risk for procedure-induced miscarriage to be approxi-
mately 1 in 450 for CVS and 1 in 900 for amniocentesis.14
Historically, prenatal testing had been considered in the con-
text of large genomic imbalances involving whole chromosomes
or large chromosomal regions that could be identified by classi-
cal cytogenetic analysis. Molecular cytogenetic techniques have
improved prenatal chromosome diagnostic capabilities to include
the detection of microdeletions and microduplications that are
not discernable by standard cytogenetic analysis. These newer
techniques include fluorescence in situ hybridisation (FISH),
quantitative fluorescence polymerase chain reaction (QF-PCR),
multiplex ligation-dependent probe amplification (MLPA) and
chromosomal microarray, with each offering unique advantages.
The reduced risk for adverse outcomes from CVS and amniocen-
tesis coupled with modern advances in the diagnosis of genomic
imbalance now provide safer, more comprehensive testing to any
woman interested in prenatal diagnosis. 
Prenatal Specimens
Prenatal specimens are primarily obtained by CVS or amniocen-
tesis. In some instances, percutaneous umbilical blood sampling
(PUBS) is performed, usually as a follow-up to a previous pro-
cedure that yielded ambiguous results. These different methods
are used during different stages of the pregnancy, and each has its
233
234 SE C T I O N 6     Prenatal Screening and Diagnosis
advantages and limitations. All specimens can be tested using clas-
sic and molecular cytogenetic techniques to identify chromosome
abnormalities in a fetus.
Chorionic Villus Sampling
Placental villi are composed of multiple layers, including the cho-
rionic membrane that makes up the outermost layer of fetal tissue
and forms the villi for vascularisation of the placenta. In CVS,
villus tissue is removed under the guidance of ultrasound by either
the transcervical or transabdominal method. Whereas the trans-
cervical method involves inserting a catheter through the vagina
and cervix, the transabdominal method uses a thin needle similar
to an amniocentesis. The position of the uterus and placenta dic-
tate which method is used. A small sample of chorionic villi is
retrieved and sent for cytogenetic analysis. Two preparations types
are possible for a CVS sample, direct and cultured. Direct prepara-
tion analyses the rapidly dividing trophoblast cells of the placenta
and may produce results in 24 to 48 hours. Cultured preparations
examine the mesenchymal cells from the villi and typically require
7 to 10 days for results. Cultured cells usually produce better
karyotype preparations and are the preferred approach for many
laboratories, although some may set up both direct and culture.
DNA can be extracted for chromosomal microarray from either
direct or cultured cell preparations, and results are generally avail-
able in 7 to 10 days for direct and 14 to 21 days for the cultures.  standard of care in the United States is to offer prenatal diagnostic
Amniocentesis
The amniotic sac surrounds the fetus and holds the amniotic fluid.
Amniotic fluid contains a variety of cell types both from the fetus
and the amnion. Amniocentesis involves removing a sample of
amniotic fluid through a thin needle inserted through the maternal
abdomen and into the gestational sac under ultrasound guidance.
Amniocentesis is the most widely used procedure for obtaining
fetal specimens. It is most commonly performed between 15 and
17 weeks’ gestation but not before 14 weeks because of the associ-
ation with talipes equinovarus and the higher risk for miscarriage.
Cells are cultured in flasks and on coverslips and may take up to 1
week before there are a sufficient number of cells for cytogenetic
analysis. The success rate for culture of amniocentesis samples is
approximately 99%, and the reliability of the cytogenetic diag-
nosis is also around 99%. Early amniocentesis (performed before
14 weeks’ gestation) does carry a 1% to 2% risk for clubfeet15;
therefore CVS is the preferred procedure for patients seeking early
prenatal diagnosis. As with CVS, DNA can be extracted directly
from the cells within the amniocentesis sample without culture
and used for chromosomal microarray analysis. Results from a
direct preparation are usually available in 7 to 10 days and in cul-
tured cells in 14 to 21 days.  of copy number variants (CNVs) with no maternal age-related risk24b
Percutaneous Umbilical Blood Sampling
Percutaneous umbilical blood sampling is an invasive procedure
that involves obtaining a fetal blood sample. PUBS may also be
referred to as cordocentesis, umbilical vein sampling and fetal
blood sampling, and it was originally developed for prenatal diag-
nosis of blood disorders such as haemoglobinopathies. PUBS
specimens are obtained through an ultrasound-guided needle
inserted transabdominally into the umbilical cord at the site of
insertion into the placenta, the intrahepatic portion of the umbili-
cal vein or rarely a free loop of cord. Several studies to assess the
safety of PUBS showed the procedure carried a 1% to 2% risk
for fetal death or spontaneous abortion, significantly higher than
CVS or amniocentesis.16,17 Recent studies have shown that the
risk may be higher in fetuses with abnormalities such as nonim-
mune fetal hydrops or intrauterine growth restriction.18,19 For
this reason, it is reserved for cases in which diagnostic informa-
tion cannot be obtained through CVS, amniocentesis and ultra-
sound or when the results of these previous diagnostic tests were
inconclusive. Occasionally, it is used in later gestations when rapid
results are important for management. Unlike CVS and amnio-
centesis, PUBS procedures are performed after 18 weeks’ gesta-
tion. Because culture time is shorter for blood samples, results are
generally available in 3 to 4 days for classic cytogenetics analysis
and 7 to 10 days for chromosomal microarray. 
Indications for Referral
Historically, advanced maternal age has been the most common
reason for referral for prenatal diagnosis of chromosome abnor-
malities. Other factors that increase the risk for a fetal chromosome
abnormality include positive first or second trimester aneuploidy
screening, fetal ultrasound anomalies, a previous pregnancy with a
chromosomal abnormality and a parent with a chromosomal rear-
rangement. The recent advances in molecular cytogenetics now
provide a reliable way to identify additional chromosome abnor-
malities without an age-related risk component. Therefore the
testing to all pregnant women regardless of age.20–22
Advanced Maternal Age
The association between increasing maternal age and Down syn-
drome was recognised as early as 1933 by Penrose.23 Advanced
maternal age is generally accepted to be 35 years or older at the
time of delivery. The age of 35 years was designated as the cutoff
because the risk for finding a chromosome abnormality and the
risk for causing a miscarriage from amniocentesis were roughly
the same. Below the age of 35 years, more miscarriages could be
expected compared with the number of liveborn children with a
chromosome aneuploidy. The increase in fetal chromosome abnor-
malities in aging women is due to an increase in nondisjunction
during meiosis. Fig. 24.1 shows the direct relationship between
maternal age and increased risk for chromosome abnormality at
the time of CVS and amniocentesis. In twin gestations, consid-
ering maternal age subgroups and twin zygosity, a significantly
lower-than-expected Down syndrome incidence is seen for both
monozygotic and dizygotic pregnancies than previous empiric cal-
culations would suggest.24a
Advances in cytogenomic technologies such as chromosomal
microarrays, noninvasive prenatal screening (NIPS), and discovery
have led to an increasing proportion of younger women (maternal
age <35 years) who are considered to be at low risk for aneuploidy to
opt for prenatal cytogenetic screening and diagnostic testing. 
Abnormal Screening Result
Although advanced maternal age increases the risk for fetal chro-
mosome abnormalities, the majority of pregnancies occur in
younger women because they are in their prime reproductive years.
As a result, the majority of liveborn children with chromosome
abnormalities are found in young mothers. To address this issue,
noninvasive screening techniques were developed as a way to iden-
tify which pregnancies have an increased risk for fetal aneuploidy
 CHAPTER 24  Prenatal Diagnosis of Chromosome Abnormalities 235

22
21
20 CVS
Amniocentesis
19
18
17
16
15
14
Frequency (%)
13
12
11
10
9
8
7
6
5
4
3
2
1
0
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Maternal age (yr)
• Fig. 24 .1  Age-related risks for trisomy at the time of chorionic villus sampling (CVS) and amniocente-
sis.105 (Data complied from Hook EB. Chromosome abnormalities: Prevalence, risks and recurrence. In:
Brock DJH, Rodeck CH, Ferguson-Smith MA, eds. Prenatal Diagnosis and Screening. Edinburg: Living-
stone, 1992: 351-392; Snijders RJ, Holzgreve W, Cuckle H et al. Maternal age-specific risks for trisomies
at 9-14 weeks gestation. Prenat Diagn 1994; 14(7): 543-552; Snijders RJ, Sundberg K, Holzgreve W
et al. Maternal age- and gestation-specific risk for trisomy 21. Ultrasound Pbstet Gynecol 1999. 13(3):
167-170.)

without incurring the risk for miscarriage from invasive CVS or
amniocentesis procedures. However, because the combined inci-
dence of fetal chromosome abnormalities (i.e. whole-chromosome
aneuploidies, submicroscopic CNVs, uniparental disomy (UPD))
is higher than the risk for miscarriage from invasive procedures
(0.11%–0.22%),17 any pregnant woman may choose diagnostic
prenatal cytogenetic testing. However, most low-risk women use
noninvasive screening to assess their risk before deciding whether
or not to proceed with a CVS or amniocentesis. Several screening
methods are currently offered to help identify pregnancies at a
higher risk for fetal chromosome abnormalities.  intermediate-risk category, the patient may proceed to second tri-
Noninvasive Screening Using Biochemical
Analytes and Assessment of Nuchal Translucency
Pregnant women have several options for noninvasive screening
in both the first and second trimesters. First trimester screen-
ing involves a combination of maternal blood testing and ultra-
sound analysis of the fetus and can be done as early as 11 weeks.
Maternal blood is tested for the levels of free β-human chorionic
gonadotropin (β-hCG) and pregnancy-associated plasma protein
A (PAPP-A) and combined with sonographic measurement of
the nuchal translucency, the space at the back of the fetal neck.
Such screening can identify 80% to 90% of fetuses with Down
syndrome. Second trimester screening analyses a different set of
maternal serum biomarkers. Quad screening using AFP, hCG,
unconjugated estriol and inhibin A identifies approximately 67%
to 76% of fetuses with Down syndrome. In all cases, the false-pos-
itive rate is around 5%. First and second trimester screening can
be done as independent tests or in combination. When both first
and second trimester screening tests are combined and presented
as a single risk assessment report, it is called integrated screening.
Sequential screening is when all screening tests are performed, but
the patient may decide if the first trimester results are elevated to
proceed to diagnostic testing or defer until all results are complete
in the second trimester. In general, the threshold for first trimester
diagnostic testing is higher (e.g. 1 in 60 to 1 in 100) than that used
later (1 in 270).
Contingent screening uses first trimester screening results to
guide the next steps of prenatal screening and diagnostic test-
ing. If a pregnancy is identified as high risk using first trimester
screening, it is recommended the patient consider invasive testing
such as CVS or amniocentesis. If the pregnancy falls within an
mester screening. Finally, if the pregnancy is deemed low risk for a
chromosome abnormality during first trimester screening, then no
further screening would be recommended. Screening via mater-
nal serum markers is limited to the most common chromosomal
aneuploidies and has a false-positive rate of 5%. In general, screen-
ing using a combination of first and second trimester markers has
a slightly higher detection rate and lower false positive rate than
single trimester screening but depending on the approach chosen
may delay diagnostic testing in screen positive cases. 
Noninvasive Prenatal Screening Using Cell-Free
Fetal DNA
Recently, the ability to analyse fetal cell-free DNA (cffDNA),
present in the maternal circulation of pregnant women, has
paved the way for the development of NIPS for fetal chromo-
somal aneuploidies (13, 18, 21 and sex chromosome anomalies)
and selected microdeletions.25,26 cffDNA is placental in origin,
and its absolute amount is a small proportion of the total cell-free
DNA (cfDNA) component of the maternal plasma, which is a
mixture of both maternal cfDNA and cffDNA (<1 μg in 20 mL of
whole blood).27-29 The current NIPS technologies do not separate
236 SE C T I O N 6     Prenatal Screening and Diagnosis
cffDNA from the maternal cfDNA. Analysis is performed on the
entire cfDNA complement in the maternal blood without extract-
ing or enriching the fetal fraction. NIPS relies on the use of next-
generation sequencing (NGS) (whole genome or targeted) that
looks for the presence of extra sequences from chromosomes of
interest such as 13, 18 and 21. If the mother is phenotypically
normal, the additional sequences are inferred to be derived from
the fetus. Two major sequencing approaches are utilised; the first
is referred to as ‘counting’ because all sequences are counted, and
the DNA sequences from the chromosomes of interest are com-
pared with a reference chromosome or chromosome set to deter-
mine whether they are in excess or missing. The second approach
uses single nucleotide polymorphisms (SNPs) to assess the geno-
type patterns that are indicative of aneuploidy.30,31 Each of these
approaches is coupled with advanced statistical algorithms and
sophisticated bioinformatics software that assesses for abnormal
amounts of chromosome-specific cfDNA in the maternal circula-
tion in pregnancies with fetal aneuploidy. cffDNA is detectable
in maternal plasma by 6 weeks’ gestation, increases during the
pregnancy at a fetal fraction rate of 0.1%/week between 10 to
21 weeks and 1%/week after 21 weeks, and is eventually cleared
from the maternal circulation within a few hours of delivery.32,33
The accuracy of any type of NIPS is limited in cases of multiple
gestations, and as such, the American College of Obstetrics and
Gynecology does not recommend NIPS for women with multiple
gestations.34 Current data suggest that NIPS-based aneuploidy
screening in singleton pregnancies has superior performance in
screening for trisomy 21 compared with all other existing methods
such as combining maternal age, first or second trimester ultra-
sound findings and first or second trimester serum biochemical
analysis.25,35,36 Therefore this screening is an attractive alterna-
tive to traditional serum screening for aneuploidy. Second, com-
pared with trisomy 21, detection rates for trisomies 18, 13 and
sex chromosome aneuploidies are lower. A recent meta-analysis
with the published studies on the performance of cfDNA test-
ing in screening for aneuploidies in singleton pregnancies revealed
weighted pooled detection rates and false-positive rates of 99.2%
and 0.09%, respectively, for trisomy 21; 96.3% and 0.13% for tri-
somy 18; 91.0% and 0.13% for trisomy 13; 90.3% and 0.23% for
monosomy X and 93.0%; and 0.14% for sex chromosome aneu-
ploidies other than monosomy X.35 NIPS was initially validated
for clinical use in high-risk patients. However, recent literature
adds to its utility in the general obstetric population.25 It is impor-
tant to note that cffDNA-based NIPS is a screening method, and
given the potential for false-positive and false-negative results, it is
recommended that screen-positive patients should be followed up
by diagnostic testing using CVS or amniocentesis.34 The current
limitations of NIPS include screening for only a few aneuploi-
dies and select microdeletions, the potential for inaccurate results
and a higher cost. Pregnant women opting for NIPS should be
appropriately counselled about its limitation to screen for only a
few aneuploidies (13, 18 and 21), about the difference between a
screening test and confirmatory diagnostic testing and about the
potential of NIPS to reveal incidental maternal findings such as
a maternal chromosome abnormality or even uncover maternal
cancer.37-39  Structural rearrangements of the chromosomes can occur in clini-
Abnormal Fetal Ultrasound Findings
Ultrasound is important for noninvasive monitoring of all preg-
nancies, from early confirmation of pregnancy to assessing fetal
status through delivery. A recent meta-analysis showed an overall
detection rate between 46% and 51% for fetal anomalies before
14 weeks.40,41 The detection rate in low risk or unselected popula-
tions was 32% and in high-risk groups around 60%.40 Neck anom-
alies, such as increased nuchal translucency or cystic hygroma,
have the highest detection rate (92%).41 Abdominal defects such
as omphalocele and gastroschisis are also detected at a high rate on
early ultrasound. Brain, spine and heart defects had around 50%
detection rate, and limb, genitourinary tract and facial defects
each had only about a 34% detection rate.41 It is also important
to note that some anomalies (e.g. agenesis of the corpus callosum,
cerebellar hypoplasia, renal agenesis, duplex kidneys) will not
become apparent until the second trimester and are not reliably
detected on early ultrasound.41 Anatomy scans between 18 and 20
weeks’ gestation are the standard of care for assessing the health of
fetuses, but with advances in early screening, many women choose
to have an ultrasound in the first trimester.
Chromosome aneuploidies are often associated with multi-
ple congenital abnormalities, which are often apparent on fetal
ultrasonography (Tables 24.1 and 24.2). In the 1990s, practitio-
ners began using sonogram measurements of the nuchal translu-
cency as a marker for Down syndrome.42 Other notable markers
for chromosomal aneuploidies include cystic hygroma, nasal
bone hypoplasia, echogenic intracardiac focus, exomphalos and
fetal growth restriction.43-45 As with serum biomarker screen-
ing, a positive finding increases the risk but does not mean the
fetus is affected with a chromosome abnormality. Generally, the
more markers identified by ultrasound, the higher the risk for an
affected fetus. 
Previous Pregnancy or Child With a
Chromosomal Abnormality
Pregnancy loss or a child born with a chromosome abnormality
can be difficult for the family, and they often seek prenatal diag-
nostic testing in subsequent pregnancies. A diagnosis of triploidy,
tetraploidy, 47,XYY or 45,X does not appear to increase the risk
for future pregnancies to be affected by chromosome abnormali-
ties. Cytogenetic findings with an increased recurrence risk for
future aneuploidies include all nonmosaic trisomies, 47,XXY,
structural rearrangements and marker chromosomes.46 Recur-
rence may be caused by advanced maternal age, as outlined earlier
in this chapter. However, gonadal mosaicism in a parent and other
factors associated with meiotic errors (e.g. translocation carrier)
also increase the risk for subsequent pregnancies with chromo-
some abnormalities. It is generally accepted that the risk increases
1.6- to 1.8-fold for trisomy in future pregnancies when trisomy
is found in a previous pregnancy or miscarriage.47 In the case of
submicroscopic CNVs, the recurrence risk depends on the mode
of inheritance. For de novo CNVs, the recurrence risk is similar to
the general population risk. However, if a parent is a carrier, the
recurrence risk increases to 50%. 
Chromosome Rearrangement or Copy Number
Variant in a Parent
cally normal people. These rearrangements may consist of bal-
anced translocations (segments of two or more chromosomes are
exchanged), inversions (a single segment of one chromosome is
flipped) or insertions. These changes do not involve any net gain
or loss of genetic material and therefore do not typically cause
health issues in the carrier. However, a carrier may face difficulties
 CHAPTER 24  Prenatal Diagnosis of Chromosome Abnormalities 237

TABLE
24.1 Incidence of Chromosome Abnormalities in Prenatal Diagnosis by indication for testing and procedure

CVS +
CVS AMNIOCENTESIS AMNIOCENTESIS

Chromosome Ultrasound All other Ultrasound All other

abnormality anomaly indications Total anomaly indications Total Overall total
Normal karyotype 51 94 86 83 97 93 89
(%)
Any chromosome 49 6 14 17 3 7 11
abnormality (%)
Study Total (n) 411 1798 2209 652 1421 2073 4282

CVS, Chorionic villus sampling.

Data from additional analysis of the 2012 NICHD microarray study dataset.24
  

TABLE Incidence and Spectrum of Chromosome Abnormalities in Prenatal Diagnosis by indication for testing and
24.2 procedure

CVS +
CVS AMNIOCENTESIS AMNIOCENTESIS

Chromosome Ultrasound All other Ultrasound All other

abnormality anomaly indications Total anomaly indications Total Overall total
Trisomy 21 (%) 21.17 3 6.38 4.14 1.41 2.27 4.39
Trisomy 18 (%) 11.19 0.61 2.58 5.37 0.07 1.74 2.17
Trisomy 13 (%) 4.38 0.17 0.95 2.30 0 0.72 0.84
45,X (%) 7.30 0.06 1.40 1.23 0 0.39 0.91
47,XXY (%) 0.97 0.17 0.32 0.15 0 0.05 0.19
47,XXX (%) 0.49 0.06 0.14 0 0.28 0.19 0.16
47,XYY (%) 0 0.06 0.05 0.31 0 0.10 0.07
69,XXX / 69,XXY (%) 1.7 0.17 0.45% 1.07 0 0.34 0.40
Struct rearrangement: 1.22 0.28 0.45% 1.84 0 0.58 0.51
unbalanced (%)
Struct rearrangement: 0.24 1.17 1% 0.31 1.13 0.87 0.93
balanced (%)
Other nonmosaic 0 0.17 0.14 0.15 0 0.05 0.09
aneuploidya (%)
Struct rearrangement: 0 0 0 0.15 0.14 0.14 0.07
markers (%)
Study total (n) 411 1798 2209 652 1421 2073 4282
aOther aneuploidies include two cases of trisomy 9 and two cases of trisomy 16.
Data from additional analysis of the 2012 NICHD microarray study dataset.24
  

with reproduction because of errors in meiotic segregation of the
rearranged chromosomes. In a reciprocal translocation involving
two chromosomes, the resulting gametes may contain a normal
chromosome complement or the balanced translocation, leading
to a clinically normal fetus. However, malsegregation can lead
to partial aneuploidies where one segment of the chromosome
is duplicated but the other is deleted. The net result is a partial
trisomy and a partial monosomy. In general, the larger the chro-
mosomal imbalance, the more likely the pregnancy will result in
a spontaneous abortion. Pericentric inversions – those involving
both arms of the chromosome – carry a similar risk for partial
aneuploidies where the regions distal to the breakpoints may be
238 SE C T I O N 6     Prenatal Screening and Diagnosis
duplicated or deleted. Paracentric inversions only involve a single
arm of the chromosome and therefore produce gametes with nor-
mal or acentric and dicentric chromosome complements. Para-
centric inversion carriers have a lower risk for birth of a child with
a chromosome abnormality because the acentric and dicentric
chromosomes will generally not be viable. Paracentric insertions
have a higher risk for children with cytogenetic abnormalities
because they may inherit unbalanced chromosomes with or with-
out the inserted genetic material.48 Of note, some inversions are
frequent in the population and considered normal genetic varia-
tions or polymorphisms with no known clinical consequences.
One example is the pericentric inversion of chromosome 9 (inv(9)
(p12q13)).
Copy number variations (CNVs) involve submicroscopic
chromosome rearrangements and may be de novo or inherited.
When a CNV is identified, it is important to do parental testing if
their carrier status is unknown. De novo CNVs are more likely to
be clinically relevant, but the recurrence risk is negligible. CNVs
inherited from a clinically normal parent are more likely to repre-
sent benign, familial variants that pose no increased risk for adverse
outcomes. However, there are known pathogenic CNVs that are
associated with phenotypic heterogeneity because of incomplete
penetrance, variable expressivity or both.49,50 One chromosomal
region that is commonly involved in inherited CNVs is the short
arm of chromosome 16. Deletions and duplications at 16p11.2
are associated with neurocognitive abnormalities and may be
inherited from a parent who is clinically normal or only mildly
affected. The recurrence risk is 50% for future pregnancies, but
the clinical outcome is difficult to predict. Genetic counselling
is an important aspect of prenatal diagnosis as we learn more
about genetic conditions and the nuances of ­genotype–phenotype
correlations.
There are many indications for referral for prenatal cytogenetic
diagnosis, and it is important to highlight the need for genetic
counselling. The risk of a fetal chromosome abnormality will vary
widely depending on the patient’s previous history and current rea-
son for referral. Genetic counsellors can integrate important clini-
cal information and communicate with the patient so that the full
scope of the inherent risk, screening results and prenatal diagnostic
results are understood. This information is crucial for the patient
as she makes decisions for her current and future pregnancies.  The sex chromosome aneuploidies can be detected by karyotype
Incidence and Spectrum of Chromosomal
Abnormalities in the Prenatal Setting
The incidence and spectrum of chromosome abnormalities observed
in the prenatal period is highly influenced by the indication for
referral as well as the gestational age at which prenatal diagnosis is
performed. The likelihood of a chromosome abnormality is signifi-
cantly increased when a fetal structural anomaly is detected. Addi-
tional data extraction from the 2012 National Institute of Child
Health and Human Development (NICHD) microarray study24
indicated that karyotype abnormalities were present in 49% and
17% of fetuses with an ultrasound anomaly detected at the time of
CVS and amniocentesis, respectively (see Table 24.1). Karyotype
abnormalities were seen in 6% and 3% of CVS and amniocentesis
samples, respectively, referred for all other indications (see Table 24.1).
In centres in which ultrasound anomalies account for roughly 25%
of referrals, the chances of finding a karyotype abnormality at the
time of prenatal diagnosis (CVS or amniocentesis) for all indica-
tions is just over 10% (see Table 24.1). Table 24.2 shows the full
incidence and spectrum of karyotype abnormalities observed in the
2012 NICHD microarray study.
Aneuploidy
The term aneuploidy is used when a cell has too many (>46) or
too few (<46) chromosomes. The gain or loss of one or several
chromosomes typically involves thousands of genes, which results
in substantial genomic imbalance. Consequently, the majority of
aneuploid conceptions are nonviable. Chromosome abnormali-
ties are observed in approximately 60% to 70% of spontaneous
abortions, and approximately 82% to 85% of them are aneu-
ploidies.51,52 Thus only a few autosomal trisomies and sex chro-
mosomes aneuploidies are routinely encountered in the prenatal
setting. The most common autosomal aneuploidies are trisomy 21
(Down syndrome), trisomy 18 (Edward syndrome) and trisomy
13 (Patau syndrome). Trisomy 21 is the most frequently observed
aneuploidy in prenatal diagnosis and accounts for approximately
one fifth of fetuses with ultrasound anomalies at the time of CVS
and only about one 25th of fetuses with structural defects at the
time of amniocentesis (see Table 24.2). Rapid laboratory meth-
ods of detecting these common aneuploidies within 24 to 48
hours include FISH, QF-PCR and MLPA. These techniques are
discussed in greater detail in the section covering diagnostic tests
for chromosome abnormalities. Table 24.2 shows the incidence
of these common aneuploidies at the time of CVS and amnio-
centesis and further stratifies their frequency according to referral
indication.
The sex chromosome aneuploidies include 47,XXY (Klinefelter
syndrome), 47,XXX (triple X syndrome), 45,X (Turner syndrome)
and 47,XYY. The 45,X and 47,XYY chromosome complements are
associated with paternal meiotic error, and the other aneuploidies
are associated with nondisjunction events caused by advanced mater-
nal age. Monosomy X is more likely to be observed in association
with specific fetal ultrasound anomalies such as cystic hygroma and
large nuchal translucency compared with the other sex chromosome
abnormalities. In the 2012 NICHD study, 7.3% of fetuses with an
ultrasound anomaly (most often enlarged NT) had monosomy X
at the time of CVS (see Table 24.2). This number is significantly
reduced to just over 1% at the time of amniocentesis (see Table 24.2).
analysis and by some of the rapid testing methods described later.
Fetuses with any of these chromosomal aneuploidies can sur-
vive to term; however, spontaneous abortion is a more likely out-
come. Consequently, there are a greater number of abnormalities
seen at the time of CVS, and this number decreases in prevalence
as the pregnancy progresses (see Fig. 24.1 and Table 24.1). Esti-
mates show about 30% of trisomy 21 pregnancies ascertained at
the time of CVS abort spontaneously before term (see Table 24.2).
Similarly, 24% of trisomy 21 pregnancies at the time of amnio-
centesis abort before term.48,53 The phenotypes for the autosomal
trisomies are well documented, making genetic counselling in
these situations fairly straightforward.
Sex chromosome abnormalities usually do not present with
severe clinical features or malformations. This is because of the
phenomenon of X inactivation, whereby in normal females, one
X chromosome undergoes a process of inactivation called lyonisa-
tion, which ‘switches off’ most of the genes.54 However, some X
chromosome genes escape inactivation and are therefore present
as two active copies in normal females. These genes include those
in the ‘pseudoautosomal regions’ (PAR1 and PAR2, at the end of
the short and long arms, respectively, of the sex chromosomes).
 CHAPTER 24  Prenatal Diagnosis of Chromosome Abnormalities
Outside PAR1 and PAR2, about 15% of genes on the X chro-
mosome escape inactivation,55 and the abnormal copy number
of these genes is very likely the cause of the abnormal phenotype
found in individuals with sex chromosome aneuploidy. Some
patients present with learning difficulties and slightly lower IQs
compared with their siblings. Infertility may be another compli-
cating factor, depending on the aneuploidy. Medical management
using hormones is beneficial in some cases, and assisted reproduc-
tive technology can be helpful in some cases with fertility issues,
although pregnancies in woman with a 45,X karyotype have sig-
nificant maternal complications.56  Partial Aneuploidy Caused by Structural
Triploidy and Hydatidiform Moles
Triploidy occurs in 1% to 3% of human conceptions, although most
of these are spontaneously aborted in early pregnancy (6%–7% of
early spontaneous abortions).52,57 Triploidy is highly correlated with
the presence of structural fetal defects and accounts for 1% to 2% of
prenatal cases with structural anomalies (see Table 24.2). Triploidy
can be accurately identified by karyotype analysis, QF-PCR and
SNP microarray analysis but cannot be detected by MLPA analysis
or array comparative genomic hybridisation microarrays that do not
contain SNPs.
Complete triploidy. The normal human chromosome comple-
ment is diploid, in which there are two sets of chromosomes—one
inherited from the mother and the other from the father. In ­triploidy,
the extra set of chromosomes may originate from either the mother
(digyny) or the father (diandry); data indicate that the phenotype of
the embryo can be correlated with the parent of origin of the extra
set of chromosomes and that the differences in phenotypes second-
ary to the parent of origin of the extra set of chromosomes may be
related to imprinting in the placenta.58 Whereas digynic triploidy
arises from the failure of the oocyte to expel the second polar body
at fertilisation, diandric triploidy is thought to be caused by disper-
mic fertilisation, although it may occasionally arise from fertilisation
with a diploid sperm. The diandric triploid state may result in a
partial molar pregnancy, but if this is avoided, the diandric or digy-
nic triploid fetus may survive well into pregnancy,59 although few
survive until term, and no nonmosaic live-born child is known to
have survived the neonatal period.  technologies.65-67 In cases with clinical sequelae, CMA and NGS
Mosaic triploidy. Triploidy may occur in mosaic form; mech-
anisms for mosaic triploidy include fusion of two zygotes, one
normal and one triploid, to give a chimeric fetus; delayed fertilisa-
tion of a zygote with a second sperm; and reincorporation of the
second polar body into the fertilised egg.60 Mosaic triploidy may
be viable to term, depending on the proportion and distribution
of the two cell lines. A case has been reported of mosaic triploidy
detected at CVS, in which the triploid cell line was only pres-
ent in extraembryonic tissues; the pregnancy resulted in a normal
infant.60  CNVs identified by microarray analysis. As more submicroscopic
Complete hydatidiform moles. These pregnancies are diploid,
but both sets of chromosomes are derived from the father. Most
are caused by fertilisation of an anucleate egg by a single sperm fol-
lowed by doubling of the paternal chromosomes; very rarely, molar
pregnancies arise after fertilisation of an anucleate egg by two dif-
ferent sperm. Molar pregnancies are generally sporadic in their
aetiology; however, recurrence in families has been documented,61
and in some cases, biparental inheritance has been demonstrated.
This is thought to be caused by mutation of a gene involved in the
pathway leading to the ‘reprogramming’ of imprinted genes in the
maternal germline, resulting in gametes with paternally imprinted
chromosomes. Complete molar pregnancies have an associated risk
239
for choriocarcinoma. Confirmation of genome-wide uniparental
paternal disomy in complete hydatidiform moles that are diploid
is possible by chromosomal microarray analysis that includes SNPs. 
Partial hydatidiform moles. Some diandric triploid concep-
tuses result in partial molar pregnancies. These are not thought to
be associated with any risk for choriocarcinoma. A partial molar
pregnancy has been described with triploidy mosaicism, in which
the triploid cell line was confined to the placenta but the diploid
fetus survived to term.62 
Rearrangements and Copy Number Variants
In contrast to whole-chromosome aneuploidies, partial aneu-
ploidy involves a smaller number of genes and a greater likelihood
of survival to birth. The size of the imbalance and gene content are
important for understanding the likelihood of abnormal clinical
features and overall prognosis. A G-banding pattern on karyotype
and FISH studies was traditionally used to identify the nature of
partial aneuploidies. Advances in chromosomal or cytogenomic
microarray analysis (CMA) now provide a faster, more accurate
assessment of the origin and nature of chromosome imbalances.
Partial aneuploidies may result when an unbalanced rearrange-
ment is inherited from a parent with a balanced translocation or
inversion. Deletions, duplications, supernumerary marker chro-
mosomes, ring chromosomes and isochromosomes also result in
partial aneuploidies.
When a partial aneuploidy is identified in the fetus, parental
chromosome studies are required to determine whether the vari-
ant is de novo or familial in nature. In general, unbalanced, de
novo changes are more often associated with adverse outcomes. De
novo and familial rearrangements that are balanced are more often
associated with a favourable prognosis. Classical cytogenetic stud-
ies have shown that about 94% of de novo apparently balanced
rearrangements are not associated with adverse clinical outcomes,
but 6% are.63 Such apparently balanced rearrangements can occur
in about 0.4% of pregnancies.64 Refinements of the risk for an
adverse outcome in cases ascertained with an apparently balanced
rearrangement have become possible using microarray and NGS
have identified submicroscopic imbalances and additional struc-
tural complexity that were not discerned at the resolution of the
standard G-banded karyotype.65-67 In contrast, unbalanced rear-
rangements are more often associated with congenital anomalies
and other adverse outcomes regardless of whether they are de novo
or inherited from a balanced parent. CNVs, like other structural
imbalances, are more likely to be deleterious if they are de novo
and benign if inherited from a phenotypically normal parent.
However, caution must be used in the interpretation of certain
balances are identified, it is apparent that a proportion of them
will be associated with phenotypic heterogeneity. Therefore the
offspring may be severely affected even when the parent carries the
same CNV and is clinically normal. Counselling must take into
account the type of imbalance and inheritance and discuss the
risk for subsequent pregnancies if the parents were not previously
known to carry a structural rearrangement or CNV. 
Long Contiguous Stretches of Homozygosity
Chromosomal microarray analysis has advanced prenatal
diagnostics by providing a method to detect submicroscopic
240 SE C T I O N 6     Prenatal Screening and Diagnosis

B C

Chromosome 6 Chromosome 7
• Fig. 24 .2 Detection of uniparental isodisomy of chromosome 7 by single nucleotide polymorphism
(SNP) microarray. A, A single long contiguous stretch of homozygosity (LCSH) is observed for chromo-
some 7 and is shown by the purple bar next to the chromosome. SNP microarray data for chromosomes
6 (B) and 7 (C) showing copy number of 2, log2 ratio of zero (consistent with a copy number of 2), allele
difference plots and presence or absence of LCSH blocks. Note the three expected alleles AA, AB and
BB for chromosome 6. Chromosome 7 demonstrates uniparental isodisomy, and only shows AA and BB
and does not have any heterozygous tracks.

chromosomal abnormalities. Copy number changes as small as
a few kilobases are detectable by copy number oligonucleotide
probes, but SNP oligonucleotide probes are necessary to deter-
mine whether a region is heterozygous or homozygous. Long
contiguous stretches of homozygosity (LCSH) observed in mul-
tiple chromosomes are suggestive of consanguinity (identity by
descent) in the parents. There is an increased risk for autosomal
recessive diseases in these LCSH regions because the genes within
these regions are now homozygous. Noting the regions of homo-
zygosity can be helpful in identifying candidate genes for further
diagnostic testing68 and expanded carrier testing of the parents is
recommended.
In some cases, two copies of a chromosome may be inher-
ited from a single parent, a phenomenon called UPD. In UPD,
there is no aneuploidy per se, but the fetus is homozygous along
the entire length of a chromosome (Fig. 24.2 and Table 24.3).
Similar to cases of consanguinity, the finding of UPD (two identi-
cal chromosomes) leads to an increased risk for autosomal reces-
sive diseases in genes in the homozygous region. If the parent
is a heterozygous carrier of a deleterious allele and both copies
of the gene are inherited from that parent, the offspring can be
affected. UPD can also cause aberrant expression of genes that
are imprinted (only expressed from one parent’s chromosome and
silenced on the other). Certain chromosomes contain imprinted
regions (chromosomes 6, 7, 11, 14, 15 and 20) and UPD for these
chromosomes can result in clinical abnormalities (Table 24.4).69
For example, UPD for chromosome 15 can lead to Prader-Willi
syndrome if both chromosomes are inherited from the mother
or Angelman syndrome if both chromosomes are inherited from
the father.
Uniparental disomy may result from different mechanisms.
When nondisjunction in the gametes leads to trisomy of a specific
chromosome in the embryo, one of the chromosomes may be lost in
a process referred to as trisomy rescue. If the two remaining ‘rescued’
chromosomes are from a single parent, the result is UPD. Similarly,
if there is monosomy for a chromosome, that chromosome may be
replicated to rescue the copy number but will also result in UPD.69
It is important to note that microarray only reliably detects isodi-
somy, inheritance of two identical homologous chromosomes from
a single parent. If a large block of homozygosity is found on a single
chromosome, it may suggest heterodisomy, two different homolo-
gous chromosomes inherited from a single parent, but additional
testing is needed for confirmation. Imprinting disorders can result
from both isodisomy and heterodisomy, but unmasking of a reces-
sive disorder will only occur in cases of isodisomy. 
Diagnostic Tests for Chromosome
Abnormalities
Karyotype
Classical cytogenetic analysis involves the identification and
organisation of a cell’s chromosomes in an ordered fashion, which
we call a karyotype. In prenatal samples, the cells may be grown
in flasks or on coverslips in specialised growth media. When the
culture has a sufficient number of dividing cells, they are subjected
to hypotonic solution, fixed and broken open to reveal a chromo-
some spread. Slides are baked and then stained with Giemsa stain
to show the characteristic banding pattern of alternating light and
dark bands (G-banding). The stained spreads are digitally imaged
TABLE Identifying Uniparental Maternal Isodisomy for Chromosome 7 by Analysis of Single Nucleotide
24.3 Polymorphism Genotypesa
Probe set ID dbSNP RS ID Chromosome Physical position Cytoband Mother Fetus Father
S-3ECAR rs73366581 6 18271814 p22.3 AA AB BB
S-3YVUO rs4714520 6 41913778 p21.1 AA AB BB
S-3VFAH rs9381707 6 48917283 p12.3 AA AB BB
S-4TELX rs435945 6 33496632 p2.31 BB AB AA
S-4NPHJ rs1321518 6 51769501 p12.3 BB AB AA
S-3YLUT rs9342564 6 67326064 q12 BB AB AA
S-4IDXW rs1636897 7 22341983 p15.3 AA AA BB
S-3EEDS rs1419767 7 22344911 p15.3 AA AA BB
S-3NWPG rs1003924 7 22609655 p15.3 AA AA BB
S-4RJFU rs2069852 7 22772260 p15.3 AA AA BB
S-3JSSN rs10270171 7 22813879 p15.3 AA AA BB
S-3TESE rs12533973 7 23831713 p15.3 BB BB AA
S-3RPWV rs78515700 7 23879102 p15.3 BB BB AA
S-4NDZW rs12667136 7 23892995 p15.3 BB BB AA
S-3BODY rs4719742 7 23925448 p15.3 BB BB AA
S-4HOWK rs17360344 7 24160169 p15.3 BB BB AA
S-4OOFE rs1474140 8 12900843 p22 AA AB BB
S-4CIVB rs454100 8 17555736 p22 AA AB BB
S-4MJRS rs7002574 8 56090880 q12.1 AA AB BB
S-4BTPP rs10957521 8 71344432 q13.3 BB AB AA
S-3HNUG rs11785802 8 80256246 q21.13 BB AB AA
S-3VXJV rs7840987 8 89929477 q21.3 BB AB AA
aA total of 51,945 single nucleotide polymorphisms (SNPs) are present on chromosome 6, 46,414 SNPs on chromosome 7 and 38,797 SNPs are present on chromosome 8. Representative informative SNPs are shown

for chromosomes 6, 7 and 8. For chromosomes 6 and 8, the fetus is always heterozygous (AB) when the parents are homozygous for the opposite alleles (i.e. mother is AA and father is BB or mother is BB and father is
AA). This is represented by the green shading. For chromosome 7, the fetus only shows homozygous genotypes identical to the mother when the parents are homozygous for the opposite alleles. This is highlighted in blue.
dbSNP RS ID, Single Nucleotide Polymorphism database reference cluster identification.
  

TABLE
24.4 Phenotypes Associated With Uniparental Disomy
UPD chromosome Parent of origin Syndrome or disorder Phenotype OMIM#
6 Paternal Transient neonatal diabetes mellitus IUGR, neonatal diabetes 601410
7 Maternal Silver-Russell syndrome IUGR/FTT, dysmorphic 180860
11 Maternal Silver-Russell syndrome IUGR/FTT, dysmorphic 180860
11 Paternal Beckwith-Wiedemann syndrome Overgrowth, haemihypertrophy, embryonal malignan- 130650
cies, dysmorphic
14 Maternal Temple syndrome IUGR, dysmorphic 616222
14 Paternal Kagami-Ogata syndrome Bell-shaped thorax, developmental retardation, dwarf- 608149
isms, dysmorphic
15 Maternal Prader-Willi syndrome Obesity, dysmorphic, ID 176270
15 Paternal Angelman syndrome ID, dysmorphic 105830
20 Maternal Growth failure, hyperactivity IUGR/FTT —
20 Paternal Pseudohypoparathyroidism 1b Pseudohypoparathyroidism 603233

FTT, Failure to thrive; ID, intellectual disability; IUGR, intrauterine growth restriction; OMIM, online Mendelian inheritance in man; UPD, uniparental disomy.
  
242 SE C T I O N 6     Prenatal Screening and Diagnosis

Trisomy 13 female Trisomy 18 male

LSI 13 LSI 13
LSI 21 LSI 21

CEP X CEP X
CEP Y CEP Y
CEP 18 CEP 18

• Fig. 24 .3  Detection of trisomy 13 in a female fetus and trisomy 18 in a male fetus by fluorescence in situ
hybridization using chromosome enumeration probes (CEP) for chromosomes X, Y and 18 (CEP X, CEP Y,
CEP18) and locus-specific identifier (LSI) probes for chromosomes 13 and 21 (LSI 13, LSI 21). Left panels,
Normal signals (2 copies) are observed for chromosomes 18 and 21, and a female hybridisation pattern is
seen for the X chromosome probe. Three signals are observed for the chromosome 13 probe, indicating
trisomy 13. Right panels, Normal signals (two copies) are observed for chromosomes 13 and 21, and a
male hybridisation pattern is seen for the X and Y chromosome probes. Three signals are observed for the
chromosome 18 probe, indicating trisomy 18.

and karyotyped using specialised computer software. Metaphase
spreads and karyotypes are analysed to identify numerical and
structural abnormalities. Standard practice dictates that multiple
cells are analysed (usually 15 colonies from in situ coverslip cul-
tures or 20 cells from flask cultures) to maximise the likelihood
(95% confidence) of detecting mosaicism at a level of 14% or
greater.70 Structural anomalies and gains or losses of chromosomal
material that are 7 to 10 megabases (Mb) or greater in size are
usually identified. However, there is a great deal of variation in the
banding resolution of each case, and some preparations that yield
karyotypes with a banding resolution around 400 may preclude
the ability to readily detect abnormalities below 10 to 20 Mb.  the FISH panel. These additional studies can also characterise
Rapid Aneuploidy Detection
Fluorescence in situ hybridisation. Molecular cytogenetic analysis,
in the clinical space, was launched with the advent of FISH.
FISH uses fluorescently tagged DNA probes to bind to unique,
complementary sequences on the chromosomes, thereby indicating
their presence/absence and relative copy number. FISH analysis
in interphase nuclei (Fig. 24.3) provides a quick diagnostic test
for uncultured chorionic villi and amniotic fluid samples.71 The
common aneuploidies involving chromosomes 13, 18, 21, X
and Y can be analysed in 1 to 2 days with a concordance of more
than 99% compared with standard G-banded karyotype analysis.
Diagnostic test parameters, such as sensitivity, specificity and
predictive values, are also greater than 99%.72 The availability of
this targeted FISH panel is advantageous for rapidly confirming
a chromosomal aneuploidy when there is an abnormal serum
screen or NIPS result and assists in making decisions regarding the
pregnancy. However, there is residual risk for low-level mosaicism,
structural rearrangements and marker chromosomes involving these
chromosomes. Additionally, no information is provided regarding
any of the other chromosomes. FISH testing should always be
followed up with karyotype, microarray or both to confirm positive
findings and rule out chromosome abnormalities not included on
the mechanism of the abnormality in some positive cases, such as
trisomy 21, caused by a translocation (10%–15% recurrence risk
if the mother is a balanced carrier) versus trisomy 21 caused by
nondisjunction (recurrence risk correlates with maternal age).
Fluorescence in situ hybridisation analysis using probes in
addition to those for chromosomes n 21, 18, 13, X and Y can
identify the origins of aberrant chromosomal material, including
rearrangements, additions, insertions and supernumerary marker
chromosomes that may be ambiguous on karyotype.73,74 The ori-
gins of such chromosomal changes are important for understand-
ing the genotype–phenotype correlation and prognosis for the
pregnancy. However, it is an expensive and time-consuming pro-
cess to sequentially test FISH probes. Chromosomal microarray
 CHAPTER 24  Prenatal Diagnosis of Chromosome Abnormalities
TABLE Most Frequent Copy Number Imbalances Observed in Patients With Fetal Anomalies Versus Without Fetal
24.5 Anomalies
FETAL ANOMALIES
CNV Frequencya (%) (deletion + duplication)
22q11.21 18.0
17q12 9.8
16p13.11 9.8
1q21.1 4.9
10q21.1 3.3
15q13.3 3.3
Single occurrence 50.8
aData from Donnelly et al.83
bData compiled from Fiorentino et al.101 Shaffer et al.102 Armengol et al.103 Lee et al.104 and Wapner et al.24
provides a single high-resolution test to identify a broad range of
chromosomal abnormalities, often in a similar time frame as tra-
ditional cytogenetics. Although interphase FISH still provides the
fastest results for the common aneuploidies, chromosomal micro-
array is now widely used for broader prenatal diagnosis.  LCSH regions. Rather than breaking open the cells to examine
Quantitative fluorescence polymerase chain reaction. Quanti
tative fluorescence polymerase chain reaction is a technique that is
used to detect trisomy in prenatal samples. The analysis involves
PCR amplification of genetic markers, called small tandem repeats
(STRs), present throughout the genome and polymorphic in the
general population. The PCR products are then separated by cap-
illary electrophoresis and chromosomal copy number is inferred
from the pattern of peaks for each chromosome. In the prenatal
setting, it is an efficient and high-throughput technique that can
rapidly confirm or exclude trisomies 13, 18 and 21, as well as sex
chromosome abnormalities.75-77 Many cytogenetics laboratories
have adopted this method as a cost-effective way to test for aneu-
ploidies. In addition, QF-PCR is more sensitive than other tech-
niques for identifying mosaicism and provides results for maternal
cell contamination, two issues described in more detail later.  much smaller imbalances. Karyotype analysis by G-banding is, at
Multiplex ligation-dependent probe amplification. Multiplex
ligation-dependent probe amplification is another PCR-based
method to detect abnormal chromosome copy number.78-80 Rather
than detecting STRs, MLPA relies on probes that target a specific
region of the genome. Each probe contains two oligonucleotides
that are ligated after they are bound to their targets in the genomic
DNA and subsequently amplify the region. Using fluorescently
labelled probes and capillary electrophoresis, the amplified regions
are analysed, and the relative quantity of each region can be deter-
mined. MLPA is one of the few techniques with the ability to
quickly and accurately identify small deletions and insertions that
may be below the resolution of FISH or CMA. However, limita-
tions in detecting mosaicism and maternal cell contamination pre-
vent MLPA from being widely applicable in the prenatal setting.  in association with or without fetal anomalies detected by ultra-
Chromosomal Microarray
Chromosomal or cytogenomic microarray analysis is a high-
resolution molecular cytogenetic diagnostic test that detects
243
ALL OTHER INDICATIONS
CNV Frequencyb (%) (deletion + duplication)
22q11.21 15.4
16p13.11 13.5
1q21.1 9.6
17p12 9.6
16p11.2 7.7
Xp21.1 7.7
Xp22.3 7.7
15q11.2 5.8
Single occurrence 23.1
unbalanced chromosomal abnormalities, including aneuploidies,
structural rearrangements, and LCSH. CMA uses copy num-
ber probes to detect any gains or losses of chromosomal mate-
rial. Some CMA platforms include SNP probes that also identify
chromosome structure as in classic cytogenetic techniques, the
DNA is isolated from the patient’s cells and hybridised to a chip
containing several million copy number and SNP probes. Soft-
ware interprets the relative fluorescent signal at each probe to
assess any gains, losses or homozygosity across the whole genome.
In the prenatal setting, CMA is used most commonly for CVS and
amniocentesis samples and may be performed on DNA extracted
from a direct preparation or cultured cells. The turnaround times
are approximately 7 to 10 days for direct preparations and 14 to
21 days for cultured cells. PUBS samples are more rarely seen but
have a similar turnaround time as direct preparations.
There are many advantages to using CMA for prenatal genetic
diagnosis of chromosomal abnormalities. The biggest advantage
over classic cytogenetic and FISH techniques is the ability to detect
best, only accurate to a resolution of approximately 5 to 10 Mb.
FISH detects smaller abnormalities but often requires clinical fea-
tures to guide probe selection, a difficult task for prenatal samples.
Rather than sequentially testing many FISH probes, CMA detects
abnormalities as small as a few kilobases anywhere in the genome in
a single test, limited only by the probes present on the chip. CMA
is 100% accurate in identifying the common aneuploidies com-
pared with karyotype and provides an increased diagnostic yield in
patients with clinical indications (advanced maternal age [AMA],
positive serum screening or ultrasound anomaly) and a normal
karyotype.24,81,82 The increase in diagnostic yield was 6%, seen in
patients with an ultrasound abnormality and a normal karyotype.24
Table 24.5 lists the most frequent copy number changes observed
sound. The 22q11.2 imbalance appears to be the most common
submicroscopic imbalance observed in cases with and without fetal
anomalies, highlighting the phenotypic heterogeneity that may be
associated with CNVs. Such clinical heterogeneity also emphasises
the inability to screen for clinically significant CNVs by ultrasound
244 SE C T I O N 6     Prenatal Screening and Diagnosis
alone. CMA provides a diagnosis for submicroscopic deletions and
duplications that are clinically relevant and are not age dependent.
Unlike nondisjunction, which increases with maternal age, micro-
deletions and duplications can occur in any conception. Moreover,
a study found that the majority of abnormal copy number changes
found on prenatal CMA were seen only once24,83 (see Table 24.5),
showing the importance of CMA in diagnosing nonrecurrent chro-
mosomal abnormalities. The increased diagnostic power gained by
CMA along with the low risk for adverse outcomes after CVS or
amniocentesis has led to the recommendation that prenatal diag-
nostic testing be offered to all pregnant women regardless of their
inherent risk for having offspring with a chromosome abnormality.
Another advantage is the ability to precisely characterise abnor-
malities identified by classical cytogenetics. A gain or loss may
be seen on a karyotype, but CMA provides the precise break-
points and the genes involved in the abnormality. This is particu-
larly important because a large deletion removing very few genes
may be less clinically relevant than a small deletion in a gene-
rich region. CMA may also reveal small gains or losses around
the breakpoints of structural rearrangements that appear balanced
by karyotype. Finally, CMA using SNP probes provides genome-
wide genotype information that uncovers UPD and identity by
descent (consanguinity).
It is important to note that CMA does have some limitations.
As with any clinical test, the sensitivity relies on the quality of
the sample and for CMA the quality of the extracted DNA. Each
CMA platform is also limited by the resolution of probe coverage
(the number of probes and their spacing) so that abnormalities
restricted to areas with low or no coverage or below the resolution
of the probe spacing may be missed. Triploidy cannot be detected
by copy number probes because the additional copy of all chro-
mosomes is masked by the normalisation procedure run by the
software analysis. SNP probes must be analysed for additional
genotypes from the extra chromosomes to identify cases of trip-
loidy. Mosaicism may also be difficult to detect and is discussed
further in the next section.
Chromosomal or cytogenomic microarray analysis does not
detect balanced rearrangements (e.g. translocations, inversions),
and a normal CMA result does not exclude the presence of single-
gene disorders. Even when the CMA result is positive, it will not
always provide the chromosomal mechanism causing the imbal-
ance. For example, CMA will detect the presence of trisomy 21
in a prenatal sample but cannot detect whether it is caused by a
translocation or nondisjunction. In such cases, classical cytoge-
netic analysis in the fetus and the parents is crucial for determin-
ing reproductive risk for future offspring. Genetic counselling is
very important for explaining the advantages and limitations of
CMA, as well as the implications of positive or negative results or
variants of uncertain significance. Parameters during CMA analy-
sis can be set to reduce the chance of finding copy number variants
of uncertain clinical significance; nevertheless, it is possible that
one will be identified and that parental testing will be needed.  mosaic trisomy 18 cases detected at CVS were confirmed in the
Special Issues for Prenatal Diagnosis of
Chromosome Abnormalities
Genotype–Phenotype Correlation
Mosaicism. Mosaicism, in the context of human genetics, refers
to the presence of more than one cell line with different genomic
complements within a person that developed from a single
fertilised egg. In constitutional cases, one cell line is likely to have
a normal 46-chromosome complement, but the other cell line has
an alteration in the number or structure of the chromosomes. For
example, genetic analysis of a person with Down syndrome may
show a karyotype in which half the cells are 46,XX and the other
half are 47,XX,+21.
For diagnosis of chromosomal mosaicism, it is imperative to
distinguish between true mosaicism and pseudomosaicism. Pseu-
domosaicism is an artefact caused by the manipulation of the
sample in the laboratory. The most common source of pseudomo-
saicism is the result of tissue culture. In such cases, an abnormal-
ity may arise in a single colony during culture growth and does
not represent the true fetal karyotype. To distinguish between true
mosaicism and pseudomosaicism in prenatal specimens, labora-
tories use three levels of classification (levels I, II and III). Level I
mosaicism is an abnormality seen in a single cell, and the result is
not confirmed after additional workup within the same culture or
separate cultures from the same patient. Level I mosaicism is not
considered clinically significant or reportable because it is most
likely a culture artefact. Level II mosaicism occurs when a single
flask from cultured CVS or an entire colony of amniocytes shows
a chromosomal abnormality, but the abnormality is not found in
other cultures after extensive workup. Level II mosaicism is usually
pseudomosaicism as well. When identified at the time of CVS, the
pregnancy most often results in a normal fetus.84,85 In amniocen-
tesis, level II mosaicism is indicative of a true fetal karyotype in
approximately 1% of cases.84-86 Level III mosaicism occurs when
the chromosomal abnormality is identified in two or more cells or
colonies across multiple cultures. Level III mosaicism is the most
likely to reflect true mosaicism rather than pseudomosaicism. If
true mosaicism is suspected but additional cultures are not avail-
able for confirmation, interphase FISH may be used to score a
large number of additional cells. In this respect, FISH is superior
to chromosomal microarray, which cannot accurately assess mosa-
icism below approximately 10% to 15%.
True mosaicism may reflect two different situations: gener-
alised mosaicism or confined placental mosaicism. Generalised
mosaicism is when the abnormality is found in both the fetus
(true fetal mosaicism) and the placenta, and confined placental
mosaicism is when the abnormality is absent from the fetus but
found in the extraembryonic tissues.69,87 Approximately 1% to
2% of CVS samples show chromosomal mosaicism, and of these,
about 87% are confined to the placenta.88 A mosaic chromosome
abnormality detected at CVS has the highest likelihood (∼37.5%)
of being confirmed in the fetus when observed in both cytotro-
phoblast and mesenchymal core and the lowest chance (∼3.7%)
when observed only in the cytotrophoblast and not in the mes-
enchyme88 (Table 24.6). The likelihood of being confirmed in
the fetus at the time of amniocentesis also differs depending on
the specific chromosome involved. In a large monocentric series
of 1001 mosaics in chorionic villi with follow-up amniocente-
sis, approximately one third of trisomy 21 mosaics and 17% of
fetus compared with 10% of trisomy 20 mosaics and only 2.4%
of trisomy 13 mosaics.88
When an abnormal result is found on CVS, it is important for
counselling purposes to consider whether the chromosome abnor-
mality may result in a liveborn child. For example, finding level II
or III mosaic trisomy 18 on CVS should result in close follow-up
care, including amniocentesis to confirm the fetal karyotype and
ultrasonography to identify any developing fetal structural anoma-
lies and amniocentesis to confirm the fetal karyotype. If the abnor-
mality is incompatible with life, such as trisomy 16, it is more likely
 CHAPTER 24  Prenatal Diagnosis of Chromosome Abnormalities
TABLE Distribution of Mosaic Cases Identified at Chorionic Villus Sampling and Subsequently Confirmed in an
24.6 Amniocentesis According to Embryonic Location of the Mosaic Abnormality
Embryonic origin MESENCHYMAL CORE +
Cytotrophoblast + CPM III
62.5%
Cytotrophoblast - CPM II
87.9%
CPM, Confined placental mosaicism; TFM, true fetal mosaicism.
Data from Malvestiti et al.88
to be confined placental mosaicism, which can be confirmed by
amniocentesis. Trisomies 2 and 7 are the most frequently detected
aneuploidies in CVS but are almost never confirmed by amnio-
centesis.88 However, with mosaicism, there are never any absolutes
as the entire phenotype in a nonviable rare trisomy can be attenu-
ated by virtue of the presence of a normal cell line. Rare autosomal
aneuploidies are observed in approximately 1 in 180 CVS and only
about 1 in 2440 amniotic fluid samples.89 The attrition in preva-
lence from the time of CVS to the time of amniocentesis indicates
that the greater majority of these are confined to the placenta. Rare
autosomal mosaic trisomies that represent true fetal mosaicism are
more likely to be associated with structural fetal anomalies, and
approximately 12.6% of chromosomally abnormal pregnancies
with ultrasound anomalies have a rare autosomal trisomy.90-93
It is important to note that abnormalities that represent con-
fined placental mosaicism may also require follow-up testing or
close clinical management. Confined placental mosaicism of tri-
somy 16 is believed to cause poor placental function, resulting in
intrauterine growth restriction of the fetus and preeclampsia in the
mother, often leading to pregnancy complications and preterm
birth.94,95 For chromosome 15 and other imprinted chromosomes,
trisomy in the placenta and normal complement in the fetus raises
the concern for UPD for that chromosome.69,94 It is possible that
the embryo was originally trisomic for chromosome 15 and had
a trisomy rescue event that allowed the fetus to survive. In such
cases, UPD testing should be performed to rule out the possibil-
ity of Prader-Willi syndrome or Angelman syndrome in the fetus.
An additional caveat is that certain chromosomal abnormalities
are observed in only a single tissue or sample type. Trisomy for
chromosome 8 may be observed on CVS but is rarely identified in
a subsequent amniocentesis.96 Patients should be counselled about
their residual risk for chromosome abnormalities despite normal
amniocentesis and ultrasound follow-up.  Technical Considerations
Phenotypic heterogeneity and variants of uncertain clinical
significance. Parental testing is an important part of assessing
genetic changes identified on prenatal testing, particularly if it is
a variant of uncertain clinical significance (VOUS). VOUSs are
genetic changes that are not commonly seen in the population and
have little or no clinical evidence available to assess pathogenicity.
VOUSs may represent benign, familial variants that produce no
clinical features or may be rare deleterious changes resulting in a
clinical phenotype. The size, gene content and inheritance pattern
can help discern whether VOUSs are more likely to be benign
or pathogenic. Chromosomal imbalances seen on karyotype are
usually fully penetrant because of changes in a large number of
genes. Therefore if the same finding is seen in a normal parent, the
prognosis is usually good for the fetus.
245
MESENCHYMAL CORE -
TFM VI CPM I TFM IV
37.5% 96.3% 3.7%
TFM V
12.1%
Interpretation of smaller copy number changes identified by
CMA can be more challenging. Public databases are available
to help identify common, benign CNVs in the population, and
every year, new reports identify recurrent pathogenic CNVs to
help with prenatal diagnosis. In general, a CNV that is inherited
from a normal parent is less likely to be clinically relevant; how-
ever, emerging data show that a growing number of CNVs have
incomplete penetrance, variable expressivity or both. Although
studies support the pathogenicity of these CNVs, the patients fall
along a spectrum of clinical outcomes, some severely affected but
others being mildly affected or normal.49,50 Many of the CNVs
that fall into this category affect neurocognitive development, and
some evidence suggests that a second hit or increased mutational
burden plays a role in the observed phenotype.49,97 Interpretation
and counselling in these situations are particularly challenging
because it is not always possible to predict the clinical outcome
for the fetus.
For phenotype–genotype correlation, in both classical and
molecular cytogenetics, it is important to remember that patients
in the postnatal setting are usually ascertained because of clini-
cal features related to their genetic condition. The prenatal set-
ting is complicated by the fact that many features are not readily
observed in a fetus or only become apparent at a later gestational
age. As CMA continues to be used widely for prenatal diagnosis,
better classification of the pathogenicity of rare copy number vari-
ants and their clinical outcomes will be possible and provide better
guidance for clinical care. Counselling patients before testing is
important so they understand the possibility of receiving results
that provide unexpected information about themselves or that do
not have clearly established or specific clinical outcomes. 
Maternal Cell Contamination
Prenatal specimens present a challenge in that there may be mater-
nal cell contamination (MCC) within the sample. Prenatal samples
that contain maternal blood, such as transplacental amniocentesis, are
more likely to have a higher percentage of MCC. CVS samples need
to be carefully cleaned to avoid contamination by maternal decidua.
Studies on cultures from amniotic fluid and CVS show a low level
of MCC, at 0.3% for amniocyte cultures and 1.8% for CVS cul-
tures.85,98,99 Molecular techniques are now very common for prenatal
testing and have the advantage of assaying specimens directly without
waiting for cultures. However, cultures tend to have less MCC so
direct analysis by FISH and microarray should be interpreted with
caution, especially if the sample contains maternal blood.
246 SE C T I O N 6     Prenatal Screening and Diagnosis
Various molecular studies can be used to rule out suspected
MCC. It is standard practice to assess MCC before running a
chromosomal microarray as presence of contamination will result
in additional SNP tracks and noisy data. A recent study showed
that MCC begins to affect the array data at approximately 10%.
For chromosomal aberrations, duplications of about 500  kb
become obscured at 20% to 30% MCC and small deletions
(75–100 kb) at 50% to 60% MCC.100 Similar to QF-PCR, STR
analysis is most commonly used to assess MCC before running
microarray analysis. The analysis involves measuring the exact
number of repeats at several loci in both the fetus and the mother
to determine the allele sizes in each. The fetal and maternal alleles
are compared, and the percentage of MCC is calculated from
maternal-specific alleles found in the fetal sample. STR analysis
is helpful for identity testing and other molecular genetic analysis
and may also be used on cytogenetic samples that may be con-
taminated (e.g. postnatal cord blood).  microdeletions and microduplications), and current recommenda-
Culture Failure
Prenatal specimens should be set up and monitored with care to
avoid culture failure. Although the rate of culture failure is gener-
ally low, it varies from laboratory to laboratory. Factors that con-
tribute to the success of prenatal cultures include the size of the
sample and the gestational age. Very small CVS samples (<5 mg)
have a higher risk for culture failure. Amniocentesis samples from
pregnancies at an advanced gestational age also have increased
failure rates because of the large number of nonviable cells pres-
ent in the amniotic fluid.46 Interphase FISH and chromosomal
microarray offer the advantage of direct analysis on the sample and
may provide results (although limited in the case of FISH) even
if the culture is unsuccessful. There is no evidence to date that the
cytogenetic status of the fetus correlates with incidence of culture
failure. If culture failure occurs, patients are offered a repeat pro-
cedure for cytogenetic or microarray analysis. 
Concluding Remarks
Prenatal cytogenetic diagnosis has undergone significant advance-
ments since it began more than 50 years ago. Common prenatal
diagnostic tests for chromosome abnormalities now include rapid
targeted techniques such as FISH, QF-PCR and MLPA, and
whole-genome assessment has expanded beyond the karyotype to
include microarray analysis. The focus of cytogenetic prenatal test-
ing has primarily been aneuploidy given the increased risk for chro-
mosomal anomalies associated with maternal age. However, recent
advances in diagnostic testing now allow for the detection of sub-
microscopic abnormalities that appear to be age independent (e.g.
tions highlight the need for all women to have access to prenatal
diagnostic testing for genomic imbalances. Despite the advances
in technology, detection of low-level mosaicism and lack of high-
resolution breakpoint data in apparently balanced de novo cytogenetic
aberrations remain challenges in clinical cytogenomic testing. These
issues are being addressed with the introduction of next generation
sequencing-based assays into the realm of prenatal diagnosis. Cur-
rent investigations of sequencing technology are aimed at assessing
their diagnostic value and clinical utility during pregnancy. Such
improvements will likely pave the way for a comprehensive copy
number and sequence-based genetic testing option during preg-
nancy by means of routine fetal sequencing.
Access the complete reference list online at ExpertConsult.com.
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References trends and outcomes. Eur J Obstet Gynecol
Reprod Biol. 2016;200:98–101.
35. Gil MM, Quezada MS, Revello R, et  al.
Analysis of cell-free DNA in maternal blood
19. Wilson RD, Gagnon A, Audibert F, et  al. in screening for fetal aneuploidies: updated
1. Jacobson CB, Barter RH. Intrauterine diag-
Prenatal diagnosis procedures and techniques meta-analysis. Ultrasound Obstet Gynecol.
nosis and management of genetic defects. Am
to obtain a diagnostic fetal specimen or tis- 2015;45(3):249–266.
J Obstet Gynecol. 1967;99(6):796–807.
sue: maternal and fetal risks and benefits. J 36. Norton ME, Jacobsson B, Swamy GK,
2. Nadler HL, Gerbie AB. Role of amniocen-
Obstet Gynaecol Can. 2015;37(7):656–670. et  al. Cell-free DNA analysis for noninva-
tesis in the intrauterine detection of genetic
20.  Committee Opinion No.682: Microarrays sive examination of trisomy. N Engl J Med.
disorders. N Engl J Med. 1970;282(11):
and next-generation sequencing technol­ogy: 2015;372(17):1589–1597.
596–599.
the use of advanced genetic diagnostic tools 37. Amant F, Verheecke M, Wlodarska I, et  al.
3. Sarto GE. Prenatal diagnosis of genetic
in obstetrics and gynecology. Obstet Gynecol. Presymptomatic identification of cancers in
disorders by amniocentesis. Wisc Med.
128(6):e262–e268. pregnant women during noninvasive prenatal
1970;69(12):255–260.
21. American College of OG. ACOG Practice testing. JAMA Oncol. 2015;1(6):814–819.
4. Steele MW, Breg Jr WR. Chromosome anal-
Bulletin No. 88, December 2007. Invasive 38. Osborne CM, Hardisty E, Devers P, et al. Dis-
ysis of human amniotic-fluid cells. Lancet.
prenatal testing for aneuploidy. Obstet Gyne- cordant noninvasive prenatal testing results in a
1966;1(7434):383–385.
col. 2007;110(6):1459–1467. patient subsequently diagnosed with metastatic
5. Gerbie AB. Amniocentesis for prenatal detec-
22. American College of O. Gynecologists Com- disease. Prenat Diagn. 2013;33(6):609–611.
tion of genetic defects. Am J Obstet Gynecol.
mittee on G. Committee opinion no. 581: 39. Vandenberghe P, Wlodarska I, Tousseyn T,
1977;127(2):158–161.
the use of chromosomal microarray analy- et  al. Non-invasive detection of genomic
6. Gerbie AB, Simpson JL. Antenatal detec-
sis in prenatal diagnosis. Obstet Gynecol. imbalances in Hodgkin/Reed-Sternberg cells
tion of genetic disorders. Postgrad Med.
2013;122(6):1374–1377. in early and advanced stage Hodgkin’s lym-
1976;59(6):129–136.
23. Penrose LS. The relative effects of paternal phoma by sequencing of circulating cell-free
7.  NICHD. Midtrimester amniocentesis for
and maternal age in mongolism. 1933. J DNA: a technical proof-of-principle study.
prenatal diagnosis. Safety and accuracy.
Genet. 2009;88(1):9–14. Lancet Haematol. 2015;2(2):e55–e65.
JAMA. 1976;236(13):1471–1476.
24a. Sparks TN, Norton ME, Flessel M, Goldman S, 40. Karim JN, Roberts NW, Salomon LJ, Papa-
8. Brambati B, Simoni G. Diagnosis of
Currier RJ. Observed rate of down syn- georghiou AT. Systematic review of first
fetal trisomy 21 in first trimester. Lancet.
drome in twin pregnancies. Obstet Gynecol. trimester ultrasound screening in detecting
1983;I(8324):586.
2016;128(5):1127–1133. PubMed PMID: fetal structural anomalies and factors affect-
9. Gosden JR, Mitchell AR, Gosden CM, et al.
27741202. ing screening performance. Ultrasound Obstet
Direct vision chorion biopsy and chromo-
24b. Wapner RJ, Martin CL, Levy B, et  al. Gynecol. 2017;50(4):429–441.
some-specific DNA probes for determination
Chromosomal microarray versus karyotyp- 41. Rossi AC, Prefumo F. Accuracy of ultra-
of fetal sex in first-trimester prenatal diagno-
ing for prenatal diagnosis. N Engl J Med. sonography at 11-14 weeks of gesta-
sis. Lancet. 1982;II(8313):1416–1419.
2012;367(23):2175–2184. tion for detection of fetal structural
10. Old JM, Ward RH, Petrou M, et  al.
25. Bianchi DW, Parker RL, Wentworth J, anomalies: a systematic review. Obstet Gyne-
First-trimester fetal diagnosis for hae-
et al. DNA sequencing versus standard pre- col. 2013;122(6):1160–1167.
moglobinopathies: three cases. Lancet.
natal aneuploidy screening. N Engl J Med. 42. Scott F, Boogert A, Sinosich M, Anderson J.
1982;II(8313):1413–1416.
2014;370(9):799–808. Establishment and application of a normal
11. Ward RH, Modell B, Petrou M, et  al.
26. Wapner RJ, Babiarz JE, Levy B, et  al. range for nuchal translucency across the first
Method of sampling chorionic villi in first
Expanding the scope of noninvasive prenatal trimester. Prenat Diagn. 1996;16(7):629–634.
trimester of pregnancy under guidance of
testing: detection of fetal microdeletion syn- 43. Filkins K, Koos BJ. Ultrasound and fetal
real time ultrasound. Br Med J (Clin Res Ed).
dromes. Am J Obstet Gynecol. 2015;212(3): diagnosis. Curr Opin Obstet Gynecol.
1983;286(6377):1542–1544.
332.e331–e339. 2005;17(2):185–195.
12. Centers for Disease Control and Prevention
27.  Ariga H, Ohto H, Busch MP, et al. Kinetics of 44. Nicolaides KH. Nuchal translucency and
ASfRM. Society for assisted reproductive
fetal cellular and cell-free DNA in the mater- other first-trimester sonographic markers
technology. Chorionic villus sampling and
nal circulation during and after pregnancy: of chromosomal abnormalities. Am J Obstet
amniocentesis: recommendations for pre-
implications for noninvasive prenatal diagno- Gynecol. 2004;191(1):45–67.
natal counseling. MMWR. 1995;44(RR-9):
sis. Transfusion. 2001;41(12):1524–1530. 45. Nyberg DA, Souter VL. Use of genetic sonog-
1–12.
28. Lo YM. Fetal DNA in maternal plasma: biol- raphy for adjusting the risk for fetal Down syn-
13. Rhoads GG, Jackson LG, Schlesselman SE,
ogy and diagnostic applications. Clin Chem. drome. Semin Perinatol. 2003;27(2):130–144.
et al. The safety and efficacy of chorionic vil-
2000;46(12):1903–1906. 46. Randolph LM. Prenatal cytogenetics. In:
lus sampling for early prenatal diagnosis of
29. Lo YM, Tein MS, Lau TK, et  al. Quanti- Gersen SL, Keagle MB, eds. The principles
cytogenetic abnormalities. N Engl J Med.
tative analysis of fetal DNA in maternal of clinical cytogenetics. Totowa, NJ: Humana
1989;320(10):609–617.
plasma and serum: implications for nonin- Press; 2005:267–321.
14. Akolekar R, Beta J, Picciarelli G, et al. Pro-
vasive prenatal diagnosis. Am J Hum Genet. 47. Warburton D, Dallaire L, Thangavelu M,
cedure-related risk of miscarriage following
1998;62(4):768–775. et  al. Trisomy recurrence: a reconsideration
amniocentesis and chorionic villus sampling:
30. Samango-Sprouse C, Banjevic M, Ryan based on North American data. Am J Hum
a systematic review and meta-analysis. Ultra-
A, et  al. SNP-based non-invasive prena- Genet. 2004;75(3):376–385.
sound Obstet Gynecol. 2015;45(1):16–26.
tal testing detects sex chromosome aneu- 48. Gardner RJM, Sutherland GR, Shaffer LG.
15. Evans MI, Wapner RJ. Invasive prenatal
ploidies with high accuracy. Prenat Diagn. Chromosome abnormalities and genetic coun-
diagnostic procedures 2005. Semin Perinatol.
2013;33(7):643–649. seling. 4th ed. New York: Oxford University
2005;29(4):215–218.
31. Zimmermann B, Hill M, Gemelos G, et al. Press; 2012.
16. Duchatel F, Oury JF, Mennesson B, Muray
Noninvasive prenatal aneuploidy testing of 49. Coe BP, Girirajan S, Eichler EE. The genetic
JM. Complications of diagnostic ultrasound-
chromosomes 13, 18, 21, X, and Y, using tar- variability and commonality of neurodevel-
guided percutaneous umbilical blood sam-
geted sequencing of polymorphic loci. Prenat opmental disease. Am J Med Genet C Semin
pling: analysis of a series of 341 cases and
Diagn. 2012;32(13):1233–1241. Med Genet. 2012;160C(2):118–129.
review of the literature. Eur J Obstet Gynecol
32. Lo YM, Zhang J, Leung TN, et  al. Rapid 50. Torres F, Barbosa M, Maciel P. Recurrent
Reprod Biol. 1993;52(2):95–104.
clearance of fetal DNA from maternal plasma. copy number variations as risk factors for
17. Hickok DE, Mills M. Percutaneous umbili-
Am J Hum Genet. 1999;64(1):218–224. neurodevelopmental disorders: critical over-
cal blood sampling: results from a multi-
33. Wang E, Batey A, Struble C, et  al. Gesta- view and analysis of clinical implications.
center collaborative registry. The Western
tional age and maternal weight effects on J Med Genet. 2016;53(2):73–90.
Collaborative Perinatal Group. Am J Obstet
fetal cell-free DNA in maternal plasma. Pre- 51. Levy B, Hirschhorn K, Kardon NB. Chro-
Gynecol. 1992;166(6 Pt 1):1614–1617; dis-
nat Diagn. 2013;33(7):662–666. mosome abnormalities in spontaneous
cussion 1617–1618.
34. Committee opinion summary no. 640: cell- abortions. In: Wells D, ed. Cytogenetics in
18. Bigelow CA, Cinelli CM, Little SE, et al. Per-
free DNA screening for fetal aneuploidy. reproductive medicine. Georgetown, TX: ­Landes
cutaneous umbilical blood sampling: current
Obstet Gynecol. 2015;126(3):691–692. Bioscience; 2007:59–66.

246.e1
246.e2 References
52. Levy B, Sigurjonsson S, Pettersen B, et  al.
Genomic imbalance in products of concep-
tion: single-nucleotide polymorphism chro-
mosomal microarray analysis. Obstet Gynecol.
2014;124(2 Pt 1):202–209.
53.  Spencer K. What is the true fetal loss rate in
pregnancies affected by trisomy 21 and how
does this influence whether first trimester detec-
tion rates are superior to those in the second tri-
mester? Prenat Diagn. 2001;21(9):788–789.
54. Lyon MF. The William Allan memorial
award address: X-chromosome inactivation
and the location and expression of X-linked
genes. Am J Hum Genet. 1988;42(1):8–16.
55. Carrel L, Cottle AA, Goglin KC, Willard
HF. A first-generation X-inactivation profile
of the human X chromosome. Proc Natl Acad
Sci U S A. 1999;96(25):14440–14444.
56. Increased maternal cardiovascular mortal-
ity associated with pregnancy in women with
Turner syndrome. Fertil Steril. 2012;97(2):
282–284.
57. Menasha J, Levy B, Hirschhorn K, Kardon
NB. Incidence and spectrum of chromosome
abnormalities in spontaneous abortions: new
insights from a 12-year study. Genet Med.
2005;7(4):251–263.
58. McFadden DE, Robinson WP. Pheno-
type of triploid embryos. J Med Genet.
2006;43(7):609–612.
59. Daniel A, Wu Z, Bennetts B, et  al. Karyo-
type, phenotype and parental origin in 19
cases of triploidy. Prenat Diagn. 2001;21(12):
1034–1048.
60. Daniel A, Wu Z, Darmanian A, et al. Three
different origins for apparent triploid/diploid
mosaics. Prenat Diagn. 2003;23(7):529–534.
61. Helwani MN, Seoud M, Zahed L, et  al.
A familial case of recurrent hydatidiform
molar pregnancies with biparental genomic
contribution. Hum Genet. 1999;105(1-2):
112–115.
62. Sarno Jr AP, Moorman AJ, Kalousek DK.
Partial molar pregnancy with fetal survival:
an unusual example of confined placental
mosaicism. Obstet Gynecol. 1993;82(4 Pt 2
suppl):716–719.
63. Warburton D. De novo balanced chromo-
some rearrangements and extra marker
chromosomes identified at prenatal diag-
nosis: clinical significance and distribu-
tion of breakpoints. Am J Hum Genet.
1991;49(5):995–1013.
64. Van Dyke DL, Weiss L, Roberson JR, Babu
VR. The frequency and mutation rate of
balanced autosomal rearrangements in man
estimated from prenatal genetic studies for
advanced maternal age. Am J Hum Genet.
1983;35(2):301–308.
65. Redin C, Brand H, Collins RL, et  al.
The genomic landscape of balanced cyto-
genetic abnormalities associated with
human congenital anomalies. Nat Genet.
2017;49(1):36–45.
66. Talkowski ME, Rosenfeld JA, Blumenthal
I, et  al. Sequencing chromosomal abnor-
malities reveals neurodevelopmental loci that
confer risk across diagnostic boundaries. Cell.
2012;149(3):525–537.
67.  Ordulu Z, Kammin T, Brand H, et al. Struc-
tural chromosomal rearrangements require
nucleotide-level resolution: lessons from next-
generation sequencing in prenatal diagnosis.
Am J Hum Genet. 2016;99(5):1015–1033.
68. Wang JC, Ross L, Mahon LW, et al. Regions
of homozygosity identified by oligonucle-
otide SNP arrays: evaluating the incidence
and clinical utility. Eur J Hum Genet.
2015;23(5):663–671.
69. Grati FR. Chromosomal mosaicism in
human feto-placental development: impli-
cations for prenatal diagnosis. J Clin Med.
2014;3(3):809–837.
70. Hook EB. Exclusion of chromosomal mosa-
icism: tables of 90%, 95% and 99% con-
fidence limits and comments on use. Am J
Hum Genet. 1977;29(1):94–97.
71. Pergament E, Chen PX, Thangavelu M, Fid-
dler M. The clinical application of interphase
FISH in prenatal diagnosis. Prenat Diagn.
2000;20(3):215–220.
72. Tepperberg J, Pettenati MJ, Rao PN, et al. Pre-
natal diagnosis using interphase fluorescence in
situ hybridization (FISH): 2-year multi-center
retrospective study and review of the literature.
Prenat Diagn. 2001;21(4):293–301.
73. Blennow E, Nielsen KB, Telenius H, et  al.
Fifty probands with extra structurally abnor-
mal chromosomes characterized by fluores-
cence in situ hybridization. Am J Med Genet.
1995;55(1):85–94.
74. Leana-Cox J, Levin S, Surana R, et  al. Char-
acterization of de novo duplications in eight
patients by using fluorescence in situ hybridiza-
tion with chromosome-specific DNA libraries.
Am J Hum Genet. 1993;52(6):1067–1073.
75. Cirigliano V, Voglino G, Ordonez E, et  al.
Rapid prenatal diagnosis of common chro-
mosome aneuploidies by QF-PCR, results of
9 years of clinical experience. Prenat Diagn.
2009;29(1):40–49.
76. Donaghue C, Roberts A, Mann K, Ogilvie
CM. Development and targeted application
of a rapid QF-PCR test for sex chromo-
some imbalance. Prenat Diagn. 2003;23(3):
201–210.
77. Hulten MA, Dhanjal S, Pertl B. Rapid and
simple prenatal diagnosis of common chro-
mosome disorders: advantages and disad-
vantages of the molecular methods FISH
and QF-PCR. Reproduction. 2003;126(3):
279–297.
78. Gerdes T, Kirchhoff M, Lind AM, et  al.
Computer-assisted prenatal aneuploidy
screening for chromosome 13, 18, 21, X and
Y based on multiplex ligation-dependent
probe amplification (MLPA). Eur J Hum
Genet. 2005;13(2):171–175.
79. Mann K, Donaghue C, Fox SP, et al. Strat-
egies for the rapid prenatal diagnosis of
chromosome aneuploidy. Eur J Hum Genet.
2004;12(11):907–915.
80. Schouten JP, McElgunn CJ, Waaijer R,
et  al. Relative quantification of 40 nucleic
acid sequences by multiplex ligation-dependent
probe amplification. Nucleic Acids Res.
2002;30(12):e57.
81. Breman A, Pursley AN, Hixson P, et al. Pre-
natal chromosomal microarray analysis in a
diagnostic laboratory; experience with >1000
cases and review of the literature. Prenat
Diagn. 2012;32(4):351–361.
82. Callaway JL, Shaffer LG, Chitty LS, et  al.
The clinical utility of microarray technolo-
gies applied to prenatal cytogenetics in the
presence of a normal conventional karyo-
type: a review of the literature. Prenat Diagn.
2013;33(12):1119–1123.
83. Donnelly JC, Platt LD, Rebarber A, et  al.
Association of copy number variants with spe-
cific ultrasonographically detected fetal anom-
alies. Obstet Gynecol. 2014;124(1):83–90.
84. Fryburg JS, Dimaio MS, Yang-Feng TL,
Mahoney MJ. Follow-up of pregnancies
complicated by placental mosaicism diag-
nosed by chorionic villus sampling. Prenat
Diagn. 1993;13(6):481–494.
85. Ledbetter DH, Zachary JM, Simpson JL,
et al. Cytogenetic results from the U.S. Col-
laborative Study on CVS. Prenat Diagn.
1992;12(5):317–345.
86. Worton RG, Stern RA. Canadian collab-
orative study of mosaicism in amniotic fluid
cell cultures. Prenat Diagn. 1984;4(spec
no):131–144.
87. Kalousek DK. Current topic: confined pla-
cental mosaicism and intrauterine fetal devel-
opment. Placenta. 1994;15(3):219–230.
88. Malvestiti F, Agrati C, Grimi B, et al. Inter-
preting mosaicism in chorionic villi: results
of a monocentric series of 1001 mosaics in
chorionic villi with follow-up amniocentesis.
Prenat Diagn. 2015;35(11):1117–1127.
89. Konialis C, Pangalos C. Dilemmas in pre-
natal chromosomal diagnosis revealed
through a single center’s 30 years’ experi-
ence and 90,000 cases. Fetal Diagn Ther.
2015;38(3):218–232.
90. Brady PD, Delle Chiaie B, Christenhusz G,
et al. A prospective study of the clinical utility
of prenatal chromosomal microarray analysis
in fetuses with ultrasound abnormalities and
an exploration of a framework for reporting
unclassified variants and risk factors. Genet
Med. 2014;16(6):469–476.
91. Daniel A, Athayde N, Ogle R, et al. Prospec-
tive ranking of the sonographic markers for
aneuploidy: data of 2143 prenatal cytoge-
netic diagnoses referred for abnormalities
on ultrasound. Aust N Z J Obstet Gynaecol.
2003;43(1):16–26.
92. Hume Jr RF, Kilmer-Ernst P, Wolfe HM, et al.
Prenatal cytogenetic abnormalities: correla-
tions of structural rearrangements and ultra-
sonographically detected fetal anomalies. Am J
Obstet Gynecol. 1995;173(4):1334–1336.
93. Nicolaides KH, Snijders RJ, Gosden CM,
et  al. Ultrasonographically detectable mark-
ers of fetal chromosomal abnormalities. Lan-
cet. 1992;340(8821):704–707.
94. Daniel A, Wu Z, Darmanian A, et al. Issues
arising from the prenatal diagnosis of some
rare trisomy mosaics—the importance
of cryptic fetal mosaicism. Prenat Diagn.
2004;24(7):524–536.
95. Neiswanger K, Hohler PM, Hively-Thomas
LB, et  al. Variable outcomes in mosaic tri-
somy 16: five case reports and literature
analysis. Prenat Diagn. 2006;26(5):454–461.
96. van Haelst MM, Van Opstal D,
Lindhout D, Los FJ. Management of prena-
tally detected trisomy 8 mosaicism. Prenat
Diagn. 2001;21(12):1075–1078.
97. Girirajan S, Rosenfeld JA, Coe BP, et  al.
Phenotypic heterogeneity of genomic disor-
ders and rare copy-number variants. N Engl J
Med. 2012;367(14):1321–1331.
98. Benn PA, Hsu LY. Maternal cell contamina-
tion of amniotic fluid cell cultures: results of
a U.S. nationwide survey. Am J Med Genet.
1983;15(2):297–305.

99. Bui TH, Iselius L, Lindsten J. European col-
laborative study on prenatal diagnosis: mosa-
icism, pseudomosaicism and single abnormal
cells in amniotic fluid cell cultures. Prenat
Diagn. 1984;4(spec no):145–162.
100. Lamb AN, Rosenfeld JA, Coppinger J,
et  al. Defining the impact of maternal cell
contamination on the interpretation of
prenatal microarray analysis. Genet Med.
2012;14(11):914–921.
101. Fiorentino F, Caiazzo F, Napolitano S, et al.
Introducing array comparative genomic
hybridization into routine prenatal diagnosis
practice: a prospective study on over 1000
consecutive clinical cases. Prenat Diagn.
2011;31(13):1270–1282.
102. Shaffer LG, Dabell MP, Fisher AJ, et  al.
Experience with microarray-based com-
parative genomic hybridization for prenatal
diagnosis in over 5000 pregnancies. Prenat
Diagn. 2012;32(10):976–985.
103. Armengol L, Nevado J, Serra-Juhe C, et  al.
Clinical utility of chromosomal microarray
References 246.e3
analysis in invasive prenatal diagnosis. Hum
Genet. 2012;131(3):513–523.
104. Lee CN, Lin SY, Lin CH, et al. Clinical utility
of array comparative genomic hybridisation
for prenatal diagnosis: a cohort study of 3171
pregnancies. BJOG. 2012;119(5):614–625.
105. Levy B, Kardon NB. Prenatal detection of
chromosome abnormalities. In: Wells D,
ed. Cytogenetics in reproductive medicine.
­Georgetown, TX: Landes Bioscience; 2007.
25
Advances in Molecular Genetics
Including Fetal Sequencing
MAGDALENA WALKIEWICZ AND IGNATIA B. VAN DEN VEYVER
KEY POINTS
• Fetal malformations can be caused by chromosomal defects
detectable by fetal karyotype and chromosomal microarray
analysis, by sequence variants (mutations) in single genes or
can be multifactorial in origin.
• Single-gene disorders can be inherited from parents (autosomal
recessive, autosomal dominant or X-linked) with substantial
recurrence risk or can be caused by de novo mutations in the
fetus with an extremely low recurrence risk.
• When a genetic disorder in the family is known, specific
single gene testing can be performed using standard se-
quencing. However, in all other circumstances, single gene
testing is limited prenatally because genetic heterogeneity
and incomplete description of the prenatal phenotype of
many single-gene disorders preclude optimal selection of
the putative disease gene to sequence.
• Next-generation sequencing, a new method that can assay
multiple genes at once, up to the entire genome, has driven
development of new genetic tests, such as disease-specific
multigene panels, whole-exome sequencing (WES) and
whole-genome sequencing.
• Multigene panels allow for testing of multiple genes in
parallel, which can be useful in prenatal diagnosis if there is a
distinct phenotype, such as skeletal dysplasia or specific brain
abnormalities.
• WES is more comprehensive than multigene panels
because it analyses the coding regions of most genes in a
single test.
• WES has improved genetic diagnosis by 16% to 45% in paedi-
atric and adult populations.
• Initial reports indicate that WES also improves detection of
a genetic aetiology for prenatally diagnosed fetal structural
abnormalities.
• Prenatal WES is usually performed as trio sequencing of
fetal, maternal and paternal DNA to facilitate more rapid
results.
• Although prenatal WES is promising, more research is
needed to address challenges related to cost and access,
clinical utility, indication for the test, interpretation of
pathogenicity of variants and issues surrounding report-
ing of variants of uncertain significance and incidental or
secondary findings.
Introduction
Three percent of all pregnancies are complicated by fetal congen-
ital anomalies identified by prenatal imaging. Standard genetic
tests, including karyotype and chromosomal microarray analysis
(CMA) performed on fetal samples, typically amniotic fluid (AF)
or chorionic villus sampling (CVS), can identify the cause in
about 30% of affected pregnancies, but for the remaining 70%,
the genetic cause remains unknown, and it is predicted that a
significant fraction of these could be explained by single-gene
disorders.1 Until recently, testing for single-gene disorders in the
prenatal setting has been difficult and limited to cases with prior
knowledge of an increased risk for a specific genetic disorder,
for example, if there is a strong family history for an inherited
autosomal dominant, autosomal recessive, X-linked condition
(Fig. 25.1). This scenario is relatively rare, and in many cases,
the affected fetus represents the first de novo presentation of the
phenotype in a particular family. In these fetuses, it is difficult
to select which gene to test for because the ultrasound findings
can be nonspecific or unexpected from known postnatal presen-
tations. In some cases, there is genetic heterogeneity, a scenario
in which mutations in several different genes can cause the same
phenotype. For others (e.g. lethal fetal anomalies), the causative
genes may not yet be known. Finally, some conditions are mul-
tifactorial with both genetic and environmental causes. Table
25.1 shows the different possible genetic and nongenetic causes
for fetal structural birth defects, their relative frequencies and
appropriate testing strategies. It is therefore difficult to choose
which gene to test after a normal karyotype or CMA result, sup-
porting the need for multiplex assays that analyse several genes
at once, but development of such assays was slowed by the limi-
tations of the older technology of Sanger sequencing. With the
development of modern next-generation sequencing (NGS),
this has now become reality. In this chapter, we focus first on the
use of NGS to find point mutations in multiple genes at once
through multigene panel sequencing and next on its applica-
tion for whole-exome sequencing (WES), a method in which the
entire ‘coding’ genome can be searched. The technology of NGS
is also the basis for other new tests highlighted elsewhere in this
textbook, such as noninvasive cell free DNA screening for cyto-
genetic conditions (see Chapter 21), noninvasive screening and
testing for single-gene disorders (see Chapter 22) and panethnic
expanded carrier screening (see Chapter 26). 
247
248 SE C T I O N 6     Prenatal Screening and Diagnosis

Autosomal Autosomal
Autosomal X-linked X-linked
dominant dominant
recessive inherited de novo
inherited de novo

Aa Aa Aa AA AA AA XY Xx XY XX
AA Aa Aa aa AA Aa AA Aa XX Xx XX Xx
1/4 1/2 1/4 1/2 1/2 rare 1/2 1/2 rare
XY xY XY XY
1/2 1/2
Single gene test:
gene must be known (rare!)     
Multigene panel: unknown
gene and strong candidate list     
(less rare but not inclusive)
Rapid identification Rapid identification Rapid identification Rapid identification Rapid identification
Whole-exome sequencing: of parental carrier of parental carrier of de novo of maternal of de novo mutation
most comprehensive status and disease status and disease mutation not carrier status not present in
allele transmission allele transmission present in parents and disease parents (father)
allele transmission
• Fig. 25.1  Patterns of inheritance with application and benefits of whole-exome sequencing testing for
each pattern of inheritance. Maternal alleles are shown in pink, paternal alleles are shown in blue. The
mutant allele is in lower case in the same colour as the parental allele for inherited mutations, and in green
for de novo mutations. A,a, autosomal; X,x, X-chromosomal; Y, Y-chromosomal.

TABLE
25.1 Causes of Structural Birth Defects with Frequencies and Recommended Testing Strategies
Condition Prevalence (%) Appropriate Diagnostic Test
Common trisomies (21, 13, 18) 0.2 Karyotype or CMA
Chromosomal abnormality other than trisomy 0.2 Karyotype or CMA, but CMA does not detect balanced rearrangements
Pathogenic copy number variants (deletion or 1.2 CMA
duplication)
Known Mendelian genetic disorders 0.4 Specific gene testing when gene is known; if not known, multigene panels or WES
(investigational)
Structural or functional congenital abnormalities 3 Karyotype or CMA to exclude chromosomal abnormalities and pathogenic CNVs
• De novo followed by WES if normal (investigational)
• New autosomal recessive disease gene
• Not genetic
Multifactorial conditions 0.2–0.1 Ultrasonography is the primary tool; genetic testing only useful to exclude other
causes; some screening possible (e.g. neural tube defects)
Teratogen exposure (e.g. warfarin, retinoid) Rare History and ultrasound are primary tools; genetic testing may be useful to exclude
other causes
Disruption (e.g. amniotic band) Rare Ultrasonography is the primary tool; genetic testing may be useful to exclude other
causes.

CMA, Chromosomal microarray analysis; WES, whole-exome sequencing.

  

What Is Next-Generation Sequencing, and
How Does It Work?
Next-generation sequencing is a relatively new technology based
on massively parallel sequencing (MPS). In MPS (Fig. 25.2), the
DNA of the sample that is being sequenced (e.g. DNA extracted
from AF or a CVS) is first sheared into small fragments and linked
to adapters to generate the ‘sequencing library’. Either the entire
library of fragments or only a selected subset of fragments of interest
is used as templates for the synthesis of millions of short and over-
lapping DNA fragments. Each nucleotide incorporated into these
fragments is labelled with a different coloured fluorescent probe so
that the sequence or ‘genetic code’ of each fragment is identifiable.
Data from all the obtained sequences are then aligned and compared
 CHAPTER 25  Advances in Molecular Genetics Including Fetal Sequencing 249

Genomic
DNA

Amniotic fluid +
or DNA Fragment Adaptor
Chorionic villi extraction ligation

Library
preparation

Capture Denature
exons and hyb to bait

Release
exons

Bind to
primer

CLUSTER GENERATION

Replication and Denature

amplification

Sequence
by synthesis

C
A
G

T
T
• Fig. 25.2  Whole-exome sequencing (WES) workflow. First, genomic DNA isolated from amniotic fluid
or chorionic villus sampling (or cultured cells from such samples) is fragmented. Linkers are added to the
DNA fragments to prepare the sequencing library. The DNA fragments are then denatured and hybridised
to a bait to ensure isolation of desired DNA fragments, such as all exons (capture), and discarding the
unwanted sequences, that is, all noncoding exons, introns and intergenic sequences (enrichment). The
baits can be selected to capture all exons for or only exons of a specific subset of genes for gene panels.
After the desired fragments are isolated, the baits are released, and sequencing can be performed. Note
that for WGS, there is no selection step with the initial denaturation, and the sequencing is done on the
entire library of fragments. The DNA fragments (sequencing library), hybridised to linkers, serve as a
template for cluster generation through binding of linkers to complementary primers, amplification and
replication, and denaturation. After the clusters are generated, sequencing is initiated. The sequencing is
based on addition of labelled nucleotides, which emit fluorescent light upon binding to the complementary
nucleotide on the single-stranded template. The emitted fluorescent light corresponding to each added
nucleotide (A, C, G or T) is then detected. Obtained sequence data are then aligned to the reference
genome sequence.
250 SE C T I O N 6     Prenatal Screening and Diagnosis

with the human genome reference sequence. Because NGS is more by the Laboratory Quality Assurance Committee of the American
error prone than traditional Sanger sequencing, each fragment is College of Medical Genetics and Genomics (ACMG). For diagnos-
sequenced multiple times, with the ultimate goal of assuring that all tic WES, a mean coverage of 100-fold for proband-only WES and
regions of the sequenced DNA are covered by multiple overlapping 70-fold coverage for trio-based tests is recommended, each with 90%
fragments. This coverage is referred to as the sequencing depth. The to 95% of the sequenced nucleotides covered at least 10-fold.2 Recent
standards for coverage when NGS is used for clinical diagnosis are set technical advances in NGS allow clinical laboratories to offer shorter
turnaround times (TATs) together with better sequencing depth.
Although NGS is a powerful new method, some limitations inher-
TABLE Whole-Exome Sequencing and Whole-Genome ent to the technology affect clinical diagnosis (Table 25.2). Some genes
25.2 Sequencing Cannot Readily Detect All Types of can be incompletely covered because of sequencing depth variation,
Mutations and it is more difficult to get accurate results from regions with ‘high
Well-Detected Mutations Poorly or Not Detected Mutations GC content’ (regions with more guanine and cytosine than adenine
and thymidine). Genes that belong to families of highly homologous
In unique genes, exons In pseudogenes, repeated exons, genes or have a pseudogene are also difficult to sequence. Certain
highly homologous genes and GC- mutation types, including triplet repeat mutations (e.g. the CGG tri-
rich sequences nucleotide repeat in fragile X syndrome), deletions and duplication
Point mutations Large rearrangements, aneuploidy that are longer than a few nucleotides, low-level mosaic mutations,
balanced and unbalanced translocations or inversions, are more diffi-
Small indels Low-level mosaic mutations cult to detect by NGS. Newer approaches to overcome some of these
Germline mutations Repeat expansions difficulties are under development. 
Only in covered regions (WES) In uncovered regions (WES)
How Are Next-Generation Sequencing Data,
Only in regions that can be In regions with low coverage or
sequenced with NGS sequencing depth Such as Whole-Exome Sequencing Results,
In regions with sufficient Epigenetic mutations Analysed and Interpreted?
coverage and depth
The interpretation of the sequencing data is the most challenging
NGS, Next-generation sequencing; WES, whole-exome sequencing. part of diagnostic genome-wide sequencing (Fig. 25.3). Because
  
PROBAND T
1% MAF cutoff
ATGAACCTTCCTCGTGGACTG
(1) (2) (3)

Previously Bona fide SNV, in-frame

reported deleterious deletion/duplication

MIM, HGMD De novo/ De novo/

ATGAACCTTCCTCGTGGACTG PubMed ClinVar novel/inherited novel/inherited
REFERENCE

Algorithm
Clinical report
predictions
Alignment to
targeted regions
in reference
genome

• Fig. 25.3 Interpretation of whole-exome sequencing (WES) results. Top left, Fragments are aligned to
the reference genome to identify their sequence content and location and any differences between the
proband’s (i.e. fetal) sequence and the reference sequence (heterozygous A/T in this example). Bottom left,
The ultimate ideal alignment provides high coverage over exons and exon–intron boundaries but no cover-
age over introns. Right, All differences between the probands and reference sequence are bioinformatically
analysed and then interpreted by the laboratory director who redacts the result report. First, variants are
filtered for a minor allele frequency (MAF) of less than 1%. Next, (1) databases such as Online Mendelian
Inheritance of Men (OMIM), Human Genome Mutation Database (HGMD), Clinical Variation database (Clin-
Var) and literature (PubMed) are searched to find previously reported disease-causing mutations or the
exome Aggregation Consortium (ExAc) for benign variants; (2) bioinformatics tools are used to determine the
functional consequence of the sequence variant to determine that it is a true (bona fide) deleterious change
(e.g. nonsense, frameshift mutations); or (3) a nonsynonymous missense single-nucleotide variant (SNV) or
in-frame deletion or duplication. It will then be determined whether potentially significant sequence variants
are inherited or de novo in the affected proband. Finally, algorithms, such as Polyphen, SIFT or Mutation
Taster, are used to predict if the sequence variants are damaging to the protein’s function.
 CHAPTER 25  Advances in Molecular Genetics Including Fetal Sequencing 251

of individual variation in DNA sequences, thousands of sequence
variants are identified with WES and sequence variants obtained
from whole-genome sequencing (WGS) amount to millions, most
of which are benign polymorphisms that are not disease causing.
To sort out and prioritise those that are clinically important, pow-
erful bioinformatics algorithms are used. Identified sequence vari-
ants that differ from the reference sequence are compared with
information in databases and interpreted for their known or pre-
dicted functional impact. They are also classified using tools that
predict the effect of the mutation on the function of the protein
coded by the gene. Information about whether sequence variants
are in known disease genes is also integrated. To ensure consis-
tency in interpretation among diagnostic laboratories, the ACMG
has provided standard guidelines for variant classification.3 Vari-
ants are classified as pathogenic, likely pathogenic, of unknown
significance, likely benign, or benign and further qualified as
in genes related to the clinical presentation or unrelated to the
clinical presentation (Table 25.3). Pathogenic or likely pathogenic
sequence variants unrelated to the proband’s clinical presentation
(phenotype) are referred to as ‘incidental findings’. In some cases,
they have well-known clinical consequences, and for a subset,
called actionable findings, there is treatment or prevention that
can improve outcome. The ACMG has published and updated a
guideline for clinical laboratories that suggests that they actively
seek and report known pathogenic and expected pathogenic vari-
ants in 59 ‘actionable’ genes (e.g. cancer or cardiac disease predis-
position genes).4 These most recent recommendations state that
patients should be counselled about this during pretest counsel-
ling and can be given the option to opt out of receiving this infor-
mation. It should be noted that this guideline does not include the
application of WES for prenatal diagnosis.  which can be considered when prenatal brain imaging reveals a
Diagnostic Sequencing Applications on
Amniotic Fluid and Chorionic Villus Sampling
Single-Gene Testing
There are a few common scenarios in which single-gene testing is
sometimes used for prenatal diagnosis. In the first, there is a known
familial mutation for which the fetus is at risk, such as when the
parents are identified to be carriers for a specific genetic disorder
through reproductive carrier screening, there is an affected child
with a known mutation or the parents themselves are affected with
a dominantly inherited or X-linked disorder. The second, much
rarer one, is when the prenatal sonographic examination uncovers
features that are highly characteristic for a well-defined specific dis-
order. An example is mutation analysis of the FGFR3 gene when
prenatal ultrasound findings are consistent with thanatophoric
dysplasia. This scenario is much rarer because it relies on accurate
genotype–phenotype correlation, which is challenging prenatally
because the prenatal phenotype of known genetic syndromes is
incompletely described or could be different from what is expected
based on how a condition presents after birth. For many genetic
disorders, there is also ‘genetic heterogeneity’, meaning that one
condition can be caused by mutations in multiple genes. Thus
choosing the most appropriate single gene to test is challenging,
and before NGS was available, ‘diagnostic odysseys’ with sequen-
tial testing of different genes was the only option. This left many
cases unresolved, especially in the prenatal setting, where there is
limited time for decision making about pregnancy management. 
Multigene Panels
Among the earlier and still expanding clinical applications of
NGS is the development of clinical diagnostic multigene sequenc-
ing panel tests. These panels contain genes in which mutations
cause categories of phenotypes, such as congenital heart disease,
skeletal dysplasias or intellectual disability. They are also devel-
oped for genes that are associated with certain phenotypes known
to be caused by mutations in one of a large number of different
genes, for example, microcephaly or Joubert syndrome panels,
small head circumference, or a molar tooth sign and cerebellar
hypoplasia. Panels generally provide better coverage of each indi-
vidual gene compared with WES. They often also include meth-
ods to identify deletions and duplications and typically avoid the
risk for incidental findings. However, their major disadvantage is
that they are based upon known disease genes, precluding detec-
tion of unsuspected and nonincluded genes. In addition, there
are frequently delays in the incorporation into the panel of newly
identified causative genes for a particular condition. Although
NGS panels are currently being used prenatally when other tests
(karyotype and CMA) yield no diagnostic results, there are no

TABLE
25.3 Classification of Sequenc Variants
Unrelated to Indication for Sequencing (Incidental
Classification Related to Indication for Sequencing Finding)
Definite pathogenic Always reported if in known disease gene (prior evidence) Typically reported if secondary finding in 59 ACMG-
Sometimes reported if in plausible gene (no or limited prior actionable genes if postnatal
evidence) Varies for other actionable genes and varies prenatally
Likely pathogenic Typically reported if in known disease gene (prior evidence). Typically reported if secondary finding in 59 ACMG-
Sometimes reported if in plausible gene (no or limited prior actionable genes if postnatally
evidence) Varies for other actionable genes and varies prenatally
Variant of uncertain significance Reporting varies Typically not reported
Likely benign Typically not reported Typically not reported
Benign Not reported Not reported

ACMG, American College of Medical Genetics and Genomics.

  
252 SE C T I O N 6     Prenatal Screening and Diagnosis
TABLE Reported Prenatal Whole-Exome Sequencing
25.4 Experiencea
Detection High-Risk
Study N Rate Change
Carss et al.18 30 3 (10%) 5 (16.7%)
Drury et al.19 24 5 (21%) 1 (4.2%)
Alamillo et al.24 7 3 (42.9%) 1 (14.3%)
Pangalos et al.21 14 6 (42.9%) — 6 (42.9%)
Wapner et al.22 168 13 (7.7%) 30 (17.9%)
Walkiewicz 61 20 (32.8%) — 20 (32.8%)
et al.23
Total 304 50 (16.5%) 37 (12.2%)
aNot all studies reported incidental or secondary findings, and not all studies differentiated
diagnostic findings from high-risk findings. There were also differences in patient selection.
  
professional guidelines concerning their use for prenatal diagnosis,
and most of them were not specifically designed for prenatal use.
One of the most commonly used ones is the Noonan syndrome
panel in euploid pregnancies with increased nuchal translucency
(NT) identified during the first trimester screening sonogram.5,6  malities. Furthermore, few of these reported studies address the
Whole-Exome Sequencing
Although WES for prenatal diagnosis is not yet recommended
outside a research setting,7 it is already well integrated in paedi-
atric and adult genetic care, where its diagnostic yield is between
16% and 45%.8-15 A few years ago, single case reports16,17 or small
series, sometimes embedded in larger reports of clinical application
of WES,8,9 that describe the utility of WES for prenatal diagnosis
began to emerge (reviewed by Van den Veyver1). Since then, there
have been other series describing the usefulness of WES specifi-
cally for prenatal diagnosis (Table 25.4), but the majority were not
on ongoing pregnancies. Carss and colleagues reported a cohort
of 30 cases with fetal anomalies identified by ultrasound screen-
ing for which trio WES analysis was performed on DNA from
various tissue types, including placenta, cord blood, CVS and cul-
tured amniocytes. They made a molecular diagnosis in three cases
(10%) with pathogenic mutations identified in FGFR3, COL2A1
and OFD1 and a possible molecular diagnosis in an additional
five.18 Drury and colleagues performed trio WES on 24 cases
from pregnancies with increased NT or other anomalies identified
by ultrasound that had a prior normal chromosomal microarray
analysis. In this cohort, a definite molecular diagnosis was made
for 5 (21%), and a probable diagnosis was made for 1 case.19 In
2, there were additional incidental findings, including a de novo
pathogenic missense variant in NF1 in a fetus with increased NT
and a ventricular septal defect and a homozygous pathogenic vari-
ant in ATP7B in a fetus with bilateral talipes, fixed flexed wrists and
echogenic hepatic foci.19 Alamillo and associates reported a posi-
tive result in 3 of 7 and likely positive result in 1 of 7 cases of WES
performed after fetal demise or termination of pregnancy for mul-
tiple fetal anomalies (total, 57%). Ellard and colleagues developed
a method for WES and interpretation for recurrent lethal fetal
anomalies that incorporates an algorithm that takes into account
likely autosomal recessive inheritance and made a molecular diag-
nosis in two families with recurrent fetal akinesia.20 Pangalos and
colleagues sequenced samples from 11 ongoing pregnancies and 3
products of conception of euploid fetuses with multiple anomalies,
using a targeted analysis of 758 genes and identified a definitive
or highly likely diagnosis in 6 (43%) of them.21 A research study
Combined presented at the 37th annual meeting of the Society for Maternal
8 (26.7%)
Fetal Medicine22 on 168 pregnancies with fetal abnormalities and a
normal karyotype or CMA demonstrated that trio WES identified
6 (25%) a diagnostic pathogenic mutation in 13 cases, for a detection rate
4 (57.1%) of 7.7%. In an additional 30 (17.9%), there were sequence variants
in genes that were considered to be strong candidates to explain
the phenotype but for which there was not enough available data
43 (25.6%) to confirm the pathogenic relationship to the fetal phenotype.
Data from the Baylor Genetics Laboratories (presented at the 2016
annual meeting of the American Society of Human Genetics and
manuscript in submission) on trio WES in 61 highly selected pre-
87 (28.6%) natal cases with fetal anomalies and normal karyotype and CMA
revealed a reported pathogenic mutation in 33%.23
In aggregate, these early results support that prenatal trio WES
is useful to resolve the cause for single or multiple fetal abnor-
malities when standard genetic testing is uninformative. However,
the wide range of detection rate from 7.7% to 57% also suggests
that there is variability in case selection and in the interpretation
of pathogenicity of sequence variants between different studies. It
further indicates that we have incomplete knowledge about the
genetic changes associated with prenatally diagnosed fetal abnor-
discovery of incidental findings in these trio samples. Professional
societies have either not commented yet on the use of prenatal
(trio) WES or have stated that it should preferentially be done
on a research basis, but that if it is used as a clinical diagnostic
test under exceptional circumstances, it should only be offered by
highly knowledgeable genetics professionals.7 
Clinical Application of Prenatal Whole-
Exome Sequencing
Based on accumulating evidence of the utility of WES for prenatal
diagnosis and recent improvement of the TAT from approximately
12 weeks to 3 weeks, a few diagnostic laboratories began offering
a trio-based WES test designed specifically for prenatal diagno-
sis.24,25 Although this has been successful, several challenges have
emerged. Sometimes limited sample sizes preclude performing
WES on DNA prepared directly from AF or CVS samples, and
culturing is often required before DNA extraction, prolonging the
time from sample submission to reporting of results by approxi-
mately two weeks. Efforts towards reducing the amount of sample
required for WES will be necessary and are already being consid-
ered. Challenges related to how the pathogenicity of variants is
interpreted and reported (see Table 25.3) are not unique to prena-
tal WES but are amplified in this setting, and thus careful pre- and
posttest counselling is paramount if WES is offered prenatally. 
Pre- and Posttest Genetic Counselling
for Prenatal Diagnostic Whole-Exome
Sequencing
Pretest Counselling
The counselling of patients undergoing diagnostic prenatal WES
is complex, lengthy and best done by trained genetic professionals
 CHAPTER 25  Advances in Molecular Genetics Including Fetal Sequencing
who can explain complicated concepts in language understandable
by patients with varying educational backgrounds. Pretest coun-
selling should address that the test is novel and still investigational,
with not yet well-defined sensitivity, specificity and clinical utility
(positive and negative predictive value) in the prenatal setting and
that diagnosis may be obtained in only a conservatively estimated
7.7% of cases but that some laboratories and studies report higher
detection rates. It should include the possibility that variants of
uncertain clinical significance are found that may cause anxiety
and difficulty with making decisions regarding pregnancy man-
agement. Because prenatal diagnostic WES is typically performed
on trios, pathogenic and likely pathogenic variants, or variants of
uncertain significance in disease genes, or pathogenic and likely
pathogenic variants in genes of uncertain functional relevance to
the phenotype can be found in the DNA from the fetus, mother
and father. Although there are general professional guidelines for
which variants should be reported with diagnostic WES, they do
not extend to its prenatal use. It is therefore important that labo-
ratories communicate their result reporting policies for prenatal
trio WES and that health care providers performing the pretest
counselling know them, including how the laboratory handles
variants of uncertain significance, incidental findings and second-
ary findings in the 59 genes that ACMG recommends reporting
on in postnatal diagnostic WES.4  lenges that need to be addressed include cost and throughput and
Posttest Counselling
Posttest counselling for a positive WES result should also be
performed by a genetics professional and should address the sig-
nificance of findings relevant to the condition that is diagnosed,
its prognosis and potential therapies, and implications for other
253
family members and future pregnancies. It should further provide
emotional and social support to help parents cope with the new
diagnosis. 
Patients and Provider Perceptions on the Use
of Prenatal Whole-Exome Sequencing
Although there is some debate in the medical genetics commu-
nity about the benefit of prenatal WES, considering the limited
data on clinical validity and utility, the results of surveys suggest
that there is strong interest from patients for this new genetic
test.26 
Conclusions
Next-generation sequencing has begun to revolutionise prenatal
diagnosis and prenatal WES has become a reality. It is not cur-
rently recommended as a routine test for prenatal care, but several
recent reports have shown benefit in selected cases with sono-
graphically identified fetal anomalies. As WES is used more fre-
quently, new phenotypes for known disease genes as well as new
causative genes for fetal abnormalities will be identified. Chal-
which types of sequence variants to report, in particular, how to
handle variants of uncertain significance and incidental or second-
ary findings in the fetal and parental samples when trio prenatal
WES is performed.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 13. Srivastava S, Cohen JS, Vernon H, et al. Clinical whole exome sequencing
in child neurology practice. Ann Neurol. 2014;76:473–483.
14. Lee H, Deignan JL, Dorrani N, et al. Clinical exome sequencing for genetic
1. Van den Veyver IB. Recent advances in prenatal genetic screening and
identification of rare Mendelian disorders. JAMA. 2014;312:1880–1887.
testing. F1000Res. 2016;5:2591.
15. Wright CF, Fitzgerald TW, Jones WD, et al. Genetic diagnosis of devel-
2. Rehm HL, Bale SJ, Bayrak-Toydemir P, et al. ACMG clinical laboratory
opmental disorders in the DDD study: a scalable analysis of genome-wide
standards for next-generation sequencing. Genet Med. 2013;15:733–
research data. Lancet. 2015;385:1305–1314.
747.
16. Filges I, Nosova E, Bruder E, et al. Exome sequencing identifies muta-
3. Richards S, Aziz N, Bale S, et  al. Standards and guidelines for the
tions in KIF14 as a novel cause of an autosomal recessive lethal fetal cili-
interpretation of sequence variants: a joint consensus recommenda-
opathy phenotype. Clin Genet. 2014;86:220–228.
tion of the American College of Medical Genetics and Genomics and
17. Talkowski ME, Ordulu Z, Pillalamarri V, et  al. Clinical diagno-
the Association for Molecular Pathology. Genet Med. 2015;17:405–
sis by whole-genome sequencing of a prenatal sample. N Engl J Med.
424.
2012;367:2226–2232.
4. Kalia SS, Adelman K, Bale SJ, et  al. Recommendations for reporting
18. Carss KJ, Hillman SC, Parthiban V, et al. Exome sequencing improves
of secondary findings in clinical exome and genome sequencing, 2016
genetic diagnosis of structural fetal abnormalities revealed by ultrasound.
update (ACMG SF v2.0): a policy statement of the American College of
Hum Mol Genet. 2014;23:3269–3277.
Medical Genetics and Genomics. Genet Med. 2017;19(2):249–255.
19. Drury S, Williams H, Trump N, et  al. Exome sequencing for prena-
5. Bakker M, Pajkrt E, Bilardo CM. Increased nuchal translucency with
tal diagnosis of fetuses with sonographic abnormalities. Prenat Diagn.
normal karyotype and anomaly scan: what next? Best Pract Res Clin Obstet
2015;35:1010–1017.
Gynaecol. 2014;28:355–366.
20. Ellard S, Kivuva E, Turnpenny P, et  al. An exome sequencing strat-
6. Pergament E, Alamillo C, Sak K, Fiddler M. Genetic assessment follow-
egy to diagnose lethal autosomal recessive disorders. Eur J Hum Genet.
ing increased nuchal translucency and normal karyotype. Prenat Diagn.
2015;23:401–404.
2011;31:307–310.
21. Pangalos C, Hagnefelt B, Lilakos K, Konialis C. First applications of a tar-
7. Committee on Genetics and the Society for Maternal-Fetal. Committee
geted exome sequencing approach in fetuses with ultrasound abnormali-
Opinion no. 682: Microarrays and Next-Generation Sequencing Tech-
ties reveals an important fraction of cases with associated gene defects.
nology: The Use of Advanced Genetic Diagnostic Tools in Obstetrics and
PeerJ. 2016;4:e1955.
Gynecology. Obstet Gynecol. 2016;128(6):e262–e268.
22. Wapner R, Petrovski S, Brennan K, et al. Whole exome sequencing in the
8. Yang Y, Muzny DM, Xia F, et  al. Molecular findings among patients
evaluation of fetal structural anomalies: a prospective study of sequential
referred for clinical whole-exome sequencing. JAMA. 2014;312:1870–
patients. Am J Obstet Gynecol. 2017;216:S5–S6.
1879.
23. Walkiewicz M, Braxton A, Liu P, et  al. The Utility of Whole Exome
9. Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing
Sequencing in Prenatal Diagnosis. 2016 Annual Meeting of the American
for the diagnosis of Mendelian disorders. N Engl J Med. 2013;369:1502–
Society of Human Genetics 23. http://www.ashg.org/2016meeting/pdf/
1511.
ASHG2016-plenary_platform-abstracts.pdf
10. Soden SE, Saunders CJ, Willig LK, et  al. Effectiveness of exome and
24. Alamillo CL, Powis Z, Farwell K, et al. Exome sequencing positively iden-
genome sequencing guided by acuity of illness for diagnosis of neurode-
tified relevant alterations in more than half of cases with an indication of
velopmental disorders. Science Transl Med. 2014;6. 265ra168.
prenatal ultrasound anomalies. Prenat Diagn. 2015;35:1073–1078.
11. Calvo SE, Compton AG, Hershman SG, et  al. Molecular diagnosis of
25. Westerfield LE, Stover SR, Mathur VS, et al. Reproductive genetic coun-
infantile mitochondrial disease with targeted next-generation sequencing.
seling challenges associated with diagnostic exome sequencing in a large
Sci Transl Med. 2012;4. 118ra10.
academic private reproductive genetic counseling practice. Prenat Diagn.
12. de Ligt J, Willemsen MH, van Bon BW, et  al. Diagnostic exome
2015;35:1022–1029.
sequencing in persons with severe intellectual disability. N Engl J Med.
26. Kalynchuk EJ, Althouse A, Parker LS, et  al. Prenatal whole-exome
2012;367:1921–1929.
sequencing: parental attitudes. Prenat Diagn. 2015;35:1030–1036.

253.e1
26
Expanded Carrier Screening
LIRAN HIERSCH AND YUVAL YARON
KEY POINTS
• This chapter describes the novel concept of expanded carrier
screening (ECS), whereby individuals are simultaneously
screened for up to 200 genetic conditions.
• Different laboratory techniques are used in ECS and include
targeted genotyping and next-generation sequencing approaches.
• The choice of conditions to be included in ECS varies among
laboratories, and no consensus presently exists.
• Various screening strategies exist, including premarital carrier
screening, cascade screening, stepwise screening and couple
screening.
• An important aspect in offering ECS is patient education.
Basic Principles of Carrier Screening
In 1968, the World Health Organization (WHO) proposed guide-
lines for establishment of screening programs (Box 26.1).1 These
initial guidelines provided general recommendations regarding
the types of disorders appropriate for screening. These recommen-
dations predated the advent of genetic testing. Thus, in 1998, the
WHO proposed modified guidelines that would specifically apply
to genetic screening2-4 (Box 26.2).
In 2006, the WHO stated that genetic prevention services are
needed to effectively reduce the burden of congenital and genetic
disorders. Yet in many countries, the level of genetic services is cur-
rently inadequate and insufficient. Frequently, these services are avail-
able only to the wealthy and well-educated. Indeed, the provision of
genetic services must be weighed responsibly and fairly against the
competing health requirements in each country. However, epidemio-
logic data regarding the prevalence of congenital and genetic disorders
and cost-effectiveness analyses of screening programs indicate that
many countries would actually benefit from incorporating preventive
genetic approaches into their health services. Unfortunately, despite
the growing number of genetic technologies that hold great promise,
these are underused in clinical practice and fail to reduce the bur-
den of inherited diseases. It is at this juncture that the introduction
of expanded carrier screening (ECS) may provide a feasible universal
solution to these shortcomings.  are based on molecular methodologies. Screening for cystic fibro-
The Evolution of Carrier Screening
Screening for Tay-Sachs Disease Using Enzyme
Analysis
One of the first successful programs has been the community-wide
screening for Tay-Sachs disease (TSD) carriers, established in the
254
1970s among Eastern European (Ashkenazi) Jews. TSD is caused
by deficiency of β-hexosaminidase A activity, which is inherited
in an autosomal recessive (AR) manner (Fig. 26.1). Unaffected
heterozygote carriers may be ascertained by detection of reduced
enzymatic activity in serum and blood lymphocytes.5-7 Wide-scale
voluntary screening programs for TSD were initiated among Ash-
kenazi Jews first in the United States, Canada and Israel but were
eventually adopted in many countries worldwide.7 In the first 30
years after the implementation of these programs, more than 1.4
million individuals were screened (mostly from North America
and Israel), and more than 1400 couples have been identified to
be at risk for offspring affected with TSD. These programs resulted
in a significant reduction in the incidence of TSD among Ashke-
nazi Jews, with the majority of affected individuals being from
other unscreened ethnicities.5 
Screening for β-Thalassemia Using Red Blood
Cell Indices
β-Thalassemia is a relatively common condition among individu-
als of Middle Eastern and Mediterranean origin. It is characterised
by deficient synthesis of β-haemoglobin chains, resulting in severe
anaemia. Heterozygote carriers have mild anaemia and reduced
red blood cell indices. Screening is based on the results of com-
plete blood count (CBC) showing a low mean corpuscular vol-
ume (MCV) and low mean corpuscular haemoglobin (MCH).
Haemoglobin electrophoresis is used to differentiate carriers from
those with iron deficiency, with high sensitivity and relatively
good correlation with phenotype.8 Because the CBC is relatively
inexpensive, easy and widely available, screening programs for
β-thalassemia are widely available. For a detailed description, see
Chapter 27. 
Genetic Screening Using Molecular Techniques
Screening methods for both TSD and β-thalassemia were ini-
tially based on nonmolecular testing (enzymatic testing and CBC,
respectively). Currently, however, most carrier screening programs
sis (CF) has become the prototype model, following the discovery
of the causative role of the CFTR gene in 1989.9 CF is an AR
disorder characterised by progressive lung disease, pancreatic dys-
function and male infertility. It is one of the most common severe
genetic diseases in caucasians, with a carrier frequency of about 1
in 25. Although more than 2000 mutations have been described
in the CFTR gene, each ethnic group may have a unique set of
mutations, with the delF508 mutation being the most prevalent
 CHAPTER 26  Expanded Carrier Screening 255

• BOX 26.1  Proposed World Health Organization
Guidelines for Screening Programs (1968)
1. Is the disease an important health problem?
2. Is there a recognisable latent or early symptomatic stage?
3. Do we know the natural history of disease?
4. Is there an effective treatment for patients with recognised
disease?
5. Is there a suitable test that will identify the disease in its early
stages?
6. Is the test acceptable to the population?
7. Do we agree on who treats the disease?
8. Are facilities for diagnosis and treatment available?
9. Case finding should be ongoing.
10. The cost of case finding (including diagnosis and treatment)
should be economically balanced in relation to possible
expenditures on medical care as a whole.
Adapted from J. M. G. Wilson, G. Jungner, Principles and practice of screening for disease.
Geneva: World Health Organization. http://www.who.int/iris/handle/10665/37650.
From World Health Organization. Genomic resource centre. http://www.who.int/genomics/public
ations/en/index1.html.
• BOX 26.2  Modified Guidelines for Genetic
Screening (2006)
• Genetic screening should be voluntary, not mandatory.
• Genetic screening should be preceded by adequate information about
the purpose and possible outcomes of the screen or test and potential
choices to be made.
• Anonymous screening for epidemiologic purposes may be conducted
after notification of the population to be screened.
• Results should not be disclosed to employers, insurers, schools or others
without the individual’s consent to avoid possible discrimination.
• In rare cases in which disclosure may be in the best interests of the
individual or of public safety, the health provider may work with the
individual towards a decision by him or her.
• Test results should be followed by genetic counselling, particularly when
they are unfavourable.
• If treatment or prevention exists or is available, this should be offered
with a minimum of delay.
• Newborn screening should be mandatory and free of charge if early
diagnosis and treatment will benefit the newborn.

Normal gene
Carrier Carrier Mutated gene
father mother

Unaffected Carrier Carrier Affected

son daughter son daughter
• Fig. 26.1  Autosomal recessive (AR) inheritance pattern. In AR inheritance, each unaffected parent car-
ries a mutation in one copy of the same gene. The risk for an affected offspring having inherited both
mutations is 25%. (Adapted from https://ghr.nlm.nih.gov/primer/inheritance/riskassessment, US National
Library of Medicine.)
256 SE C T I O N 6     Prenatal Screening and Diagnosis
and accounting for about 70% of CF alleles. In 2001 the Ameri-
can College of Obstetricians and Gynecologists (ACOG) and the
American College of Medical Genetics (ACMG) set guidelines for
prenatal and preconception CF carrier screening.10 Initial guide-
lines recommended limiting screening to caucassian individu-
als or those with a family history of CF. However, in 2011, the
guidelines were updated, stating that it has become increasingly
difficult to classify individuals with CF into distinct ethnic cat-
egories. Thus the Committee agreed that offering CF screening
to all couples planning a pregnancy is reasonable.6,11 For couples
in whom both partners are carriers of mutations in the same AR
gene, there is a reproductive risk of 25% for affected offspring.
Knowing this before delivery provides an opportunity for genetic
counselling during which reproductive options such as prenatal
diagnosis by chorionic villus sampling or amniocentesis may be
discussed. Knowing the carrier status before pregnancy also pro-
vides the option of preimplantation genetic diagnosis of embryos
attained by in vitro fertilisation.  significant disease burden. A study conducted by Mount Sinai
Panethnic Screening
Although many genetic disorders have an ethnic predilection,
only a few are universally frequent in diverse populations. One
such example is spinal muscular atrophy (SMA), a severe AR
neuromuscular disease caused by degeneration of motor neurons
in the spinal cord, resulting in progressive muscle weakness and
paralysis. It is for this reason that the ACMG recommends that
carrier screening be offered to all couples, regardless of race or
ethnicity.12,13 The same is also true for some CF mutations (e.g.
delF508 mutation), which account for approximately 70% of
CF alleles worldwide. This mutation is thought to have occurred
more than 52,000 years ago, in a population genetically distinct
from any present European group.14 Panethnic screening for CF
is currently recommended by ACOG and ACMG.11,13 Similarly,
fragile X syndrome (FXS), a condition associated with intellectual
disability (ID) and autistic behaviour, is prevalent in most popula-
tions. Indeed, it is the most common cause of inherited mental
retardation. The prevalence of the fragile X syndrome is estimated
to be about 1 in 4000 males and about 1 in 7000 females.15 The
clinical phenotype in males includes varying degrees of ID with
mild dysmorphic features such as relative macrocephaly, large ears,
a prominent jaw and macro-orchidism after puberty. Affected
females usually present with a less severe phenotype and may
exhibit only subtle cognitive impairment. The disorder is caused
by a CGG triplet repeat expansion mutation in the FMR1 gene
on chromosome Xq27.3. Unaffected individuals usually have less
than 55 CGG repeats. Individuals with the fragile X syndrome
have more than 200 CGG repeats, a full mutation. Individuals
with 55 to 200 CGG repeats are considered to have a premuta-
tion. Female premutation carriers are at risk for having offspring
with further CGG expansion, which may ultimately result in a
full mutation (>200 repeats). There is a strong correlation between
the number of CGG repeats in the carrier mother and the risk for
expansion to full mutation in the offspring.16 Population-based
carrier screening for fragile X has been widely used in Israel since
the mid-1990s.17,18 It is now included in most basic screening
panels for all ethnicities.  to reduce the overall burden of these Mendelian disorders, many
Screening Panels for Multiple Disorders
In the 1990s and 2000s an increasing number of gene dis-
coveries have led to a growing number of conditions available
for screening. This has been particularly the case in the rather
homogeneous Ashkenazi Jewish population in Israel and North
America. These included Canavan disease, Gaucher disease,
familial dysautonomia, Fanconi anaemia group C, Niemann-Pick
disease type A, mucolipidosis type IV, Bloom syndrome and
many others.
These discoveries ushered in the introduction of what has
become known as the Ashkenazi Jewish Genetic Panel (AJGP).
The ACOG initially recommended that individuals of East-
ern European Jewish (Ashkenazi) ancestry be offered carrier
screening for TSD and CF as part of routine obstetric care.19
The current recommendations of ACOG and ACMG for the
AJGP include only 4 or 9 diseases, respectively.20,21 However,
the content of AJGPs in many laboratories has continued to
expand, and some current AJGP offerings include several dozen
conditions.22 Although many of the diseases included in the
AJGP are individually rare, cumulatively, they do amount to a
Laboratory demonstrated that additively, the risk for being a
carrier of at least one condition on a 16-disease AJGP is at least
30%.23
Although the AJGP has a high yield in the Ashkenazi Jewish
population, with a few exceptions, it is not very effective among
non-Ashkenazi Jews and even less so in non-Jewish populations.24
This is because most mutations in this panel are unique founder
mutations, likely the result of genetic drift following a bottleneck
of this ethnic group.25
The increase in the number of genetic conditions amenable to
screening, the complexity of designing ethnic-specific screening
panels and dramatic technological innovations have highlighted
the fact that current screening strategies are inadequate and may,
in fact, become obsolete. This is further complicated by the fact
that the rapid population fluxes characteristic of this era result
in increasing ethnic admixtures to the point where individuals
have become unaware of their precise ethnic background. These
considerations have resulted in the paradigm shift that is ECS. In
this approach, hundreds of conditions are screened for simultane-
ously using a uniform platform. The test is offered to all, regardless
of stated ancestry.26 In this chapter, different aspects of ECS are
discussed. 
Expanded Carrier Screening
In 2005 the ACOG updated its recommendation to justify
panethnic screening for specific disorders such as CF because
‘it is becoming increasingly difficult to assign a single ethnicity
to individuals’.27 In fact, approximately 12% of newborns diag-
nosed with β-haemoglobinopathies by newborn screening dur-
ing the 1990s in California were not from the groups allocated
in the ACOG carrier screening guidelines.28 This reasoning
called for extending the screening programs for haemoglobin-
opathies, and potentially other conditions, to more ethnically
diverse populations.29
It is estimated that 18% to 20% of infant hospitalisations and
deaths can be attributed to Mendelian diseases. Offering screen-
ing for only a limited number of conditions, if at all, does little
of which could be averted by offering ECS to the entire popula-
tion.30 The rapidly expanding list of disorders that can now be
identified makes ECS a reasonable option, especially as novel
sequencing technologies enable relatively inexpensive analysis of
many AR diseases simultaneously.31,32

These assumptions have already been confirmed in several
studies. Lazarin and coworkers described a targeted mutation
panel which includes 417 mutations associated with 108 genetic
diseases.33 In their cohort of more than 23,000 patients of mixed
ethnicities, a carrier status of at least one mutation was identified
in 24% of cases. Despite the rarity of each individual disorder
included in the panel, the overall risk for having an affected off-
spring was calculated at 1 in 280.24 The authors argue that this
risk exceeds that of having an offspring with a neural tube defect
(NTD), for which screening is universally accepted.34 Compa-
rable findings were found in an even larger study analysing the
results of 322,484 patients undergoing routine carrier testing by
ECS using a panel of 88 severe or profound recessive diseases in
which the frequency of affected pregnancies was approximately 1
in 550. They noted that the number of patients needed-to-screen
to detect one affected case was lower with ECS compared with
standard screening for Down syndrome or NTDs.26,35
In 2017 ACOG reviewed the role of ECS and suggested ‘that
ethnic-specific, panethnic, and ECS are acceptable strategies
for prepregnancy and prenatal carrier screening’.36 Each obste-
trician–gynaecologist or other health care provider or practice
should establish a standard approach that is consistently offered
to and discussed with each patient, ideally before pregnancy. After
counselling, a patient may decline any or all carrier screening. If
a patient requests a screening strategy other than the one used by
the obstetrician–gynaecologist or other health care provider, the
requested test should be made available to her after counselling on
its limitations, benefits and alternatives.
The European Society of Human Genetics has developed and
published recommendations regarding responsible implementa-
tion of ECS.37 It confirms that the primary goal of carrier screen-
ing is to facilitate informed reproductive decision making by
identifying couples at risk for having an affected child with an
(autosomal or X-linked) recessive disorder. ECS allows testing of
all individuals regardless of ancestry or geographic origin (‘pan-
ethnic’ or ‘universal’), which in this respect increases equity and
reduces the chance of stigmatisation. The best time to offer screen-
ing is during the preconception period because identifying carrier
couples before pregnancy allows the greatest number of options
with more time to make an informed decision. However, respon-
sible implementation of expanded genetic carrier screening raises
many technical, ethical, legal and social questions. 
Laboratory Methodologies
Two main methodologies are used for ECS: targeted array-based
genotyping and sequence analysis by next-generation sequenc-
ing (NGS). Each have specific advantages and disadvantages
(Table 26.1).
Targeted Array–Based Genotyping
Targeted genotyping is based on analysis of a preselected list
of pathogenic variants (mutations), known to be causative of a
particular disease.38 Such systems are often based on develop-
ment of multiple fluorescently labelled sequences tags, each cor-
responding to a specific sequence variant.39,40 Most variants are
straightforward biallelic single nucleotide polymorphisms (SNPs).
However, specific modifications are also made to allow detec-
tion of more complex variants, such as triallelic SNPs, insertions
and deletions, copy number variants and so on.39,41,42 Targeted
array–based genotyping platforms enable the simultaneous
CHAPTER 26  Expanded Carrier Screening 257
TABLE Comparison of Targeted Array–Based
26.1 Genotyping to Next-Generation Sequencing
Targeted Array–Based Next-Generation
Genotyping Sequencing
Pros • Cheaper • More comprehensive
• Detects only known • Fits all ethnic groups
causative mutations • Detects rare mutations
• Easier pre- and posttest
counselling
Cons • Will miss rare mutations • Detects variants of
• Needs constant modification unknown significance
of mutations • Requires bioinformat-
• Different panel needed for ics support
different ethnicities • May be difficult
posttest counselling
  
analysis of hundreds of known pathogenic variants.38,39 Several
validation studies, including those comparing a microarray-
based platform to single-gene based methods, found similar ana-
lytical performance.39,42 Moreover, this method was found to be
100% concordant in a group of known mutation carriers, dem-
onstrating that high-throughput targeted genotyping assays can
accurately identify carriers in the tested population.43 Because
targeted analysis includes only well-curated mutations with a
known genotype–phenotype correlation, posttest counselling is
relatively simple and uncomplicated for those detected as car-
riers. Conversely, targeted mutation analysis would miss rare
pathogenic variants that have yet to be included in the panel.
As a result, constant modifications may be required any time a
novel pathogenic variant in a specific disease gene is discovered
or when new genes need to be included in the panel. In addi-
tion, for many conditions, the pathogenic variants have been
described only in a specific population. Thus targeted mutation
analysis should be considered as comprehensive only for the eth-
nicities for which it was initially designed. It is for this reason
that referral to targeted panels as ‘universal screening’ should be
discouraged. 
Next-Generation Sequencing
Next-generation sequencing is based on the ability to sequence,
in parallel, millions of DNA fragments, and introduction of NGS
technology has resulted in a dramatic increase in speed and con-
tent of sequencing at a fraction of the cost.44 Described briefly,
first a DNA library is prepared from the patient’s sample by frag-
mentation, purification and amplification of the DNA sample.
Individual fragments are then physically isolated by attachment to
solid surfaces or small beads. The sequence of each of these frag-
ments is resolved simultaneously by such techniques as sequenc-
ing by synthesis. The resulting sequence data are computationally
aligned against a ‘normal reference’ genome.45 This enables the
detection of many sequence alterations in a single reaction.
Next-generation sequencing–based screening has been shown
to have high clinical sensitivity in the assayed genes.46-48 Mutation
detection has been shown to have about 95% sensitivity and 100%
specificity for a variety of alterations such as SNPs, insertions and
deletions, splicing mutations and gross deletions.46,48 Proponents
of NGS-based carrier screening claim that it shows high accuracy,
precision, reproducibility and robustness for clinical use compared
258 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Terminology Used for the Classification of Variants Identified Using Genetic Sequencing Techniques, Based
26.2 on American College of Medical Genetics Recommendations
Variant Class Variant Classification Definition
1 Pathogenic Variants that have been previously reported in individuals with disease and/or are strongly suspected of being
pathogenic based on clinical studies
2 Likely pathogenic Variants that are likely to be implicated in disease pathogenesis but for which conclusive evidence of
pathogenicity is not yet available
3 Variants of uncertain Variants that have some features suggestive of possible functional consequence; however, there is insufficient
significance evidence for either a pathogenic or benign role
3 Likely benign Variants for which only weak data may be available in the medical literature supporting pathogenicity but for
which the majority of evidence suggests the effect of the variant is benign
4 Benign Genetic variants not predicted to alter gene expression or function

From Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics
and the Association for Molecular Pathology. Genet Med 17:405–424, 2015.
  

with the targeted mutation analysis.47 Because sequencing is per-
formed throughout the genes of interest, unrecognised or rare
pathogenic variants, not included in any targeted arrays, may
be detected. This allows the implementation of carrier screen-
ing across a wider range of ethnically diverse populations, more
closely approximating the term ‘universal’.
The cost of NGS based carrier screening, which in the past
has been a major deterrent, is also gradually decreasing. Because
most relevant sequence variants within a gene are detected, con-
stant modifications are not required. Modelling a population of
1,000,000 couples that is representative of the US population
would result in detection of 83,421 mutation carriers. It has been
estimated that NGS-based screening would avert 21 additional
affected births compared with screening by targeted genotyping.
Cost saving would amount to approximately $13 million. Com-
pared with no screening at all, NGS-based carrier screening would
avert 223 additional affected births. The results are sensitive to
assumptions regarding mutation detection rates and carrier fre-
quencies in multiethnic populations.49
Next-generation sequencing–based approaches have several
shortcomings: some of the novel variants detected by NGS may
have no clinical significance. For some variants, no clear genotype–
phenotype correlation exists. Therefore this methodology requires
robust bioinformatic capabilities that will allow accurate determina-
tion of the pathogenicity of each detected variant using a variety of
in silico analyses as well as literature reviews. Laboratories usually
limit their reports to include variants in classes 1 and 2 only, but for
some variants, it may be difficult to make a call (Table 26.2). This
may put a strain on laboratory personnel, genetic counsellors and
physicians alike. In such circumstances, the importance of pre- and
posttesting counselling cannot be overemphasised.50  that the risk for TSD in the offspring be included among the fac-
Screening Strategies
Several different approaches to screening have been developed,
each catering to specific requirements and limitations characteris-
tic of the screened population.
Premarital Carrier Screening Programs
The high burden of β-thalassemia in Cyprus motivated the local
Ministry of Health in 1973 to initiate a prevention program
aimed at identifying carriers before marriage.51 Since 1983, the
Greek Orthodox Church requires a certificate attesting that the
couple underwent premarital screening and received genetic
counselling before being issued a marriage license. The certificate
does not record the results of the tests because the intention is
to make the couples aware of the existing reproductive options
before marriage. Programs include not only carrier screening but
also genetic counselling and prenatal diagnosis.52 The effective-
ness of these programs has resulted in a significant reduction in
the incidence of β-thalassemia major in Cyprus from 1 in 250
births to 1 in 4000 births.51 The obligation to have genetic testing
before marriage may be viewed by some as an infringement on
personal rights. However, proponents argue that it is an effective
means of providing information and reproductive options before
marriage. In a number of Muslim countries such as Lebanon, Iran,
Saudi Arabia, Tunisia, United Arab Emirates, Bahrain and Qatar,
the national premarital screening programs are mandatory and are
actually aimed at preventing the marriage and subsequent child-
bearing among disease carriers.53–54
Another successful premarital carrier screening program is Dor
Yeshorim, which was initially aimed to prevent TSD among ultra-
Orthodox Jews. Despite the increased incidence of TSD among
Ashkenazi Jews, the ultra-Orthodox community did not partici-
pate in the already-established screening programs because Jewish
law forbids termination of pregnancy after 40 days of concep-
tion. The program was developed in the early 1980s, after having
obtained support of the major religious leaders. The program is
based on the community’s custom of marriages though match-
making55 in which the decision about the suitability of a possible
match is made according to various factors. Dor Yeshorim proposed
tors taken into consideration. Blood samples are taken from ultra-
Orthodox high school students by Dor Yeshorim representatives,
and each individual is given a unique number. The test results are
not reported to the individuals or their physicians but are stored in
the Dor Yeshorim database. When a certain match is proposed, the
numbers of the prospective partners are checked and compared.
The match is approved if both are not carriers of the same genetic
disease. The number of conditions screened for by this program
has expanded over time as more disease genes and mutations
have been discovered. This program has averted births of poten-
tially affected individuals, but the precise magnitude can only be
 CHAPTER 26  Expanded Carrier Screening 259

Screen one
partner

Carrier of Not a carrier of

disease(s) any disease

Test other partner

No further action
for same disease(s)

Carrier Not a carrier

Prenatal No further
testing/PGD action

• Fig. 26.2 The stepwise screening model for autosomal recessive conditions. PGD, Preimplantation
genetic diagnosis.

Screen both
partners

Both partners carry

Only one partner Both partners are
mutations in the
is a carrier screen negative
same gene

Prenatal testing/
Inform/counsel No further action
PGD

• Fig. 26.3 The couple screening model for autosomal recessive conditions. PGD, Preimplantation
genetic diagnosis.

speculated. Despite its success, the program does have some draw-
backs. Because individuals are not aware of their potential carrier
status, no effort is made in detecting other potential carriers in the
family (see Cascade Screening later). If only one partner is found
to be a carrier and the other does not carry any of the common
mutation, there remains a residual reproductive risk for affected
offspring, which can be detected by NGS or gene sequencing.
Finally, fragile X is not included in the panel for obvious reasons
because female carriers would not be matched.  Couple Screening
Stepwise Screening
The most common model adopted for population-based screening
is the stepwise screening approach (Fig. 26.2). Initially, only one
partner is tested. If no mutation is detected, no further action is
taken. If a mutation is detected, ideally, genetic counselling is pro-
vided in which the implications of the carrier state are discussed,
and testing is recommended for the second partner. If both part-
ners are found to be carriers of the same condition, reproductive
options are discussed and offered. This method has been the most
widely used by laboratories since the 1990s. It is relatively cost-
efficient because not all members of the population are tested.
Because screening tests are often performed during an ongoing
pregnancy and because fragile X screening is done only in females,
it is most often the female partner who is the first to be tested.
In this stepwise model, there is an inherent lag time between the
identification of the first partner as a carrier and obtaining final
results of the second partner. Even though the latter is, in most
cases, negative, stress and anxiety are often observed during this
lag period.56–57 As more conditions are included in the screening
test, the chance for such an occurrence increases. In addition, as
more conditions are added, the call-back rate also increases to the
point where this approach becomes ineffective and cumbersome. 
This is also known as concurrent screening or tandem screening.
In this model, both partners are screened simultaneously for the
same list of AR conditions (Fig. 26.3). Positive results are con-
sidered only if both partners are found to be carriers of a patho-
genic variant in the same gene (not necessarily the same variant)
because only these are at a significant risk (25%) for affected off-
spring. Genetic counselling is provided to couples wherein both
are carriers and reproductive options are presented, as previously
discussed. If only one partner is found to be a carrier, the informa-
tion is provided, but no further action is usually taken. Couple
screening may be more costly than stepwise screening. However,
when dealing with ECS, simultaneously screening both partners
becomes more efficient than stepwise screening. Couple screen-
ing eliminates the anxiety of waiting for the results of the second
partner.58 In addition, it may be the method of choice for ECS
because when screening for numerous diseases is undertaken, the
260 SE C T I O N 6     Prenatal Screening and Diagnosis
chance for an individual being screen positive for at least one con-
dition may be as high as one in three.  panels include diseases that have mild to moderate implications or
Cascade Screening
In this approach, screening is not offered equally to all members of
the population. Rather, screening is initiated after the identifica-
tion of an affected individual or a carrier in the family. Immediate
family members are offered genetic counselling and testing only
for the specific condition detected. In those who are screen nega-
tive, no further action is taken. For those screened positive, testing
is extended to other unscreened immediate family members and
so on. Most important, partners of the newly identified carriers
are also screened to detect couples at risk for affected offspring.
This strategy has been most commonly used for CF screening.59,60
The main disadvantage of cascade screening is that it is often ini-
tiated only after the birth of at least one affected individual. In
addition, this mode has lower inclusion rates than population-
based screening and is therefore less efficacious at averting affected
births in the entire population. To demonstrate this, let us posit a
theoretical population with a carrier frequency of 4% for a certain
disease, testing all siblings and first cousins of all identified carriers
would require locating and testing only 2% of the entire popula-
tion but would detect only 15% of all new cases. Likewise, for a
condition with a carrier frequency of 1%, testing all siblings and
first cousins of all identified carriers would require locating and
testing only 0.1% of the entire population but would detect only
3% of all new cases. The detection rate increases with increasing
carrier frequency, family size and extending the testing to second
cousins of identified carriers but at the cost of greater increases in
the proportion of the population located and tested.61 Thus the
performance of cascade testing is too poor to justify its introduc-
tion into practice as an effective screening test for any AR disorder.  facts surrounding the decision to be made. The second is that the
Determining the Content of Expanded
Screening Panels
The ability to integrate large numbers of conditions into expanded
screening panels resulted in a systematic effort to prioritise dis-
eases for inclusion. In addition, the fact that these ECS panels
include numerous genetic disorders makes it virtually impossible
to describe each one in any detail during pre-test counselling. This
requires the categorisation of the tested conditions into groups
of diseases with similar characteristics. This approach may serve
as a practical method of simplifying counselling and decision-
making regarding the conditions for which patients would opt to
be screened for. As disease severity is a major criterion, classifica-
tions of the conditions screened in ECS by severity or other major
characteristics seems most appropriate.62–64 Different classifica-
tions systems have been proposed (Table 26.3). Such classification
systems may be used for pre-test patient education regarding the
expected effect of a certain disease on lifespan and quality of life.
In addition, they may facilitate patient’s choice regarding catego-
ries of information they wish to receive.
Laboratories performing ECS differ in the type and number
of diseases tested with a range of several dozen up to several hun-
dreds of different conditions in a single test.62 The 2008 ACMG
Guidelines proposed criteria for disease screening, indicating that
diseases should carry a potential for significant morbidity or mor-
tality, have a carrier frequency of at least 1% and be testable by
an assay with sensitivity of 90% or greater.65 However, current
ECS panels do not all adhere to the ACMG guidelines. Some ECS
adult onset even though the utility of screening for such diseases
has not been established by professional societies.66–68 It should
be stressed that the inclusion of mild or late-onset diseases in ECS
panels have some potential drawbacks because they pose both
counselling challenges for health care providers and ethical dilem-
mas for patients faced with making reproductive choices. At the
very least, such conditions should be made optional to the patient
or referring health care provider.66,67,69
The 2017 ACOG guidelines on screening suggest that ‘Given
the multitude of conditions that can be included in ECS panels,
the disorders selected for inclusion should meet several of the fol-
lowing consensus-determined criteria: have a carrier frequency of
1 in 100 or greater, have a well-defined phenotype, have a detri-
mental effect on quality of life, cause cognitive or physical impair-
ment, require surgical or medical intervention, or have an onset
early in life. Additionally, screened conditions should be able to be
diagnosed prenatally and may afford opportunities for antenatal
intervention to improve perinatal outcomes, changes to delivery
management to optimise newborn and infant outcomes, and edu-
cation of the parents about special care needs after birth’.36 
Patient Education
Pretest Counselling
An informed consent has been described as one in which a capable
person makes a reasonable choice based on the benefits and risks
of the decision to be made and his or her personal values.70 With
regard to informed consent, two basic elements have been identi-
fied: The first is that the patient fully comprehends the medical
patient is able to make his or her own choice without coercion.63,71
The main limitation of pretest education of ECS is the fact that
it is impractical to fully discuss all the diseases and conditions
included in the panel. This is in contrast to pretest counselling for
classical carrier screening programs, which includes information
regarding the natural history, detection rates, a priori and poste-
rior carrier probabilities of a limited number of diseases. Thus the
use of ECS necessitates modification of this model.26
In 1994, Elias and Annas addressed the limited ability to inform
those offered screening or testing for reproductive purposes about
all the genetic information that can be obtained and the implica-
tions of that information in cases of routine multidisease screening
program.72 Instead, they offered an approach for genetic counsel-
ling which is based on a generic consent. In the course of such
counselling, important factors common to all genetic screening
tests would be highlighted, including the limitations of screening
tests; the possible need for additional tests to establish a definitive
diagnosis; the reproductive options that might have to be con-
sidered, such as prenatal diagnosis, adoption, gamete donation,
abortion or acceptance of risks; the costs of screening; issues of
confidentiality; and the possibility of social stigmatisation, includ-
ing discrimination in health insurance and employment. If carrier
status is detected in one partner, it must be emphasised that the
other should also be screened.72
However, a study addressing patients’ preference regarding
informed consent scenarios for screening of multiple genetic
conditions found that there was significant variability in what
patients considered ideal informed consent, although the majority
preferred a more detailed consent and agreed that generic rather
 CHAPTER 26  Expanded Carrier Screening 261

TABLE
26.3 Classification Systems for Disease in Expanded Carrier Screening Panels
Classification System Description
Severity Based62
Profound Diseases associated with intellectual disability and infant or childhood life expectancy
Severe Diseases with intellectual disability or shortened life expectancy: (early adulthood); other symptoms possible, such as physical
malformations or impaired mobility
Moderate May include some (but not all) symptoms such as physical malformation, limited mobility, deafness or blindness; normal
lifespan and intellect are expected
Mild Adult-onset and mild or treatable condition; many individuals may never experience symptoms
Characteristics Based63
Group 1 Diseases are deadly, cause frequent sickness and pain, are associated with poor growth, include significant physical impair-
ment (walking, feeding), have a lifespan of less than a few years and have no effective treatment or cure
Group 2 Diseases are progressive, cause frequent sickness and pain, are associated with deterioration of already learned skills, have a
lifespan into early adulthood; some have treatments available to delay symptoms from developing and to manage
symptoms when they do develop, and they have no cure
Group 3 Chronic conditions that cause medical problems throughout a person’s life; episodes during which a person experiences
symptoms, have treatments and therapies available to prevent episodes from happening as often, but a person must be
treated for his or her entire life, might not cause shorter life expectancy, especially if the condition is well managed, living
at least into late adulthood and have no cure
Group 4 Conditions that cause mental retardation; do not affect lifespan or cause significant health problems; may have better
outcomes when children get help early but still likely to cause problems learning or living independently and have no cure.
Taxonomy Based64
Shortened lifespan Most patients do not live past early childhood even with medical interventions
Serious medical problems Most patients will have medical problems requiring regular medical visits, daily medications, carefully monitored diets or
surgeries or will have serious problems with learning, vision, hearing or mobility
Mild medical problems Most patients will have medical problems that require occasional extra medical visits, occasional medications, a slightly
modified diet, surgery; will have mild problems with learning, vision, hearing or mobility
Unpredictable medical The outcome is difficult to predict for many children; some children will have more serious versions, but others will have mild
outcomes or be asymptomatic
Adult-onset conditions Few have any symptoms as children, but medical, behavioural, vision or hearing problems may begin as adults

than disease specific detailed counselling would provide appropri-
ate information to make an informed decision.63 Because detailed
pretest education by genetic counsellor before each ECS may not
always be feasible, pretest information may be delivered through a
nongenetics-trained provider, print, video or internet. Addressing
this issue, a Joint Statement of the American College of Medi-
cal Genetics and Genomics, ACOG, National Society of Genetic
Counsellors, Perinatal Quality Foundation and Society for Mater-
nal-Fetal Medicine suggests that during pretest counselling for
ECS, several components of consent should be included69:
1. Carrier screening of any nature is voluntary, and it is reasonable
to accept or decline.
2. Results of genetic testing are confidential and protected in
health insurance and employment by the Genetic Information
Non-Discrimination Act of 2008.
3. Conditions included on ECS panels vary in severity. Many are
associated with significant adverse outcomes such as cognitive
impairment, decreased life expectancy and need for medical or
surgical intervention.
4. Pregnancy risk assessment depends on accurate knowledge of
paternity. If the biologic father is not available for carrier screening,
accurate risk assessment for recessive conditions is not possible.
5. A negative screen does not eliminate risk to offspring.
6. Because ECS includes a large number of disorders, it is com-
mon to identify carriers for one or more conditions. In most
cases, being a carrier of an AR condition has no clinical conse-
quences for the individual carrier. If each partner is identified
as a carrier of a different AR condition, offspring are not likely
to be affected.
7. In some instances, an individual may learn that she or he
has two pathogenic variants for a condition (homozygous
or compound heterozygous) and thus learns through car-
rier screening that she or he has an AR condition that could
affect their personal health. Some expanded screening panels
screen for selected autosomal dominant and X-linked condi-
tions, and likewise an individual may learn that she or he has
one of these conditions that might affect her or his health.
Referral to an appropriate specialist for medical management
and genetic counselling is indicated in such circumstances to
review the inheritance patterns, recurrence risks and clinical
features.
Consultation before performing ECS should lead to either
obtaining an informed consent or declining testing. Either deci-
sion should be documented in the medical record. 
Posttest Counselling and Management
As previously mentioned, as many as one in three individuals
screened by ECS is identified as a carrier of at least one disease.
This rate is expected to rise as more conditions are included and
262 SE C T I O N 6     Prenatal Screening and Diagnosis
as testing methodologies improve.33 Thus there are practical con-
cerns on the feasibility of a thorough follow-up of all screen-posi-
tive cases. If generic pretest counselling is provided, more detailed
counselling should be provided upon discovery of a carrier state.
Although it is preferred that the delivery of the result of ECS will
be made by genetic counsellors,73 personalised genetic counselling
for every positive result may not be attainable given the current
availability of genetic counsellors. Therefore alternative models
for high-volume results distribution must be taken into consid-
eration, including online results disclosure with patient acknowl-
edgement functionality74 or telephonic genetic counselling.75 In
addition, supplementary posttest genetic counselling by laborato-
ries performing ECS may also help address the needs and dynam-
ics of the local environment.33  discrepancies can partially be attributed to the relative novelty of
Introducing Expanded Carrier Screening to
Clinical Care
Despite the aforementioned advantages of ECS, a controversy
exists regarding the recommendation for its implantation. Some
studies have indicated that current ECS offerings were believed
by genetics professionals to have major limitations and that it was
not ready for routine use in reproductive health care.32 Such views
reflected previously raised concerns regarding an expected increase
in the likelihood of clinically ambiguous findings, particularly as
the number of conditions tested increases.76
In 2012 an anonymous online survey assessing knowledge
and attitudes of genetic counsellors toward ECS was distributed
to all 3039 participating members of the National Society of
Genetic Counselors (NSGC) via email with a total of 337 (11%)
genetic counsellors completing the survey.77 The results demon-
strated general agreement with ECS as a concept. For instance,
most agreed that carrier screening should include the disorders
described in the current guidelines. However, there were disagree-
ments expressed regarding appropriate pre- and posttest counsel-
ling and practical implementations, including the amount of time
available for follow-up care and other options for the delivery
of the content (e.g. the use of print, video or internet media).
The positive attitude for ECS was also reflected by another sur-
vey conducted among approximately 200 participants, 61% of
whom were medical doctors.78 In this survey, 77% of participants
declared they would prefer to be tested for a larger number of
disorders compared with a smaller number, assuming the cost
was the same. The importance of posttest consultation was also
noticed because a majority of participants believed that a posttest
consultation with a genetic counsellor would be helpful (83.7%),
if not essential (78.1%).
The increasing acceptance of ECS among health care providers
was mirrored by a similar trend among patients.79 Nonetheless,
actual use of ECS is still sluggish. Only 15% of ACOG Fellows
offer ECS to all of their patients, and approximately three quarters
indicate that it should only be offered based on race or ethnicity,
family history or as recommended by ACOG.73 A similar pat-
tern was also found among genetic counsellors: Although 80%
declared that they would personally elect ECS for themselves, only
a few were offering it to all of their patients.77 Although these
ECS, it may take more time and proof before the paradigm shift
will occur. 
Conclusions
Expanded carrier screening offers testing for multiple genetic dis-
orders of all individuals, regardless of ancestry. It is facilitated by
new molecular technologies that enable the expansion of screen-
ing without a concomitant increase in cost. The optimal timing
to offer screening is during the preconception period because
identifying a carrier couple before pregnancy provides more
reproductive options and more time to make an informed deci-
sion. There are still several challenges to the implementation of
ECS, including low genetic literacy among patients and medical
practitioners, cost and reimbursement issues, variability among
different national and regional health services, lack of profes-
sional guidelines and a shortage of professionals skilled in genetic
counselling. These issues will require time and effort to resolve
before ECS can be widely used. Education of the public as well
as the medical professionals will be required. Cost-effectiveness
analyses may be needed to demonstrate to decision makers not
only the obvious medical benefits but also the financial rewards.
Innovative solutions will have to be developed to deal with pre-
and posttest education. Despite these shortcomings, it may
be safe to assume that ECS has already become a valid clinical
option.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References Prenatal and preconceptional carrier screen-
ing for genetic diseases in individuals of East-
38. Lazarin GA, Haque IS. Expanded carrier
screening: a review of early implementation and
ern European Jewish descent. Obstet Gynecol. literature. Semin Perinatol. 2016;40:29–34.
1. Wilson JM, Jungner YG. Principles and prac-
2004;104:425–428. 39. Srinivasan BS, Evans EA, Flannick J, et al. A uni-
tice of mass screening for disease. Bol Oficina
20. ACOG Committee on Genetics. ACOG versal carrier test for the long tail of Mendelian
Sanit Panam. 1968;65:281–393.
committee opinion no. 442: preconception disease. Reprod Biomed Online. 2010;21:537–
2. World Health Organization. Genomic
and prenatal carrier screening for genetic 551.
resource centre. http://www.who.int/genomi
diseases in individuals of Eastern European 40. Absalan F, Ronaghi M. Molecular inversion
cs/publications/en/index1.html.
Jewish descent. Obstet Gynecol. 2009;114: probe assay. Methods Mol Biol. 2007;396:315–
3. Godard B, ten Kate L, Evers-Kiebooms
950–953. 330.
G, et  al. Population genetic screening pro-
21. Gross SJ, Pletcher BA, Monaghan KG. 41. Daly TM, Dumaual CM, Miao X, et al. Mul-
grammes: principles, techniques, practices, and
Professional Practice and Guidelines Com- tiplex assay for comprehensive genotyping of
policies. Eur J Hum Genet. 2003;11:S49–S87.
mittee. Carrier screening in individuals genes involved in drug metabolism, excretion,
4. Khoury MJ, McCabe LL, McCabe ER. Popu-
of Ashkenazi Jewish descent. Genet Med. and transport. Clin Chem. 2007;53:1222–1230.
lation screening in the age of genomic medi-
2008;10:54–56. 42. Wang Y, Moorhead M, Karlin-Neumann G,
cine. N Engl J Med. 2003;348:50–58.
22. https://geneaware.clinical.bcm.edu/GeneAwa et  al. Analysis of molecular inversion probe
5. Kaback MM. Population-based genetic screen-
re/Providers/PanelList.aspx. performance for allele copy number determi-
ing for reproductive counseling: the Tay-Sachs
23. Scott SA, Edelmann L, Liu L, et  al. Experi- nation. Genome Biol. 2007;8:R246.
disease model. Eur J Pediatr. 2000;159(Suppl. 3):
ence with carrier screening and prenatal diag- 43. Klugman S, Schreiber-Agus N, Nazareth S,
S192–S195.
nosis for 16 Ashkenazi Jewish genetic diseases. et  al. Detection of carriers in the Ashkenazi
6. Nazareth SB, Lazarin GA, Goldberg JD.
Hum Mutat. 2010;31:1240–1250. Jewish population: an objective comparison
Changing trends in carrier screening for genetic
24. Haque IS, Lazarin GA, Kang HP, et  al. of high-throughput genotyping versus gene-
disease in the United States. Prenat Diagn.
Modeled fetal risk of genetic diseases identi- by-gene testing. Genet Test Mol Biomarkers.
2015;35:931–935.
fied by expanded carrier screening. JAMA. 2013;17:763–767.
7. Kaback M, Lim-Steele J, Dabholkar D, et al.
2016;316(7):734–742. 44. Drmanac R, Sparks AB, Callow MJ, et  al.
Tay-Sachs disease—carrier screening, prenatal
25. Bray SM, Mulle JG, Dodd AF, et al. Signatures Human genome sequencing using unchained
diagnosis, and the molecular era. an interna-
of founder effects, admixture, and selection in base reads on self-assembling DNA nanoar-
tional perspective, 1970 to 1993. the inter-
the Ashkenazi Jewish population. Proc Natl rays. Science. 2010;327:78–81.
national tsd data collection network. JAMA.
Acad Sci U S A. 2010;107:16222–16227. 45. Bamshad MJ, Ng SB, Bigham AW, et  al.
1993;270:2307–2315.
26. Lazarin GA, Goldberg JD. Current con- Exome sequencing as a tool for Mende-
8. Cao A. Carrier screening and genetic coun-
troversies in traditional and expanded Car- lian disease gene discovery. Nat Rev Genet.
selling in beta-thalassemia. Int J Hematol.
rier screening. Curr Opin Obstet Gynecol. 2011;12:745–755.
2002;2:105–113.
2016;28:136–141. 46. Bell CJ, Dinwiddie DL, Miller NA, et  al.
9. Riordan JR, Rommens JM, Kerem B, et  al.
27. Committee on Genetics. American College of Carrier testing for severe childhood recessive
Identification of the cystic fibrosis gene: clon-
Obstetricians and Gynecologists. ACOG diseases by next-generation sequencing. Sci
ing and characterization of complementary
committee opinion. number 325, 2005. Transl Med. 2011. 3:65ra4.
DNA. Science. 1989;245:1066–1073.
Update on carrier screening for cystic fibrosis. 47. Hallam S, Nelson H, Greger V, et al. Validation
10. American College of Medical Genetics. Amer-
Obstet Gynecol. 2005;106:1465–1468. for clinical use of, and initial clinical experience
ican College of Obstetricians and Gynecologists.
28. Shafer FE, Lorey F, Cunningham GC, et al. with, a novel approach to population-based
Preconception and Prenatal Carrier Screening
Newborn screening for sickle cell disease: 4 carrier screening using high-throughput, next-
for Cystic Fibrosis: Clinical and Laboratory
years of experience from California’s newborn generation DNA sequencing. J Mol Diagn.
Guidelines. Washington, DC: ACOG; 2001.
screening program. J Pediatr Hematol Oncol. 2014;16:180–189.
Bethesda (MD): ACMG.
1996;18:36–41. 48. Kobelka CE. Sequencing: the next genera-
11. American College of Obstetricians and Gyne-
29. Ross LF. A re-examination of the use of eth- tion. moving beyond population-based reces-
cologists Committee on Genetics. ACOG
nicity in prenatal carrier testing. Am J Med sive disease carrier screening. Clin Genet.
committee opinion No. 486: update on car-
Genet A. 2012;158:19–23. 2011;80:25–26.
rier screening for cystic fibrosis. Obstet Gyne-
30. Kingsmore S. Comprehensive carrier screen- 49. Azimi M, Schmaus K, Greger V, et  al. Car-
col. 2011;117:1028–1031.
ing and molecular diagnostic testing for reces- rier screening by next-generation sequencing:
12. Prior TW. Carrier screening for spinal muscu-
sive childhood diseases. PLoS Curr. 2012. health benefits and cost effectiveness. Mol
lar atrophy. Genet Med. 2008;10:840–842.
e4f9877ab8ffa9. Genet Genomic Med. 2016;4:292–302.
13. Gitlin JM, Fischbeck K, Crawford TO, et al.
31. Jackson L, Pyeritz RE. Molecular technolo- 50. Richards S, Aziz N, Bale S, et al. Standards and
Carrier testing for spinal muscular atrophy.
gies open new clinical genetic vistas. Sci Transl guidelines for the interpretation of sequence
Genet Med. 2010;12:621–622.
Med. 2011;3:65. variants: a joint consensus recommendation
14. Morral N, Bertranpetit J, Estivill X, et al. The
32. Cho D, McGowan ML, Metcalfe J, et  al. of the American College of Medical Genetics
origin of the major cystic fibrosis mutation
Expanded carrier screening in reproductive and Genomics and the Association for Molec-
(delta F508) in European populations. Nat
healthcare: perspectives from genetics profes- ular Pathology. Genet Med. 2015;17:405–424.
Genet. 1994;7:169–175.
sionals. Hum Reprod. 2013;28:1725–1730. 51. Angastiniotis MA, Hadjiminas MG. Pre-
15. Turner G, Webb T, Wake S, et al. Prevalence
33. Lazarin GA, Haque IS, Nazareth S, et  al. vention of thalassaemia in Cyprus. Lancet.
of fragile X syndrome. Am J Med Genet.
An empirical estimate of carrier frequencies 1981;1:369–371.
1996;12:196–197.
for 400+ causal Mendelian variants: results 52. Cao A. Results of programmes for antenatal
16. Fu YH, Kuhl DP, Pizzuti A, et  al. Varia-
from an ethnically diverse clinical sample detection of thalassemia in reducing the incidence
tion of the CGG repeat at the fragile X site
of 23,453 individuals. Genet Med. 2013;15: of the disorder. Blood Rev. 1987;1:169–176.
results in genetic instability: resolution of
178–186. 53. Samavat A, Modell B. Iranian national thalassae-
the Sherman paradox. Cell. 1991;67:1047–
34. Cheschier N. ACOG committee on practice mia screening programme. BMJ. 2004;329:1134–
1058.
bulletins-obstetrics. ACOG practice bulletin. 1137.
17. Berkenstadt M, Ries-Levavi L, Cuckle H, et al.
neural tube defects. number 44, July 2003. 54. Inati A, Zeineh N, Isma’eel H, et  al. Beta-
Preconceptional and prenatal screening for
Int J Gynaecol Obstet. 2003;83:123–133. thalassemia: the Lebanese experience. Clin
fragile X syndrome: experience with 40,000
35. Hale JE, Parad RB, Comeau AM. Newborn Lab Haematol. 2006;28:217–227.
tests. Prenat Diagn. 2007;27:991–994.
screening showing decreasing incidence of cys- 55. Zlotogora J. Population programs for the detec-
18. Toledano-Alhadef H, Basel-Vanagaite L,
tic fibrosis. N Engl J Med. 2008;358:973–974. tion of couples at risk for severe monogenic
Magal N, et  al. Fragile-X carrier screening
36.  Committee on Genetics. Committee opinion genetic diseases. Hum Genet. 2009;126:247–
and the prevalence of premutation and full-
no. 690: carrier screening in the age of genomic 253.
mutation carriers in Israel. Am J Hum Genet.
medicine. Obstet Gynecol. 2017;129(3):e35–e40. 56. Miedzybrodzka ZH, Hall MH, Mollison J,
2001;69:351–360.
37. Henneman L, Borry P, Chokoshvili D, et  al. et al. Antenatal screening for carriers of cystic
19. ACOG Committee on Genetics. ACOG
Responsible implementation of expanded carrier fibrosis: randomised trial of stepwise v couple
committee opinion. Number 298, 2004.
screening. Eur J Hum Genet. 2016;24:e1–e12. screening. BMJ. 1995;310:353–357.

262.e1
262.e2 References
57. Mennie ME, Gilfillan A, Compton M, et al.
Prenatal screening for cystic fibrosis. Lancet.
1992;340:214–216.
58. Wald NJ, Morris JK, Rodeck Ch, et al. Cystic
fibrosis: selecting the prenatal screening strategy
of choice. Prenat Diagn. 2003;23:474–483.
59. Krawczak M, Cooper DN, Schmidtke J.
Estimating the efficacy and efficiency of cas-
cade genetic screening. Am J Hum Genet.
2001;69:361–370.
60. Super M, Schwarz MJ, Malone G, et  al.
Active cascade testing for carriers of cystic
fibrosis gene. BMJ. 1994;308:1462–1467.
61. Morris JK, Law MR, Wald NJ. Is cascade test-
ing a sensible method of screening a popula-
tion for autosomal recessive disorders?. Am J
Med Genet A. 2004;128. 27127–27125.
62. Lazarin GA, Hawthorne F, Collins NS, et al.
Systematic classification of disease severity for
evaluation of expanded carrier screening pan-
els. PLoS One. 2014;9:e114391.
63. Reeves A, Trepanier A. Comparison of
informed consent preferences for multiplex
genetic carrier screening among a diverse pop-
ulation. J Genet Couns. 2016;25:166–178.
64. Leo MC, McMullen C, Wilfond BS, et  al.
Patients’ ratings of genetic conditions validate a
taxonomy to simplify decisions about precon-
ception carrier screening via genome sequenc-
ing. Am J Med Genet A. 2016;170:574–582.
65. Gross SJ, Pletcher BA, Monaghan KG. Pro-
fessional practice and guidelines committee.
carrier screening in individuals of Ashkenazi
Jewish descent. Genet Med. 2008;10:54–56.
66. Wienke S, Brown K, Farmer M, et al. Expanded
carrier screening panels-does bigger mean bet-
ter? J Community Genet. 2014;5:191–198.
67. Henneman L, Borry P, Chokoshvili D, et al.
Responsible implementation of expanded
carrier screening. Eur J Hum Genet.
2016;24:e1–e12.
68. Rose NC. Expanded carrier screening:
too much of a good thing? Prenat Diagn.
2015;35:936–937.
69. Edwards JG, Feldman G, Goldberg J, et  al.
Expanded carrier screening in reproductive
medicine-points to consider: a joint statement
of the American College of Medical Genetics
and Genomics, American College of Obstetri-
cians and Gynecologists, National Society of
Genetic Counselors, Perinatal Quality Foun-
dation, and Society for Maternal-Fetal Medi-
cine. Obstet Gynecol. 2015;125:653–662.
70. Bekker H, Thornton JG, Airey CM, et  al.
Informed decision making: an annotated bib-
liography and systematic review. Health Tech-
nol Assess. 1999;3:1–156.
71. ACOG Committee on Ethics. ACOG com-
mittee opinion no. 439: informed consent.
Obstet Gynecol. 2009;114:401–408.
72. Elias S, Annas GJ. Generic consent for genetic
screening. N Engl J Med. 1994;330:1611–1613.
73. Benn P, Chapman AR, Erickson K, et  al.
Obstetricians and gynecologists’ practice and
opinions of expanded carrier testing and
noninvasive prenatal testing. Prenat Diagn.
2014;34:145–152.
74. Matheny ME, Gandhi TK, Orav EJ, et  al.
Impact of an automated test results manage-
ment system on patients’ satisfaction about
test result communication. Arch Intern Med.
2007;167:2233–2239.
75. Deene EW, Gilats M, Lazarin GA, et  al. A
unique service delivery model for genetic
counseling services. J Genet Counsel. 2015;24:
1059.
76. Kohane IS, Masys DR, Altman RB. The inci-
dentalome: a threat to genomic medicine.
JAMA. 2006;296:212–215.
77. Lazarin GA, Detweiler S, Nazareth SB, et al.
Genetic counselors’ perspectives and prac-
tices regarding expanded carrier screening
after initial clinical availability. J Genet Couns.
2016;25:395–404.
78. Ready K, Haque IS, Srinivasan BS, et al. Knowl-
edge and attitudes regarding expanded genetic
carrier screening among women’s healthcare
providers. Fertil Steril. 2012;97:407–413.
79. Holtkamp KC, van Maarle MC, Schouten
MJ, et  al. Do people from the Jewish com-
munity prefer ancestry-based or pan-ethnic
expanded carrier screening? Eur J Hum Genet.
2016;24:171–177.
27
Prenatal Screening for Thalassemias
KWOK YIN LEUNG, PATRICK AU AND MARY TANG
KEY POINTS
• Homozygous α0-thalassemia and β-thalassemia major are
global autosomal disorders.
• Different α- and β-thalassemia genotypes may be associated
with variable phenotypes.
• Universal screening is preferred for high-prevalence areas and
countries with migrants from high-prevalence areas.
• Screening by mean corpuscular volume or mean corpuscular
haemoglobin with or without haemoglobin (Hb) pattern is
feasible.
• Workup for screen-positive couples includes Hb and molecu-
lar studies.
• Molecular diagnosis of α- and β-thalassemia is useful for di-
agnosis of carriers with borderline haematologic parameters
and for prenatal diagnosis.
• Ultrasound exclusion of homozygous α0-thalassemia can
effectively reduce the need for invasive testing in the majority
of unaffected pregnancies.
• Early screening is preferred. For populations with a high prev-
alence of α-thalassemia carriers, screening is still advisable in
late gestation in view of severe maternal risks associated with
homozygous α0-thalassemia.
• Education of health care professionals and community, and
counselling with informed consent are essential for effective
screening programs.
Introduction
Haemoglobin (Hb) is a tetrameric protein made up of two α-like
(α or ζ) and two β-like (ε, γ, δ or β) globin chains (Fig. 27.1).
Thalassemias are characterised by the reduced synthesis of the glo-
bin chains of Hb. The two most severe forms of thalassemias are
homozygous α0-thalassemia with deletion of four α-globin genes
and β-thalassemia major with defects of both β-globin genes
(Tables 27.1 and 27.2), which cause major public health prob-
lems.1,2 Thalassemia is common in Mediterranean area, India,
Southeast Asia and sub-Saharan Africa because of positive selec-
tion due to falciparum malaria. Nowadays, it is a global health
problem because of population migration.3,4
Thalassemia is an autosomal recessive disorder. If the couple
carry the same (α or β) thalassemia trait, their offspring have a one
in four risk for thalassemia major. Prevention programs for severe
thalassemia have been implemented in many countries,5,6 mostly
by prenatal or premarital screening and diagnosis and rarely by
preimplantation genetic diagnosis; however, this is not feasible in
developing countries.7,8 Although the cost-effectiveness of prena-
tal screening program for β-thalassemia has been demonstrated,
there is limited evidence on screening for α-thalassemia. 
Thalassemias
α-Thalassemia
In a normal individual, there are four α-globin genes. α-Thalassemia
is characterised by a deficiency of α-globin chain synthesis caused
by a defect in one or more of the four α-globin genes. Its clini-
cal presentation varies, depending on the number of the defective
α-globin genes and the function of the remaining α-globin genes
(from zero to three) (see Table 27.1). 
Two or Three Functional α-Globin Genes
Individuals with α-thalassemia trait are asymptomatic. The muta-
tions are denoted as α0- or α+-thalassemia (Table 27.3). In α0-
thalassemia, deletions involve both α-globin genes with or without
ζ- globin genes. The gene defect in α+-thalassemia are mostly dele-
tional such as the 3.7-kb deletion (-α3.7) or the 4.2-kb deletion
(-α4.2) and less commonly nondeletional such as Hb Quong Sze
or Hb Constant Spring. 
No Functional α-Globin Genes: Hb Bart Disease
or Homozygous α0-Thalassemia
Because all four α-globin genes are deleted, a fetus affected by
­homozygous α0-thalassemia cannot synthesise any α-globin to pro-
duce fetal haemoglobin (Hb F [α2γ2]) in utero or Hb A (α2β2) after
birth. An affected fetus develops anaemia starting from 8 weeks’
gestation when there is a switch from the ζ to the α gene (see Fig.
27.1), with production of the abnormal Hb Bart (γ 4), which does
not release oxygen as effectively as the Hb F, and a small amount
of Hb Portland (ζ2γ2), resulting in hypoxia, hydrops fetalis, still-
birth or neonatal death. Severe maternal complications, including
preeclampsia-like ‘mirror’ syndrome or postpartum haemorrhage,
or rarely maternal death, can occur. Long-term survivors after in
utero and postnatal transfusion and bone marrow transplant have
been reported, some with congenital anomalies, delay in cogni-
tive and motor function, and problems of iron overload. In cases
of --FIL/--FIL in which deletion involve both α- and ζ-globin genes,
miscarriage may occur before 8 weeks. 
263
264 SE C T I O N 6     Prenatal Screening and Diagnosis

100
F—α2γ2

80

Haemoglobins (% of total)
A
60

40
F
ζ2€2
20
A—α2β2
ζ2γ2
α2€2 A2—α2δ2
0

50 α
Globin subunits (% of total)

40
αγ β
30

20 ζ Aγ
10 €
β
δ
0
0 10 20 30 10 20 30 40 weeks
Birth
Crown–rump
3 5 10 15 20 = cm

Fetus Newborn
• Fig. 27.1  Changes in haemoglobin tetramers (top panel) and in globin subunits (bottom panel) during
human development from embryo to early infancy. (From Bunn HF and Forget BG. Hemoglobin: molecu-
lar, genetic and clinical aspects. Philadelphia, PA: Saunders; 1986:68, with permission.)

TABLE a
27.1 Genotypes and Phenotypes of α-Thalassemias
Number of Functional α-Globin
Genes Disorders or Normal Genotype Phenotype
0 Homozygous α0-thalassemia (--/--) Hydrops fetalis
1 Haemoglobin H disease (--/-α) or (--/αTα) Moderate anaemia, usually not
transfusion dependent
2 Homozygous for α+-thalassemia or (-α/-α), (αTα/αTα) or (--/αα) No clinical problems
heterozygous for α0-thalassemia Low MCV and MCH
3 α+-Thalassemia (-α/αα), (αTα/αα) No clinical problems
Slightly reduced MCV and MCH
4 Normal (αα/αα) Normal MCV and MCH
aα0-Thalassemia: deletions of both α-globin genes in cis in the same chromosome (--); α+-thalassemia: deletion of one of the two α-globin genes (-α), or a nondeletion defect (αTα) on one chromosome.
MCH, Mean corpuscular haemoglobin; MCV, mean corpuscular volume.
  

One Functional α-Globin Gene: Hb H Disease (genotypes --/αTα). The latter type can have a more severe phe-
notype than the former. In general, patients with Hb H dis-
In Hb H disease, three of four α-globin genes are defective, and ease can have relatively normal lives and are not transfusion
only one α-globin gene is functional. There are two types of ­dependent. They have moderate anaemia, splenomegaly and may
Hb H disease: deletional (genotype --/- α ) and nondeletional require transfusions or hospitalisation during stress or infection. 
 CHAPTER 27  Prenatal Screening for Thalassemias 265

TABLE
27.2 Genotypes and Phenotypes of β-Thalassemias and Related Disorders
Disorder Genotype Phenotype
Homozygous β0-thalassemia (β0/β0) Thalassemia major
or (β0/Hb Lepore or severe β+-thalassemia)
Compound heterozygous for β0-thalassemia/Hb
Lepore or severe β+-thalassemia
Compound heterozygous for β0- (β0/β+) Variable: thalassemia intermedia to major depend-
thalassemia/β+-thalassemia (β+/β+) ing on type of β+ mutation (mild or severe)
or (Hb E/β0 or β+)
Homozygous β+-thalassemia or (δβ0-thalassemia/β0 or β+)
Compound heterozygous for Hb E/β0 or severe
β+-thalassemia or
Compound heterozygous for δβ0-
thalassemia/β0 or severe β+-thalassemia
Compound heterozygous for Hb O-Arab/β0- (Hb O-Arab/β0-thalassemia) Severe thalassemia intermedia
thalassemia
Homozygous δβ0-thalassemia (δβ0/δβ0) Thalassemia intermedia
Compound heterozygous for δβ0-thalassemia/ (δβ0-thalassemia/β+) Mild thalassemia intermedia
mild β+-thalassemia or (ααα/β0 or β+ )
Compound heterozygous for ααα/β0 or severe
β+-thalassemia
Compound heterozygous for Hb C/β0 or severe (Hb C/β0 orβ+-thalassemia) Variable: β-thalassemia trait to intermedia
β+-thalassemia
Heterozygous for β0-thalassemia or β+- (β0/β) or Variable: normal to thalassemia trait
thalassemia (β+/β) Low MCV and MCH
Homozygous or heterozygous for HPFH (HPFH/HPFH) No clinical problems
(HPFH/β) Low MCV and MCH
Homozygous or heterozygous for Hb E (Hb E/Hb E) No clinical problems
(Hb E/β) Normal or low MCV and MCH
Normal (β/β) Normal MCV and MCH

β, Normal β-globin genotype; Hb, haemoglobin; HPFH, hereditary persistence of fetal haemoglobin; MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume.
  

β-Thalassemia
In a normal fetus, there are two β-globin genes. β-Thalassemia is
characterised by a deficiency of β-globin chain synthesis caused
by a defect of one or both β-globin genes, which results in
β-thalassemia trait or homozygous β-thalassemia, respectively.  [α2β2]).
β-Thalassemia Trait
Individuals with β-thalassemia trait are asymptomatic. Most
β-thalassemia mutations are nondeletional. The mutations are
denoted as either β+ type, which is associated with a reduction of
the expression of the β-globin gene, or β0 type, which is associ-
ated with the complete absence of β-globin (see Table 27.3).
Of these β+ or β0-type mutations, the majority are associated
with a severe phenotype in homozygotes. Patients with a milder
phenotype have an increased amount of β-gene expression or Hb
F production.  heterozygosity for β0-thalassemia/β+ (mild) -thalassemia or
Homozygous β-Thalassemia
In contrast to Hb Bart disease, a fetus affected by homozy-
gous β-thalassemia will not develop anaemia in utero because
of the presence of Hb F (α2γ2). Anaemia will become appar-
ent several months after birth when switching from γ to β
and δ genes occurs (see Fig. 27.1), and there are no functional
β-globin genes to produce normal adult haemoglobin (Hb A
β-Thalassemia major can be caused by homozygous β0-
thalassemia, compound heterozygosity for β0-thalassemia/β+
(severe) -thalassemia or homozygous β+ (severe) -thalassemia
(see Table 27.2). Affected individuals are transfusion dependent,
have iron overload from repeated transfusion and may die in the
second or third decade of life from cardiac failure, although some
may reach 40 years of age in good health, and have children.
With the availability of a human leukocyte antigen–matched
sibling or relative, bone marrow transplantation can successfully
cure this disorder.9
β-Thalassemia intermedia can be caused by compound
homozygous β+ (mild) -thalassemia (see Table 27.2). The clini-
cal presentation can be variable. In general, affected individu-
als have a milder clinical condition, present later, receive fewer
blood transfusions and have less iron overload than individuals
with thalassemia major. 
266 SE C T I O N 6     Prenatal Screening and Diagnosis

TABLE Gene Deletion or Mutation in and Distribution β+-thalassemia may lead to thalassemia major or intermedia (Fig.
27.3 of α-, β-Thalassemia and δβ0-Thalassemia 27.2).
Prediction of phenotype on the basis of a known β-thalassemia
Gene genotype may not always be accurate. Although homozygosity for
Deletions or β+ (mild) -thalassemia usually results in intermedia, compound het-
Thalassemia Mutations Distribution erozygote for β+(severe) -thalassemia/β+ (mild) -thalassemia may
result in thalassemia major in some cases, thalassemia major geno-
α+-Thalassemia -α3.7 Worldwide
Common in Africa, Mediter-
type will be associated with thalassemia intermedia phenotype if
ranean countries, the Middle there is coinheritance of α-thalassemia or a high Hb F determinant.
East, the Indian subcontinent In general, prenatal diagnosis is not required for Hb H dis-
and Melanesia ease because an affected individual can have a normal life. Occa-
-α4.2 Worldwide sionally, a nondeletional type Hb H disease may present with
Common in Southeast Asia and hydrops fetalis, and at-risk couples may request prenatal diag-
Pacific regions nosis. Prenatal ultrasonographic features of anaemia with raised
-αT Mostly found in Mediterranean middle cerebral artery peak systolic velocity (MCA-PSV) have
area, Africa and Southeast Asia been reported. If prenatal diagnosis is required, screening for
α0-Thalassemia --SEA Southeast Asia and South China both α0- and α+-thalassemia is needed. An alternative is new-
--THAI Thailand and South China born screening.
--FIL Philippines Compound heterozygosity for Hb S and β0/severe β+-thalassemia
-(α)20.5 Mediterranean countries such results in sickle cell disease, which is outside the scope of this chapter. 
as Greece, Cyprus and Turkey
--MED Mediterranean countries such
as Greece, Cyprus and Turkey Screening by Mean Corpuscular Volume or Mean
--SA Rare in India Corpuscular Haemoglobin
--BRIT Rare in the United Kingdom
Both MCV and MCH are useful for screening for α- and
β+-Thalassemia β+ Mediterranean countries, β-thalassemia. MCH is preferred if analysis of blood samples
Southeast Asia, Africa and cannot be performed shortly after blood taking because red cells
United Kingdom will swell over time and thus affect MCV values. An MCV cutoff
β0-Thalassemia β0 South Asia, Indonesia of 80 fl or MCH cut-off of 27 pg has been shown to detect all
SEA deletion carriers and β-thalassemia carriers.13 Some labo-
δβ0-Thalassemia δβ0 Mediterranean countries, ratories may use a higher cutoff (82 fl) for MCV. Screening by
Vietnam and South China
MCV/MCH alone may not detect α+-thalassemia and Hb E
-(α)20.5,20.5-kb Deletion involving α2-globin gene and the 5’ end of α1-globin gene; -α3.7, because the MCV/MCH can be normal (between 80 and 85
3.7-kb deletion of α2-globin gene; -α4.2, 4.2-kb deletion of α2-globin gene; -αT, other dele- fl) and thus may miss the prenatal diagnosis of Hb H disease or
tion of α2-globin gene; --BRIT, British; --FIL, Filipino; -- MED, Mediterranean; --SA, South compound heterozygote for Hb E/β-thalassemia if the partner
African; -- SEA, Southeast Asian; --THAI, Thai. carries α0-thalassemia and β-thalassemia, respectively. Coinheri-
   tance of α- and β-thalassemia mutations may have a higher mean
values of MCV and MCH and a lower HbA2 level than sim-
ple β-thalassemia.14 Thus when a near-normal range of MCV/
MCH is encountered, the possibility of coinheritance of α- and
Prenatal Screening β-thalassemia mutations should be considered. 
Universal or Selective Screening
The prenatal screening strategy depends on the prevalence and types
Haemoglobin H Inclusion Bodies
of severe thalassemia disorders in a local area. Whereas universal pre- The presence of Hb H inclusion bodies on incubating the red
natal screening is preferred for high-prevalence areas, selective screen- blood cells with brilliant cresyl blue suggests the diagnosis of
ing based on ethnic origin of the pregnant woman and her partner is α0-thalassemia trait. On the other hand, absence of Hb H inclu-
used in low prevalence areas. High-prevalence areas include Africa, sion bodies does not exclude the diagnosis, and DNA analysis is
the Mediterranean, the Middle East, the Indian subcontinent, South- required for the diagnosis.
east Asia and the Pacific region.10 Genetic information of thalassemia The presence of a large amount of Hb H inclusion suggests the
mutations in various populations can be found online.11  diagnosis of Hb H disease because of the excess β-chains in the
reticulocyte. 
How to Screen Haemoglobin Electrophoresis and High-
To detect the two most severe thalassemia disorders, homozygous Performance Liquid Chromatography or
α0-thalassemia and β-thalassemia major (see Table 27.2), screen-
ing for the carrier states for α0-thalassemia, β0/β+–thalassemia is
Capillary Electrophoresis
performed, mostly by measurement of mean corpuscular volume Haemoglobin electrophoresis provides qualitative analysis of Hbs
(MCV), mean corpuscular haemoglobin (MCH) or both. (A, F, A2 and others), but high-performance liquid chromatogra-
In at-risk areas, screening for δβ-thalassemia, Hb C, Hb O-Arab, phy (HPLC) or capillary electrophoresis (CE) allows quantitation
Hb E and Hb Lepore12 by Hb electrophoresis is also needed because of the Hb fractions. If the aim is to detect β-thalassemia alone, Hb
compound heterozygosity for one of these mutation and β0/severe analysis by HPLC or CE (Fig. 27.3) can be selectively added when
 CHAPTER 27  Prenatal Screening for Thalassemias 267

δβ Thalassemia
α0 Thalassemia
α+ Thalassemia

β Thalassemia

Hb Lepore

Hb DPunjab
Hb OArab
Carrier of:

carrier
Not a
HPFH
Hb S

Hb C
Hb E
α+ Thalassemia

α0 Thalassemia

Hb S

β Thalassemia

δβ Thalassemia

Hb Lepore

Hb E

Hb OArab

Hb C

Hb DPunjab

HPFH

Not a
carrier

Serious risk

Less serious risk

Possible hidden risk for α0-thalassemia

No risk

• Fig. 27.2  Summary of the main genetic risks for couples with thalassemia or haemoglobinopathy that
can result in an affected pregnancy. Hb, Haemoglobin; HPFH, hereditary persistence of fetal haemoglobin.
(Adapted from Old JM. Screening and genetic diagnosis of haemoglobinopathies. Scand J Clin Lab Invest
67:71–86, 2007 and Taylor & Francis Ltd., www.tandfonline.com, with permission.)

MCV or MCH is low. β-Thalassemia carriers are diagnosed by the
presence of low MCV and raised Hb A2. The latter is usually high
(3.5%–7.0%) except in coinheritance with δ-thalassemia trait, or
very rarely, in carriers of silent β-thalassemia or normal Hb A2β-
thalassemia. A very high level of Hb A2 (7.0%–9.0%) suggests the
presence of a deletion mutation affecting the promoter region of
the β-globin gene.
For β-thalassemia carriers with iron deficiency, the Hb A2
level may be normal. Thus it is essential to exclude iron deficiency
before excluding β-thalassemia carrier state based on the finding
of low MCV and normal Hb A2. Repeat blood investigations 4
weeks after iron therapy may help.
In at-risk areas where δβ-thalassemia, Hb C, Hb O-Arab, Hb E or
Hb Lepore is found,12 performing Hb electrophoresis irrespective of
MCV or MCH values is required because MCV/MCH can be normal.
In areas where silent β-thalassemia (normal A2) is found,
β-globin genotyping can be performed in a couple if one partner is
a β-thalassemia carrier irrespective of the MCV/MCH and HbA2
levels of the other partner.15
Rarely, when an elevated level of Hb F is found, either δβ-
thalassemia or deletion types of hereditary persistence of fetal
haemoglobin (HPFH) is suspected. 
Workup for Screen-Positive Couples
Screen-positive individuals are those whose MCV or MCH falls
below the cutoff value. α0-Thalassemia trait, β-thalassemia trait
and iron deficiency need to be considered. Laboratory tests to look
for the presence of Hb H inclusion bodies and elevation in Hb A2
level and an iron study should be performed. The presence of Hb
H inclusion bodies and elevation in Hb A2 (>3.5%) are diagnostic
of α0-thalassemia trait and β-thalassemia, respectively. Iron defi-
ciency may account for the microcytosis and hypochromasia, but
coexistence of β-thalassemia cannot be totally excluded.
Workup on the partner should begin with MCV, MCH
or both. A positive screening result in the partner necessitates
laboratory tests for the presence of Hb H inclusion bodies and
elevation in Hb A2 level and an iron study. The purpose is to
268 SE C T I O N 6     Prenatal Screening and Diagnosis

45.0

37.5

% of Haemoglobins
30.0

22.5

Hb A2: 3.62 min

Hb A: 2.46 min
Hb F: 1.05 min
15.0

7.5
*

0.0

0 1 2 3 4 5 6
A Time (min)

Z15 Z14 Z13 Z12 Z11 Z10 Z9 Z8 Z7 Z6 Z5 Z4 Z3 Z2 Z1

Hb A

Hb F

Hb A2

B
• Fig. 27.3  High-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Common
automated methods in measuring haemoglobin (Hb) A2 and Hb F. A, Quantitation of Hb F and Hb A2
by HPLC. B, Quantitation of Hb F and Hb A2 by CE. (From Greene DN, Pyle AL, Chang JS, et al. Com-
parison of Sebia Capillarys Flex capillary electrophoresis with the BioRad Variant II high pressure liquid
chromatography in the evaluation of haemoglobinopathies. Clin Chim Acta 413(15-16):1232–1238, 2012,
with permission.)

identify whether the couple has the same type of thalassemia
(i.e. whether they are an α-α or β-β couple). In regions with
high prevalence of both α- and β-thalassemia, up to 7% of
β-thalassemia carriers are compound α- and β-thalassemia het-
erozygotes. Couples discordant for α- and β-thalassemia (α-β
couples) should therefore be offered a DNA study to exclude
coexistent α-Thalassemia in the partner with β-thalassemia. Less
commonly, when an individual is a carrier of δβ-thalassemia, Hb
C, Hb O-Arab, Hb E or Hb Lepore12 while the partner is a carrier
of β0/severe β+-thalassemia, the fetus is at 25% risk for thalassemia
major or intermedia.
Molecular Diagnosis of Thalassemias
Molecular testing is used to exclude coexisting α-thalassemia
in β-thalassemia carrier and in prenatal diagnosis of α- and
β-thalassemia for at-risk couples. Molecular diagnosis is also used
to identify the nucleotide changes resulting in Hb variants 
Detection of αo- and α+-Thalassemia Deletion
Each local population has its own characteristic spectrum of α thalas-
semia mutations (see Table 27.3).16 It is essential to know the ethnic
origin of the individual or the partner to enable an appropriate and the
quick identification of the underlying deletions or mutational defects.
Gap-PCR (polymerase chain reaction) can be used first to
screen for the seven most common deletion alleles, including
-α3.7, -α4.2, -- FIL, -- THAI, -- MED, -(α)20.5 and -- SEA. PCR primers
flanking the common deletions are designed to detect the muta-
tions in a multiplex PCR reaction (Fig. 27.4). PCR primers for
the amplification of normal α2-globin gene and a control gene for
monitoring the PCR reaction are included (Fig. 27.5).
 CHAPTER 27  Prenatal Screening for Thalassemias 269

ladder
Mother Father Fetus If the results are negative, multiplex ligation–dependent probe
DNA
--SEA/-- SEA

αα/αα
αα/-- α3.7
αα/-- α4.2
αα/-- SEA

αα/-- THAI
αα/-- FIL
amplification (MLPA) can be used to detect rare deletion. MLPA
using probes distributed along the α-globin gene cluster can detect
various different deletions within the region. The interpretation of
MLPA result may be complicated by the occurrence of duplications
with deletions in the α-globin gene cluster. 
Control
gene
-α3.7
Detection of Nondeletion α+-Thalassemia
Mutations
α2–globin
gene
-α4.2

--SEA
The common nondeletion α+-thalassemia mutations such as Hb
--FIL Constant Spring and Hb Quong Sze, which result in more severe Hb
H disease when compounded with αo-thalassemia deletion, can be
--THAI
detected by allele-specific hybridisation, multiplex fluorescent minise-
quencing, allele-specific PCR or PCR/restriction enzyme analysis.
For multiplex fluorescent minisequencing, sequencing primers spe-
• Fig. 27.4 Multiplex gap polymerase chain reaction for common dele- cific for the mutations to be detected are designed. There are only fluo-
tions in α0 thalassemia (--SEA, --FIL, --THAI) and α+ thalassemia (-α3.7, -α4.2) rescently labelled dideoxynucleotides in the sequencing reaction so that
found in a Southeast Asian population. Both the mother and the father are there will be incorporation of a single dideoxynucleotide corresponding
heterozygous carriers of α0-thalassemia (--SEA) deletion, and the fetus has to the normal or mutant allele at the site. The minisequencing products
a normal αα/αα genotype.

Check maternal blood for MCV/MCH +/– Hb pattern

Maternal MCV <80 fL or MCH < 27 pg Maternal MCV ≥80 fL and MCH ≥27 pg
or abnormal Hb pattern and normal Hb pattern (if available)

Check paternal MCV and Hb pattern Fetus usually not at risk

Maternal Hb pattern (if not yet done)
Both iron profile

Couple whose haematologic

results indicated thalassemia

Refer to prenatal diagnostic and counselling centre

for counselling and DNA analysis

Couple: β-thalassemia Couple: α-thalassemia

risk risk

Offer CVS/amnio
(if mutation is found)
Accept an invasive Accept serial ultrasound
test (CVS or amnio) examinations

Presence of fetal cardiomegaly or placentomegaly

Yes No

Invasive test (CVS or amnio) offered No invasive test

• Fig. 27.5 The process of antenatal screening and follow up for thalassemia. amnio, Amniocentesis;
CVS, chorionic villus sampling; Hb, haemoglobin; MCH, mean corpuscular haemoglobin; MCV, mean
corpuscular volume.
270 SE C T I O N 6     Prenatal Screening and Diagnosis

are separated and detected by capillary electrophoresis, and the specific
mutation can be identified from the analysis of result (Fig. 27.6).
If the screening result is negative, PCR sequencing for α1- and
α2-globin gene can be used to detect rare mutations.  Molecular Diagnosis of Haemoglobin Variants
Detection of β-Thalassemia Mutations
Although more than 200 point mutations have been reported,
for each place, there are a small number of specific mutations
which account for the majority. When the ethnicity of an indi-
vidual is known, the most prevalent mutations in that group are
screened using techniques such as hybridisation of allele-specific
oligonucleotide probes (ASOs) for single mutation, reverse dot
hybridisation (Fig. 27.7), multiplex fluorescent minisequencing
(Fig. 27.8) for multiple mutations, microarrays for simultaneous
detection of large number of mutations or amplification refrac-
tory mutation system (ARMS) for quick screening of common
mutations. Besides, gap-PCR can be used for diagnosis of the
deletion mutations. When the ethnic origin is not known, PCR
sequencing of the β-globin gene is preferred. High-resolution
melting, a powerful technology for scanning sequence alteration,
is potentially useful for rapid and high-throughput platforms
for screening and prenatal diagnosis of common β-thalassemia
mutations.17
Normal and mutant oligonucleotide probes specific for
β-thalassemia mutations are immobilised on nylon membrane
and hybridised with biotinylated PCR fragments of β-globin
gene. Subsequent colour development resulted from binding of
streptavidin-enzyme conjugate followed by conversion of added
substrate to colored precipitates.  effectively reduce the need for invasive testing in the majority of
Detection of δβ-thalassemia deletions
Gap-PCR can be used to detect common δβ−thalassemia dele-
tions found in the specific ethnic group (Fig. 27.9). MLPA with
probes distributed along the β-globin gene cluster can be used to
detect rare deletions in the region. 
For unknown Hb variants, mass spectrometry (MS) is an effective
method of characterising variant Hbs.18
The nucleotide change that resulted in the formation of Hb
variant can also be identified by PCR sequencing of the α1- and
α2-globin gene or β-globin gene (Fig. 27.10). 
At-Risk Couples
When couples at risk for major α- or β-thalassemia are identified,
counselling and prenatal testing, invasive or noninvasive, should
be carried out by personnel and laboratories with experience in
prenatal diagnosis.
However, unusual cases of homozygous α0-thalassemia or
β-thalassemia major caused by maternal uniparental disomy or
nonpaternity, in which a woman’s MCV is low but her partner’s
MCV is normal, have been reported.19 Midtrimester scan can
detect these unusual cases of homozygous α0-thalassemia but not
β-thalassemia major.
Ultrasound Exclusion of Homozygous α0-
Thalassemia
A noninvasive approach consisting of serial two-dimensional
ultrasound examinations starting from 12 weeks’ gestation can
unaffected pregnancies (Fig. 27.11).20,21 In an affected pregnancy,
the ultrasonographic measurements of the fetal cardiothoracic ratio
(CTR) and placental thickness (PT) are increased because of severe
fetal anaemia (Fig. 27.12 and Video 27.1). Because α-globin–
dependent Hb F is the major Hb of a fetus from 8 weeks’ gestation

33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65
20,000
16,000 Heterozygous for Hb QS
Hb WM
12,000
8000 α2 Cd 30
Hb CS α2 Cd 59
4000 Hb QS
0
33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65
10,000
8000 Heterozygous for Hb CS
Hb WM
6000
4000 Hb QS
α2 Cd 30
2000 Hb CS α2 Cd 59
0
33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65
12,000
10,000 Hb WM Heterozygous for α2 Cd30
8000
6000
4000 Hb QS Hb CS
2000 α2 Cd 30 α2 Cd 59
0
33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65
10,000
8000 Heterozygous for Hb WM
Hb WM
6000
Hb QS α2 Cd 30 Hb CS
4000
α2 Cd 59
2000
0

• Fig. 27.6 Multiplex fluorescent minisequencing for five common nondeletion mutations in α2-globin
gene found in a Southern Chinese population. Arrows indicate primer peaks resulting from mutant alleles.
Sequencing primers specific for the mutations to be detected are designed. The presence of a specific
mutation is identified by the incorporation of a single dideoxynucleotide corresponding to the normal or
mutant allele. α2 Cd 30, Codon 30 (delGAG) in α2-globin; α2 Cd 59, codon 59 (GGC GAC) in α2-globin;
Hb CS, haemoglobin Constant Spring; Hb QS, haemoglobin Quong Sze; Hb WM, haemoglobin Westmead.
 CHAPTER 27  Prenatal Screening for Thalassemias 271

onwards, anaemia can occur in an affected fetus after this week of chorionic villus sampling (CVS) or amniocentesis for fetal DNA
gestation (see Fig. 27.1). analysis will be offered for confirmation of homozygous α0-
For all women at risk for carrying fetuses with homozygous α0- thalassemia.21 In areas where resources for molecular studies are
thalassemia, the option of a noninvasive approach can be offered as limited, cordocentesis can be performed to collect a fetal blood
an alternative to avoid invasive procedures in unaffected pregnancies. sample for Hb study, but the complication rate is higher than for
Serial two-dimensional ultrasound examinations are performed at 12 amniocentesis or CVS. With the use of quantitative PCR, a rapid
to 15, 18 to 20 and 30 weeks’ gestation.20,21 The fetal CTR is a ratio report can be available within 1 or 2 days after amniocentesis or
of the fetal transverse cardiac diameter taken at the level of the atrio- CVS. The reporting time is comparable to fetal Hb pattern study
ventricular valves between the epicardial surfaces at diastole to the after cordocentesis. 
transverse fetal thoracic diameter. PT is a measurement of the maxi-
mal PT, with the transducer placed perpendicularly to the placenta
and measurements taken in the longitudinal and transverse sections. Benefits and Limitations of Ultrasound
If there is fetal cardiomegaly (CTR ≥0.50, 0.52 and 0.59 at Exclusion
12–15, 18 and 30 weeks’ gestation respectively), or placentomeg-
aly (>18 mm at 12 weeks’ or >30 mm at 18 weeks’ gestation), The overall sensitivity and specificity of this ultrasound approach
were reported to be 100% and 95.6%, respectively.21 The major
benefit in the use of this noninvasive approach was the avoidance
14/15 -28 17 HbE 27/28 -29
of an invasive test in about 75% of patients, while the cost saving
Normal was relatively small in comparison with the cost of the whole pre-
natal screening program for thalassemia.20 This approach is appli-
Mutant cable in singleton as well as twin pregnancies and can be used to
Mother
confirm normality in pregnancies conceived after preimplantation
Cd41/42 - βA
Normal genetic diagnosis.
However, there are several limitations of this noninvasive approach.
Mutant First, there is a risk for delaying the diagnosis of an affected pregnancy
because of either lack of experience in the examination of the fetal
43 41/42 71/72 654 15 IVS-I-5 heart or suboptimal image resolution obtained at early second trimes-
ter.21 The use of this approach demands an accurate measurement
of the fetal CTR. Adequate training and subsequent quality control
are essential. When the image quality of the fetal heart at 12 weeks’
gestation is suboptimal, even with the use of transvaginal scan, rescan
14/15 -28 17 HbE 27/28 -29 in 2 or 3 weeks is a reasonable option if a woman still prefers ultra-
sound monitoring to an invasive testing.21 Of note, the placenta of
Normal an affected pregnancy may be large but not thick. The risk for delay-
ing the diagnosis of an affected pregnancy until the second trimester
Mutant Father and the disadvantages of second trimester termination of an affected
Cd17/ βA pregnancy should be balanced against the risk for an invasive testing.
Normal Second, the false-positive rate of this noninvasive approach was
about 3% because disorders such as intrauterine growth restriction,
Mutant
congenital heart disease21 and Hb H disease can present with car-
43 41/42 71/72 654 15 IVS-I-5 diomegaly, placentomegaly or both. In addition, a focal myometrial
contraction or oblique measurement of the PT may overestimate
the PT. So, an invasive test is still required to confirm or exclude
the diagnosis of an affected fetus. Third, two-dimensional ultraso-
nography is not predictive of affected pregnancies before 12 weeks’
14/15 -28 17 HbE 27/28 -29 gestation. The usefulness of measurement of MCA-PSV at 12 to 13
weeks’ gestation has not been proven because of extensive overlap of
its values between affected and unaffected fetuses at such early gesta-
Normal
tion. Fourth, the predictive values of the fetal CTR decrease with
gestational age.21 In advanced gestation, hydropic signs, including
Mutant Fetus
ascites or pleural effusion, are more apparent than cardiomegaly in
Cd41/42 - Cd17
affected pregnancies.21 Measurement of the fetal middle cerebral
Normal
artery peak systolic velocity (MCA-PSV) may be a more sensitive
Mutant sonographic parameter in identifying affected fetuses.22 

43 41/42 71/72 654 15 IVS-I-5

• Fig. 27.7  Reverse dot blot hybridisation using allele-specific oligonucle-
otide probes for 12 common mutations in β−thalassemia found in a South-
ern Chinese population. The mother is a heterozygous carrier of Cd 41/42
mutation, the father is a heterozygous carrier of Cd 17 mutation and the
fetus is a thalassemia major, compound heterozygous for Cd 41/42 and
Cd 17 mutations.
Other Noninvasive Testing for Homozygous
α0-Thalassemia
If a woman opts for first trimester combined Down syndrome
screening, normal maternal serum-free β-human chorionic gonad-
otrophin (β-hCG) and pregnancy-associated placental protein A
272 SE C T I O N 6     Prenatal Screening and Diagnosis

35 37 39 41 43 45 47 49 51 53 57 59 61 63 65 67 69
4000
Cd 17 Cd 41/42
3000 Hb E
Mother
2000 IVS-II-654
-28 Cd 71/72
1000

0
35 37 39 41 43 45 47 49 51 53 57 59 61 63 65 67 69
4000
Cd 41/42
3000 Cd 17
2000 Hb E
Father
IVS-II-654 -28 Cd 71/72
1000

0
35 37 39 41 43 45 47 49 51 53 57 59 61 63 65 67 69
4000

3000 Cd 17 Cd 41/42
Hb E
2000 Fetus
IVS-II-654 -28 Cd 71/72
1000

• Fig. 27.8  Multiplex fluorescent minisequencing for six common mutations in β-thalassemia found in a
Southern Chinese population. The mother is a heterozygous carrier of Cd 41/42 mutation, the father is a
heterozygous carrier of Cd 17 mutation and the fetus is a thalassemia major, compound heterozygous for
Cd 41/42 and Cd 17 mutations. (Arrows indicate primer peaks resulted from mutant alleles.)
DNA ladder

Mother

Father

Fetus

C S F A

? Hb J

Normal
fragment
Deletion TA T G GA CAA CC C T A A G GT G AA G GC
fragment 400 405 410 415 420
[Greek beta]-Globin
1200 Codon 56, GGC → GAC Hb J-Bangkok
1000 (Gly) (Asp)
800 G/A
600
400
200

B 4850 4900 4950 5000 5050

• Fig. 27.9  Detection of Chinese Gγ(Aγδβ)o thalassemia by gap-polymerase
chain reaction. The mother is negative for the deletion, and the father and
the fetus are heterozygous for the deletion.
(PAPP-A) at 11 to 14 weeks ‘ gestation is a reassuring sign of nor-
mality for fetuses at risk for homozygous α0-thalassemia.
Three-dimensional placental volumetry is not predictive of
affected pregnancies before 12 weeks’ gestation, although the vol-
ume is larger in affected than unaffected pregnancies.23
Immunofluorescence staining of fetal erythrocytes in mater-
nal blood is a potentially useful but labour-intensive noninvasive
technique in the first trimester. In affected pregnancies, positive
staining with fluorescence-labeled monoclonal anti-zeta but not
• Fig. 27.10  Identification of Hb variant. A, Haemoglobin (Hb) electropho-
resis at a pH of 8.6 using cellulose acetate membrane showed a fast-
moving Hb J variant constituted by β-globin chain variant. B, Polymerase
chain sequencing of β-globin gene identified the variant as Hb J-Bangkok
with anti–α-globin antibodies can identify fetal nonnucleated red
blood cells in maternal blood.
Noninvasive Prenatal Diagnosis for Thalassemia
More recently, noninvasive testing for affected fetuses became fea-
sible by examination of cell-free DNA in maternal plasma. Details
are discussed in another chapter. 

A B
19cm/s
Voluson
E8
-19cm/s
45
C 30
15
cm/s
• Fig. 27.11  Ultrasound image of a pregnancy unaffected by homozygous
α0-thalassemia in the second trimester showing a normal fetal cardiotho-
racic ratio (A), normal placental thickness (B) and normal middle cerebral
artery peak systolic velocity (C).
Timing of Screening
Early screening allows time for workup, counselling of at-risk
couples and an earlier relief of maternal anxiety in an unaffected
pregnancy. In case termination of pregnancy is considered, timely
diagnosis of thalassemia major in the fetus is desirable. For popu-
lations with a high prevalence of α-thalassemia carriers, antenatal
thalassemia screening should be offered to all pregnant women
regardless of gestational age, in view of maternal risks associated
with pregnancies with homozygous α0-thalassemia.  sickle cell disease, Hb E or other haemoglobinopathies.
Education and Counselling
Education of health care professionals, information for couples
and informed consent are required before screening. Information
for couples may be provided through various means, including
pamphlets, video display, posting on a website or explanation in
person. The prognosis of affected infants, maternal risks associ-
ated with affected pregnancies, available support and therapy for
affected infants, available invasive and noninvasive options for
prenatal tests, test accuracy, limitation of prenatal tests in prenatal
diagnosis, screening workflow and need for confirmation testing
at delivery all need to be discussed with the at-risk couples.  majority of pregnancies unaffected by homozygous α0-thalassemia.
Conclusion
• Thalassemia is an autosomal recessive disorder, and thus if both
couple carries the same (α- or β-) thalassemia trait, their off-
spring will be at one in four risk for having thalassemia major.
• Prevention programs of severe thalassemia have been implemented
in many countries, mostly by prenatal screening and diagnosis.
CHAPTER 27  Prenatal Screening for Thalassemias 273
A B
19cm/s
Voluson
E8
-19cm/s
C 45
30
15
cm/s
• Fig. 27.12  Ultrasound image of a pregnancy affected by homozygous
α0-thalassemia in the first trimester showing fetal cardiomegaly (A), plac-
entomegaly (B) and high middle cerebral artery peak systolic velocity (C).
• Homozygous α0-thalassemia, deletion of four α-genes, is asso-
ciated with hydrops fetalis, stillbirth or neonatal death, and
severe maternal complications or rarely death.
• β-Thalassemia major can be caused by homozygous β0- or
β+- (severe) thalassemia, or compound heterozygote for β0-
thalassemia/β+-thalassemia (severe), and affected individuals
are transfusion dependent.
• Universal prenatal screening is preferred for high-prevalence
areas, and selective screening based on ethnic origin of the preg-
nant woman and her partner is used for low prevalence areas.
• Both MCV and MCH are useful for screening α-and
β-thalassemia, and Hb pattern can be added in areas at risk for
• The presence of Hb H inclusion bodies and elevation in
Hb A2 (>3.5%) are diagnostic of α0-thalassemia trait and
β-thalassemia, respectively.
• Each local population has its own characteristic spectrum of thalas-
semia mutations. Therefore it is essential to know the ethnic origin
of a patient or her partner to select an appropriate DNA test.
• When couples at risk for major α- orβ-thalassemia are identi-
fied, counselling and prenatal testing should be carried out by
personnel and laboratories with experience in prenatal diagnosis.
• A noninvasive approach consisting of serial two-dimensional ultra-
sound examinations of fetal CTR and PT starting from 12 weeks’
gestation can effectively reduce the need for invasive testing in the
• Early screening allows time for workup, counselling of at-risk
couples, earlier relief of maternal anxiety in an unaffected preg-
nancy and earlier management of an affected pregnancy.
• Education of health care professionals and patients’ informed
consent before prenatal screening are required.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References thalassemia major. Hematol Oncol Clin North
Am. 2014;28:1187–1200.
17. Prajantasen T, Fucharoen S, Fucharoen G.
High resolution melting analytical platform
10. Higgs DR, Thein SL, Wood WG. In distribu- for rapid prenatal and postnatal diagnosis of
1. Rund D. Thalassemia 2016: modern medi-
tion and population genetics of the thalassae- β-thalassemia common among Southeast Asian
cine battles an ancient disease. Am J Hematol.
mias. In: Weatherall DJ, Clegg JB, eds. The population. Clin Chim Acta. 2015;20:56–62.
2016;91:15–21.
thalassaemia syndromes. London: Blackwell Sci- 18. Griffin M, Eyre J, Dack S, Wright J. Mass spec-
2. Piel FB, Weatherall DJ. The α-thalassemias. N
ence; 2001:237–284. trometric analysis of unknown haemoglobin
Engl J Med. 2014;371:1908–1916.
11. Patrinos GP, Giardine B, Riemer C, et  al. fractions identified by high-performance liquid
3. Crighton G, Wood AE, Scarborough R, et al.
Improvements in the HbVar database of chromatography in an antenatal screening pro-
Haemoglobin disorders in Australia: where are
human hemoglobin variants and thalassemia gramme. J Clin Pathol. 2015;68:388–390.
we now and where will we be in the future?
mutations for population and sequence varia- 19. Kou KO, Lee H, Lau B, et  al. Two unusual
Intern Med J. 2016;46(7):770–779.
tion studies. Nucl Acids Res. 2004;32:D537– cases of haemoglobin Bart’s hydrops fetalis due
4. Sayani FA, Kwiatkowski JL. Increasing preva-
D541 (Database issue). to uniparental disomy or non-paternity. Fetal
lence of thalassemia in America: implications
12. Weatherall DJ, Clegg JB. The thalassemia syn- Diagn Ther. 2014;35:36–308.
for primary care. Ann Med. 2015;47:592–604.
dromes. 4th ed. Oxford, United Kingdom: 20. Leung KY, Lee CP, Tang MHY, et  al. Cost
5. Moafi A, Vallian R, Vallian S, et  al. The pros
Blackwell Scientific Publications; 2001. effectiveness of prenatal screening for thal-
and cons of the fourth revision of thalassaemia
13. Ma ESK, Chan AYY, Ha SY, et al. Thalassae- assemia in Hong Kong. Prenat Diagn.
screening programme in Iran. J Med Screen.
mia screening based on red cell indices in the 2004;24:899–907.
2017;24(1):1–5.
Chinese. Haematologica. 2001;86:1286–1287. 21. Leung KY, Liao C, Li QM, et al. A new strat-
6. Choudhuri S, Sen A, Ghosh MK, et al. Effec-
14. Saleh-Gohari N, Khademi Bami M, Nikbakht egy for prenatal diagnosis of homozygous
tiveness of prenatal screening for hemoglobin-
R, Karimi-Maleh H. Effects of α-thalassaemia alpha-thalassaemia. Ultrasound Obstet Gynecol.
opathies in a developing country. Hemoglobin.
mutations on the haematological param- 2006;28:173–177.
2015;39:380–383.
eters of β-thalassaemia carriers. J Clin Pathol. 22. Leung KY, Cheong KB, Lee CP, et al. Ultra-
7. Li J, Yan JM, Xie XM, et al. Consequences of
2015;68:562–566. sonographic prediction of homozygous alpha0-
delayed prenatal diagnosis of β-thalassemia in
15. Gorivale M, Sawant P, Mehta P, et  al. Chal- thalassemia using placental thickness, fetal
mainland China. Hemoglobin. 2016;1:1–3.
lenges in prenatal diagnosis of beta thal- cardiothoracic ratio and middle cerebral artery
8. Ngim CF, Ibrahim H, Lai NM, Ng CS.
assaemia: couples with normal HbA2 in one Doppler: alone or in combination? Ultrasound
A single centre study on birth of children
partner. Prenat Diagn. 2015;35:353–1357. Obstet Gynecol. 2010;35:149–154.
with transfusion-dependent thalassaemia in
16. Moghadam M, Karimi M, Dehghani SJ, et al. 23. Chen M, Leung KY, Lee CP, et  al. Placental
Malaysia and reasons for ineffective prevention.
Effectiveness of β-thalassemia prenatal diag- volume measured by three-dimensional ultra-
Prenat Diagn. 2015;35:51–59.
nosis in Southern Iran: a cohort study. Prenat sound in the prediction of fetal αο-thalassemia:
9. Mathews V, Srivastava A, Chandy M.
Diagn. 2015;35:1238–1242. a preliminary report. Ultrasound Obstet Gyne-
Allogeneic stem cell transplantation for
col. 2006;28:166–172.

273.e1
28
Sonography of the Fetal Central
Nervous System
LUC DE CATTE, BART DE KEERSMAECKER, LUC JOYEUX AND MICHAEL AERTSEN

KEY POINTS Ultrasound examination in the second trimester as screening

• Prenatal ultrasound of the fetal brain in the second and third
trimester is described, from basics to advanced neurosonog-
raphy.
• Classification of central nervous system anomalies is dis-
cussed. The neurodevelopment stage determines some of the
pathological conditions; others are related to external factors
interfering with normal brain development.
• The diagnosis of neural tube defects deserves attention in the
light of potential fetal surgery.
• Analysis of the anterior and posterior complex aids in the
diagnosis of ventral induction disorders.
• The size and position of the vermis is key in the differential
diagnosis of posterior fossa anomalies.
• The diagnosis and differentiation of cortical developmen-
tal anomalies remains challenging, but fetal magnetic
resonance imaging overcomes some of the sonographic
diagnostic challenges.
• Destructive lesions are linked to intracranial bleeding,
congenital infections, ischemic and vascular lesions and
arteriovenous malformations.
• Fetal cystic lesions and cerebral tumours may cause neurologic
impairment because of the mass effect rather than the type
of the lesion.
• Some lesions are borderline but frequently encountered;
usually if isolated, the prognosis is fairly good.
Introduction
tool for the detection of structural brain lesions mainly relies
on three historical planes: the transthalamic plane, the ven-
tricular plane and the plane though the posterior fossa (Figs.
28.1 to 28.3)2 The proper use of these planes in midtrimester
ultrasound screening enables the detection of most of the major
CNS malformations. Fetuses in which there is a suspicion of
brain anomalies should be evaluated by dedicated fetal medi-
cine specialists imaging the brain in the different orthogonal
planes, transabdominally and transvaginally whenever possible
(Figs. 28.4 to 28.6). Additional imaging by three-dimensional
(3D) ultrasonography may facilitate fetal brain exploration (Fig.
28.7). Recently, automated algorithms have allowed for the rec-
ognition of the major essential landmarks in normal developing
fetal brains.3,4 Novel and complementary magnetic resonance
imaging (MRI) techniques after 24 weeks of gestation may
enrich the detailed exploration of the fetal brain by ultrasound
in some conditions.5,6
The International Society of Ultrasound in Obstetrics and
Gynecology published practice guidelines about the performance
of fetal MRI.7 
TABLE Total Number and Prevalence of Major Groups
28.1 of Congenital Malformations in Europe
Group of Prevalence Percent
Congenital Total Per 10,000 of Genetic Percent
Malformation Number Births Conditions Live Births
Heart defects 22,709 76.46 15.0 87.6

Congenital anomalies (CAs) of the central nervous system Urinary tract 10,082 33.95 6.6 84.5
anomalies
(CNS)1 account for 10.2% of all registered CAs and represent
the fourth most common group of congenital malformations Limb 12,817 43.16 9.5 86.7
(Table 28.1). The prevalence for all cases is 25.97 per 10,000 anomalies
births. As many as 8.5% of CNS anomalies are related to a Nervous 7712 25.97 15.0 44.1
genetic condition. In contrast with the leading causes of CA, system
however, the number of live births is only about 44%. The anomalies
majority of cases end in a termination of pregnancy (TOP)
(52.4%) or a fetal death (3.5%). In addition, more than 10% Adapted from Garne E, Dolk H, Loane M, Boyd PA. Eurocat. EUROCAT website data on prena-
of infant deaths secondary to CA worldwide are related to tal detection rates of congenital anomalies. J Med Screen. 2010;17(2):97–98.

the CNS.   

275
276 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Fig. 28.1  Transthalamic axial view of the fetal brain at 22 weeks of ges-
tation. The most common structures identified are the thalami (asterisk),
cavum septi pellucidi (^), falx cerebri (arrow), corpus callosum (- - - - -) and
sylvian sulcus (°).
• Fig. 28.2  Axial view through the posterior fossa, illustrating the cerebel-
lum (asterisk), cisterna magna (arrow), remnants of the Blake pouch (^)
and cerebral peduncles (°).
Central Nervous System Anomalies
Ventriculomegaly
Fetal ventriculomegaly (VM) is an enlargement of the lateral cere-
bral ventricle caused by an excess of cerebrospinal fluid (CSF). The
incidence of VM ranges from 0.3 to 1.5% life births. Unilateral
VM occurs in 60% of the cases, leaving 40% bilateral. There is a
male predominance (70%). VM may result from obstructive mal-
formations, destructive lesions and abnormal development of the
brain (Table 28.2).
The diagnosis is made with the measurement of the lateral
ventricle in a strict sagittal plane on ultrasonography. The mea-
surement is performed opposite to the internal parieto-occipital
sulcus, putting the callipers on the inner wall at its widest part
and aligned to the long axis8 (Fig. 28.8). An atrial width between
Voluson
E10
• Fig. 28.3  The brain can be extensively examined in the coronal and sag-
ittal planes transabdominally or transvaginally. The incorporation of multi-
planar three-dimensional investigation may be of great help in identifying
structures that in regular planes are hardly visible or recognisable. Shown
are the thalami (asterisk), cavum septi pellucidi (^), falx cerebri (arrow),
corpus callosum (- - - - - -) and sylvian sulcus (°)
3
2
1 4 9
6 5
7
10
8
• Fig. 28.4  Midsagittal view illustrates the most important element in the
midbrain formation: the (1) cavum septum pellucidi, (2) corpus callosum,
(3) cingulate gyrus, (4) thalamus, (5) cerebellar vermis, (6) fastigium, (7)
pons, (8) medulla oblongata, (9) tentorium and (10) cisterna magna.
10 and 15 mm is considered mild VM; a width greater than 15
mm constitutes severe VM (Figs. 28.9 and 28.10). Fetal MRI is
helpful to diagnose additional abnormalities in 5% to 50% of the
cases.9,10
Fetal VM is often associated with CNS anomalies (agenesis of
the corpus callosum [CC], spina bifida [SB]). In 30% of cases,
severe VM is associated with non-CNS anomalies. Chromosomal
anomalies are detected in more than 15% of cases when VM is
associated with other fetal anomalies.11 In mild VM, structural
anomalies range from 10% to 76%. However, even in apparently
isolated VM, malformations are found in 13% of the cases after
birth.12
 CHAPTER 28  Sonography of the Fetal Central Nervous System 277

Ventriculomegaly associated with other brain anoma-
lies carries a high mortality rate (60%–70%). Early detec-
tion and progression of the VM are bad prognostic factors.
Isolated mild VM has a poor outcome in 20% of the cases
with perinatal death in 1.4%. In 3% of cases with isolated
mild VM chromosomal abnormalities are seen (mainly T21:
9 times more).12 Prenatal stabilisation in size or regression is
a favourable sign. Recently, isolated mild VM documented by
a normal fetal MRI has been associated with a normal neu-
rodevelopment outcome when evaluated between 18 and 36
months of age by the Vineland Adaptive Behavior Scales. The
same study demonstrated a similar result for moderate VM
but on a rather small sample size and should be confirmed on
a larger population.13 

1
3

• Fig. 28.5  Parasagittal scanning plane allows the evaluation of the lateral • Fig. 28.6  Colour Doppler flow of the cerebral vascularisation is particu-
larly helpful in the identification of normal vascularisation and the presence
ventricle including the temporal horn. In addition, some information on the
of arteriovenous malformations. This image reveals the details of the peri-
cerebral gyration is visualised: the (1) choroid plexus, (2) temporal horn of
callosal arterial supply.
the lateral ventricle, (3) posterior horn and (4) subarachnoid space.

A
• Fig. 28.7  (A) Acquisition of a three-dimensional (3D) volume allows for multiplanar analysis or tomo-
graphic (B) evaluation of the brain by interrogation of an acquired 3D volume in a fetus with an encepha-
locele. Volume contrast imaging (C) showing three parallel axial slices through a sagittally acquired 3D
volume of a normal fetal brain demonstrating (1) the upper axial plane of the lateral ventricles and midline,
(2) the plane through the anterior horns of the lateral ventricles and cavum septi pellucidi and (3) the axial
plane through the thalami.
278 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

C
Fig. 28.7, cont’d.

neurulation results in closed NTD. Causes of NTD are multifac-

Anomalies Related to Dorsal Induction Failure
Introduction. Neural tube defects (NTDs) are caused by a failure
of closure of the neural tube before the end of the sixth week of
gestation at the two most vulnerable locations (i.e., the rostral and
caudal neuropores). Dorsal induction failures are divided into
open and closed defects and classified according to the level of
the lesion.
Failure of the rostral and caudal neuropores to close during
the primary neurulation results, respectively, in cranial and spi-
nal open NTD. Failure of the canalisation during the secondary
torial, involving genetic and mainly epigenetic risk factors along
with complex mechanisms.
At the spinal (caudal) level, an NTD is called spinal dysra-
phism or SB.
When a spinal column defect remains covered (closed SB)
rather than open (open SB), fetuses do not develop the same
severe sequelae. In its open form, SB presents as a progressive
disease explained by the two-hit pathogenesis.14,15 First, there
is the failed closure of the neural tube by the sixth week of
gestation. Second, from 16 weeks onwards, there is secondary
 CHAPTER 28  Sonography of the Fetal Central Nervous System 279

TABLE Fetal Conditions Associated With

28.2 Ventriculomegaly
Leading Cause of Ventriculomegaly Anomaly
Abnormal turnover of Obstructive Aqueduct stenosis
cerebrospinal fluid
Spina bifida and
cephaloceles
Intracranial haemorrhage
Arachnoid cyst ^
Tumours
Infections
Nonobstructive Papilloma of the choroid
plexus Vp
Structural Agenesis of the corpus
malformations callosum
Holoprosencephaly
Destructive lesions Infections • Fig. 28.8 Measurement of the lateral ventricle (Vp) should be stan-
Porencephaly dardised to incorporate the following structures: the cavum septi pellu-
Vascular insults cidi (asterisk), the ambient cistern (°) and the atrium of the lateral ventricle
Syndromes Trisomy 21, 13 or 18 (arrow). Measurement is preformed from the inner to the outer border at
Miller-Dieker the level of the sulcus parieto-occipitalis medialis (^), comparable with the
syndrome technique of nuchal translucency evaluation in the first trimester.
Smith-Lemli-Opitz
syndrome
Aicardi syndrome
Migration Lissencephaly
Schizencephaly
Megalencephaly
Microcephaly
  

damage to the exposed spinal cord and nerves caused by direct

trauma and neurotoxic agents in the amniotic fluid,16-18 as well
as to the brain. The latter is evidenced by the development of
VM and Chiari II malformation (CM) caused by CSF leakage
at the level of the defect, leading to a ‘suction gradient’.19 A
similar two-hit pathogenesis could explain the other types of
NTD. For anencephaly, animal studies and case reports have
shown that there is progression from acrania to exencephaly
and finally anencephaly caused by secondary degeneration of
the brain.
A study of long-term trends in prevalence of NTDs in Europe
• Fig. 28.9 Mild ventriculomegaly without apparent associated central
from 1991 to 2011 found that total prevalence fluctuates slightly nervous system anomalies.
but without an obvious downward trend.20 SB is the most frequent
NTD with a total prevalence of 4.9 in 10,000 in Europe from
2008 to 2012 according to the European Concerted Action on
Congenital Anomalies and Twins (EUROCAT) registry,1,20 3.17 detection rate of 84%.26 Detection rates depend on the presence
in 10,000 in the United States,21 1.4 in 10,000 in Brazil,22 and of standardised screening programs, the gestational age at which
15.0 in 10,000 in Northern China.23 Anencephaly and encepha- screening is performed and the type of lesion. 
locele have, respectively, a total prevalence of 5.40 and 1.06 per
10,000 births in Europe.24 Thanks to primary prevention in Cranial Neural Tube Defects
North America using folic acid food fortification, the total preva- Anencephaly and Exencephaly. The three phases in the
lence rate of SB in the United States declined by 31% from the development of anencephaly are (i) a defective development of
pre- (1995–1996; 5.04 in 10,000) to the postfortification period the cranial vault resulting from a failed closure of the rostral part
(1998–2006; 3.49 in 10,000). In addition, preconception folic of the neural groove, (ii) exposure to the amniotic fluid and invol-
acid supplementation might also reduce the severity of NTD.25 untary fetal movements changing the developing brain to amor-
Prenatal screening for NTD by ultrasound in Europe showed phous neurovascular tissue (exencephaly) and (iii) a disintegration
detection rates ranging from 25% to 94%, with an overall of the brain tissue resulting in anencephaly.1,16,19
280 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Fig. 28.10  Severe ventriculomegaly at 19 weeks’ gestation resulting in
macrocephaly. Note the thin cortex and the dangling choroid plexus in the
lateral ventricle. This fetus presented with severe muscle atrophy of the
lower legs bilaterally.
• Fig. 28.11  In exencephaly, the fetal brain tissue (arrowheads) bulges out
of the skull as an irregularly shaped structure. The absence of the cranium
is evident in this 12-week fetus.
Sonographically, exencephaly is recognised by the bulging brain
above the orbits (the Mickey Mouse Face or the French bonnet
sign). Early in gestation, therefore, the diagnosis may be missed
because the crown–rump length (CRL) may be normal. Complete
disintegration of the brain subsequently results in a short CRL for
gestational age and the typical frog-like appearance of the fetal
face caused by the absence of any structure above the orbits (Figs.
28.11 and 28.12). Differential diagnoses are large encephalocele
and amniotic band syndrome (Fig. 28.13). Sonographic detection
of anencephaly reaches 100%, most of which occurs now in the
first trimester.27 Associated anomalies are present in 25% to 50%
• Fig. 28.12  Three-dimensional image of a fetus with anencephaly.
Posterior
Anterior
• Fig. 28.13  Large encephalocele (arrows) containing the midbrain (°) and
the occipital lobes (asterisk) on the axial plane.
of the cases. In isolated cases, the risk for chromosomal defects is
low.28
Anencephaly is fatal prenatally, intrapartum or within 48 hours
of birth.29
Cephaloceles result from a defect in the skull and dura mater
with protrusion of meninges (cranial meningocele) or hernia-
tion of brain tissue covered by meninges (encephalocele). Their
pathogenesis resulting directly from failure of neural tube forma-
tion has been contested. However, at present, these lesions are still
considered postneurulation disorders.30 Cephaloceles are usually
covered by skin or a thin epithelium layer, occur most frequently
at the midline and may involve the occipital (75%–85%), frontal
(12%), eccentric parietal (13%–15%) and interparietal regions.31
The size of the lesion may vary considerably; occasionally, the size
 CHAPTER 28  Sonography of the Fetal Central Nervous System 281

TABLE
28.3 Encephalocoele: Associated Malformations

Group Anomalies
Central nervous Corpus callous agenesis, intracranial cysts,
system tectorial malformations, Dandy-Walker,
cerebellar vermis agenesis, ventriculomegaly,
hydrocephaly, grey matter heterotopia
Nonsyndromic Ventricular septal defect, aortic coarctation
Cleft palate
Tracheoesophageal fistula, gastroschisis,
diaphragmatic hernia
Costal malformations, talipes
Pyelectasis, ureteral agenesis
Chromosomal Trisomy 13, trisomy 18, mosaic trisomy 20,
abnormality 13q-, monosomy X
Syndromic forms Meckel-Gruber syndrome
Walker-Warburg syndrome
Knobloch syndrome
Apert syndrome
Fronto-facio-nasal dysplasia
Oculo-encephalo-hepato-renal syndrome • Fig. 28.14 Axial T2 half-Fourier acquisition single-shot turbo spin-echo
Vascular Vertical positioned straight sinus, elongation of (HASTE) image of the brain at 27 weeks and 4 days’ gestation. The small
the vein of Galen, fenestration of the superior cystic structure in the left parietal area with tapering towards the bone (arrow)
sagittal sinus suggests a connection with the subarachnoid space: a small encephalocele.

of the encephalocele is larger than the fetal head, which, because

of the herniation, becomes microcephalic.32
Infants with encephaloceles, especially giant encephaloceles,
present with additional cerebral33 and extracerebral malformations
in 20% to 36.8% of the cases34,35 (Table 28.3). Syndromic forms
such as Merkel-Gruber syndrome and Walker-Warburg syndrome
carry an increased recurrence risk. Chromosomal abnormalities
are rare.35 Prenatal ultrasound has great potential in the early
detection of encephaloceles, even in the first trimester.35 Efforts
should be taken to highlight the associated malformations and
offer invasive prenatal testing, particularly in nonisolated cases.
Differential diagnosis includes amniotic band syndrome, inien-
cephaly, cystic hygroma and scalp cysts (Fig. 28.14).
In isolated encephaloceles (73%–80%), the prognosis depends
on the site, the size and the content of the lesion.36 In noniso-
lated cases, the mortality rate is as high as 79%. Surviving neo-
nates present with neurologic impairment in 75% to 80% of the
cases, including seizures and significant developmental delays.37,38
In giant encephaloceles, TOP may be offered. Microcephaly and
hydrocephaly are good indicators of an impaired outcome.
Small lesions without brain tissue may have surgical correction • Fig. 28.15  Cervical rachischisis with hyperextension of the fetal head as
with good outcome. Caesarean section is performed for the larger seen in iniencephaly. However, iniencephaly is a closed spina lesion.
lesions containing brain tissue to prevent trauma. 
Sonographic appearance showed a fixed flexion of the head
Craniospinal Defects with an upturned face (stargazer appearance). Cervical and
Craniorachischisis. Only 3% of NTDs display the most extreme thoracic vertebrae show incomplete formation or closure; the
form of primary neurulation failure: absent development of the cranial occipital bones seem fused with the cervicothoracic vertebrae.
vault and defective closure of the vertebrae and skin. Sonographically, Associated anomalies have been reported in more than 80% of
this lethal condition translates into an anencephaly with prolongation cases.
into a myeloschisis.30 Most cases end in early fetal loss (Fig. 28.15).  Iniencephaly should be distinguished from extreme hyperex-
Iniencephaly. The aetiology of this lethal condition is tension of the fetal head, which may resolve spontaneously, and
unknown. Iniencephaly is a combination of a deficient occiput Klippel-Feil syndrome.39 Because of the hyperextension of the
and inion, a rachischisis of the cervicothoracic spine and an head, dystocia may occur. Early induction of labour should be
extreme retroflexion of the head. considered to avoid Caesarean section.40 
282 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Open spina bifida
Spinal dysraphism
Closed spine bifida
• Fig. 28.16  Classification of spinal dysraphism.
Open Spinal Dysraphism
The classification of spinal dysraphism has been revised recently as
shown in Fig. 28.16.41
Meningomyelocele. Open spinal dysraphism (OSD) con-
sists of vertebral defects without skin coverage, exposing the
neural tissue either directly to the amniotic fluid (myelocele
or myeloschisis) or with coverage by a meningeal membrane
(meningomyelocele).
Cerebrospinal fluid leaks into the amniotic cavity and may
be responsible for the scalloping of the frontal bones (lemon
sign on ultrasound). In addition, the cerebellum often her-
niates through the foramen magnum (Arnold-Chiari malfor-
mation type 2); obstructs the circulation of CSF, which may
result in VM; and leads to reversal of the lemon sign after 24
weeks’ gestation41 (Fig. 28.17). Reduced head circumference
(HC) on ultrasonography is frequently part of the clinical
spectrum of open NTD with 53% and 74% of the fetuses
presenting with an HC below the 3rd and 10th centiles,
respectively.42
Screening for open NTDs by ultrasound relies on these cra-
nial markers rather than on the direct features of the spinal lesion.
In 99% of cases, at least one cranial marker is present before 24
weeks’ gestation (Table 28.4). Other cerebral signs in the second
trimester include the pointed deformity of the posterior horn,43
tectal beaking or elongation of the tectum44 or the presence of an
interhemispheric cyst.45
The identification and evaluation of the extent of the lesion
demand sonographic exploration of the spine in all three planes
Meningomyelocele
Myelocele
Myeloschisis
Meningocele
Lipomyelomeningocele
Myelocystocele
Lipoma
Diastematomyelia
Caudal regression
(see Fig. 28.17). The 12th rib may serve as reference point to
describe the level, although about 6% of fetuses display an abnor-
mal number of ribs.46 Multiplanar 3D exploration of the spine
and thoracic cage offers a standardised method of exploring and
defining each vertebral level (Fig. 28.18). Moving in the sagittal
A plane, the B plane shows the axial view of the vertebral bodies
and the overlying skin, which is the optimal view to identify the
spinal defect. However, the bony defect does not always correlate
with the functional level, prenatal prediction of the neurologic
disability and functionality may be inaccurate.
Recently, detection of open SB at the 11- to 13-week scan
by visualisation of the intracranial translucency (IT) and pos-
terior brain in the midsagittal plane gained increased inter-
est.47-49 However, the sensitivity of these markers is low.50,51
In the hands of experts, the sensitivities of an absent IT and
cisterna magna were 18% and 64%, respectively but increased
significantly with the use of specific cutoff values to 45% and
73%, respectively.51 Other ultrasound features may enable a
shift to first trimester diagnosis such as the reduction in the
amount of intracranial CSF.52
Fetal MRI has little additional value in the determining the
level of the spinal defect, evaluation of the neural placode or
detection of CM compared with experienced neurosonogram
with high-resolution probes. This is due to the low spatial reso-
lution of MRI compared with ultrasound, although this can
be compensated with the high-contrast resolution.41,53,54 How-
ever, MRI is of value in the preoperative setting because of the
essential anatomical information for the neurosurgeon before
 CHAPTER 28  Sonography of the Fetal Central Nervous System 283

E10

A B

Cranial
MMC

Angulated spine
Caudal
C D
• Fig. 28.17  A–D, Characteristics of myelomeningocele (MMC): lemon sign (A), banana sign (B), transverse
section of the spine with MMC (C) and sagittal section (D) showing angulation of the spine and MMC.

Closed Spinal Dysraphism. Closed NTD are characterised by

TABLE Frequency of Cranial Markers in Open Neural
28.4 Tube Defects
vertebral defects covered by skin. In the classification proposed
by Tortori-Donati lesions are subcategorised into defects with
<24 wk (%) >24wk (%) Overall (%) a subcutaneous mass and lesions without a subcutaneous mass.
In the absence of a subcutaneous mass, lesions are often missed
Lemon sign 97–98 13 53
prenatally.41
Banana sign (small 96–97 96 96 Tethered Cord Syndrome. The spinal cord is fixed in the ver-
cerebellum) tebral canal because of adhesions, resulting in a limited or absent
Effaced cisterna 93 93 ascent of the tip of the medullar cone. During fetal development,
magna the vertebral spine grows faster than the spinal cord, so that the
medullar cone ascends from L3 and L4 at weeks 13 to 18, to L2
Ventriculomegaly 75 75–81 between 20 and 24 weeks.56,57
Small biparietal 61–74 Tethered cord syndrome can be congenital or acquired due to a
diameter complication of spinal injury or in relation to an NTD.
The conus medullaris presents on ultrasound as a needle-shaped
Ventricular point >75
triangular hypoechogenic structure between two echogenic lines
at the caudal end of the spine.58 Some anatomical landmarks can
be reference points to determine the level of the medullar cone
such as the upper pole of the kidney (T11) and the most infe-
surgical planning.55 More subtle abnormalities such as callo- rior rib (T12).56 In tethered cord, the medullar cone is positioned
sal dysgenesis, periventricular nodular heterotopia, cerebellar below L2 (Fig. 28.19). 
dysplasia, syringohydromyelia, diastematomyelia and destruc- Split Cord Malformations. Diastematomyelia or split cord
tive lesions are more easily identified on prenatal MRI.41 The malformation describes the splitting of the lower thoracic or upper
neurologic impairments associated with open NTDs are shown lumbar spinal cord into two hemicords by a bony or cartilaginous
in Table 28.5.  spur. The diagnosis is suggested by widening of the spinal canal
284 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

T12

L5

• Fig. 28.18  Three-dimensional multiplayer examination of the spine allows for accurate identification of
the level of the spinal defect.

TABLE
28.5 Neurologic Impairment Associated With Open Neural Tube Defect
Open NTD Lethal Neurologic Impairment at Cranial Level Neurologic Impairment at Spinal Level
Anencephaly Yesa Absence of cerebral hemispheres function Absence of spinal cord function
Encephalocele No Mild (anterior EC) to severe (posterior EC) cerebral Absent or mild (anterior EC) to severe (posterior EC)
dysfunction: spinal cord dysfunction:
• Developmental delay • Spastic quadriparesis (sensory-motor limb deficits)
• Cognitive dysfunction • Bladder, bowel and sexual dysfunction
• Seizures • Orthopaedic disabilities
• Hydrocephalus sometimes Requiring CSF shunting
• Ataxia
• Vision impairments
• Rarely, Chiari 2 malformation, and Dandy-Walker
malformation
Open SB (MMC and No Absent to moderate cerebral dysfunction: Mild to severe spinal cord dysfunction:
myeloschisis) • Hydrocephalus sometimes requiring CSF shunting • Spastic paraplegia (sensory-motor lower limb
• Chiari 2 malformation leading to hindbrain dysfunc- deficits)
tion • Bladder (continence), bowel (continence) and
• Rarely, cognitive dysfunction, seizures, ataxia, vision sexual (erectile) dysfunction
impairments • Tethered cord syndrome
• Orthopaedic disabilities (clubfoot, scoliosis)
aLethal prenatally, intrapartum or ≤48 hours of birth.
CSF, Cerebrospinal fluid; EC, encephalocele; MMC, myelomeningocele; NTD, neural tube defect; SB, spina bifida.
  

in the coronal plane and an echogenic bony fragment underlying Anomalies of Prosencephalic Development
the intact skin (Fig. 28.20). Other spinal malformations are often Introduction. The development of the prosencephalon or
associated with this condition. The prognosis seems favourable in forebrain into the telencephalic vesicles and the diencephalon
isolated cases.59  results from a series of inductive processes starting at the fifth
 CHAPTER 28  Sonography of the Fetal Central Nervous System 285

TABLE Anomalies Resulting From Defective

28.6 Prosencephalic Development

Developmental Failure Anomalies

Prosencephalic formation Aprosencephaly
Atelencephaly
Prosencephalic cleavage Holoprosencephaly (involves
diencephalon and telencephalon)
Holotelencephaly (involves telencephalon)
Prosencephalic midline Agenesis or dysgenesis of the corpus
development callosum
Agenesis of septum pellucidum
Septo-optic dysplasia

TABLE
• Fig. 28.19  Occult spina bifida with myelolipoma (asterisk) and tethering
28.7 Potential Causes of Holoprosencephaly
of the spinal cord (arrowheads). Notice the small skin appendage (°).
Group Anomalies
Syndromal anomalies Smith-Lemli-Opitz syndrome
(18%–25%)
Pseudo trisomy 13
Meckel syndrome
Steinfeld syndrome
Chromosomal anomalies Trisomy 13
(35%–45%)
Trisomy 18
Secondary to gene point SHH on 7q36
mutations
ZIC2 on 13q32
SIX3 on 2p21
TGIF on 18p11.3
PCTH
TDGF1
GLI2
FOXH1
NODAL
DISP1
• Fig. 28.20  Diastematomyelia is a closed spinal defect characterised by GAS1
diplomyelia and a bony spur (arrowhead). FGF8
  

Holoprosencephaly often presents with a variable degree of

postovulatory week. The process of ventral induction responsible
for the formation, cleavage and midline development of the
prosencephalon also takes part in the formation of the midface.
Defective development results in a variety of malformations often
associated with facial involvement (Table 28.6).  The diagnosis of semilobar and alobar HPE is feasible from the
Holoprosencephaly. Holoprosencephaly (HPE) is a complex
and heterogeneous forebrain malformation. As a result of a ventral
induction failure, the prosencephalic vesicle fails to divide into
the telencephalic and diencephalic vesicles. In addition, the failed
outgrowth of the frontonasal process is responsible for a variable
degree of midfacial maldevelopment.
The incidence of holoprosencephaly is estimated at 1 in
6000 to 1 in 16,000 live births and at 1 in 250 in early concep-
tions. There is no male-to-female preponderance, nor a higher
prevalence related to ethnicity. Table 28.7 lists the potential
aetiologies.
According to the degree of forebrain separation, four different
types can be distinguished: alobar, semilobar, lobar and interhemi-
spheric variant (Table 28.8).
midfacial maldevelopment, ranging from cyclopia and proboscis,
severe hypotelorism and ethmocephaly, to arhinencephaly and a
normal-appearing face.60,61 Occasionally, the sole indicator might
be a single incisor.
first trimester of pregnancy by either transabdominal or transvagi-
nal ultrasound62,63 (Figs. 28.21 and 28.22).
Three-dimensional ultrasound, inversion mode rendering
or 3D sono-automated volume count (AVC) of the ventricular
system and sonographic tomographic imaging may be helpful
in defining the severity of the malformation and characteris-
ing facial malformations.64 MRI is of great additional value in
depicting the features that differentiate abnormal brain forma-
tion and aids in the diagnosis and counselling of these cleavage
disorders, which have a different prognosis depending on the
severity of failure of ventral induction.65
Because of its frequent association with chromosomal mal-
formations, fetal karyotyping or comparative genomic hybridi-
sation (CGH) should be offered routinely. The most frequently
286 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
28.8 Ventricular and Midline Characteristics in Different Types of Holoprosencephaly (HPE)
Type of HPE Ventricles Midline Structures
Alobar Single primitive ventricle • Fused thalami
Residual ‘brain mantle’ • Absence of the midline structures
• Pancake type • Falx cerebri
• Cup type • Interhemispheric fissure
• Ball type • Third ventricle
• Septi pellucidi
• Corpus callosum
• Dorsal cyst: present
Semilobar Rudimentary lateral ventricles • Partial interhemispheric fissure posteriorly with development of temporal
Rudimentary third ventricle horns
• Partially fused thalami
• Corpus callosum dysgenesis of agenesis
• Absent cave septi pellucidi
• Dorsal cyst: rare
Lobar Partial fusion of anterior horns with • Shallow frontal area with, possible small, continuity of the frontal cortex
squared borders and flattened roof • Dysgenesis or agenesis of corpus callosum
• Absent cave septi pellucidi
• Dorsal cyst: rare
Middle interhemispheric • Separated anterior horns and • Normal anterior and posterior interhemispheric fissure
(syntelencephaly) posterior horns • Abnormal separation of thalami
• Fusion of the middle portion of • Normal separation of the basal ganglia and hypothalamus
the lateral ventricular systems • Corpus callous abnormal: genu and splenium of corpus callosum can be
• Third ventricle separates hypothalamus present but body is absent
and lentiform nuclei • Dorsal cyst: 0%

  

Posterior

Anterior Fused lateral ventricles

• Fig. 28.22  Interhemispheric holoprosencephaly.

• Fig. 28.21  In alobar holoprosencephaly, the prosencephalic vesicle per-
sists as a large single brain vesicle, overlying the fused thalami.
encountered chromosomal malformations are trisomy 13, del
(13q), del (18p), trisomy 18, triploidy, dup (3p) and del (7)
(pter+q32).) The high risk for syndromic HPE demands a search
for additional structural anomalies; therefore a pathological inves-
tigation is recommended.
The empiric recurrence risk for HPE is 5% to 6%. If HPE
occurs in the context of a chromosomal anomaly; the recurrence
risk is usually less than 1%. In syndromic forms, the recurrence
rates may increase up to 25% to 50%. Dominant inheritance with
reduced penetrance explains the inheritance pattern of familial
HPE.
The outcome of the different types of HPE has been sum-
marised in Table 28.9.66,67 TOP is usually offered for alobar, semi-
lobar and interhemispheric forms of HPE. 
Corpus Callosum Dysgenesis. The CC is the major com-
missure between the two cerebral hemispheres and plays an
important role in the integration of information between the
hemispheres. At about 12 weeks’ gestational age, the CC starts
 CHAPTER 28  Sonography of the Fetal Central Nervous System
TABLE
28.9 
Outcome of Holoprosencephaly (HPE)
Mortality • 50% of alobar HPE die by 5 mo of age
• >30% live beyond 1 yr
• >50% of semi- and lobar HPE >1 yr
Neurologic • No individuals with alobar HPE can sit or speak
impairment • 50% of individuals with lobar HPE able to walk,
have mildly impaired hand function and speak
single words
• Mild interhemispheric variant: walking with assis-
tance, speak, function with mild impairment
Morbidity • Ventriculoperitoneal shunts: 17%
• Anticonvulsant therapy: 40%
• Cerebral palsy
• Swallowing problems, chronic lung disease
caused by aspiration
• Poor gastric emptying, reflux, constipation
• Hypothalamic dysfunction: sleeping problem,
temperature regulation, endocrine disorders
Adapted from Stashinko EE, Clegg NJ, Kammann HA, et al. A retrospective survey of perina-
tal risk factors of 104 living children with holoprosencephaly. Am J Med Genet A 128A(2):
114–119, 2004.
  
to develop from the lamina terminalis as a bundle of fibres that
connects the two hemispheres. The development of the CC is
closely related to the normal appearance of the septa pellucida.68
Anterior to the foramina of Monroe, the space between the septa
is called the cavum cave septi pellucidi (CSP); posterior to this
structure, it is referred to as the cavum vergae (CV) The CC,
which is composed of four segments, starts developing from
the 11th week of gestation with the genu, and subsequently the
body, isthmus and splenium form. The rostrum, the most ante-
rior part, develops later. Completion of the CC is achieved by 18
to 20 weeks of gestation. At the median surface of the cerebrum,
the gyrus cinguli creates a partial girdle around the CC and fol-
lows the callosal curve.
Normal sonographic development of the anterior and poste-
rior complex in the axial plane indicate adequate midline develop-
ment; however, morphologic abnormality in both complexes is a
strong indicator for midline abnormalities and cortical malforma-
tions.69 The midsagittal and the coronal views are the best planes
to directly visualise the CC. In a midsagittal two-dimensional
view, the CC appears as a thin anechoic space, delineated supe-
riorly and inferiorly by two smooth echogenic lines. The com-
plete visualisation and measurement of the CC is feasible from 18
weeks’ gestation onwards. Normative charts can be used to evalu-
ate the length and thickness of the CC. Transvaginal ultrasound,
multiplanar 3D and three-dimensional volume contrast imaging
in the C-plane (VCI-C) imaging facilitate the proper identifica-
tion.70,71 The midsagittal plane reveals the CC with all its neigh-
bouring structures; the CSP, the CV, the cavum veli interpositi
and the cingulate gyrus. Using colour-flow Doppler, the anterior
cerebral and pericallosal arteries with their branches and the vein
of Galen are easily displayed in the early second trimester.
The terminology to describe CC dysgenesis includes complete
and partial agenesis, hypoplasia (thinning of the CC), hyperpla-
sia (thickening of the CC) and morphologically oddly shaped
CC.72 A prevalence of 1.4 and 0.4 per 10,000 live births for ACC
and hypoplasia of the CC has been suggested, respectively. These
287
figures may be an underestimate as a proportion of asymptomatic
ACC patients escape detection.73
Absence of the Corpus Callosum. Complete absence of the
CC (ACC) results in an abnormal induction of medial cerebral
convolutions determining a radiate arrangement of the cerebral
sulci around the roof of the third ventricle. With ACC, the semi-
circular loop of the pericallosal artery is lost, and branches of
the anterior cerebral artery ascend linearly with a radial arrange-
ment. ACC has been associated with other cranial anomalies,
such as abnormalities of the posterior fossa and interhemispheric
cyst and neuronal migration disorders. Because the development
of the CC coincides with cortical development, lissencephaly,
heterotopia, polymicrogyria (PMG) and schizencephaly are
often present. The rate of chromosomal anomalies is estimated
at about 17.8%, including microdeletions for which array com-
parative genomic hybridisation might be considered. In addi-
tion, ACC-linked syndromes show an autosomal dominant,
recessive or X-linked mode of inheritance.74 An OMIM (Online
Mendelian Inheritance in Man) search results in more than 200
entries, of which Aicardi and Andermann syndromes are the
more common. Occasionally, a metabolic aetiology is found,
such as hyperglycemia without ketose, a pyruvate dehydrogenase
deficiency or phenylketonuria.75
Indirect sonographic features are often helpful when screen-
ing for the absence of the CC. The absent CSP should raise
suspicion,68,76 and dilation of the atria and occipital horns
(colpocephaly) shaping the lateral ventricles like teardrops is
very suggestive for CCA (Fig. 28.23A). There is, however, no
progressive VM.77 On a coronal view, the lateral ventricles
are displaced laterally because of the failure of the bundles of
Probst to cross the midline, making the anterior parts of the
lateral ventricles look like bull’s horns. Often the third ven-
tricle is enlarged and extends posterior and cranially. Because
of the lack of communicating fibres, there is an increased dis-
tance between both hemispheres (Fig. 28.23B), which shows
in the axial plane as three parallel lines: the falx cerebri and
the medial borders of the two hemispheres. However, at the
time of midtrimester ultrasound screening, indirect signs may
be either absent or barely visible but may appear more clearly
later in gestation. In a sagittal view, ACC results in an abnor-
mal induction of the medial cerebral convolutions, leading to a
radial arrangement of the sulci, strengthened by the hypoplasia
or absence of the cingulate gyrus. By colour-flow Doppler, the
pericallosal arteries display an irregular radiant vascular pattern
(Figs. 28.24 and 28.25). 
Partial Agenesis of the Corpus Callosum. In partial ACC, the
CSP is usually present but is abnormally shaped.78,79 Only the
midsagittal views of the fetal brain allows for the differentiation
among complete agenesis, hypoplasia or partial formation of the
CC80,81 (Fig. 28.26). In hypoplasia of the CC, often the posterior
portion is affected. Only a handful of cases have been detected
prenatally.82
The pericallosal artery follows the anterior part of the CC but
loses its normal course posteriorly. 
Corpus Callosum Dysgenesis. A thick CC is identified in
5% of CC abnormalities. It can be associated with macrocephaly,
macrocephaly-capillary syndrome83 and Cohen syndrome.84
Hyperechogenicity of the CC is characteristic of pericallosal
lipoma which is often isolated. Sometimes it is part of a fronto-
nasal dysplasia, Goldenhar syndrome or Pai syndrome.85 Because
of the association with chromosomal abnormalities of various
kinds, array CGH is highly recommended. In addition to the fetal
288 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B
• Fig. 28.23  Indirect sonographic signs suggesting complete absence of the corpus callosum. A, Colpo-
cephaly with a teardrop-shaped lateral ventricle. B, Three-layer sign.

• Fig. 28.24  Abnormal irregular radial vascularisation pattern of the peri-
callosal artery in complete absence of the corpus callosum.
neurosonogram, a detailed examination of all fetal organ systems,
especially the fetal heart, the genitourinary system and the skel-
eton, should be performed.
The identification by means of fetal MRI of more discrete
CNS lesion (≤22.5%) such as abnormal gyration, heterotopia
and migration anomalies in association with CCA enables refine-
ment of the diagnosis and prognosis.86,87 MR is less accurate in
measuring thickness of the CC because of the low spatial reso-
lution of MRI.88 Subjective callosal thickening should alert the
specialist, and additional abnormalities should be looked for.88,89
In addition, new MRI techniques such as fibre tracking and func-
tional MRI may help to differentiate isolated cases with usually a
good prognosis from those with additional cerebral lesions and an
adverse outcome.90  a fluid-filled box on an axial plane between the frontal horns, the CC
Neurologic Outcome. Significant neurodevelopmental
delay may occur in 15% to 36% of the cases of isolated
CCA.80,91 The presence of other cerebral and extracerebral mal-
formations worsens the neurodevelopmental outcome.92 A sys-
tematic review assessing the of neurodevelopmental outcome
• Fig. 28.25 Sagittal T2 HASTE image of the brain in a fetus at 31 weeks.
Absence of the corpus callosum with visualisation of the fornices (circle) and
typical sunburst radiation of the gyri.
in 132 fetuses revealed a normal outcome in 74.3% with com-
plete CCA and in 65.5% of partial CCA. Moderate and severe
disability was reported in 14.3% and 11.4% of CCA and in
6.9% and 27.6% of partial CCA, respectively.86 Subtle per-
ceptual, neuropsychological and motor defects may arise later
in life.
Of major importance, an additional 15% of prenatally isolated
ACC cases were found to have associated problems after birth.75
At the age of 10 years, 75% of the children have a normal intel-
ligence but frequently with mild learning difficulties.93,94 
Absence of the Cavum Septi Pellucidi. Absence of the CSP occurs
in about 0.2 to 0.3 per 10,000 pregnancies.95,96 The CSP appears as
and the thalami. In a sagittal plane, it is localised under the anterior
part of the CC. The CSP is square in 73% and triangular in 27%;
however, in cases with CV, the appearance is rectangular.69 The mean
width at midtrimester is 3.4 mm. The CSP progressively decreases in
size from 26 weeks of gestation. By term, the closure of the CSP is
 CHAPTER 28  Sonography of the Fetal Central Nervous System 289

seen in 97% of fetuses, although occasionally, this cavity remains open
until adulthood.68 Failure to visualise the CSP between 18 and 37
weeks is highly indicative of cerebral anomalies. Although it may occur
as a normal variant if isolated,76 absence may be associated with the
HPE spectrum, septo-optic dysplasia (SOD), callosal dysgenesis and
hypogenesis, chronic severe hydrocephalus resulting from aqueductal
stenosis or CM, schizencephaly, porencephaly or hydranencephaly and
basilar encephaloceles. Genetic causes are rare. The persistence of an
enlarged CSP (>1cm) beyond infancy has been associated with cerebral
dysgenesis.  absent CSP is usually good.98 In nonisolated cases, the prognosis
Septo-Optic Dysplasia. Septo-optic dysplasia can be suspected
when the CSP is absent in an otherwise normal brain (Fig. 28.27).
The frontal horns are fused at the midline, and the CC is fre-
quently thin. VM can be present. In experienced hands, sono-
graphic 3D visualisation and measurement of the chiasma and
optic nerves may confirm the diagnosis.97 Fetal MRI is generally
more suited to exclude optic tract hypoplasia. Additional endo-
crinologic evaluation and visual assessment are mandatory to dif-
ferentiate from isolated absent CSP. The prognosis of an isolated
is related to the associated conditions. The recurrence risk for iso-
lated absent CSP is low, but in a few cases, Mendelian inheritance
is suggested. 

Posterior Fossa Anomalies

• Fig. 28.26 Partial agenesis of the corpus callosum: the rostrum and
genu are absent (arrowhead). Note the presence of a well-developed
cavum septi pellucidi (asterisk).
Introduction. By the fifth or sixth gestational week, the pontine
flexure creates the plica choroidea and divides the rhombencephalon
into the metencephalon and the myelencephalon. Invagination
divides the roof of the fourth ventricle into an anterior and a
posterior membranous area. The anterior membranous area
gives rise to the cerebellar vermis. Cerebellar vermian growth
forces the posterior membranous area of the roof of the fourth
ventricle to invaginate posteriorly below the vermis, resulting in
the Blake pouch. The median fenestration of the Blake pouch by
the foramen of Magendie leads to its subsequent disappearance.
The foramina of Luschka open later, around the fourth month.
In 1% to 2% of healthy subjects, the foramen of Magendie is
absent; the communication between the fourth ventricle and the
subarachnoid space is established when the foramina of Luschka
open.99 The cerebellum results from the development of two
lateral primordia that fuse subsequently across the midline to
form the vermis, starting at the end of the sixth week. At the 11th
week, the cerebellum covers the fourth ventricle and subsequently

• Fig. 28.27  Fusion of the anterior horns and absence of the septum pellucidum has been observed as
an isolated finding in association with optic atrophy in septo-optic dysplasia and in the presence of lobar
holoprosencephaly.
290 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
starts subdividing into fissures directed perpendicularly to the
longitudinal axis of the brainstem. The development of the
cerebellar cortex results in the formation of folia.100
Routine sonographic evaluation of the posterior fossa occurs in
the axial suboccipital bregmatic view, displaying the two cerebellar
hemispheres connected with the vermis and the cisterna magna
as the space between the vermis and the inner layer of the occipi-
tal bone. The cerebellum appears as a butterfly-shaped structure
formed by the round cerebellar hemispheres joined in the middle
by the slightly more echogenic cerebellar vermis. The cerebellar
hemispheres are well depicted on transverse and coronal images
and are hypoechoic with a more echogenic lining. By the end of
the second trimester, the increased development of the folia leads
to an increased echogenicity characterising the striped appearance.
Two retrocerebellar septa, perpendicular to the cerebellum, can be
visualised in a transverse view in the cisterna magna in the second
and third trimesters in 84% to 92% of fetuses, respectively, and
are considered to be remnants of the walls of the Blake pouch.101
A more detailed examination of the vermis can be performed
using the midsagittal plane depicting the triangular fourth ven-
tricle (fastigium), the pons, the posterior fossa fluid space and the
tentorium cerebelli. The vermis appears markedly hyperechoic,
and the primary fissure is constantly observed on the midsagittal
image from 24 weeks’ gestation as a transverse more echoic line.
Reliable interpretation of the normal development and growth of
the cerebellar hemispheres and vermis is possible from 18 weeks
onwards.102,103  in 11% to 29% of cases. In syndromic conditions, the prognosis
Dandy-Walker Complex. Posterior fossa malformations have
recently been grouped as the Dandy-Walker continuum because
of new insights in the embryologic development of this region.
This continuum includes isolated enlarged cisterna magna, the
Blake pouch cyst, vermian hypoplasia and the Dandy-Walker mal-
formation (DWM). The sonographic categorisation of posterior
fossa fluid collections depends on the assessment of the position
of the torcular and the integrity of the cerebellar vermis. Pitfalls in
the diagnosis of posterior fossa anomalies have been attributed to
confusion in terminology describing vermian pathology, the gesta-
tional age at diagnosis, the incorrect assessment of the midsagittal
plane of the cerebellum and the late development of some patho-
logical conditions.104 More detailed examination by transvaginal
ultrasound includes the proper identification of the fastigium and
its relation to the vermis by assessment of the angle between ver-
mis or brainstem and tentorium and the appearance of the pri-
mary fissure.70,103,105,106 Multiplanar imaging of a standard 3D
volume and the use of tomographic ultrasound imaging or VCI
(volume contrast imaging) facilitates the visualisation and biom-
etry of the midsagittal structures of the posterior fossa, especially
the vermis and the fastigium.70,105
Fetal MRI offers a clearer view of the torcular, and the assess-
ment of the integrity of the vermis remains difficult particularly
in midgestation.107 Current fetal MRI, particularly in early
gestation before 24 gestational weeks, has limitations in accu-
rately predicting postnatal MRI abnormalities. In 41%, fetal and
postnatal MRI diagnoses disagree; postnatal MRI reversed fetal
MRI diagnoses in 15% and revealed additional anomalies in
26%.108,109 MRI in early gestation (before 18 weeks), although
presumed safe, results in a higher false-positive rate because
of fetal motion, small anatomic structures (limited spatial and
tissue resolution) and rapid growth of the cerebellum later in
pregnancy (second and third trimesters).110 T2-weighted images
provide structural information and can be complemented with
T1-weighted and echo planar imaging to detect haemorrhage.
Normative charts of the posterior fossa and its structures are
available. 
Isolated Mega Cisterna Magna. The cisterna magna is a fluid-
filled space posterior to the cerebellum. In the second half of ges-
tation, the anteroposterior diameter of the cisterna magna is stable
and measures between 2 and 10 mm. Early in gestation as the
cerebellar vermis does not completely cover the fourth ventricle,
it may give the false impression of a defect of the vermis. A CM
of more than 10 mm without associated anomalies suggests the
diagnosis of isolated mega cisterna magna (MCM).
Isolated MCM is defined as a distance between the vermis of
the cerebellum and the inner border of the occipital bone of more
than 10 mm on an axial plane through the CSP and the vermis.
The vermis is intact, the fourth ventricle is normal and there is no
VM nor displacement of the torcular. The prevalence is estimated
at 2%. Isolated MCM is usually an incidental finding and may be
secondary to a temporary distention of the Blake pouch without
displacement of the vermis.
The condition should be differentiated from the Dandy-Walker
complex, cerebellar hypoplasia and posterior fossa arachnoid
cyst.111
Adults with isolated MCM have normal cognitive function
but with reduced memory and verbal fluency. Children with
an enlarged cisterna magna are at risk for mild developmen-
tal delay.112 Most studies report a good prognosis.113 The non-
isolated cases of MCM have abnormal developmental function
depends on the underlying condition.114 
Blake Pouch Cyst. If the Blake pouch fails to perforate to form
the midline aperture of the foramen of Magendie, the accumula-
tion of CSF results in a cystlike structure projecting into the cis-
terna magna. The Blake pouch cyst remains in communication
with the fourth ventricle. The position of the tentorium cerebelli
is intact, but an upward and posterior rotation (<45 degrees) of
a normally developed vermis with a normal cisterna magna is
characteristic of a Blake pouch cyst. On an axial oblique plane,
such a Blake pouch cyst may be misinterpreted as partial ver-
mian agenesis (Fig. 28.28). The diagnosis can be made in the
second trimester of pregnancy. Differential diagnosis with infe-
rior vermian hypoplasia remains difficult even with the help of
prenatal MRI. The fluid content of the Blake pouch cyst shows
a more translucent echogenicity than that of the cisterna magna.
On fetal MRI, the same findings will be visible, most evident in
the sagittal plane, with a complete but rotated vermis, normal
torcular herophili and a large cyst in communication with the
fourth ventricle.
The overall neurologic outcome of Blake pouch cyst is good
because the cerebellum and vermis have developed completely.
A delayed fenestration between 24 and 26 weeks’ gestation may
occur in 50% of cases.115 In case of secondary VM, postnatal
ventriculoperitoneal shunting is mandatory. In a retrospective
evaluation of 19 cases of Blake pouch cyst, 10 fetuses presented
without associated malformations, 7 of which presented a nor-
mal outcome. In 4 cases of the 10, a late fenestration occurred.
Nine cases of 19 showed additional malformations: 5 of 9 had a
cardiac anomaly, 1 of which had trisomy 21.115 The recurrence
risk is low. 
Dandy-Walker Malformation. Dandy-Walker malformation
ranges from 1 in 25,000 to 1 in 35,000 live births to 1 in
5000116 and is characterised by the absence or severe dyspla-
sia of the cerebellar vermis, a cystic enlargement of the poste-
rior fossa in communication with the fourth ventricle and an
 CHAPTER 28  Sonography of the Fetal Central Nervous System 291

A B

• Fig. 28.28  Blake pouch cyst. A, Axial view of the posterior fossa, revealing a cystic structure in associa-
tion with the cerebellum. B, Sagittal imaging allows easy differentiation form other posterior fossa anoma-
lies (asterisk indicates the upward-rotated vermis; ° indicates Blake pouch).

upward displacement of the tentorium cerebelli. This condition

is often associated with other CNS anomalies (in 50%–60%
of cases): the VM(36%–67%), agenesis of the CC (5%–50%)
and HPE. Associations with congenital heart defects, urinary
tract anomalies and facial clefts have been described, often
in the context of chromosomal anomalies (50%–70% of the
cases: T13, T18, T21, 45,X) and genetic syndromes(Walker-
Warburg syndrome, Aicardi syndrome, Neu-Laxova syndrome
and Meckel-Gruber syndrome). Maternal diabetes, excessive
alcohol consumption and early in utero infection may predis-
pose to the condition.
On ultrasound, DWM is characterised by a large commu-
nication between ‘the fourth ventricle’ and the cisterna magna,
without clear visualisation of the vermis on the axial transcerebel-
lar section. On sagittal imaging, the vermis is absent or severely
hypoplastic, rotated counterclockwise and positioned behind the
quadrigeminal plate. The tentorium cerebelli is elevated by an
infratentorial cyst increasing the angle between the brainstem
and tentorium to more than 40 degrees.117 VM is present in
68% of the cases despite the absence of associated malforma-
tions or an abnormal karyotype.118 Prenatal array CGH and in • Fig. 28.29  Sagittal T2 HASTE image demonstrating the characteristic find-
particular the exclusion of subtelomeric deletions (e.g., 6p) is ings in Dandy-Walker malformation consisting of enlarged posterior fossa with
recommended. elevated torcular (thick arrow), cystic enlargement of the fourth ventricle (thin
Fetal MRI provides detailed information about neuroanat- arrow) and hypoplastic vermis with abnormal rotation (and increased teg-
omy and can identify additional malformations, such as brain- mento-vermian angle indicated by the lines). In addition, there is a dysplastic
corpus callosum (freeform).
stem abnormalities, heterotopia or abnormal gyration, which
are typically hard to find on ultrasound. MRI improves the
diagnostic accuracy up to 88% compared with 65% for ante- recurrence rate in nonsyndromic DWM is sporadic (1%–5%);
natal ultrasound (Fig. 28.29). The overall effect of the fetal in case of chromosomal anomalies, it is less than 1% and in
MRI on clinical management is significant in 35% of cases.119 syndromic forms (e.g., Walker-Warburg, Meckel-Gruber, Leu-
Developmental delay, including hypotonia, cerebellar dysfunc- Laxova syndromes) up to 25%. 
tion and hemiparesis have been reported in 40% to 60% of Cerebellar Hypoplasia. Cerebellar hypoplasia is character-
children with DWM.120 The outcome is extremely variable ised by a reduced cerebellar volume due to the maldevelop-
ranging from severely delayed motor development (30.4%), ment of one or both hemispheres and a small but normally
hypotonia, ataxia and seizures. Intellectual development is shaped vermis. This heterogeneous condition is associated with
normal in 35% to 50% of the cases depending on the verm- trisomies 9, 13 and 18, congenital disorders of glycosylation,
ian development and absence of supratentorial malformations. anticonvulsant drugs (valproic acid) or cocaine.122 It occurs in
Partial agenesis of the vermis with a normal or almost normal isolation in genetic cerebellar hypoplasia and pontocerebellar
lobulation, although difficult to evaluate because of the mass hypoplasia, disruption in congenital cytomegalovirus (CMV)
effect of the posterior fossa cyst, has a better prognosis.121 The infection or a posterior fossa haemorrhage.123 The spectrum
292 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
of cerebellar disruption includes cerebellar agenesis, unilat-
eral agenesis and hypoplasia.124 Overall, cerebellar hypopla-
sia should be suspected when cerebellar biometry is below 2
standard deviation (SD) for gestational age (GA). Typically, in
unilateral cerebellar hypoplasia or aplasia, an asymmetric pons
is present with contralateral volume reduction. It has been
reported as early as 20 to 24 weeks of gestation.125 Cerebel-
lar hypoplasia is frequently a progressive disorder, requiring
serial ultrasound evaluation. Genetic testing is available for
some conditions associated with cerebellar hypoplasia. Con-
genital CMV infection should be ruled out, and fetal MRI is
recommended.
The prognosis is poor.126 Absence of the cerebellum is associated
with neonatal death, delayed neuromotor development, deficient
movement coordination and mental retardation.123 However, in
unilateral cerebellar hypoplasia, the amount of surface loss of the
cerebellar hemisphere is indicative of poor outcome. The presence
of a normal vermis in association with unilateral cerebellar hypo-
plasia is often associate with a normal neurologic outcome.127 The
recurrence risk is low.  sis. The presence and size of the vermis can easily be assessed
Joubert Syndrome and Related Disorders. Joubert syn-
drome and related disorders feature a developmental defect of
the cerebellar vermis and a malformed brainstem, which result
in a ‘molar tooth’ sign on axial MRI scans. The ‘molar tooth’
sign, however, is not exclusive to Joubert syndrome. To date,
more than 30 genes have been identified in Joubert syndrome.
The causative gene is identified in 83% to 94% of the fami-
lies.128,129 This disorder is considered part of the general group
of ciliopathies.
Prenatal ultrasound diagnosis is extremely difficult and has
only been reported in a very limited number of cases.128,130 Ultra-
sound diagnosis is possible from 21 weeks onwards. The vermis
has an hypoplastic appearance: the superior part is present, but
the inferior part is absent, producing a midline cleft connecting
the fourth ventricle to the cisterna magna. The fourth ventricle is
abnormal in all cases: it is umbrella shaped or rounded with loss
of the characteristic triangular shaped fastigium in the axial plane.
The most common associated CNS anomaly is the dysgenesis of
the CC. Extra-CNS anomalies may include renal cysts, facial cleft,
occipital encephalocele and polydactyly.131 However, the diagno-
sis of this anomaly is based on the pathognomonic ‘molar tooth’
sign on ultrasound or axial MRI scans.
Joubert syndrome is predominantly inherited in an autosomal
recessive manner; however, the mutation of OFD1 is inherited in
an X-linked manner. For pregnancies at known increased risk for
Joubert syndrome, prenatal diagnosis by ultrasound examination
with or without fetal MRI has been successful.
The poor prognosis is characterised by periodic seizures, abnor-
mal eye movements, hypotonia, ataxia, developmental delay and
mental retardation.  spine; musculoskeletal anomalies; and cardiovascular, respi-
Hypoplasia of the Vermis. In most patients, the superior part
remains normal, and only the inferior vermis becomes attenuated.
The overall appearance of the vermis is normal, but the biom-
etry is small. The vermian hypoplasia is identified by an upward
displacement of a small vermis. The cisterna magna and torcular
herophili are normal.
Current theories suggest that this malformation results
from a global developmental defect affecting the area mem-
branacea of the roof of the rhombencephalon, which explains
the association with a variable degree of cerebellar dyspla-
sia. Cerebellar hypoplasia may be observed in the setting of
aneuploidy (particularly trisomy 21 and 18), congenital infec-
tion (CMV) and certain metabolic disorders and syndromes
(e.g., PHACE [posterior fossa abnormalities, haemangioma,
arterial lesions, cardiac abnormalities or aortic coarctation,
and eye anomalies] syndrome). It may also be associated with
brainstem hypoplasia (pontocerebellar hypoplasia). When iso-
lated, it may be asymptomatic, but precise risk figures are not
available.132
Prenatal MRI in the late second or third trimester can more
easily differentiate hypoplasia from partial genesis. 
Partial Agenesis of the Vermis. This is mostly a sporadic con-
dition. Typical postnatal MR signs are an absent posterior lobe,
hypoplasia of the anterior lobe and a cleft separating the hemi-
spheres with a fourth ventricle deformity. This condition was
originally defined as Dandy-Walker variant, a term no longer
in use. It has been associated with chromosomal anomalies in
up to 30% of the cases. The diagnosis of inferior vermian agen-
esis remains questionable until 24 weeks of gestation. Vermian
biometry and structural appearance are critical for the diagno-
by a 3D sweep of the posterior fossa along the axial plane with
subsequent multiplanar analysis. The vermis appears as an oval
echogenic structure, interposed between the fourth ventricle and
the cisterna magna. A communication between the ventricle and
cisterna magna at any level is suspicious of a partial vermian
agenesis or a hypoplastic vermis. A sagittal view of the posterior
fossa allows the differentiation between these entities.117 Further
differentiation has to be made with Blake pouch cyst, Joubert
syndrome and MCM. 
Rhombencephalosynapsis. Rhombencephalosynapsis (RES)
is a rare midline brain malformation characterised by vermian
agenesis with fusion of the cerebral hemispheres and peduncles.
RES occurs isolated or in combination with other malformations.
The most common congenital syndrome associated with RES is
Gomez-Lopez-Hernandez syndrome.133 The aetiology and the
prevalence of RES remain unknown, but more than 90 cases have
been reported.134
On imaging, the posterior fossa appears small and associ-
ated supratentorial abnormalities (absence of cavum septi
pellucidi, abnormal gyration and hydrocephalus) may be pres-
ent. On the axial transcerebellar view, the vermis is absent,
and the hypoplastic cerebellar hemispheres are fused on the
midline (Fig. 28.30). The fourth ventricle shows a rounded or
diamond-shaped appearance. Cerebellar hypoplasia and VM,
with or without the absence of the septum pellucidum, should
prompt careful evaluation for hindbrain fusion.135 Additional
cerebral findings are fusion of the inferior colliculi, cerebral
peduncles and the thalami, and midline defects. Non-CNS
findings include segmentation and fusion anomalies of the
ratory and renal anomalies.135,136 In a minority of families,
evidence points towards an autosomal recessive cause. Infants
with isolated RES have ataxia, muscular hypotonia, attention
deficit, hyperactivity disorders and impaired cognitive func-
tions.137 The recurrence rate is unknown. 
Disorders of Cortical Development
Disorders of cortical development can be divided into malfor-
mations secondary to abnormal neuronal and glial prolifera-
tion, malformations due to abnormal neuronal migration and
 CHAPTER 28  Sonography of the Fetal Central Nervous System 293

malformations secondary to abnormal postmigrational develop-
ment.138 Because of the overlap between these developmental
stages, disorders of cortical development may present with com-
plex malformations.
The recent update of the classification reflects the advances in
embryology and of molecular biology of normal and abnormal
cortical development. In addition, the importance of many genes
has been associated with different phenotypes of malformations of
cortical development (MCDs).139
The complexity of this topic does not allow for a compre-
hensive elaboration. Instead, we will focus on some of the
anomalies in each subgroup (Fig. 28.31).
Calcification of the cranium and the limited access to the sur-
face of the brain through small interosseous acoustic windows
renders prenatal diagnosis of MCD difficult. Ultrasound features
encompass premature abnormal sulci, irregular and thin cortical
mantle, wide abnormal overdeveloped gyri and nodular projec-
tions in the lateral ventricle. High-resolution multiplanar MRI is
more accurate in differentiation between grey and white matter,
in analysing the white matter formation and in characterising the
development of gyri and sulci.140,141

Cereb Abnormal Neuronal and Glial Proliferation or Apoptosis

• Fig. 28.30 The small cerebellum (Cereb) shows no separate hemi-
spheres and no interposed vermis: rhombencephalosynapsis.
Microcephaly. The diagnosis of microcephaly varies consider-
ably because of the different diagnostic criteria applied and the use of
growth charts to populations with different ethnic backgrounds. The
estimated prevalence of nongenetic microcephaly in Europe is 1.53 in
10,000 births (1.16 to 1.96). An additional 23% to 31% have a genetic
condition causing in microcephaly.142,143 The aetiology of fetal micro-
cephaly, defined as a prenatal HC below the third percentile,144 rarely
includes isolated autosomal recessive familial disorders, which usually

Malformations of cortical development

Abnormal neuronal and glial Abnormal postmigrational

proliferation or apoptosis Abnormal neural migration development

Polymicrogyria and
Microcephaly Heterotopia
schizencephaly

• Predominantly anterior or diffuse

• Predominantly posterior

Megalencephaly Lissencephaly Inborn errors of metabolism

• Normal cortex • Predominantly anterior or diffuse • Mitochondrial disorders

• Periventricular nodular heterotopia • Predominantly posterior • Peroxisomal disorders
• Polymicrogyria • X-linked, callosal agenesis
• Reelin type

Cortical dysgenesis with

Subcortical heterotopia Focal cortical dysplasia
abnormal cell proliferation

• Diffuse cortical dysgenesis

• Hemimegalencephaly
• Focal cortical dysplasia type II
• Tuberous sclerosis

Postmigrational
Cobblestone malformation
microcephaly

• Fig. 28.31  Classification of cortical developmental disorders.

294 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
progress rapidly in the first year of life. In fact, most prenatal cases are
part of a syndrome or chromosomal abnormality or are associated with
primary cerebral organisation disorders or with destructive events.
Therefore an unexplained decreased HC (<P3) should lead to
a detailed and systematic analysis of the fetal brain, taking into
account the steps shown in Table 28.10.144  brain and characterised by abnormalities in the width of the gyri
Megalencephaly. Megalencephaly refers to an increased brain
volume in the absence of hydrocephaly and is defined as an HC
greater than 2 standard deviations (SDs) above the mean for ges-
tational age. The increased growth of cerebral structures relates to
benign familial occurrence, metabolic conditions (e.g., lysosomal
storage diseases) or developmental or anatomical causes (e.g.,
mamalian Target of Rapamycine [mTOR], RAS-mitogen activated
protein kinase [RAS/MAPK] or sonic hedgehog [SHH] pathways)
and can present as unilateral or bilateral.145,146 Benign or idio-
pathic megalencephaly refers to children who have an abnormal
large head without neurologic impairment. Often one or both of
the parents have an increased HC. Some inborn errors of metab-
olism manifest with megalencephaly. The diagnosis is based on
specific neurologic features. A familial history of similar disorders
with a recessive hereditary pattern either autosomal or sex-linked
is suggestive. The clinical course is progressive and other organ
structures are involved, including the heart, eye, liver and spleen.
The group of anatomic megalencephaly manifest with develop-
mental megalencephaly linked to a single gene mutation involving
early brain cellular growth, migration or replication.147,148  matter interdigitations and shallow Sylvian fissures.
Abnormal Neural Migration. Failure of initial neuronal migra-
tion results in periventricular heterotopia. Disorders of later migra-
tion cause disruption of the normal six-layered cortex (classical
TABLE
28.10 Fetal Microcephaly Investigation
DETAILED AND SYSTEMATIC EXPLORATION OF THE FETAL
BRAIN IN CASE OF HC <3 SD
Explore the context Environmental factors, ethnic origin, consanguinity
and extracephalic fetal biometry
Determine the Deviation of the head measurements from the
severity normal; organise a longitudinal follow-up
Examine the Enlarged distance between brain and the
pericerebral internal surface of the skull may reflect more
space correctly the underdevelopment of the fetal
brain. MRI of brain volume measurement
may more accurately predict the correct
brain size.
Sylvian fissure Morphology may reveal gyration disorders.
Smooth cerebral Fetal MRI can differentiate between true lissen-
surface cephaly and a simplified gyral pattern, which
are genetically completely different entities.
Search for Such as congenital infection (e.g., Zika, CMV),
destructive vascular lesions or intracranial bleeding
lesions
Exclude midline Investigation of the anterior and posterior
anomalies complexes
Explore the
posterior fossa
CMV, Cytomegalovirus; HC, head circumference; MRI, magnetic resonance imaging; SD,
standard deviation.
  
lissencephaly) and subcortical band heterotopia. However, neu-
rons that fail to stop at their intended cortical destination and
continue to migrate onto the cortical surface result in cobblestone
malformation.138
Lissencephaly (type I). Lissencephaly is defined as a smooth
and the thickness of the cortex. The neocortex of lissencephalic
patients lacks normal cortical lamination and contains two to four
layers instead of six.149 The degree of agyria is graded. The com-
plete form is characterised by a smooth surface of the entire brain.
In the incomplete form, which is the more common type, there is
a gradient in severity from anterior to posterior depending on the
genetic defect.
Lissencephaly type 1 and Miller-Dieker syndrome are usually
diagnosed in utero after 27 to 30 weeks’ gestation. Mild VM and
delayed Sylvian fissure development are the earliest signs and can
be seen by 23 weeks of gestation in fetuses at risk. Furthermore,
dysgenesis of the Sylvian fissure, delayed sulcal appearance, cal-
losal abnormality and cortical thickening may be present. The
abnormal opercular formation is responsible for a ‘figure-eight’–
shaped brain.150
On postnatal MRI, classical lissencephaly shows an hourglass
configuration of the brain with large ventricles, a smooth thick-
ened cortex (>10 mm) separated from a deep layer of neurons by a
‘cell sparse layer’, thin subcortical white matter, lack of grey-white
Lissencephaly is usually symmetric. Agenesis of the CC or cer-
ebellar hypoplasia may be associated. 
Cobblestone Malformation. Cobblestone malformation, for-
merly called type 2 lissencephaly, is characterised by a nodular
cortical surface accompanied by ocular anomalies and congenital
muscular disorders.151
Cobblestone malformations have been divided into three dif-
ferent groups based on severity. Fukuyama congenital muscular
dystrophy is the mildest form and muscle-eye brain disease being
the moderate form. Walker-Warburg syndrome is the most severe
form. Congenital muscular dystrophy is a key feature.
The structural abnormalities result from lack of connection of
the radial glia to the pial limiting membrane and neuronal overmi-
gration through pial gaps.138 A first trimester diagnosis of Walker-
Warburg syndrome is usually associated with a cephalocele. Other
suggestive findings are early enlargement of the lateral ventricles,
abnormal vermis, retinal detachment, cataract, abnormal sulca-
tion, kinked brainstem and bifid pons.152 
Periventricular Heterotopia. Heterotopia occurs when neu-
rons originating in the periventricular region fail to migrate,
leaving tracks or nodules of normal neurons in abnormal
locations adjacent to the ependymal lining or in subcortical
topography.153 Based on MRI scans, there are three types of
heterotopia: periventricular nodular, focal subcortical and
band heterotopia.
Periventricular nodular heterotopia should be considered
when prenatal ultrasound shows an irregular lateral ventricu-
lar wall with indentations of periventricular tissue (Fig. 28.32).
The sonographic diagnosis is difficult, mainly when the lateral
ventricle width is normal. MRI demonstrates multiple small
nodular subependymal foci of low signal intensity, isointense to
the germinal matrix, similar to that of the grey matter, located
in the margins of the lateral ventricles (Fig. 28.33). Periven-
tricular nodular heterotopia has a significantly higher incidence
of associated hippocampal, cerebellar and brainstem anoma-
lies compared with anterior or diffuse periventricular nodular
 CHAPTER 28  Sonography of the Fetal Central Nervous System
Voluson
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• Fig. 28.32  Irregularly shaped border of the lateral ventricle with nodular
projections. Image of periventricular heterotopia (arrowhead). In addition,
polymicrogyria is present.
• Fig. 28.33  T2 HASTE image in the sagittal plane at a gestational age of
27 weeks. Irregular hypointense lining of the lateral ventricle (circles) with
irregular cortical folding in keeping with migrational abnormalities consist-
ing of subependymal heterotopia and polymicrogyria. The findings were
confirmed at autopsy after the parents decided for termination of preg-
nancy.
heterotopia, which may be present with agenesis of the CC and
mega cisterna magna.151  The seroprevalence, which is about 50% in industrialised
Abnormal Postmigrational Development
Polymicrogyria. Polymicrogyria is caused by an interruption in
normal cerebral cortical development in the late neuronal migra-
tion or early postmigrational development periods. It is a spectrum
of cortical malformations with the common feature being exces-
sive gyration. All have in common a derangement of the normal
six-layered lamination of the cortex, an associated derangement of
sulcation and fusion of the molecular layer across sulci.154,155 The
cortical changes of PMG take place late in pregnancy and appear
295
as localised or generalised absence of normal sulcation with mul-
tiple abnormal infoldings of the affected cortex.156
In early gestation (<24 weeks), the identification of this cor-
tical malformation is quite difficult, and the manifestations on
both ultrasound and MRI are subtle. They include presence of
sulci that are not expected according to the gestational age; abnor-
mal opercular development, an irregular surface of the brain and
absence of the normal signal of the cortical ribbon. The most fre-
quent prenatal MRI presentation demonstrates mild ventricular
dilatation associated with numerous sulci in the perisylvian area,
with irregular cortex–white matter junction and a prominent sub-
arachnoid space overlying the cortical malformation. The most
common cause of fetal PMG is congenital CMV infection. In
these cases, PMG is associated with other signs of brain infection
such as VM, abnormal echogenic ventricular lining and adhe-
sions, periventricular pseudocysts, temporal cysts, calcifications
and cerebellar anomalies. 
Schizencephaly. These are clefts which transverse the full
thickness of the hemisphere, connecting the ventricle to the sub-
arachnoid space. In type I or closed-lip schizencephaly, most fre-
quently a unilateral finding, the walls of the cleft are opposed. In
type II or open-lip schizencephaly, which is more often bilateral,
CSF separates the walls.
They often occur in the perisylvian area. Heterozygous muta-
tions of the gene EMX2 are suspected to play a role. Depending
on the degree of severity and associated anomalies, severe mental
and motor retardation have been observed. The prenatal detection
rate is low.157 The majority of the cases are diagnosed after 28 ges-
tational weeks, MRI being superior to ultrasound. 
Congenital Infections
Introduction. Ultrasound markers such as microcephaly, VM,
periventricular calcifications and visceral lesions may suggest
intrauterine infection. These markers may represent a poor
prognosis. Nevertheless, the sensitivity and predictive value of
these markers on routine ultrasound scanning is poor. In cases
with a single ultrasound marker in the brain, there was no
significant difference in the prevalence of recent toxoplasmosis
or CMV infection; however, the prevalence depended largely on
the type of malformation. These data indicate that brain and
visceral markers suggestive for congenital infections have a low
sensitivity.158 
Congenital Cytomegalovirus Infection. Cytomegalovirus
infection is by far the most common prenatal and perinatal infec-
tion, causing more perinatal mortality and long-term morbidity
than all other congenital infections put together.159
Cytomegalovirus is a neurotropic double-stranded DNA virus
that after primary infection becomes latent and resides in neurons.
Infected individuals shed the virus in urine, saliva and nasal secre-
tions, but CMV may also be transmitted by sexual contact.
countries, rises to 90% in developing regions around the world.
International guidelines do not favour generalised prenatal
screening because serologic testing is difficult, the presence of
maternal immunoglobulin G does not exclude the possibility
of reactivation or reinfection and effective prenatal treatment
is lacking. In addition, recurrent CMV infections in pregnant
women with preexisting antibodies cause most of the sensorineu-
ral hearing impairment in children with congenital cytomegalo-
virus (cCMV) infection.160 Vertical transmission rates increase
with advancing gestational age, but mortality and morbidity
296 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

gradually decrease. Severe morbidity is rarely encountered in TABLE Fetal Sonographic Brain Lesions Related to
cCMV infections occurring after 16 to 20 weeks of gestation. 28.11 Cytomegalovirus Infection164
Only 1 in 10 newborns infected in utero have obvious and often
severe signs of cCMV; in addition, 10% to 15% of the asymp- Hyperechogenic lining of the lateral ventricle(s)
tomatic ones at birth will develop neurodevelopmental sequelae Anechogenic cavity at posterior horn
later in life. Intraventricular adhesions
Screening for cCMV by ultrasound has a very poor sensitivity Corpus callosum dysgenesis
Vermian hypoplasia
and specificity and should not be advocated.161 Serologic screen-
Cerebral and cerebellar calcifications
ing performed in the early first trimester and repeated before 20 Lenticulostriate vasculopathy
weeks may detect most cases of primary infection.162 Prenatal Microcephaly
serologic screening might also reduce the number of pregnant Ventriculomegaly
women acquiring the infection in the first place by introducing Periventricular cysts
hygienic measures.163 Increased arachnoid space
Fetal infection is diagnosed by a positive polymerase chain Periventricular calcifications
reaction (PCR) for CMV on amniotic fluid as a result of viral Polymicrogyria
shedding into the fetal urine after infection. At this stage, serial Subependymal cysts
ultrasound evaluation enables the detection of cCMV-related Irregular ventricular wall, nodular heterotopia
abnormalities in the brain and other organ systems.161,164   
Although all cell types may be infected, CMV shows a high tro-
pism for progenitor cells located in the periventricular region. In
early gestation, at the time of neurogenesis and neuronal migra-
tion, cCMV results more often in cortical plate anomalies and
microcephaly by direct cytotoxic effect as well as inflammation
and microglial activation. Polymicrogyria does not result from
cell migration disorder but rather is related to a complex reactive
inflammatory process damaging the radial glial scaffold. CMV
infection of the amygdala and of the parahippocampal–temporal
germinal matrix zone causes temporal lobe cysts and may occur
in the absence of cortical lesions.
In the periventricular regions, the high viral density and the
presumed lower functionality of the cellular immunity contrib-
ute to the cytotoxic and lytic lesions and calcifications.165 Infec-
tion of the placenta enhances the destructive action of CMV in
the brain through hypoxemia. In general, ultrasound lesions have
been found in 37.7% to 43.5% of cCMV-infected fetuses.164,166
Even in infants with normal ultrasound evaluation, autopsy or
postnatal clinical investigation revealed CMV-related anomalies
in 55% of cases.166
In symptomatic fetuses, a large variety of CMV-related brain • Fig. 28.34  Congenital cytomegalovirus-related anomalies: severe cere-
lesions have been detected on ultrasound in more than 50% of bral atrophy associated with ventricular wall calcification.
cases.164 The type and the number of the lesions may evolve dur-
ing gestation. Although the sonographic brain lesions in cCMV
infection are nonspecific, not diagnostic and unreliable to predict asexual replication starts after passage in the intestinal tract. These
symptomatic cCMV infection, they become potent prognostic tachyzoites are responsible for the haematogenous spread and for
markers in association with a positive PCR on amniotic fluid.161 the vertical transmission to the fetus. After development of an
The different sonographic brain lesions related to CMV are listed immune response, Toxoplasma remains present in the body as tis-
in Table 28.11 and illustrated in Fig. 28.34 and 28.35. sue cysts containing slowly dividing parasites: bradyzoites. Inges-
Adverse neonatal outcome has been associated with cerebral tion of uncooked meat by humans can activate these bradyzoites
ultrasound anomalies,167 in particular, microcephaly, polymi- and start the sexual replication.
crogyria and periventricular intraparenchymal cystic lesions.168 In Europe, the seroprevalence hovers around 50%. The actual
Dedicated neurosonography is comparable to fetal MRI in the seroconversion rate for toxoplasmosis in pregnancy dropped sig-
diagnosis of fetal brain anomalies,168 although fetal MRI is supe- nificantly because of the rigorous introduction of hygienic mea-
rior in the detection of cortical abnormalities.169 Isolated subtle sures and is considered to be around 0.1%.172 Fetal infection
MRI lesions may carry a limited prognostic value, not justifying depends on the transmission rates, which increases with gestational
TOP.170 Nevertheless, MRI has a high negative predictive value age. However, the probability of severe intracranial or eye lesions
of MRI in predicting sensorineural hearing loss and neurologic decreases significantly as transmission occurs later in gestation. As
impairment from 28 weeks onward.171  with CMV, general serologic screening is not recommended but
Congenital Toxoplasma Infection. After ingestion of uncooked is mainly performed in the first trimester to implement hygienic
meat containing bradyzoites, Toxoplasma gondii starts a sexual measures, which seems very effective to prevent the acquisition of
reproduction cycle in the intestinal tract of the cat. Cat faeces the parasite.
contain oocytes filled with infective sporozoites and are spread on Fetal infection is diagnosed by PCR on amniotic fluid (sensitiv-
vegetables, soil, water and grass. After ingestion of these oocytes, ity, 87%; specificity, 99%) up to 5 weeks after maternal diagnosis
 CHAPTER 28  Sonography of the Fetal Central Nervous System
• Fig. 28.35  Coronal T2 HASTE image in a 25-week-old fetus after proven
cytomegalovirus seroconversion. Unilateral temporal cyst (thick arrow) and
contralateral dilation of the temporal horn (dashed arrow).
or after 18 weeks of gestation if seroconversion occurred in the
first trimester.173,174
Sonographic intracranial findings suggestive for toxoplasmo-
sis consist of VM, echogenic nodular foci, calcifications, diffuse
periventricular echogenicity, cysts and callosal dygenesis.175 This
is in accordance with less detailed studies from the 1990s in
which VM, intracranial calcifications and nodular parenchymal
lesions were reported as major findings.176 Occasionally, in severe
cases and with modern ultrasound equipment, retinal involve-
ment may be observed. The ultrasound signs frequently appear
late in gestation and therefore suggest that the sequelae require
a prolonged time to develop.177 In the absence of VM, detailed
sonographic evaluation of the fetal brain is indicated even in
cases in which maternal seroconversion occurred at midgesta-
tion or later. Furthermore, upon maternal seroconversion and a
negative amniotic fluid PCR for Toxoplasma, careful sonographic
follow-up might be indicated because transmission of tachyzo-
ites from mother to fetus might be delayed. Rapid development
of severe sequelae after late transmission could be related to par-
ticular virulent strains of toxoplasmosis.177 The improvement of
ultrasound resolution and the addition of MRI in the prenatal
management has led to better visualisation of the common brain
abnormalities in toxoplasmosis: VM, echogenic densities, cysts
and abscesses.175 The brain lesions are not restricted to the peri-
ventricular zone.  ventricle (Fig. 28.36). Retraction of the clot may decrease the VM.
Congenital Zika Infection. Infection with this flavivirus is
caused by the bites of mosquitoes (Aedes aegypti and Aedes albop-
ictus). However, the virus can also be transmitted by semen,
blood transfusion and possibly saliva and breastfeeding, but this
has not been confirmed so far. Often the infectious course is
asymptomatic, but nonspecific flulike symptoms and fever may
occur.
The Zika virus has a particular affinity for neural progenitor
cells, therefore causing severe anomalies in the fetal brain. In a
review by Sarno and colleagues, the ultrasound findings of 52
cases of microcephaly related to Zika virus were summarised.178
In a retrospective case series of 19 fetuses, the importance of fetal
297
severe microcephaly (HC < 3SD) (14 of 19) was revealed,179
and the presence of other additional CNS lesions (17 of 19),
including VM, increased cisterna magna, parenchymal calcifi-
cations, ocular calcifications, DWM, increased subarachnoid
space, cerebellar hypoplasia and cortical atrophy, was demon-
strated. In addition to these CNS anomalies, Zika virus is asso-
ciated with early and late miscarriages, stillbirths, intrauterine
growth restriction and hydrops fetalis.180 On fetal MRI, the
most common prenatal abnormalities were a reduced HC, VM,
calcifications and neuronal migration anomalies, consisting of
lissencephaly, pachygyria, polymicrogyria or opercular anoma-
lies.180 In 94% of Zika virus cases, volume changes in association
with abnormal cortical development occur. Additional anoma-
lies are CC dysgenesis and VM.181 
Destructive Lesions
Intracranial Haemorrhage. Although in neonates, intraventricular
haemorrhage (IVH) and germinal matrix bleeding (GMB) are
quite frequent and occur more frequently with early prematurity
(25%–45% at <1500 g), the incidence in fetal life remains
low.182,183 Reports from referral centres indicate figures for ICH
ranging from 1 in 10,000 to 0.46 in 1000 deliveries.182 However,
small GMB IVH may go undetected, therefore underestimating
the true incidence.
Typical locations of intracerebral haemorrhage in the fetus are
the ganglionic eminence, the choroid plexus and the cortical plate.
In addition, they are often late in appearance and might only be
detected after targeted diagnostic imaging in patients with predis-
posing conditions.184
Germinal Matrix Intraventricular Haemorrhage. Damage or
rupture of the fragile blood vessels in the germinal matrix between
24 and 32 weeks of gestation related to sudden changes in blood
pressure or perinatal asphyxia can initiate germinal matrix and
intraventricular bleeding. The highest risk is between 26 and 28
weeks of gestation. Subsequent terminal vein obstruction may
result in haemorrhagic venous infarction and leukomalacia. Addi-
tionally, obstruction to CSF flow, obliterative arachnoiditis and
reactive CSF production results in accumulation of CSF and VM.
Numerous causes associated with IVH are listed in Table
28.12. Most frequently, alloimmune thrombocytopenia, hypoxia-
related conditions and maternal trauma are held responsible. Neo-
natal classification into four grades correlates well with neurologic
outcome.185
A recent haemorrhage shows as a homogeneous hyperecho-
genic area in the parenchyma or the ventricle without posterior
shadowing. After a few days to 1 week, liquefaction turns the
blood clot into a complex heterogeneous mass, with irregular
echogenic lining and some degree of enlargement of the ipsilateral
Cases with an intraparenchymal bleed with venous infarction will
result in destruction of brain tissue and porencephalic cysts forma-
tion over a period of a few weeks.186 Small IVH, however, may
undergo complete resolution.
On MRI, methaemoglobin is hyperintense on T1-weighted
images, as are calcifications. Additionally, T2-weighted and
echoplanar sequences are particularly helpful in depicting small
haemorrhages or blood-breakdown products because these
appear like strong hypointense structures when mainly con-
sisting of deoxyhaemoglobin.187 The signal intensity of haem-
orrhage on diffusion-weighted sequences is variable because of
the stage of involution188 (Fig. 28.37). Haemorrhages may no
298 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
28.12 Aetiology of Intracranial Bleeding

Maternal trauma
Hypoxia
Congenital infection cCMV, toxoplasmosis, parvovirus B19
Congenital vascular defects
Use of anticoagulant drugs Warfarin, aspirin
Coagulation disorders
Maternal complications of Placental abruption, preeclampsia,
pregnancy hypertension, severe hypotension,
seizures
Maternal vitamin K depletion
TTTS
Thrombosis of the umbilical
cord
Alloimmune thrombocytopenia
Unexplained

cCMV, congenital cytomegalovirus; TTTS, twin-to-twin transfusion syndrome.

   • Fig. 28.37  Axial T2 image of a 32-week-old fetus demonstrating bilat-
eral ventriculomegaly of 16 mm and 19 mm. In the largest left ventricle,
there is an irregular T2 isohypointense structure (circle) corresponding to
blood products after a germinal matrix haemorrhage with intraventricular
extension.

transfusion.192 The association with VM, hydrocephaly and cer-

• Fig. 28.36  Axial view of the brain showing an old intraventricular haem-
orrhage with residual small clots (asterisk) at the wall of the lateral ven-
tricles, echogenic lining (arrowheads) and dilation of the ventricular system
(°) and foramina of Monro.
longer be visible after several weeks because of active transport
of blood-breakdown products.189  with ischemic and haemorrhagic insult than in cases with no pro-
Cerebellar Bleeding. Bleeding involving the posterior fossa
and the cerebellum are frequent findings at autopsy and in pre-
term infants up to 1550 g (3%). Small cerebellar haemorrhage
has been observed in 20% of preterm infants190 and significantly
impacts neurodevelopmental outcome in survivors.191 It is fre-
quently associated with supratentorial bleeding. Most often pre-
term neonates with cerebellar haemorrhagic injury are critically ill
with impaired vascular autoregulation and often require intensive
supportive treatment.191
Cerebellar bleeding remains a rare condition in fetal life,
although the advances in fetal MRI have increased its detection.192
Most often, it occurs between 21 to 25 weeks of gestation and is
located in the germinal matrix of the subpial and subependymal
external granular layer. Pathogenesis has been associated with focal
vascular pathology (e.g., haemangioma), drug abuse (e.g., cocaine),
birth trauma, sepsis, congenital infection (CMV), preeclamp-
sia, severe fetal anaemia (parvovirus B19) and after intrauterine
ebellar hypoplasia has been described.
Ultrasound shows a hyperechogenic area in the posterior fossa
or in the cerebellum. A dedicated neurosonographic survey of the
posterior fossa detects the majority of the cerebellar malformations.
MRI offers a high resolution of acute or chronic haemorrhagic
insults, especially after 24 to 26 weeks of gestation. Involvement
of the caudal portion of the vermis seems to be present on MRI in
two thirds of the cases and more often than estimated by prenatal
ultrasound alone. Evolution towards abnormal cerebellar foliation
has been observed.193 On fetal MRI, the haemorrhage looks simi-
lar to the signal intensity of haemorrhage in different locations in
the brain. The use of advanced MR sequences, such as diffusion-
weighted imaging (DWI) and susceptibility-weighted imaging,
helps in the differentiation of haemorrhagic lesions from other
destructive lesions.192 Progressive resorption may finally result in
cerebellar or vermian hypoplasia or complete disruption of the
cerebellum (empty posterior fossa). However, in cases of cerebellar
hypoplasia, progression of the lesion was more often associated
gression.127 Nevertheless, resorption of the haematoma can also be
complete without cerebellar damage. 
Subdural and Subarachnoid Haemorrhage. In preterm
infants, subdural haemorrhage has been attributed mainly to
perinatal trauma in 3% to 18%.194 Sonographic differentiation
between subarachnoid and epidural bleeding is difficult. How-
ever, in epidural (extradural) haemorrhage, the dura mater is
displaced away from the interior lining of the skull. Most often,
subdural haemorrhages are located over the cerebral hemisphere
and rarely infratentorial.195 The true incidence of subdural or
subarachnoid bleeding is unknown. Cases have been reported
sporadically. They occur in association with maternal or birth
trauma, coagulation disorders, drugs,183 vascular anomalies196
or maternal medical conditions. Occasionally, no aetiology is
found. Because of compression of the cerebral cortex, VM may
appear. Other associated anomalies include polyhydramnios and
fetal hydrops (Fig. 28.38).
 CHAPTER 28  Sonography of the Fetal Central Nervous System
Voluson
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• Fig. 28.38  Subdural bleeding in a 34-week-old fetus; the image shows
a huge retracted clot (asterisk) and an important mass effect on the ipsilat-
eral hemisphere with ventriculomegaly.
Ghi and associates197 summarised 19 cases of subdural haema-
toma. Two ended in TOP and 1 in an intrauterine death. Of the
16 liveborn cases, follow-up was available in 9 cases, 6 of which
showed a normal neurologic outcome. The prognosis of subdural
haemorrhage varies considerably from in utero demise to complete
resolution before delivery with normal neurodevelopment.198
After prenatal diagnosis, history should focus on maternal drug
intake, maternal medical conditions and recent trauma. Labo-
ratory testing should focus on maternal platelet count, platelet
antibodies, parental human platelet antigen-1a (HPA-1a) and
coagulation disorders (factor V Leiden, factor X deficiency pro-
thrombin G20210A mutation, protein C or S deficiency, and
antiphospholipid antibody syndrome).
Fetal blood sampling may be indicated to assess fetal blood
cell count, platelet count and coagulation factors. Further genetic
investigation of coagulation disorders on stored fetal DNA may
be indicated.  MRI has allowed us to detect small hypoxic-ischemic lesions.
Neonatal Outcome. Neonatal outcome of in utero intracranial
haemorrhages varies from fetal death to normal neurologic devel-
opment. However, reports on outcome date back to 10 years ago.
Ghi and colleagues197 and Elchalal and colleagues182 showed that
in surviving infants (25 of 49), normal neurologic outcome was
more often observed in grade 2 bleeding (5 of 7) than in grade 3
and 4 bleedings (1 of 3 and 1 of 11, respectively).
Tiller and colleagues199 documented a multicentre series of
43 intracranial haemorrhages caused by fetal and neonatal allo-
immune thrombocytopenia (FNAIT), of which 54% occurred
before 28 weeks of gestation. The first-born child was affected in
63%. Poor outcome was characterised by a 35% to 59% neona-
tal death rate200 within 4 days after birth and a severe neurologic
impairment in 53% of the survivors. Only 12% (5 of 43) were
discharged without sequelae. Classification of the lesions revealed
more complex bleeding (IVH or parenchymal) before 28 weeks.
The majority of cases with cerebellar bleeding ended in a TOP.
Severe bleeding often results in a dismal prognosis with impaired
cognitive function, subtle motor deficits, and language and social
behavioural problems.192 Progressive cerebellar hypoplasia related
to posterior fossa haemorrhage has been reported without neuro-
logic or clinical compromise.197 Recently, Hayashi and colleagues
summarised the reported cases of prenatal cerebellar haemor-
rhage.192 Of the 18 pregnancies, 7 ended in a TOP, 3 presented
299
with fetal death beyond 22 weeks, and 2 neonates died at 18 and
46 days. Of the remaining 6 survivors, 2 had a developmental
delay.
Termination of pregnancy might be offered if the cerebel-
lum is affected. Diagnosis of intracranial bleeding should lead
to multidisciplinary counselling. Without legal constraints,
late TOP may be offered in IVH III to IV and subdural haem-
orrhages. No treatment for the index case is available. How-
ever, in patients with FNAIT and a previous prenatal case of
ICH, the recurrence rate is as high as 80%,201 weekly pro-
phylactic treatment with intravenous immunoglobulin should
be initiated from 20 weeks onwards to prevent midsecond
trimester bleeding. Prenatal invasive treatment with repeti-
tive fetal blood sampling and in utero platelet transfusions
has been abandoned largely because of the high rate of com-
plications, and management strategies have been introduced
based on risk stratification.202,203 In addition, noninvasive
fetal genotyping of HPA-1a by PCR has been developed to
determine the risk in subsequent pregnancies after a diagnosis
of FNAIT.204 Caesarean section does not significantly improve
neurologic outcome in cases of ICH and should be considered
individually.205 
Congenital Porencephaly. Secondary to fetal cerebral hypo-
perfusion in the second trimester, the fetal brain may undergo
liquefaction with subsequent resorption of destroyed brain paren-
chyma creating porencephalic cysts (Table 28.13). These are
round and irregular-shaped cysts in communication with the ven-
tricular system.
On ultrasound, a porencephalic cyst appears as an avascular
cystic lesion, which can be in communication with the ventric-
ular system.186 The lesions may evolve over time (Fig. 28.39).
Careful and complete fetal exploration should be performed
to rule out potential causes. Investigations include screening
for infectious disease, bleeding and platelet disorders, and a
fetal MRI.
Magnetic resonance imaging may help to visualise the extent
of destruction and localisation. The development of DWI in fetal
Expanding PC may cause mass effect on the surrounding paren-
chyma.206 No communication between the porencephalic cyst
and the ventricular system is needed.
Neurodevelopmental outcome is usually poor with severe delay
and seizures. Occasionally, neonatal surgery with cyst fenestration
may improve the hemiparesis and reduce the number and severity
of the seizures. 
Hydranencephaly. Defined as a complete destruction of the
cerebellar hemisphere with presence of the falx, the midbrain and
the posterior fossa are usually not involved. The fetal head has
a variable size. Vascular occlusion, infection, hypotension and
haemorrhage are the main aetiologies.207 Differential diagnosis
with hydrocephaly has to be considered. 
Periventricular Leukomalacia. This degenerative disorder
of the white matter, most frequently seen in premature infants,
carries a poor prognosis. The cystic variety can be visualised by
ultrasound as multiple cysts adjacent to the lateral ventricles.
They present 7 to 10 days after a hypoxic-ischemic or infec-
tious event as periventricular hyperechoic lesion replaced by
cysts after 2 weeks.208 The differential diagnoses are periven-
tricular cysts which are located in the germinal matrix, the cau-
dothalamic groove or the caudate nucleus. Histologically, they
are not covered by epithelium and usually have a favourable
outcome.209 
300 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
TABLE
28.13 
Aetiology of Congenital Porencephaly
Fetal cerebral hypoperfusion Fetal demise in monochorionic twin
pregnancy
Low-output cardiac failure caused by
steal effect in tumours or AVM
Incomplete laser ablation in TTTS
Fetal surgery
Peripartum events in premature
infants
Maternal events Hypotension caused by hypovolemic
shock
Prolonged hypoxia
Drug abuse (e.g., cocaine)
Infection Toxoplasma, CMV, parvovirus B19,
varicella
Chorioamnionitis
Inborn errors of metabolism
Alloimmune thrombocytopenia Intracranial haemorrhage type IV
evolving to porencephalic cysts
COL4A1/2 mutations Association with cataract
AVM, Arteriovenous malformation; CMV, cytomegalovirus; TTTS, twin-to-twin transfusion
syndrome.
  
Vascular Malformations
Arteriovenous Fistula. Abnormal arterial to venous connection
without an intervening capillary system (arteriovenous malforma­
tion [AVM]) results in dilation of the vascular junction. In the
brain, these AVMs most frequently occur supratentorial, but 15%
are found in the posterior fossa. On ultrasound, they appear as cystic
structures with a high-velocity, low-resistance arterial flow causing
high-output cardiac failure, fetal hydrops and ischemic brain
lesions caused by steal effect, hypoperfusion, direct compression or
venous thrombosis. Often dilation of the draining venous system
is present.210 In large lesions, platelet consumption may lead to the
Kasabach-Merritt sequence.211
Arteriovenous malformation has to be differentiated from vein
of Galen malformation, which is a specific AVM in the midline,
vascular tumours and intracranial cysts.
Fetal MRI may provide additional information on the impact of the
AVM on the surrounding structures (e.g., involved vascular structures,
brain lesions). Postnatal treatment consists of embolization or open sur-
gery. In about 37% of cases, a multimodality approach is needed.212,213  final treatment at about 4 to 6 months after delivery. Transfemoral
Vein of Galen Aneurysmal Malformation. Vein of Galen AVM
can be found in 1 in 10,000 to 1 in 25,000 live births. This is a
sporadic condition with a low recurrence rate. Between the 6th and
11th weeks of gestation, an arteriovenous (AV) connection between
the primitive choroidal vessels and the median prosencephalic vein
of Markowski214 leads to abnormal flow and prevents the embry-
onic vein from involuting. Frequently, occlusion of the dural
sinuses of the posterior fossa, especially the sigmoid sinuses occur
in conjunction, giving rise to an engorgement of the vein of Galen,
the straight sinus, the confluence and the transverse sinuses.215
Prenatal diagnosis is usually made in the third trimester by
ultrasound revealing a cerebral cystic lesion located in the mid-
line and posterior to the third ventricle, extending posteriorly
into a dilated tubular structure pointing towards the occiput. Dif-
ferential diagnosis includes arachnoid cyst, porencephalic cyst
• Fig. 28.39  At 31 weeks, this fetus presented with ventriculomegaly and
a porencephalic cyst (asterisk).The parasagittal image shows the anterior
location.
and intracranial teratoma. However, colour Doppler investigation
typically shows aliasing turbulent blood flow. In addition, VM or
hydrocephalus may be present because of the direct compression of
the aqueduct or decreased resorption of the CSF because of malfunc-
tion of the Pacchionian granulations secondary to venous hyperten-
sion. High-output cardiac failure due to the AV anastomosis may lead
to congestive heart failure, tricuspid insufficiency, cardiomegaly, peri-
cardial effusion and finally fetal hydrops. In addition, the increased
vascular flow may result in regional brain damage.216 3D imaging
may help to identify the number of feeding vessels, which may be of
prognostic importance at the time of embolization217 (Fig. 28.40).
Serial ultrasound scans are essential for evaluation of cardiac function,
the mass effect of the aneurysm and the development of cerebral corti-
cal lesions. Antenatal MRI has been used to identify injury to the grey
and white matter, termed ‘melting brain’.218 Parenchymal volume loss
can also be identified with MRI. More recently, the Yuval criteria for
good perinatal outcome have been simplified to the absence of cardiac
and neurologic defects.219
Pregnancies complicated with a Vein of Galen Aneurysmal
malformation (VGAM) should deliver in a tertiary care centre,
necessitating an intense neonatal follow-up to manage the conges-
tive heart failure and to optimise the neonatal condition for the
arterial embolization results in complete occlusion in fewer than
60% of cases. Additional procedures to significantly reduce the
blood flow in the AVM may be needed but might increase the
risk for intracerebral haemorrhage and venous thrombosis.220 The
long-term survival rate in children with VGAM managed multi-
disciplinary reaches 85% to 90%. However, a good outcome was
observed in only 60% to 68% of the cases.220,221 
Thrombosis of Torcular Herophili, Dural Sinus Malformation
and Dural Arteriovenous Shunt. In the paediatric literature, three
types of dural arteriovenous shunt (DAVS) have been recognised:
the dural sinus malformation (DSM), the infantile type of DAVS
and the adult type of DAVS.222 In the prenatal literature, the con-
dition is mainly known as dural sinus malformation or thrombosis
of the torcular herophili. On prenatal ultrasound, a large cysti-
clike dilation of the posterior fossa is observed with a rounded
 CHAPTER 28  Sonography of the Fetal Central Nervous System
2
1
3
• Fig. 28.40 Three-dimensional colour-rendered image of the arteriove-
nous malformation of the vein of Galen: (1) arteriovenous malformation
with feeding arterial vessels, (2) straight sinus, (3) confluence, (4) superior
sagittal sinus and (5) transverse sinus draining to the jugular veins.
echogenic mass (thrombus) near the tentorium. Colour Doppler
reveals pulsatile flow before thrombosis (Fig. 28.41); the flow
becomes absent upon complete thrombosis of the confluence.
Rarely, additional brain lesions are seen. Spontaneous regression
even before birth has been documented.
On MRI, the intensity of the thrombus, can vary on T1 and
T2 according to the different stages of the blood products that
are present in the thrombus.223 Fetal MRI is also valuable in
the detection of secondary brain abnormalities caused by altered
perfusion.
Favourable prognostic factors appear to be a normal brain
with decreasing mass and mass effect during monitoring, the
presence of alternative venous drainage (via cavernous sinuses or
persistent occipital sinuses), the absence of brain parenchymal
lesions and thrombosis of the deep venous system.223,224 Occa-
sionally, mild neurodevelopment and speech disabilities have
been reported.
Two recent reports summarise the prenatal diagnostic experi-
ence and outcome of DSM223,225 (Table 28.14).  cysts and DWM.
Intracranial Masses: Cysts and Tumours
Introduction. Intracranial cysts are relatively common findings
on prenatal ultrasound, most of which are benign and remain
clinically silent. If isolated, the prognosis is mostly favourable.
Cysts are classified according to their location in the brain:
extraaxial (arachnoid cysts), intraventricular (choroid plexus cysts
[CPCs]) or intraparenchymal (porencephalic cyst).
Congenital brain tumours present about 0.5% to 1.5% of all
paediatric brain tumours. An incidence of 3.6 per 100,000 births
has been reported.226 The exact cause for the development of
tumours has not been elucidated yet.  of the cyst.234 The presence of a normal CC, absence of extra-CNS
Intracranial Cysts
Arachnoid Cysts. Arachnoid cysts result from an abnor-
mal splitting of the arachnoid web and the inner layer the
301
• Fig. 28.41  Huge dilation of the torcular with whirlpool effect. On colour
Doppler flow imaging, the feeding artery is highlighted.
pia mater and subsequent entrapment of CSF. They account
for about 1% of all intracranial masses in children.227 Most
of the arachnoid cysts are located supratentorially; 50% to
60% are located in the middle cranial fossa. They are also
found in the quadrigeminal cistern (5%–10%), suprasellar
cistern (5%–10%), cerebral convexity (5%) or posterior fossa
(5%–10%).227,228
On ultrasound, they appear as well-delineated, unilocular, reg-
ularly shaped hypoechoic masses without colour Doppler signal
located on the surface of the brain.
In 25% of the cases, the diagnosis of arachnoid cysts is made in
the second trimester; the remaining 75% are diagnosed between
28 and 34 weeks of gestation. Supratentorial cysts are picked up
later in gestation than the ones in the posterior fossa.229
The differential diagnosis includes porencephalic cysts, which
are usually unilateral and communicating with the lateral ven-
tricular system.230 Additionally, CPCs are common intraven-
tricular findings, and glioependymal cysts are located in the
parenchyma of the brain.231,232 Aneurysms of the vein of Galen
and other vascular anomalies are easily differentiated by colour
Doppler analysis. Rare conditions to differentiate are Rathke
cleft cysts, schizencephaly, teratoma and IVH. In the posterior
fossa, the differential diagnosis includes MCM, Blake pouch
Fetal MRI does not modify the diagnosis in the majority of
cases,141 but it is able to differentiate the AC from other cystic
lesions.229 In addition, MRI may be more precise in locating
the lesion and determining its relationship with the surround-
ing structures, especially when located in the posterior fossa(Fig.
28.42). Finally, MRI more easily detects additional anomalies
such as heterotopias and CC dysgenesis.233 Postnatal workup
mainly consists of a neurosonogram to confirm the location, size
and number of the lesions and MRI to exclude associated cere-
bral anomalies.
The prognosis of a fetus with an arachnoid cyst depends partic-
ularly on the brain integrity rather than on the volume or location
anomalies, a slowly growing cyst, the absence of VM and the loca-
tion near the Sylvian fissure are favourable variables. Outcome of
isolated arachnoid cysts is generally good. Neurologic symptoms
reflect the anatomical localisation of the AC and the effect on CSF
302 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Outcome of Prenatally Diagnosed Thrombosis

28.14 of Torcular Herophili
Rayssiguier
Corral et al225 et al223 Total
Number of patients 8 8 16
Median gestational 24 (22–32) 24 (22–33) 24
age (wk) at diag-
nosis (range)
Prenatal sonographic 8/8 DSM 5/8 DSM 13/16
finding thrombus, 1/8: cerebral
­dilatation of the tumour
torcular and 2/8: arach-
SSS in all cases noid cysts
TOP 0/8 3/8 3/16
Complete or near to 5/8 0/8 5/16
complete sponta-
neous resolution
Decrease in size 7/8 5/8 12/16 • Fig. 28.42  Large cyst (arrow) in the posterior fossa with complete ver-
mis. Moderate mass effect of the cyst on the posterior fossa structures.
Live birth 8/8 5/8 13/16 There is no secondary ventriculomegaly.
Caesarean section 8/8 NA
Additional intervention 0/8 0/5 0/13
TABLE
Normal neurological 4/8 5/5 9/13 28.15 
Arachnoid Cysts and Associated Malformations
outcome
Chromosomal
Mild disability 3/8 0/5 3/13 anomalies (6%)
Severe disability 1/8 0/5 1/13 Central nervous Agenesis of corpus callosum
system anomalies
Other complications 0/8 0/5 0/13
Absent cavum sept pellucidi
DSM, dural sinus malformation; TOP, termination of pregnancy; SSS, superior sagittal sinus. Arnold Chiari type 1 malformation
   Cortical development anomalies
Arteriovenous malformations
Single-gene Xq22
flow. Associated underlying abnormalities can give rise to symp- mutations
toms, not directly related to the cyst itself (Table 28.15). 9q22
14q32.3
The natural history of AC varies from regression, stabilisation, slow
11p15
growth towards acute enlargement of cyst, subdural effusion after rup-
ture of cyst and subdural or intracystic bleeding, with or without trauma. Part of a syndrome Aicardi syndrome
There is a debate in the literature between shunting and open Chudley McCullough syndrome
microsurgical or endoscopic fenestration with cystoventriculos- Neurofibromatosis type 1
tomy or cystocisternostomy. In a prospective long-term survey, a Marfan syndrome
restrictive attitude to surgery for intracranial AC in the absence Autosomal dominant polycystic kidney disease
Glutaric aciduria type 1
of objectively verified symptoms or signs of obstructions was
advocated. 
  
Choroid Plexus Cysts. Choroid plexus cysts are frequently
observed on midtrimester routine ultrasound scans (≤3.6%). They may be mistaken for VM. CPCs have to be differentiated from
result from entrapment of CSF into the choroid plexus. If iso- choroid plexus papilloma (CPP) and IVH.
lated, they are transient in nature and usually regress in the second In isolated cases, the neurologic outcome is excellent with or
half of the second trimester or early third trimester of gestation. If without prenatal regression of the cysts.237 
seen in conjunction with other significant markers of aneuploidy Periventricular Pseudocysts. Periventricular pseudocysts
or structural defects, karyotyping should be offered to exclude tri- (PVPCs) are found frequently in newborns (1%–5%) and espe-
somy 18. There is no increased risk for trisomy 21.235 Multiple cially in preterm neonates. They are located in the wall of the lat-
and bilateral cysts do not increase the risk for trisomy 18; however, eral ventricles and result from lysis of undifferentiated germinal
a diameter larger than 10 mm might do so.236 matrix cells, which because of the high mitotic index are particu-
Upon diagnosis, extensive ultrasound examination for other larly vulnerable. Because of the lack of an ependymal lining, they
markers and structural defects is mandatory. In the absence of are called pseudocysts. They can be of infectious, vascular, meta-
any other signs, the patients should be reassured about the benign bolic or chromosomal origin. A list of causative factors is displayed
course and without the need for intense follow-up. Large CPCs in Table 28.16.
 CHAPTER 28  Sonography of the Fetal Central Nervous System
TABLE
28.16 Aetiology of Periventricular Pseudocysts
Viral infections: cytomegalovirus, rubella
Haemorrhage: grade I bleeding
Focal hypoxic ischemic damage
Toxins: e.g., cocaine
Metabolic disease: Zellweger syndrome, cerebro-hepato-renal syn-
drome, generalised peroxisomal disorder, holocarboxylase deficiency,
molybdenum cofactor deficiency
Mitochondrial diseases
Chromosomal abnormalities
Periventricular pseudocysts can be divided into connatal cysts
(frontal horn cysts) and subependymal pseudocysts more fre-
quently located posterior to the caudothalamic notch238 (Fig.
28.43). Clear differentiation has to be made with periventricu-
lar leukomalacia. Upon ultrasound diagnosis, a workup should
include maternal TORCH serology, a detailed fetal ultrasound
evaluation with fetal echocardiography and extended neurosono-
gram, amniocentesis for array CGH and PCR for CMV, and
fetal MRI. On MRI, these cysts are hyperintense on T2-weighted
images and hypointense on T1-weighted images and are mostly
located in the caudothalamic groove and lateral to the fron-
tal horns. When atypical in location or morphology, MRI is of
increasing importance because of its ability to identify additional
CNS anomalies.239
Isolated PVPCs have a good prognosis. However, in associa-
tion with CMV, the developmental quotient at 18 months of age
was significantly lower than in PVPC without CMV infection.240
Other factors associated with a less favourable outcome are sum-
marised in Table 28.17.239  pler flow differentiates teratoma’s from haemorrhagic lesions.242
Intracranial Tumors. The incidence of tumours of the CNS is
0.34 per million live births. They represent about 1% of all pae-
diatric CNS tumours and 10% of all antenatal neoplasms. Their
aetiology remains unclear, but maternal exposure to exogenous fac-
tors such as drugs, ionising radiation and pesticides has been sug-
gested.241 Intracranial tumours are usually diagnosed in the third
trimester. Differential diagnosis by means of prenatal ultrasound
is challenging. Fetal MRI has allowed for more detailed evaluation
of the invasive growth and compression of the remaining brain
tissue by T2-, T1 - and T2*-weighted sequences.242 DWI allows
identification of hypoxic-ischemic parenchymal lesions secondary
to the mass effect of the intracranial tumour.243 The diagnosis of a
fetal brain tumour should be suspected in case of a solid or solid or
cystic mass causing compression of the CSF circulation with VM
or hydrocephaly, macrocrania, intracranial haemorrhage, polyhy-
dramnios and high-output cardiac failure. The final diagnosis is
only confirmed after histologic investigation of the tumour. The
most frequently encountered tumours are teratoma followed by
astrocytoma and craniopharyngioma.244 The overall perinatal out-
come is poor, with a high stillbirth rate and a survival rate of only
15%. Most tumours are inoperable because of their mass effect
involving large areas of the brain.
Fetal Teratoma. Fetal teratoma is by far the most common
intracranial mass. It accounts for nearly 50% of all brain tumours
in prenatal life. Most often it is localised supratentorially (70%).
Its complex and heterogeneous appearance on ultrasound is
caused by a variable degree of cystic and solid components,
which tend to grow rapidly from the midline (Fig. 28.44). It
may fill the entire cranium and because of its fast growth may
303
• Fig. 28.43  Large bilateral periventricular pseudocysts on an axial plane
(asterisks). There were no additional malformations in this fetus.
TABLE Factors Associated With Unfavourable Outcome
28.17 in Periventricular Pseudocysts
Posterior to the caudal-thalamic groove
Adjacent to temporal or occipital horn
Atypical morphology
Great axis ≥9 mm
  
cause macrocrania and even rupture of the skull.245 Colour Dop-
In a recent review, diagnosis was made at a mean of 32 weeks
(21–41 weeks) for an average tumour size of 10 cm (3.5–23 cm).
Fewer than 8% of the fetuses survived, and nearly half of them
died in utero.246
On MRI, a teratoma is seen as a heterogenous mass on T2-
and T1-weighted images containing solid and cystic components
with variable signal intensity of the cysts ranging from T2 hyper-
intense and T1 hypointense to T2 hyperintense and T1 hyperin-
tense depending on the presence of haemorrhage. MRI will also
evaluate the extension of the tumour and the mass effect on the
surrounding structures in detail.242 
Astrocytoma (Glioma). This solid tumour arises as a sono-
graphic echogenic mass in the cerebral hemispheres, causing a
midline shift. It is usually detected in the third trimester. Often
macrocephaly, hydrocephaly and intracranial haemorrhage are
associated. If low-grade, astrocytomas may grow slowly. However,
anaplastic astroblastoma with intracranial haemorrhage may need
tumour debulking and multiple courses of chemotherapy.247 Pro-
ton therapy has also been proposed.248 A broad range of survival
(20%–90%) has been reported, largely depending on the histo-
logic type and grading.249 Nearly 10% end in stillbirth.
On MRI, these tumours appear isointense on T1-weighted
images and hyperintense on T2-weighted images.250 
Craniopharyngioma. This benign tumour in the midline of
the suprasellar region originates from remnants of the squamous
cells originating from Rathke pouch. Their echogenic aspect makes
them difficult to distinguish from teratomas. Because of their
localisation, these tumours may compress the optic chasm and the
optic tract or produce hypothalamic or pituitary dysfunction and
304 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Fig. 28.44  A fast-growing intracranial teratoma occupied the centre of
the brain and resulted in severe hydrocephaly and destruction of the brain
architecture over a few weeks.
hydrocephalus. Surgical removal is often incomplete, and although
the tumour is benign, survival rates of prenatal cases are poor.
On MRI, the T1 signal intensity depends on the content of
the lesion (cholesterol, keratin and methaemoglobin) and can be
bright to hypointense in signal. On T2-weighted images, these
tumours are heterogeneous with high signal intensity.250  by fetal MRI256; conventional T1- and T2-weighted sequences,
Choroid Plexus Papilloma. Choroid plexus papilloma devel-
ops most frequently in the choroid plexus of the lateral and occa-
sionally in the third or fourth ventricle.251 Detection is often late
in gestation and mainly because of associated unilateral or bilateral
VM. Its echogenic intraventricular appearance resembles recent
intraventricular haemorrhage from which it can de differenti-
ated by showing its vascularisation. Occasionally, the papilloma
is cystic in nature with small echogenic projections on the wall.
(Fig. 28.45). On fetal MRI, the tumour is seen as a solid mass
with T1-isointense and T2-isohyperintense signal. T2*-weighted
sequences may help in the determination of intratumoral haem-
orrhage or differentiate the mass from haemorrhage. Single-voxel
spectroscopy may help in distinguishing between papilloma and
the less frequent carcinoma.252,253
The prognosis of CPP is fairly good with surgical removal,
which is possible in 96% of cases, although it may be complicated
by severe uncontrolled bleeding. Degeneration into carcinoma has
been observed in about 20% of cases. The differential diagnosis
can only be made by histopathologic examination.243  probes, transvaginal scanning and 3D imaging have signifi-
Metabolic Conditions
Although inborn errors of metabolism (IEMs) are rare, they repre-
sent a heterogenous group with more than 500 entities described
in current literature.254 Many IEMs are associated with struc-
tural brain defects, and they are present in up to 14% of patients
with congenital disease. Most of the IEM have a postnatal onset
because the placenta often clears the toxic metabolites; however,
IEMs can be apparent prenatally.255
Prenatal imaging manifestations of diseases such as Zellweger
syndrome, nonketotic hyperglycinaemia, pyruvate dehydrogenase
• Fig. 28.45 In the coronal plane, there is a small T2 hypointensity in
the wall of the ventricle. This presumably was part of the wall but cor-
responded on ultrasound with a small mural nodule (arrow). Fetal MRI
showed increased T2 signal intensity of the periventricular white matter
in the parietal area (dashed arrows), suggesting the diagnosis of a cystic
choroid plexus papilloma.
deficiency and glutamic aciduria type I can also be discovered
DWI and diffusion tensor imaging are used to study microstruc-
ture variance in white matter tracts, and MR spectroscopy is used
to measure brain metabolism in static and dynamic models. MR
spectroscopy is a noninvasive technique capable of producing
information on a large number of chemicals.257
Children with cortical grey matter involvement present with
seizures and encephalopathy.
Infants with deep grey matter involvement typically manifest
extrapyramidal findings of dystonia, chorea, athetosis or other
involuntary movement disorders. White matter disorders mani-
fest pyramidal signs such as spasticity and hyperreflexia and visual
impairment. Involvement of the cerebellum or its connecting
tracts leads to ataxia. 
Conclusion
Prenatal investigation of the fetal CNS remains a complex task.
However, advanced sonographic imaging by high-frequency
cantly improved the diagnostic potential for fetal CNS anoma-
lies. Complementary use of prenatal MRI further improves the
morphologic and most probably in the near future the func-
tional investigation of the normal and malformed fetal brain.
Furthermore, rapid technological development in the field of
molecular genetics opens new diagnostic potentials. In the
end, only long-term standardised follow-up of isolated brain
anomalies may provide adequate information for parental
counselling.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 18. Sival DA, Guerra M, den Dunnen WF, et al.
Neuroependymal denudation is in progress
36. Kiymaz N, Yilmaz N, Demir I, Keskin
S. Prognostic factors in patients with
in full-term human foetal spina bifida aperta. occipital encephalocele. Pediatr Neurosurg.
1. Garne E, Dolk H, Loane M, Boyd PA. Euro-
Brain Pathol. 2011;21(2):163–179. 2010;46(1):6–11.
cat. EUROCAT website data on prenatal
19. Adzick NS, Thom EA, Spong CY, et  al. A 37. Joo JG, Papp Z, Berkes E, et  al. Non-
detection rates of congenital anomalies. J Med
randomized trial of prenatal versus postnatal syndromic encephalocele: a 26-year experi-
Screen. 2010;17(2):97–98.
repair of myelomeningocele. N Engl J Med. ence. Dev Med Child Neurol. 2008;50(12):
2. Salomon LJ, Alfirevic Z, Berghella V, et  al.
2011;364(11):993–1004. 958–960.
Practice guidelines for performance of the
20. Khoshnood B, Loane M, de Walle H, et  al. 38. Da Silva SL, Jeelani Y, Dang H, et al. Risk fac-
routine mid-trimester fetal ultrasound scan.
Long term trends in prevalence of neural tube tors for hydrocephalus and neurological deficit
Ultrasound Obstet Gynecol. 2011;37(1):
defects in Europe: population based study. in children born with an encephalocele. J Neu-
116–126.
BMJ. 2015;351:h5949. rosurg Pediatr. 2015;15(4):392–398.
3. Rizzo G, Capponi A, Pietrolucci ME, et al. An
21. Canfield MA, Mai CT, Wang Y, et  al. The 39. Saker E, Loukas M, Oskouian RJ, Tubbs RS.
algorithm based on OmniView technology to
association between race/ethnicity and major The intriguing history of vertebral fusion
reconstruct sagittal and coronal planes of the
birth defects in the United States, 1999–2007. anomalies: the Klippel-Feil syndrome. Childs
fetal brain from volume datasets acquired by
Am J Public Health. 2014;104(9):e14–e23. Nerv Syst. 2016;32(9):1599–1602.
three-dimensional ultrasound. Ultrasound
22. Orioli IM, Lima do Nascimento R, 40. Ayachi A, Mourali M. [An unexpected cause
Obstet Gynecol. 2011;38(2):158–164.
Lopez-Camelo JS, Castilla EE. Effects of folic of face presentation: iniencephaly]. Pan Afr
4. Rizzo G, Capponi A, Persico N, et  al. 5D
acid fortification on spina bifida prevalence Med J. 2016;25:188.
CNS+ software for automatically imaging
in Brazil. Birth Defects Res A Clin Mol Teratol. 41. Ben-Sira L, Garel C, Malinger G, Constantini
axial, sagittal, and coronal planes of normal
2011;91(9):831–835. S. Prenatal diagnosis of spinal dysraphism.
and abnormal second-trimester fetal brains. J
23. Liu J, Zhang L, Li Z, et  al. Prevalence and Childs Nerv Syst. 2013;29(9): 1541–1452.
Ultrasound Med. 2016;35(10):2263–2272.
trend of neural tube defects in five counties 42. Cuppen I, de Bruijn D, Geerdink N, et  al.
5. Paladini D, Quarantelli M, Sglavo G, et  al.
in Shanxi province of Northern China, 2000 Small biparietal diameter and head circum-
Accuracy of neurosonography and MRI in
to 2014. Birth Defects Res A Clin Mol Teratol. ference are part of the phenotype instead of
clinical management of fetuses referred with
2016;106(4):267–274. independent prognostic markers in fetuses
central nervous system abnormalities. Ultra-
24. Fleurke-Rozema JH, Vogel TA, Voskamp BJ, with spinal dysraphism. Fetal Diagn Ther.
sound Obstet Gynecol. 2014;44(2):188–196.
et al. Impact of introduction of mid-trimester 2015;37(2):135–140.
6. Rossi AC, Prefumo F. Additional value of fetal
scan on pregnancy outcome of open spina 43. Callen AL, Filly RA. Supratentorial abnor-
magnetic resonance imaging in the prenatal
bifida in The Netherlands. Ultrasound Obstet malities in the Chiari II malformation, I:
diagnosis of central nervous system anomalies:
Gynecol. 2014;43(5):553–556. the ventricular ‘point’. J Ultrasound Med.
a systematic review of the literature. Ultra-
25. Bergman JE, Otten E, Verheij JB, de Walle 2008;27(1):33–38.
sound Obstet Gynecol. 2014;44(4):388–393.
HE. Folic acid supplementation influences 44. Callen AL, Stengel JW, Filly RA. Supraten-
7. Prayer D, Malinger G, Brugger PC, et  al.
the distribution of neural tube defect sub- torial abnormalities in the Chiari II malfor-
ISUOG Practice Guidelines: performance of
types: a registry-based study. Reprod Toxicol. mation, II: tectal morphologic changes. J
fetal magnetic resonance imaging. Ultrasound
2016;59:96–100. Ultrasound Med. 2009;28(1):29–35.
Obstet Gynecol. 2017;49(5):671–680.
26. Boyd PA, Devigan C, Khoshnood B, et  al. 45. Wong SK, Barkovich AJ, Callen AL, Filly RA.
8. Guibaud L. Fetal cerebral ventricular mea-
Survey of prenatal screening policies in Europe Supratentorial abnormalities in the Chiari II
surement and ventriculomegaly: time for
for structural malformations and chromosome malformation, III: the interhemispheric cyst. J
procedure standardization. Ultrasound Obstet
anomalies, and their impact on detection and ter- Ultrasound Med. 2009;28(8):999–1006.
Gynecol. 2009;34(2):127–130.
mination rates for neural tube defects and Down’s 46. Hershkovitz R. Prenatal diagnosis of isolated
9. Salomon LJ, Ouahba J, Delezoide AL, et  al.
syndrome. BJOG. 2008;115(6):689–696. abnormal number of ribs. Ultrasound Obstet
Third-trimester fetal MRI in isolated 10- to
27. Salvador J, Arigita M, Carreras E, et al. Evo- Gynecol. 2008;32(4):506–509.
12-mm ventriculomegaly: is it worth it?
lution of prenatal detection of neural tube 47. Chaoui R, Nicolaides KH. From nuchal trans-
BJOG. 2006;113(8):942–947.
defects in the pregnant population of the lucency to intracranial translucency: towards
10. Morris JE, Rickard S, Paley MN, et  al. The
city of Barcelona from 1992 to 2006. Prenat the early detection of spina bifida. Ultrasound
value of in-utero magnetic resonance imag-
Diagn. 2011;31(12):1184–1188. Obstet Gynecol. 2010;35(2):133–138.
ing in ultrasound diagnosed foetal isolated
28. Yazici LE, Malatyalioglu E, Sakinci M, et  al. 48. Chaoui R, Nicolaides KH. Detecting open
cerebral ventriculomegaly. Clin Radiol.
Chromosomal anomalies and additional spina bifida at the 11-13–week scan by assess-
2007;62(2):140–144.
sonographic findings in fetuses with open ing intracranial translucency and the posterior
11. Gaglioti P, Oberto M, Todros T. The sig-
neural tube defects. Arch Gynecol Obstet. brain region: mid-sagittal or axial plane? Ultra-
nificance of fetal ventriculomegaly: etiology,
2012;286(6):1393–1398. sound Obstet Gynecol. 2011;38(6):609–612.
short- and long-term outcomes. Prenat Diagn.
29. Obeidi N, Russell N, Higgins JR, O’Donoghue 49. Meller C, Aiello H, Otano L. Sonographic
2009;29(4):381–388.
K. The natural history of anencephaly. Prenat detection of open spina bifida in the first tri-
12. Melchiorre K, Bhide A, Gika AD, et al. Counsel-
Diagn. 2010;30(4):357–360. mester: review of the literature. Childs Nerv
ing in isolated mild fetal ventriculomegaly. Ultra-
30. Copp AJ, Stanier P, Greene ND. Neu- Syst. 2017;33(7):1101–1106.
sound Obstet Gynecol. 2009;34(2):212–224.
ral tube defects: recent advances, unsolved 50. Maruotti GM, Saccone G, D’Antonio F, et al.
13. Bar-Yosef O, Barzilay E, Dorembus S, et  al.
questions, and controversies. Lancet Neurol. Diagnostic accuracy of intracranial trans-
Neurodevelopmental outcome of isolated
2013;12(8):799–810. lucency in detecting spina bifida: a system-
ventriculomegaly: a prospective cohort study.
31. Nayak A, Sharma S, Vadher RK, et al. Congeni- atic review and meta-analysis. Prenat Diagn.
Prenat Diagn. 2017;37(8):764–768.
tal interparietal encephalocele: a case report. J 2016;36(11):991–996.
14. Coleman BG, Langer JE, Horii SC. The
Clin Diagn Res. 2015;9(4):PD09–PD10. 51. Chen FC, Gerhardt J, Entezami M, et  al.
diagnostic features of spina bifida: the role
32. Mahapatra AK. Giant encephalocele: a Detection of spina bifida by first trimester
of ultrasound. Fetal Diagn Ther. 2015;37(3):
study of 14 patients. Pediatr Neurosurg. screening—results of the prospective mul-
179–196.
2011;47(6):406–411. ticenter Berlin IT study. Ultraschall Med.
15. Mirsky DM, Schwartz ES, Zarnow DM.
33. Gao Z, Massimi L, Rogerio S, et  al. Ver- 2017;38(2):151–157.
Diagnostic features of myelomeningocele: the
tex cephaloceles: a review. Childs Nerv Syst. 52. Loureiro T, Ushakov F, Maiz N, et al. Lateral
role of ultrafast fetal MRI. Fetal Diagn Ther.
2014;30(1):65–72. ventricles in fetuses with aneuploidies at 11-13
2015;37(3):219–225.
34. Stoll C, Dott B, Alembik Y, Roth MP. Asso- weeks’ gestation. Ultrasound Obstet Gynecol.
16. McLone DG, Dias MS. The Chiari II malfor-
ciated malformations among infants with 2012;40(3):282–287.
mation: cause and impact. Childs Nerv Syst.
neural tube defects. Am J Med Genet A. 53. Aaronson OS, Hernanz-Schulman M,
2003;19(7-8):540–550.
2011;155A(3):565–568. Bruner JP, et  al. Myelomeningocele: prena-
17. Stiefel D, Copp AJ, Meuli M. Fetal spina
35. Sepulveda W, Wong AE, Andreeva E, et  al. tal evaluation–comparison between trans-
bifida in a mouse model: loss of neural func-
Sonographic spectrum of first-trimester fetal abdominal US and MR imaging. Radiology.
tion in utero. J Neurosurg. 2007;106(suppl
cephalocele: review of 35 cases. Ultrasound 2003;227(3):839–843.
3):213–221.
Obstet Gynecol. 2015;46(1):29–33.

304.e1
30
30
44
.e
e
.2 References
54. Appasamy M, Roberts D, Pilling D, Bux-
ton N. Antenatal ultrasound and magnetic
resonance imaging in localizing the level of
lesion in spina bifida and correlation with
postnatal outcome. Ultrasound Obstet Gynecol.
2006;27(5):530–536.
55. Glenn OA, Barkovich J. Magnetic reso-
nance imaging of the fetal brain and spine:
an increasingly important tool in prenatal
diagnosis: part 2. AJNR Am J Neuroradiol.
2006;27(9):1807–1814.
56. Zalel Y, Lehavi O, Aizenstein O, Achiron R.
Development of the fetal spinal cord: time of
ascendance of the normal conus medullaris as
detected by sonography. J Ultrasound Med.
2006;25(11):1397–1401; quiz 402–403.
57. Perlitz Y, Izhaki I, Ben-Ami M. Sonographic
evaluation of the fetal conus medullaris at
20 to 24 weeks’ gestation. Prenat Diagn.
2010;30(9):862–864.
58. Blondiaux E, Katorza E, Rosenblatt J, et  al.
Prenatal US evaluation of the spinal cord using
high-frequency linear transducers. Pediatr
Radiol. 2011;41(3):374–383.
59. Has R, Yuksel A, Buyukkurt S, et al. Prenatal
diagnosis of diastematomyelia: presentation of
eight cases and review of the literature. Ultra-
sound Obstet Gynecol. 2007;30(6):845–849.
60. Dubourg C, Bendavid C, Pasquier L, et  al.
Holoprosencephaly. Orphanet J Rare Dis.
2007;2:8.
61. Volpe P, Campobasso G, De Robertis V, Rem-
bouskos G. Disorders of prosencephalic devel-
opment. Prenat Diagn. 2009;29(4):340–354.
62. De Catte L, De Keersmaeker B, Claus F.
Prenatal neurologic anomalies: sonographic
diagnosis and treatment. Paediatr Drugs.
2012;14(3):143–155.
63. Engels AC, Joyeux L, Brantner C, et al. Sono-
graphic detection of central nervous system
defects in the first trimester of pregnancy. Pre-
nat Diagn. 2016;36(3):266–273.
64. Timor-Tritsch IE, Monteagudo A, Santos R.
Three-dimensional inversion rendering in the
first- and early second-trimester fetal brain: its
use in holoprosencephaly. Ultrasound Obstet
Gynecol. 2008;32(6):744–750.
65. Pulitzer SB, Simon EM, Crombleholme
TM, Golden JA. Prenatal MR findings of
the middle interhemispheric variant of holo-
prosencephaly. AJNR Am J Neuroradiol.
2004;25(6):1034–1036.
66. Plawner LL, Delgado MR, Miller VS, et  al.
Neuroanatomy of holoprosencephaly as pre-
dictor of function: beyond the face predicting
the brain. Neurology. 2002;59(7):1058–1066.
67. Stashinko EE, Clegg NJ, Kammann HA, et al.
A retrospective survey of perinatal risk factors
of 104 living children with holoprosencephaly.
Am J Med Genet A. 2004;128A(2):114–119.
68. Winter TC, Kennedy AM, Byrne J, Woodward
PJ. The cavum septi pellucidi: why is it impor-
tant? J Ultrasound Med. 2010;29(3):427–444.
69. Vinals F, Correa F, Goncalves-Pereira PM.
Anterior and posterior complexes: a step
towards improving neurosonographic screen-
ing of midline and cortical anomalies. Ultra-
sound Obstet Gynecol. 2015;46(5):585–594.
70. Pilu G, Segata M, Ghi T, et al. Diagnosis of
midline anomalies of the fetal brain with the
three-dimensional median view. Ultrasound
Obstet Gynecol. 2006;27(5):522–529.
71. Merz E. Targeted depiction of the fetal cor-
pus callosum with 3D-ultrasound. Ultraschall
Med. 2010;31(5):441.
72. Hanna RM, Marsh SE, Swistun D, et  al.
Distinguishing 3 classes of corpus callosal
abnormalities in consanguineous families.
Neurology. 2011;76(4):373–382.
73. Glass HC, Shaw GM, Ma C, Sherr EH.
Agenesis of the corpus callosum in California
1983–2003: a population-based study. Am J
Med Genet A. 2008;146A(19):2495–2500.
74. Richards LJ, Plachez C, Ren T. Mechanisms
regulating the development of the corpus cal-
losum and its agenesis in mouse and human.
Clin Genet. 2004;66(4):276–289.
75. Santo S, D’Antonio F, Homfray T, et  al.
Counseling in fetal medicine: agenesis of the
corpus callosum. Ultrasound Obstet Gynecol.
2012;40(5):513–521.
76. Malinger G, Lev D, Oren M, Lerman-Sagie
T. Non-visualization of the cavum septi pel-
lucidi is not synonymous with agenesis of the
corpus callosum. Ultrasound Obstet Gynecol.
2012;40(2):165–170.
77. Griffiths PD, Batty R, Connolly DA, Reeves
MJ. Effects of failed commissuration on
the septum pellucidum and fornix: impli-
cations for fetal imaging. Neuroradiology.
2009;51(5):347–356.
78. Shen O, Gelot AB, Moutard ML, et  al.
Abnormal shape of the cavum septi pellu-
cidi: an indirect sign of partial agenesis of the
corpus callosum. Ultrasound Obstet Gynecol.
2015;46(5):595–599.
79. Karl K, Esser T, Heling KS, Chaoui R. Cavum
septi pellucidi (CSP) ratio: a marker for partial
agenesis of the fetal corpus callosum. Ultra-
sound Obstet Gynecol. 2017;50(3):336–341.
80. Volpe P, Paladini D, Resta M, et al. Characteris-
tics, associations and outcome of partial agenesis
of the corpus callosum in the fetus. Ultrasound
Obstet Gynecol. 2006;27(5):509–516.
81. Ghi T, Carletti A, Contro E, et  al. Prenatal
diagnosis and outcome of partial agenesis and
hypoplasia of the corpus callosum. Ultrasound
Obstet Gynecol. 2010;35(1):35–41.
82. Chadie A, Radi S, Trestard L, et al. Neurode-
velopmental outcome in prenatally diagnosed
isolated agenesis of the corpus callosum. Acta
Paediatr. 2008;97(4):420–424.
83. Conway RL, Pressman BD, Dobyns WB,
et  al. Neuroimaging findings in macroceph-
aly-capillary malformation: a longitudinal
study of 17 patients. Am J Med Genet A.
2007;143A(24):2981–3008.
84. Schupper A, Konen O, Halevy A, et al. Thick
corpus callosum in children. J Clin Neurol.
2017;13(2):170–174.
85. Dobrocky T, Ebner L, Liniger B, et  al. Pre-
and postnatal imaging of Pai syndrome with
spontaneous intrauterine closure of a fron-
tal cephalocele. Pediatr Radiol. 2015;45(6):
936–940.
86. Sotiriadis A, Makrydimas G. Neurodevel-
opment after prenatal diagnosis of isolated
agenesis of the corpus callosum: an integrative
review. Am J Obstet Gynecol. 2012;206(4):337.
e1–e5.
87. Craven I, Bradburn MJ, Griffiths PD. Ante-
natal diagnosis of agenesis of the corpus cal-
losum. Clin Radiol. 2015;70(3):248–253.
88. Koob M, Weingertner AS, Gasser B, et  al.
Thick corpus callosum: a clue to the diagno-
sis of fetal septopreoptic holoprosencephaly?
Pediatr Radiol. 2012;42(7):886–890.
89. Lerman-Sagie T, Ben-Sira L, Achiron R, et al.
Thick fetal corpus callosum: an ominous sign?
Ultrasound Obstet Gynecol. 2009;34(1):55–61.
90. Mitter C, Kasprian G, Brugger PC, Prayer D.
Three-dimensional visualization of fetal white-
matter pathways in utero. Ultrasound Obstet
Gynecol. 2011;37(2):252–253.
91. Fratelli N, Papageorghiou AT, Prefumo F,
et al. Outcome of prenatally diagnosed agen-
esis of the corpus callosum. Prenat Diagn.
2007;27(6):512–517.
92. Shevell MI. Clinical and diagnostic profile of
agenesis of the corpus callosum. J Child Neu-
rol. 2002;17(12):896–900.
93. Moutard ML, Kieffer V, Feingold J, et  al.
Isolated corpus callosum agenesis: a ten-year
follow-up after prenatal diagnosis (how are the
children without corpus callosum at 10 years
of age?). Prenat Diagn. 2012;32(3):277–283.
94. D’Antonio F, Pagani G, Familiari A, et  al.
Outcomes associated with isolated agenesis of
the corpus callosum: a meta-analysis. Pediat-
rics. 2016;138(3): pii: e20160445.
95. Belhocine O, Andre C, Kalifa G, Adams-
baum C. Does asymptomatic septal agenesis
exist? A review of 34 cases. Pediatr Radiol.
2005;35(4):410–418.
96. Malinger G, Lev D, Kidron D, et al. Differ-
ential diagnosis in fetuses with absent sep-
tum pellucidum. Ultrasound Obstet Gynecol.
2005;25(1):42–49.
97. Bault JP, Salomon LJ, Guibaud L, Achiron
R. Role of three-dimensional ultrasound mea-
surement of the optic tract in fetuses with
agenesis of the septum pellucidum. Ultrasound
Obstet Gynecol. 2011;37(5):570–575.
98. Damaj L, Bruneau B, Ferry M, et al. Pediatric
outcome of children with the prenatal diag-
nosis of isolated septal agenesis. Prenat Diagn.
2010;30(12-13):1143–1150.
99. Robinson AJ, Blaser S, Toi A, et al. The fetal
cerebellar vermis: assessment for abnormal
development by ultrasonography and mag-
netic resonance imaging. Ultrasound Q.
2007;23(3):211–223.
100. Ten Donkelaar HJ, Lammens M. Develop-
ment of the human cerebellum and its disor-
ders. Clin Perinatol. 2009;36(3):513–530.
101. Robinson AJ, Goldstein R. The cisterna magna
septa: vestigial remnants of Blake’s pouch and
a potential new marker for normal develop-
ment of the rhombencephalon. J Ultrasound
Med. 2007;26(1):83–95.
102. Achiron R, Kivilevitch Z, Lipitz S, et  al.
Development of the human fetal pons: in
utero ultrasonographic study. Ultrasound
Obstet Gynecol. 2004;24(5):506–510.
103. Zalel Y, Yagel S, Achiron R, et  al. Three-
dimensional ultrasonography of the fetal
vermis at 18 to 26 weeks’ gestation: time of
appearance of the primary fissure. J Ultrasound
Med. 2009;28(1):1–8.
104. Malinger G, Lev D, Lerman-Sagie T. The fetal
cerebellum. Pitfalls in diagnosis and manage-
ment. Prenat Diagn. 2009;29(4):372–380.
105. Paladini D, Volpe P. Posterior fossa and ver-
mian morphometry in the characterization of
fetal cerebellar abnormalities: a prospective
three-dimensional ultrasound study. Ultra-
sound Obstet Gynecol. 2006;27(5):482–489.
106. Tepper R, Kidron D, Hershkovitz R. Sono-
graphic measurements of the fetal fastigium
between 20 and 40 weeks’ gestation. J Ultra-
sound Med. 2009;28(12):1657–1661.
107. Gandolfi Colleoni G, Contro E, Carletti A,
et al. Prenatal diagnosis and outcome of fetal
posterior fossa fluid collections. Ultrasound
Obstet Gynecol. 2012;39(6):625–631.

108. Prayer D, Brugger PC, Kasprian G. The pedi-
atric posterior fossa: fetal MRI. Neuroradiol J.
2007;20(4):403–409.
109. Limperopoulos C, Robertson Jr RL, et  al.
How accurately does current fetal imaging
identify posterior fossa anomalies? AJR Am J
Roentgenol. 2008;190(6):1637–1643.
110. Chapman T, Mahalingam S, Ishak GE, et al.
Diagnostic imaging of posterior fossa anoma-
lies in the fetus and neonate: part 1, normal
anatomy and classification of anomalies. Clin
Imaging. 2015;39(1):1–8.
111. Volpe P, Contro E, De Musso F, et al. Brain-
stem-vermis and brainstem-tentorium angles
allow accurate categorization of fetal upward
rotation of cerebellar vermis. Ultrasound
Obstet Gynecol. 2012;39(6):632–635.
112. Zimmer EZ, Lowenstein L, Bronshtein
M, et  al. Clinical significance of isolated
mega cisterna magna. Arch Gynecol Obstet.
2007;276(5):487–490.
113. Liu Z, Han J, Fu F, et al. Outcome of isolated
enlarged cisterna magna identified in utero:
experience at a single medical center in mainland
China. Prenat Diagn. 2017;37(6):575–582.
114. Long A, Moran P, Robson S. Outcome of
fetal cerebral posterior fossa anomalies. Prenat
Diagn. 2006;26(8):707–710.
115. Paladini D, Quarantelli M, Pastore G, et  al.
Abnormal or delayed development of the pos-
terior membranous area of the brain: anatomy,
ultrasound diagnosis, natural history and out-
come of Blake’s pouch cyst in the fetus. Ultra-
sound Obstet Gynecol. 2012;39(3):279–287.
116. Parisi MA, Dobyns WB. Human malforma-
tions of the midbrain and hindbrain: review
and proposed classification scheme. Mol Genet
Metab. 2003;80(1-2):36–53.
117. Ghi T, Contro E, De Musso F, et  al. Nor-
mal morphometry of fetal posterior fossa at
midtrimester: brainstem-tentorium angle
and brainstem-vermis angle. Prenat Diagn.
2012;32(5):440–443.
118. D’Antonio F, Khalil A, Garel C, et al. System-
atic review and meta-analysis of isolated poste-
rior fossa malformations on prenatal ultrasound
imaging (part 1): nomenclature, diagnostic
accuracy and associated anomalies. Ultrasound
Obstet Gynecol. 2016;47(6):690–697.
119. Griffiths PD, Brackley K, Bradburn M, et  al.
Anatomical subgroup analysis of the MERID-
IAN cohort: posterior fossa abnormalities. Ultra-
sound Obstet Gynecol. 2017;50(6):745–752.
120. D’Antonio F, Khalil A, Garel C, et al. System-
atic review and meta-analysis of isolated poste-
rior fossa malformations on prenatal imaging
(part 2): neurodevelopmental outcome. Ultra-
sound Obstet Gynecol. 2016;48(1):28–37.
121. Klein O, Pierre-Kahn A, Boddaert N, et  al.
Dandy-Walker malformation: prenatal diag-
nosis and prognosis. Childs Nerv Syst. 2003;19
(7-8):484–489.
122. Main SL, Kulesza RJ. Repeated prenatal
exposure to valproic acid results in cer-
ebellar hypoplasia and ataxia. Neuroscience.
2017;340:34–47.
123. Poretti A, Limperopoulos C, Roulet-Perez
E, et  al. Outcome of severe unilateral cer-
ebellar hypoplasia. Dev Med Child Neurol.
2010;52(8):718–724.
124. Poretti A, Boltshauser E, Huisman TA. Pre-
natal cerebellar disruptions: neuroimaging
spectrum of findings in correlation with likely
mechanisms and etiologies of injury. Neuroim-
aging Clin North Am. 2016;26(3):359–372.
125. Malinger G, Zahalka N, Kidron D, et al. Fatal
outcome following foetal cerebellar haemor-
rhage associated with placental thrombosis.
Eur J Paediatr Neurol. 2006;10(2):93–96.
126. Bolduc ME, Limperopoulos C. Neurodevel-
opmental outcomes in children with cerebellar
malformations: a systematic review. Dev Med
Child Neurol. 2009;51(4):256–267.
127. Massoud M, Cagneaux M, Garel C, et al. Pre-
natal unilateral cerebellar hypoplasia in a series
of 26 cases: significance and implications for
prenatal diagnosis. Ultrasound Obstet Gynecol.
2014;44(4):447–454.
128. Pugash D, Oh T, Godwin K, et  al. Sono-
graphic ‘molar tooth’ sign in the diagnosis of
Joubert syndrome. Ultrasound Obstet Gynecol.
2011;38(5):598–602.
129. Vilboux T, Doherty DA, Glass IA, et  al.
Molecular genetic findings and clinical corre-
lations in 100 patients with Joubert syndrome
and related disorders prospectively evaluated
at a single center. Genet Med. 2017;19(8):
875–882.
130. Parisi MA. Clinical and molecular features
of Joubert syndrome and related disor-
ders. Am J Med Genet C Semin Med Genet.
2009;151C(4):326–340.
131. Quarello E, Molho M, Garel C, et al. Prena-
tal abnormal features of the fourth ventricle
in Joubert syndrome and related disorders.
Ultrasound Obstet Gynecol. 2014;43(2):
227–232.
132. Limperopoulos C, Robertson RL, Estroff JA,
et al. Diagnosis of inferior vermian hypoplasia
by fetal magnetic resonance imaging: potential
pitfalls and neurodevelopmental outcome. Am
J Obstet Gynecol. 2006;194(4):1070–1076.
133. Sukhudyan B, Jaladyan V, Melikyan G, et al.
Gomez-Lopez-Hernandez syndrome: reap-
praisal of the diagnostic criteria. Eur J Pediatr.
2010;169(12):1523–1528.
134. Ishak GE, Dempsey JC, Shaw DW, et  al.
Rhombencephalosynapsis: a hindbrain malfor-
mation associated with incomplete separation
of midbrain and forebrain, hydrocephalus and
a broad spectrum of severity. Brain. 2012;135
(Pt 5):1370–1386.
135. Napolitano M, Righini A, Zirpoli S, et  al.
Prenatal magnetic resonance imaging of
rhombencephalosynapsis and associated brain
anomalies: report of 3 cases. J Comput Assist
Tomogr. 2004;28(6):762–765.
136. McAuliffe F, Chitayat D, Halliday W, et  al.
Rhombencephalosynapsis: prenatal imaging
and autopsy findings. Ultrasound Obstet Gyne-
col. 2008;31(5):542–548.
137. Poretti A, Alber FD, Burki S, et  al. Cog-
nitive outcome in children with rhomb-
encephalosynapsis. Eur J Paediatr Neurol.
2009;13(1):28–33.
138. Barkovich AJ, Guerrini R, Kuzniecky RI, et al.
A developmental and genetic classification
for malformations of cortical development:
update 2012. Brain. 2012;135(Pt 5):1348–
1369.
139. Poretti A, Boltshauser E, Huisman TA. Con-
genital brain abnormalities: an update on
malformations of cortical development and
infratentorial malformations. Semin Neurol.
2014;34(3):239–248.
140. Malinger G, Lev D, Lerman-Sagie T. Normal
and abnormal fetal brain development dur-
ing the third trimester as demonstrated by
neurosonography. Eur J Radiol. 2006;57(2):
226–232.
References 304.e3
141. Malinger G, Kidron D, Schreiber L, et al. Pre-
natal diagnosis of malformations of cortical
development by dedicated neurosonography.
Ultrasound Obstet Gynecol. 2007;29(2):178–
191.
142. von der Hagen M, Pivarcsi M, Liebe J, et al.
Diagnostic approach to microcephaly in
childhood: a two-center study and review
of the literature. Dev Med Child Neurol.
2014;56(8):732–741.
143. Morris JK, Rankin J, Garne E, et  al. Preva-
lence of microcephaly in Europe: population
based study. BMJ. 2016;354:i4721.
144. Guibaud L, Lacalm A. Diagnostic imaging
tools to elucidate decreased cephalic biometry
and fetal microcephaly: a systematic analysis of
the central nervous system. Ultrasound Obstet
Gynecol. 2016;48(1):16–25.
145. Winden KD, Yuskaitis CJ, Poduri A. Mega-
lencephaly and macrocephaly. Semin Neurol.
2015;35(3):277–287.
146. Pavone P, Pratico AD, Rizzo R, et al. A clinical
review on megalencephaly: a large brain as a
possible sign of cerebral impairment. Medicine
(Baltimore). 2017;96(26):e6814.
147. Mirzaa GM, Conway RL, Gripp KW, et al. Mega-
lencephaly-capillary malformation (MCAP)
and megalencephaly-polydactyly-polymicro-
gyria-hydrocephalus (MPPH) syndromes: two
closely related disorders of brain overgrowth
and abnormal brain and body morpho-
genesis. Am J Med Genet A. 2012;158A(2):
269–291.
148. De Keersmaecker B, Van Esch H, Van Schou-
broeck D, et al. Prenatal diagnosis of MPPH
syndrome. Prenat Diagn. 2013;33(3):292–
295.
149. Guerrini R, Dobyns WB. Malforma-
tions of cortical development: clinical fea-
tures and genetic causes. Lancet Neurol.
2014;13(7):710–726.
150. Fong KW, Ghai S, Toi A, et  al. Prenatal
ultrasound findings of lissencephaly associ-
ated with Miller-Dieker syndrome and com-
parison with pre- and postnatal magnetic
resonance imaging. Ultrasound Obstet Gynecol.
2004;24(7):716–723.
151. Abdel Razek AA, Kandell AY, Elsorogy LG,
et  al. Disorders of cortical formation: MR
imaging features. AJNR Am J Neuroradiol.
2009;30(1):4–11.
152. Strigini F, Valleriani A, Cecchi M, et al. Pre-
natal ultrasound and magnetic resonance
imaging features in a fetus with Walker-
Warburg syndrome. Ultrasound Obstet Gyne-
col. 2009;33(3):363–365.
153. Andrade CS, Leite Cda C. Malformations of
cortical development: current concepts and
advanced neuroimaging review. Arq Neurop-
siquiatr. 2011;69(1):130–138.
154. Barkovich AJ. Current concepts of poly-
microgyria. Neuroradiology. 2010;52(6):
479–487.
155. Leventer RJ, Jansen A, Pilz DT, et al. Clinical
and imaging heterogeneity of polymicrogyria:
a study of 328 patients. Brain. 2010;133(Pt
5):1415–1427.
156. Guibaud L. Contribution of fetal cerebral
MRI for diagnosis of structural anomalies.
Prenat Diagn. 2009;29(4):420–433.
157. Howe DT, Rankin J, Draper ES. Schizen-
cephaly prevalence, prenatal diagnosis and
clues to etiology: a register-based study.
Ultrasound Obstet Gynecol. 2012;39(1):
75–82.
30
30
44
.e.4 References
158. Beke A, Latkoczy K, Nagy GR, et  al. Com-
parison of prevalence of toxoplasma and
cytomegalovirus infection in cases with fetal
ultrasound markers in the second trimester
of pregnancy. Prenat Diagn. 2011;31(10):
945–948.
159. Manicklal S, Emery VC, Lazzarotto T,
et  al. The ‘silent’ global burden of congeni-
tal cytomegalovirus. Clin Microbiol Rev.
2013;26(1):86–102.
160. de Vries JJ, van Zwet EW, Dekker FW, et al.
The apparent paradox of maternal seroposi-
tivity as a risk factor for congenital cyto-
megalovirus infection: a population-based
prediction model. Rev Med Virol. 2013;23(4):
241–249.
161. Guerra B, Simonazzi G, Puccetti C, et  al.
Ultrasound prediction of symptomatic con-
genital cytomegalovirus infection. Am J Obstet
Gynecol. 2008;198(4):380.e1–e7.
162. Adler SP. Screening for cytomegalovirus
during pregnancy. Infect Dis Obstet Gynecol.
2011;1–9:2011.
163. Adler SP, Nigro G. Prevention of maternal-
fetal transmission of cytomegalovirus. Clin
Infect Dis. 2013;57(suppl 4):S189–S192.
164. Picone O, Teissier N, Cordier AG, et  al.
Detailed in utero ultrasound description of 30
cases of congenital cytomegalovirus infection.
Prenat Diagn. 2014;34(6):518–524.
165. Teissier N, Fallet-Bianco C, Delezoide AL,
et al. Cytomegalovirus-induced brain malfor-
mations in fetuses. J Neuropathol Exp Neurol.
2014;73(2):143–158.
166. Leyder M, Vorsselmans A, Done E, et al. Pri-
mary maternal cytomegalovirus infections:
accuracy of fetal ultrasound for predicting
sequelae in offspring. Am J Obstet Gynecol.
2016;215(5):638.e1–e8.
167. Benoist G, Salomon LJ, Jacquemard F, et al.
The prognostic value of ultrasound abnor-
malities and biological parameters in blood of
fetuses infected with cytomegalovirus. BJOG.
2008;115(7):823–829.
168. Malinger G, Lev D, Zahalka N, et  al. Fetal
cytomegalovirus infection of the brain: the
spectrum of sonographic findings. AJNR Am
J Neuroradiol. 2003;24(1):28–32.
169. Doneda C, Parazzini C, Righini A, et  al.
Early cerebral lesions in cytomegalovirus
infection: prenatal MR imaging. Radiology.
2010;255(2):613–621.
170. Lipitz S, Yinon Y, Malinger G, et al. Risk of
cytomegalovirus-associated sequelae in rela-
tion to time of infection and findings on
prenatal imaging. Ultrasound Obstet Gynecol.
2013;41(5):508–514.
171. Cannie MM, Devlieger R, Leyder M, et  al.
Congenital cytomegalovirus infection:
contribution and best timing of prenatal
MR imaging. Eur Radiol. 2016;26(10):
3760–3769.
172. Breugelmans M, Naessens A, Foulon W. Pre-
vention of toxoplasmosis during pregnancy–
an epidemiologic survey over 22 consecutive
years. J Perinat Med. 2004;32(3):211–214.
173. Avci ME, Arslan F, Ciftci S, et  al. Role
of spiramycin in prevention of fetal toxo-
plasmosis. J Matern Fetal Neonatal Med.
2016;29(13):2073–2076.
174. de Oliveira Azevedo CT, do Brasil PE, Guida
L, et al. Performance of polymerase chain reac-
tion analysis of the amniotic fluid of pregnant
women for diagnosis of congenital toxoplas-
mosis: a systematic review and meta-analysis.
PLoS One. 2016;11(4).e0149938.
175. Malinger G, Werner H, Rodriguez Leonel
JC, et  al. Prenatal brain imaging in congeni-
tal toxoplasmosis. Prenat Diagn. 2011;31(9):
881–886.
176. Abboud P, Harika G, Saniez D, et al. [Ultra-
sonic signs of fetal toxoplasmosis. Review of
the literature]. J Gynecol Obstet Biol Reprod
(Paris). 1995;24(7):733–738.
177. Gay-Andrieu F, Marty P, Pialat J, et al. Fetal
toxoplasmosis and negative amniocentesis:
necessity of an ultrasound follow-up. Prenat
Diagn. 2003;23(7):558–560.
178. Sarno M, Aquino M, Pimentel K, et al. Pro-
gressive lesions of central nervous system in
microcephalic fetuses with suspected congeni-
tal Zika virus syndrome. Ultrasound Obstet
Gynecol. 2017;50(6):717–722.
179. Carvalho FH, Cordeiro KM, Peixoto AB, et al.
Associated ultrasonographic findings in fetuses
with microcephaly because of suspected Zika
virus (ZIKV) infection during pregnancy. Pre-
nat Diagn. 2016;36(9):882–887.
180. Vouga M, Baud D. Imaging of congenital
Zika virus infection: the route to identifi-
cation of prognostic factors. Prenat Diagn.
2016;36(9):799–811.
181. 
Soares de Oliveira-Szejnfeld P, Levine D,
et  al. Congenital brain abnormalities and
Zika virus: what the radiologist can expect
to see prenatally and postnatally. Radiology.
2016;281(1):203–218.
182. Elchalal U, Yagel S, Gomori JM, et  al. Fetal
intracranial hemorrhage (fetal stroke): does
grade matter? Ultrasound Obstet Gynecol.
2005;26(3):233–243.
183. Bauder F, Beinder E, Arlettaz R, et al. Intra-
uterine subdural hemorrhage in a preterm
neonate possibly associated with maternal
low-molecular weight heparin treatment. J
Perinatol. 2009;29(7):521–523.
184. Carletti A, Colleoni GG, Perolo A, et al. Pre-
natal diagnosis of cerebral lesions acquired
in utero and with a late appearance. Prenat
Diagn. 2009;29(4):389–395.
185. Papile LA, Burstein J, Burstein R, Koffler H.
Incidence and evolution of subependymal and
intraventricular hemorrhage: a study of infants
with birth weights less than 1,500 gm. J Pedi-
atr. 1978;92(4):529–534.
186. Williams T, Wilkinson AG, Kandasamy J,
et al. Antenatal diagnosis of intracranial haem-
orrhage and porencephalic cyst. BMJ Case Rep.
2015;2015:pii: bcr2014209130.
187. Prayer D, Brugger PC, Kasprian G, et al. MRI
of fetal acquired brain lesions. Eur J Radiol.
2006;57(2):233–249.
188. Manganaro L, Bernardo S, La Barbera L,
et al. Role of foetal MRI in the evaluation of
ischaemic-haemorrhagic lesions of the foe-
tal brain. J Perinat Med. 2012;40(4):419–
426.
189. Johansson PA, Dziegielewska KM, Liddelow
SA, Saunders NR. The blood-CSF barrier
explained: when development is not immatu-
rity. Bioessays. 2008;30(3):237–248.
190. Steggerda SJ, De Bruine FT, van den Berg-
Huysmans AA, et  al. Small cerebellar hem-
orrhage in preterm infants: perinatal and
postnatal factors and outcome. Cerebellum.
2013;12(6):794–801.
191. Haines KM, Wang W, Pierson CR. Cerebel-
lar hemorrhagic injury in premature infants
occurs during a vulnerable developmental
period and is associated with wider neu-
ropathology. Acta Neuropathol Commun.
2013;1:69.
192. Hayashi M, Poretti A, Gorra M, et al. Prena-
tal cerebellar hemorrhage: fetal and postnatal
neuroimaging findings and postnatal out-
come. Pediatr Neurol. 2015;52(5):529–534.
193. Martino F, Malova M, Cesaretti C, et  al.
Prenatal MR imaging features of isolated
cerebellar haemorrhagic lesions. Eur Radiol.
2016;26(8):2685–2696.
194. Hagmann CF, Schmitt-Mechelke T, Caduff
JH, Berger TM. Fetal intracranial injuries in
a preterm infant after maternal motor vehicle
accident: a case report. Pediatr Crit Care Med.
2004;5(4):396–398.
195. Paladini D, Sglavo G, Quarantelli M, et  al.
Large infratentorial subdural hemorrhage
diagnosed by ultrasound and MRI in a sec-
ond-trimester fetus. Ultrasound Obstet Gyne-
col. 2005;26(7):789–791; discussion 791.
196. Strigini FA, Nardini V, Carmignani A, Val-
leriani AM. Second-trimester diagnosis of
intracranial vascular anomalies in a fetus
with subdural hemorrhage. Prenat Diagn.
2004;24(1):31–34.
197. Ghi T, Simonazzi G, Perolo A, et al. Outcome of
antenatally diagnosed intracranial hemorrhage:
case series and review of the literature. Ultrasound
Obstet Gynecol. 2003;22(2):121–130.
198. Barozzino T, Sgro M, Toi A, et al. Fetal bilat-
eral subdural haemorrhages. Prenatal diag-
nosis and spontaneous resolution by time of
delivery. Prenat Diagn. 1998;18(5):496–503.
199. Tiller H, Kamphuis MM, Flodmark O, et al.
Fetal intracranial haemorrhages caused by fetal
and neonatal alloimmune thrombocytopenia:
an observational cohort study of 43 cases from
an international multicentre registry. BMJ
Open. 2013;3(3).
200. Delbos F, Bertrand G, Croisille L, et al. Fetal
and neonatal alloimmune thrombocytopenia:
predictive factors of intracranial hemorrhage.
Transfusion. 2016;56(1):59–66;quiz 58.
201. Radder CM, Brand A, Kanhai HH. Will it
ever be possible to balance the risk of intra-
cranial haemorrhage in fetal or neonatal allo-
immune thrombocytopenia against the risk of
treatment strategies to prevent it? Vox Sang.
2003;84(4):318–325.
202. Kamphuis MM, Oepkes D. Fetal and neonatal
alloimmune thrombocytopenia: prenatal inter-
ventions. Prenat Diagn. 2011;31(7):712–719.
203. Pacheco LD, Berkowitz RL, Moise Jr KJ,
et  al. Fetal and neonatal alloimmune throm-
bocytopenia: a management algorithm
based on risk stratification. Obstet Gynecol.
2011;118(5):1157–1163.
204. Scheffer PG, Ait Soussan A, Verhagen OJ, et al.
Noninvasive fetal genotyping of human platelet
antigen-1a. BJOG. 2011;118(11):1392–1395.
205. van den Akker E, Oepkes D, Brand A, Kan-
hai HH. Vaginal delivery for fetuses at risk
of alloimmune thrombocytopenia? BJOG.
2006;113(7):781–783.
206. Abergel A, Lacalm A, Massoud M, et  al.
Expanding porencephalic cysts: prenatal
imaging and differential diagnosis. Fetal Diagn
Ther. 2017;41(3):226–233.
207. Pavone P, Pratico AD, Vitaliti G, et al. Hydra-
nencephaly: cerebral spinal fluid instead of
cerebral mantles. Ital J Pediatr. 2014;40:79.
208. van Gelder-Hasker MR, van Wezel-Meijler
G, de Groot L, et al. Peri- and intraventricu-
lar cerebral sonography in second- and third-
trimester high-risk fetuses: a comparison with
neonatal ultrasound and relation to neurologi-
cal development. Ultrasound Obstet Gynecol.
2003;22(2):110–120.

209. Malinger G, Lev D, Ben Sira L, et al. Congeni-
tal periventricular pseudocysts: prenatal sono-
graphic appearance and clinical implications.
Ultrasound Obstet Gynecol. 2002;20(5):447–451.
210. McInnes M, Fong K, Grin A, et al. Malforma-
tions of the fetal dural sinuses. Can J Neurol
Sci. 2009;36(1):72–77.
211. O’Rafferty C, O’Regan GM, Irvine AD, Smith
OP. Recent advances in the pathobiology and
management of Kasabach-Merritt phenom-
enon. Br J Haematol. 2015;171(1):38–51.
212. Appaduray SP, King JA, Wray A, et al. Pediat-
ric dural arteriovenous malformations. J Neu-
rosurg Pediatr. 2014;14(1):16–22.
213. Zaidi HA, Kalani MY, Spetzler RF, et  al.
Multimodal treatment strategies for com-
plex pediatric cerebral arteriovenous fis-
tulas: contemporary case series at Barrow
Neurological Institute. J Neurosurg Pediatr.
2015;15(6):615–624.
214. Gupta AK, Varma DR. Vein of Galen malforma-
tions: review. Neurol India. 2004;52(1):43–53.
215. Raybaud C. Normal and abnormal embry-
ology and development of the intracranial
vascular system. Neurosurg Clin North Am.
2010;21(3):399–426.
216. Nuutila M, Saisto T. Prenatal diagnosis of vein
of Galen malformation: a multidisciplinary chal-
lenge. Am J Perinatol. 2008;25(4):225–227.
217. Dobrosavljevic A, Dobrosavljevic B, Razna-
tovic SJ, Vranes B. The significance of 3D
power Doppler in prenatal diagnosis and
the evaluation of the anatomical structure
of vein of Galen aneurysmatic malforma-
tion: case report. Clin Exp Obstet Gynecol.
2013;40(2):300–303.
218. Wagner MW, Vaught AJ, Poretti A, et al. Vein
of Galen aneurysmal malformation: prognos-
tic markers depicted on fetal MRI. Neuroradiol
J. 2015;28(1):72–75.
219. Paladini D, Deloison B, Rossi A, et  al. Vein
of Galen Aneurysmal malformation (VGAM)
in the fetus. A retrospective analysis of peri-
natal prognostic indicators in a two-center
series of 49 cases. Ultrasound Obstet Gynecol.
2017;50(2):192–199.
220. Yan J, Wen J, Gopaul R, et al. Outcome and
complications of endovascular embolization
for vein of Galen malformations: a system-
atic review and meta-analysis. J Neurosurg.
2015;123(4):872–890.
221. Khullar D, Andeejani AM, Bulsara KR.
Evolution of treatment options for vein of
Galen malformations. J Neurosurg Pediatr.
2010;6(5):444–451.
222. Barbosa M, Mahadevan J, Weon YC, et  al.
Dural sinus malformations (DSM) with
giant lakes, in neonates and infants. Review
of 30 consecutive cases. Interv Neuroradiol.
2003;9(4):407–424.
223. Rayssiguier R, Dumont C, Flunker S, et  al.
Thrombosis of torcular herophili: diagnosis,
prenatal management, and outcome. Prenat
Diagn. 2014;34(12):1168–1175.
224. Byrd SE, Abramowicz JS, Kent P, et  al.
Fetal MR imaging of posterior intracranial
dural sinus thrombosis: a report of three
cases with variable outcomes. Pediatr Radiol.
2012;42(5):536–543.
225. Corral E, Stecher X, Malinger G, et  al.
Thrombosis of the torcular herophili in the
fetus: a series of eight cases. Prenat Diagn.
2014;34(12):1176–1181.
226. Cavalheiro S, Moron AF, Hisaba W, et al. Fetal
brain tumors. Childs Nerv Syst. 2003;19(7-
8):529–536.
227. Di Rocco F, Andre A, Roujeau T, et al. [Diag-
nosis, evolution and prognosis of prenatally
diagnosed suprasellar cysts]. Neurochirurgie.
2016;62(6):300–305.
228. Hayward R. Postnatal management and
outcome for fetal-diagnosed intra-cerebral
cystic masses and tumours. Prenat Diagn.
2009;29(4):396–401.
229. De Keersmaecker B, Ramaekers P, Claus
F, et  al. Outcome of 12 antenatally diag-
nosed fetal arachnoid cysts: case series and
review of the literature. Eur J Paediatr Neurol.
2015;19(2):114–121.
230. Chen CP. Prenatal diagnosis of arach-
noid cysts. Taiwan J Obstet Gynecol.
2007;46(3):187–198.
231. Tange Y, Aoki A, Mori K, et al. Interhemispheric
glioependymal cyst associated with agenesis of
the corpus callosum—case report. Neurol Med
Chir (Tokyo). 2000;40(10):536–542.
232. Muhler MR, Hartmann C, Werner W, et al.
Fetal MRI demonstrates glioependymal cyst
in a case of sonographic unilateral ventriculo-
megaly. Pediatr Radiol. 2007;37(4):391–395.
233. Youssef A, D’Antonio F, Khalil A, et al. Out-
come of fetuses with supratentorial extra-axial
intracranial cysts: a systematic review. Fetal
Diagn Ther. 2016;40(1):1–12.
234. Al-Holou WN, Yew AY, Boomsaad ZE,
et al. Prevalence and natural history of arach-
noid cysts in children. J Neurosurg Pediatr.
2010;5(6):578–585.
235. Sonek J, Croom C. Second trimester ultra-
sound markers of fetal aneuploidy. Clin Obstet
Gynecol. 2014;57(1):159–181.
236. Beke A, Barakonyi E, Belics Z, et al. Risk of
chromosome abnormalities in the presence
of bilateral or unilateral choroid plexus cysts.
Fetal Diagn Ther. 2008;23(3):185–191.
237. DiPietro JA, Cristofalo EA, Voegtline KM,
Crino J. Isolated prenatal choroid plexus
cysts do not affect child development. Prenat
Diagn. 2011;31(8):745–749.
238. Cooper S, Bar-Yosef O, Berkenstadt M, et al.
Prenatal evaluation, imaging features, and
neurodevelopmental outcome of prenatally
diagnosed periventricular pseudocysts. AJNR
Am J Neuroradiol. 2016;37(12):2382–2388.
239. Esteban H, Blondiaux E, Audureau E, et  al.
Prenatal features of isolated subependymal
pseudocysts associated with adverse preg-
nancy outcome. Ultrasound Obstet Gynecol.
2015;46(6):678–687.
240. Cevey-Macherel M, Forcada Guex M, Bickle
Graz M, Truttmann AC. Neurodevelop-
ment outcome of newborns with cerebral
subependymal pseudocysts at 18 and 46
months: a prospective study. Arch Dis Child.
2013;98(7):497–502.
References 304.e5
241. Kunkle B, Bae S, Singh KP, Roy D. Increased
risk of childhood brain tumors among chil-
dren whose parents had farm-related pesti-
cide exposures during pregnancy. JP J Biostat.
2014;11(2):89–101.
242. Cassart M, Bosson N, Garel C, et  al. Fetal
intracranial tumors: a review of 27 cases. Eur
Radiol. 2008;18(10):2060–2066.
243. Anselem O, Mezzetta L, Grange G, et  al.
Fetal tumors of the choroid plexus: is differ-
ential diagnosis between papilloma and car-
cinoma possible? Ultrasound Obstet Gynecol.
2011;38(2):229–232.
244. Isaacs H. Fetal brain tumors: a review of 154
cases. Am J Perinatol. 2009;26(6):453–466.
245. Bolat F, Kayaselcuk F, Tarim E, et  al. Con-
genital intracranial teratoma with massive
macrocephaly and skull rupture. Fetal Diagn
Ther. 2008;23(1):1–4.
246. Isaacs Jr H. Fetal intracranial teratoma. A
review. Fetal Pediatr Pathol. 2014;33(5-
6):289–292.
247. Iruretagoyena JI, Heiser T, Iskandar B, Shah
D. A rare malignant fetal brain tumor. Fetal
Diagn Ther. 2016;39(4):311–314.
248. Shin HJ, Kwon YJ, Park HJ, et al. An infant
with prenatally diagnosed congenital ana-
plastic astrocytoma who remains disease-
free after proton therapy. J Korean Med Sci.
2013;28(9):1394–1398.
249. Isaacs Jr H. Perinatal (fetal and neonatal)
astrocytoma: a review. Childs Nerv Syst.
2016;32(11):2085–2096.
250. Vazquez E, Castellote A, Mayolas N,
Carreras E, et al. Congenital tumours involv-
ing the head, neck and central nervous system.
Pediatr Radiol. 2009;39(11):1158–1172.
251. Severino M, Schwartz ES, Thurnher MM,
et al. Congenital tumors of the central nervous
system. Neuroradiology. 2010;52(6):531–548.
252. Vazquez E, Mayolas N, Delgado I, Higueras
T. Fetal neuroimaging: US and MRI. Pediatr
Radiol. 2009;39(suppl 3):422–435.
253. Bekiesinska-Figatowska M, Jurkiewicz E,
Duczkowski M, et al. Congenital CNS tumors
diagnosed on prenatal MRI. Neuroradiol J.
2011;24(4):477–481.
254. Prasad AN, Malinger G, Lerman-Sagie T. Pri-
mary disorders of metabolism and disturbed
fetal brain development. Clin Perinatol.
2009;36(3):621–638.
255. Brassier A, Ottolenghi C, Boddaert N,
et  al. [Prenatal symptoms and diagnosis of
inherited metabolic diseases]. Arch Pediatr.
2012;19(9):959–969.
256. Barkovich AJ. A magnetic resonance approach
to metabolic disorders in childhood. Rev Neu-
rol. 2006;43(suppl 1):S5–S16.
257. Cakmakci H, Pekcevik Y, Yis U, et al. Diag-
nostic value of proton MR spectroscopy and
diffusion-weighted MR imaging in child-
hood inherited neurometabolic brain diseases
and review of the literature. Eur J Radiol.
2010;74(3):e161–e171.
29
The Heart
CHRISTOPH WOHLMUTH AND HELENA M. GARDINER
KEY POINTS
• Current evidence suggests that prenatal diagnosis of con-
genital heart disease (CHD) reduces morbidity and mortality,
gives expectant parents time to prepare and allows planning
for delivery in a tertiary care centre.
• About 85% of babies born with CHD are born to mothers that
are ‘low risk’, suggesting that screening of the entire pregnant
population is essential to optimise detection.
• A comprehensive fetal heart examination protocol, such as
the standardised five transverse views, should be incorpo-
rated into routine second trimester anatomy screening.
• Suspicion of a cardiac defect should result in referral to a
specialist in fetal cardiology
• Management of CHD requires a multidisciplinary team includ-
ing paediatric cardiology, fetal medicine, obstetrics, neonatol-
ogy and nursing. Delivery in a tertiary care centre is required
for fetuses with duct dependent cardiac lesions or multiple
anomalies to coordinate delivery and postnatal interventions
• In almost all pregnancies with CHD, vaginal delivery can be
attempted unless there are maternal/obstetric indications
for caesarean section. Contraindications to vaginal delivery
include congenital heart block and poor cardiac function.
Induction of labour may be required if the mother does not
live near the tertiary centre or to coordinate perinatal services
such as equipment or surgical expertise.
• In selected cases of aortic or pulmonary valve stenosis
or intact atrial septum, fetal cardiac intervention may be
considered.
Overview
Introduction
Congenital heart disease (CHD) affects about 6 to 9 per 1000 live-
born infants; however, the prenatal prevalence is higher because
some affected pregnancies result in spontaneous fetal demise, and
others will be terminated.1,2 About half have major CHD defined
as requiring intervention within the first year of life, and just under
half of these have important right or left ventricular outlet or arch
obstruction and are duct dependent (often called critical CHD),
requiring early intervention soon after birth as the arterial duct
closes. The unique features of the prenatal circulation (placenta,
ductus venosus (DV), foramen ovale (FO), arterial duct) allow
compensation for structural malformations of the heart in utero,
and most fetuses with isolated CHD survive to delivery without
hydrops. It is estimated that 85% of babies born today with CHD
will survive to adult life. The population of adults with CHD is
larger than children and is growing rapidly. However, approxi-
mately one third of fetuses diagnosed with CHD also have either
additional malformations or aneuploidy. Therefore a combined
prenatal approach is essential to appreciate the extent of the prob-
lems that will require treatment in the perinatal period. Caution
should be exercised in perinatal planning because the full extent
of a fetus’ malformations, particularly those affecting the upper
airways or oesophagus, may not always be recognised prenatally. 
Screening for Congenital Heart Disease
Most national screening programs now offer routine population
screening, and this is a significant improvement on previous years
where CHD screening was only offered to the 15% of pregnancies
that were identified through patient history as ‘high risk’.3-6
When there is a history of major CHD, fetal echocardiography
at 11 to 14 weeks’ gestation may be diagnostic or reassuring, but
interpretation of first trimester heart scans requires special exper-
tise and caution. Some types of major CHD will not be detected
at 11 to 14 weeks, particularly semilunar valvar abnormalities,
atrioventricular septal defect (AVSD) (because the atrioventricu-
lar (AV) septum is still developing) and anomalies of pulmonary
venous connection.7 The increased demand for early fetal echo
arising from the nuchal translucency programme (see Chapter
19) is likely to recur from the newer tests such as cell-free DNA
testing with expanded panels (see Chapter 22). The appropriate
management of screen-positive results requires careful planning.
The optimum time to perform fetal echocardiography will remain
during the second trimester anatomy scan. We recommend all
women undergo competent cardiac screening as part of their anat-
omy scan, and if the findings suggest a structural or functional
abnormality, referral should be made to a specialist who can per-
form a full fetal echocardiogram (defined later). The individual
may have a fetal medicine or cardiology background. Although
specific counselling for cardiac disease is best done by a cardiolo-
gist, a team approach is encouraged to provide optimal advice and
streamline pregnancy and perinatal management.
In countries where the five transverse view protocol has been
supplemented with practical teaching support, there has been
an encouraging increase in the prenatal diagnosis of CHD over
the past decade. Many series now report an overall 60% prenatal
detection rate, but it still tends to be lower for lesions identified
predominantly by outflow tract abnormalities such as complete
transposition of the great arteries (TGA).8 It remains higher
(∼85%) in centres co-located with fetal medicine facilities
305
306 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
where confirmation of suspected abnormality and practical
teaching are more readily available to the screening team.9 The
majority of important cardiac defects are detectable during
pregnancy, but not all are obvious in the mid-second trimester.
Valvular abnormalities such as aortic and pulmonary stenosis
may be progressive during pregnancy and manifest during the
third trimester, so it is wise to examine the heart at follow-
up scans for fetal growth and placental lie. Isolated anoma-
lous pulmonary venous connection is notoriously difficult
to detect.  The discussion should cover the need for additional tests to
Effects of Prenatal Congenital Heart Disease
Screening
An effective screening program should provide evidence of ben-
efit. Prenatal detection of CHD confers several advantages:
1. Prenatal diagnosis allows parents to understand the nature of
the cardiac lesion and to discuss available treatment options
and the prognosis to ultimately come to an informed decision.
2. The options include expectant management, transfer of care
to a specialised centre, invasive diagnostic procedures or ter-
mination of pregnancy. It has been shown that parents pre-
fer comprehensive information to make an informed choice,
particularly including information about the quality of
life.10-12
3. The identification of extracardiac anomalies or a chromosomal
or genetic abnormality may significantly alter the progno-
sis.13,14 In selected patients, fetal cardiac intervention may alter
the natural history and improve surgical options.15-18
4. The most important reason to optimise prenatal screening is its
direct impact on postnatal outcome. A reduction in mortality,
morbidity and preoperative brain injury has been observed in
TGA, outflow tract obstruction and coarctation of the aorta
(CoA).19-23  perinatal events and cardiac surgery is changing. Neurocogni-
Examination of the Fetal Heart
In 2001, Yagel and colleagues proposed a standardised sono-
graphic approach to simplify routine prenatal cardiac screening
based on five transverse planes: abdominal situs, four-chamber,
left ventricular outflow tract (LVOT), right ventricular outflow
tract (RVOT) and three-vessel and tracheal (3VT) views.6 (Fig.
29.1). This approach has been broadly accepted by the boards
of international societies providing ultrasound guidelines.3-5 A
checklist is provided in Table 29.1. Incorporation of colour Dop-
pler in the four-chamber and 3VT views provides important addi-
tional information at routine screening.
When an abnormality is suspected in these views, the preg-
nancy should be referred to a team that can perform a compre-
hensive fetal echocardiogram, which is intended to be diagnostic.
It builds on the five transverse views and includes additional imag-
ing planes: short-axis views of the ventricles and great arteries will
demonstrate the morphology and arrangement of the four cardiac
valves; coronal views will profile the atrial appendages, atrial sep-
tum and systemic venous connections and sagittal planes to clarify
suspected abnormality of the aortic arch, such as coarctation or
interruption detected in the 3VT. The addition of colour and
pulse-wave Doppler evaluation of cardiac flows and interrogation
of the systemic and pulmonary venous systems complete the com-
prehensive diagnostic fetal echocardiogram. Most fetal medicine
specialists also routinely incorporate umbilical cord and middle
cerebral Doppler studies.  criteria have been developed and subsequently modified but have
Management of Pregnancies with Congenital
Heart Disease
After a cardiac defect and the presence of additional defects or
chromosomal abnormalities are confirmed, the family will be
offered counselling with a multidisciplinary team. Evaluation in
a fetal medicine centre provides the optimal setting for pregnancy
management (of the pregnant woman and her fetus) and perinatal
planning.
help confirm the diagnosis or diagnose suspected chromosomal
associations, pregnancy options such as termination of pregnancy
or comfort care after delivery, within local and national laws. For
ongoing pregnancies, serial sonographic evaluation, every 4 to 6
weeks, is usually arranged, with complementary imaging such as
magnetic resonance imaging (MRI) and consultation with the
postnatal surgical teams (cardiovascular and paediatric surgery) as
appropriate. At each visit, it is wise to reevaluate the anatomy,
the presence of extracardiac malformations, fetal growth and well-
being and to check for haemodynamic instability. The majority of
fetuses with congenital heart disease can be delivered vaginally at
term.
Fetuses with critical CHD or who have additional malforma-
tions (or uncertain findings) should be delivered in a facility where
the appropriate specialists are co-located to provide the expertise
required to evaluate and treat the baby in the first hours after
delivery and to avoid separation of the mother and baby. 
Neurodevelopmental Delay
An increased risk for neurodevelopmental delay has been recog-
nised for many years in children with CHD. The initial percep-
tion that this was entirely postnatal in origin, associated with
tive deficits are seen commonly in children with hypoplastic
left heart syndrome (HLHS) but are also reported in those with
TGA. Prenatal diagnosis was rare in these series, and neurocog-
nitive deficits appear to be less prevalent and less severe in chil-
dren with a prenatal diagnosis of TGA.24 Studies are ongoing,
and brain abnormalities described on prenatal and presurgical
imaging such as functional MRI may not necessarily correlate
with postnatal functional abnormality.25
Further information is required to provide guidance for
­counselling families about the likelihood of important neurode-
velopmental deficit in their fetuses with CHD.26
Important lesion-specific issues are outlined in the section on
perinatal management. 
Prenatal Therapy
Certain CHD lesions will promote discussion with families
whether prenatal therapy is indicated. Aortic stenosis (AoS) and
pulmonary stenosis (PS) and a restrictive FO have been treated
prenatally with mixed results.16,27-29 The rationale of fetal therapy
is to restore near-normal circulation and thus prevent ventricular
involution and maintain a two-ventricle circulation postnatally
(for AoS and PS) and to protect the pulmonary bed in HLHS and
enable a more successful Fontan procedure. Experience suggests
there is a more limited role for fetal aortic or pulmonary valvulo-
plasty than was originally anticipated, in part because case selec-
tion and timing are difficult to assess.15,30 Single-centre selection
 CHAPTER 29  The Heart 307

Ductal arch
Pulmonary artery
V5
(Three-vessel Transverse aortic arch
trachea view)
Superior vena cava
Trachea
SVC Main pulmonary artery
Ao Ascending aorta
Duct V4 Superior vena cava
RPA
Ao LPA (pulmonary
Left Right pulmonary artery
artery)
RPV pulmonary Descending aorta
artery Spine
LA LPV
Aortic valve
Right
V3 ventricle
FO Left atrium
(aortic
RA Descending aorta
root) Left
LV ventricle
Spine
RV Right
ventricle Tricuspid valve
Left Left atrium
IVC V2 ventricle
(four chambers) Pulmonary vein
Mitral Descending aorta
Placenta
AoD valve
Spine
Pulmonary vein
Umbilical vein

V1 Stomach
(situs) Inferior vena cava
Descending aorta
Spine

• Fig. 29.1  Diagram illustrating the five transverse scanning planes through the fetal body. V1, Abdominal situs: fetal lie must be determined so the
left and right sidedness of structures can be assessed to diagnose complex cardiac malformations accurately. In normal situs, the aorta (Ao) lies to
the left and the inferior vena cava (IVC) to the right of the spine. V2, Four-chamber view: allows assessment of morphology and symmetry. The left
atrium (LA) is characterised by the coronary sinus and the left atrial appendage and the right ventricle (RV) by the offsetting of the tricuspid valve
with attachments to the septum and the moderator band. V3 and V4, Great arterial crossover: the Ao arises first, sweeping to the fetal right, and the
pulmonary artery crosses over. The great arteries are usually easiest to differentiate by confirming early bifurcation, characteristic of the pulmonary
artery. V5, The three-vessel and trachea view enables a comparison of the transverse aortic arch and ductal arch. They should be of similar sizes.
Additional vessels such as a persistent left superior caval vein or aberrant right subclavian artery may be identified at this level. AoD, descending
aorta; FO, foramen ovale; LPA, left pulmonary artery; LPV, left pulmonary vein; LV, left ventricle; RA, right atrium; RPA, right pulmonary artery; RPV,
right pulmonary vein; SVC, superior vena cava.

not been independently verified in other populations.17,18,31,32 Pathophysiology. In almost all cases, the mitral valve is either
In countries with established fetal valvuloplasty programs, a new stenotic or imperforate. Thus flow through the FO is reversed,
diagnosis of AoS of PS could be discussed with the family and, being predominantly left to right. The ascending aorta is usually
if agreed, evaluated with the experienced interventional team to hypoplastic, and the arterial duct supplies blood to the upper half
decide whether a procedure is likely to make a lasting difference. of the body. Appreciation of the atrial communication is critical in
At present, there is no evidence from a trial to inform the discus- the presence of left heart obstruction because an intact or restric-
sion further.  tive atrial septum causes abnormally high pressures in the pulmo-
nary vasculature and results in ‘arterialisation’ of the pulmonary
veins and lymphatic dilation (lymphangiectasia).34,35 
Specific Lesions
Associated anomalies
Lesion with Abnormal Four-Chamber View • HLHS includes the spectrum of features of left heart hypoplasia.
Hypoplastic left heart syndrome • Extracardiac malformations occur in about 30%. Growth
Overview. Hypoplastic left heart syndrome is not a specific restriction is overrepresented in HLHS and affects about 20%
malformation but rather describes a spectrum of left heart hypo- of all fetuses.36
plasia occurring in about 3.5% of all babies born with CHD.33 • Chromosomes are abnormal in 10% to 15%, including Turner
The classical definition is aortic atresia or stenosis with mitral syndrome (which is common in the early first trimester, when
atresia or stenosis; usually there is an intact ventricular septum or pregnancy loss occurs), trisomies 18 and 13; and Holt-Oram,
small ventricular septal defect (VSD).  Noonan, Jacobsen and 22q11 syndromes.36,37 
308 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
29.1 Checklist for ‘Screening’ Echocardiography
Transverse
view Items
RV
Situs • Aorta: left of spine
• IVC: anterior and right RA
• Stomach: left LV IAS

Four- • Normal ribs rFO

chamber • Normal lungs
• Size and position of the heart LA
• Angle of the apex (45 ± 20 degrees towards left side)
• No pericardial effusion, no pleural effusion
EFE Ao
• Two atria, roughly equal in size
• Two ventricles, roughly equal in size
• LV apex forming
• Two separate AV valves
• AV concordance (LV on left side; RV on right side):
• Normal offsetting (TV more towards the apex
than MV)
• Septal attachments (no septal attachments of MV) • Fig. 29.2  Hypoplastic left heart syndrome with restrictive foramen ovale
• Moderator band in the RV at 37 weeks of gestation. The interatrial septum is thickened and bows
• Normal atrial septum primum into the right atrium (RA). The left ventricle (LV) appears rounded and
• Interventricular septum intact with echo-bright endocardium (endocardial fibroelastosis). Ao, Descend-
• Foramen flap in LA ing aorta; EFE, endocardial fibroelastosis; IAS, interatrial septum; LA, left
• Normal coronary sinus, not dilated atrium; rFO, restrictive foramen ovale; RV, right ventricle.
• Pulmonary venous drainage into LA
• At least one vein from left lung
• At least one vein from right lung the four-chamber view with the septum perpendicular to the ultra-
• Colour Doppler: forward flow through both AV valves, sound beam (Fig. 29.2 and Video 29.1) or from a short-axis view of
no aliasing or regurgitation the heart in the coronal plane. Pulsed-wave Doppler of the pulmo-
LVOT • Aorta arising from the LV nary veins may demonstrate reversal of the A wave with restrictive
• Septo-aortic continuity (‘ballerina’s foot’) atrial septum.38 Severe tricuspid valve (TV) regurgitation may indi-
• Patent, ‘thin’ aortic valve cate right ventricular dysfunction and indicates a poor prognosis. 
• Colour Doppler: forward flow through aortic valve, no
aliasing or regurgitation Specific features to check at follow-up and perinatal
RVOT • Pulmonary artery arising from RV management
• Patent, ‘thin’ pulmonary valve • Check for flow across mitral and aortic valves.
• From left to right: pulmonary artery (dividing into two • Measure left heart dimensions (mitral valve, LV, aortic valve,
equal-sized branch PAs), aorta, SVC ascending aorta, transverse aortic arch) and obtain gestational
• PA biggest, Ao smaller, SVC smallest age–adjusted z-scores.39,40
• No LSVC • Check appearance and mobility of the atrial septum.
• Colour Doppler: forward flow through pulmonary • Check flow across the FO.
valve, no aliasing or regurgitation
• Check the TV for regurgitation
3VT • Aortic arch passing to left of the trachea • Check the direction of flow in the transverse arch.
• Arterial duct left of the aortic arch • Offer invasive diagnosis.
• Aortic arch and arterial duct roughly equal in size • Consider referral for prenatal atrial stenting in cases with
• No LSVC restrictive FO.
• Normal-sized thymus • Plan delivery in a tertiary care centre to maintain ductal patency
• Colour Doppler: unaliased forward flow in both arches
with prostaglandin E (PGE); prepare for urgent balloon atrial
Ao, Aorta; AV, atrioventricular; IVC, inferior vena cava; LA, left atrium; LSVC, left superior septoplasty or surgery in cases with restrictive or closed FO. 
vena cava; LV, left ventricle; LVOT; left ventricular outflow tract; PA, pulmonary artery; RV,
right ventricle; RVOT, right ventricular outflow tract; SVC, superior vena cava; 3VT, three- Treatment and outcome. Hypoplastic left heart syndrome is a
vessel-and-tracheal view.
duct-dependent lesion; PGE is given after birth to maintain ductal
   patency, and the three-staged ‘Norwoods’ procedure or a hybrid
is performed (Fig. 29.3). Stage 1 consists of LVOT reconstruction
Ultrasound findings. The four-chamber view is abnormal and systemic-to-pulmonary shunt placement between the subcla-
showing a hypoplastic left ventricle (LV). The mitral valve appears vian and the branch pulmonary artery (Blalock-Taussig shunt) or
atretic or is absent with a thick band of fibrous tissue separating the the right ventricle and the pulmonary artery (Sano shunt) during
left atrium (LA) from the LV. In the LVOT view, the aortic valve is the first days of life. Stage 2 (Glenn operation) includes redirec-
thickened. The ascending aorta is hypoplastic, and reversal of flow tion of venous return from the superior vena cava (SVC) into the
in the transverse aortic arch can be seen using colour Doppler in pulmonary artery. Stage 3 (Fontan completion) is redirection of
the 3VT. The size of the FO and flow across it can be assessed from blood from the inferior vena cava (IVC) to the pulmonary artery.
 CHAPTER 29  The Heart 309

SVC
SVC
Ao Ao

LPA LPA
RPA RPA

Fenestration
External RV
RV conduit

TV
PV TV

A IVC B IVC
• Fig. 29.3  A, The stage II procedure: the cavopulmonary shunt. B, The stage III procedure: the total cavo-
pulmonary connection. The technique uses an extracardiac conduit to direct the inferior vena cava flow into
the pulmonary arteries. Ao, Aorta; IVC, inferior vena cava; LPA, left pulmonary artery; PV, pulmonary valve;
RPA, right pulmonary artery; RV, right ventricle; SVC, superior vena cava; TV, tricuspid valve. (From Barron
DJ, Kilby MD, Davies B, et al. Hypoplastic left heart syndrome. Lancet 374(9689):551–564, 2009.)

The early survival rate for the Norwood stage 1 operation may be velocities through the aortic valve; however, with poor LV function,
90% in fetuses without risk factors,41 but the overall survival rate the velocities may be normal or low. In severe cases, mitral valve
to school age is about 50% to 60%. The mortality rate is higher inflow becomes monophasic, and severe regurgitation may be seen.
with restrictive FO or poor right ventricular function.41,42 Prena- The ascending aorta may show poststenotic dilation beyond the aor-
tal diagnosis significantly improves neonatal survival.21 HLHS has tic valve or become hypoplastic. Aortic arch flow is often reversed. 
been related to an increased risk for neurodevelopmental delay,
and parents should be counselled accordingly. Although this was Specific features to check at follow-up and perinatal
formerly mainly attributed to cardiac surgery, recent studies sug- management
gest that brain abnormalities are developmental.25  • Check left heart dimensions (mitral valve, LV, aortic valve,
ascending aorta, transverse aortic arch, isthmus) and obtain
Critical aortic stenosis gestational age–adjusted z-scores.39,40
Overview. The severity of AoS varies with a range of outcome • Check the direction and velocity of flow at the FO.
from biventricular to functionally univentricular. The early signs • Check mitral valve Doppler and its duration in the cardiac cycle.
of AoS may be subtle and are easily missed at second trimester • Check the velocity of flow across the aortic valve.
screening. Valvular aortic stenosis is the most common type. • Check the direction of flow in the transverse arch and ascend-
The LV may continue to have relatively normal growth, become ing aorta.
dilated or involute, progressing to HLHS.  • Check diastolic flow velocities in the isthmus (associated CoA).
• Consider referral for prenatal balloon valvuloplasty in selected
Pathophysiology. The initial response to AoS may be left ven- patients.
tricular dilatation followed by its involution and secondary dam- • Arrange for interventional cardiology and cardiovascular surgi-
age caused by reduced coronary perfusion and subsequent fibrosis cal consultations prenatally.
(endocardial fibroelastosis), resulting in poor function.  • Plan delivery in a tertiary care centre. Severe cases will require
PGE. 
Associated Anomalies
• AoS may be associated with VSD, bicuspid aortic valve and Treatment and outcome. About 45% of fetuses with aortic
CoA. Extracardiac anomalies are rare. stenosis will progress to a functionally univentricular circulation,
• Chromosomal abnormalities are rare with valvular AoS, but requiring palliative surgery after birth.31 In selected fetuses, prenatal
supravalvular AOS is associated with William syndrome.  balloon valvuloplasty may be considered.43 The postnatal manage-
ment pathway depends on left heart morphology and function: In
Ultrasound findings. A dilated LV with a hyperechogenic endo- mild cases, postnatal balloon valvuloplasty may suffice. However,
cardium is seen in the four-chamber view; the contractility is often many children will need more complex surgery after birth, placing
noticeably reduced. The LA may be dilated if there is severe mitral the pulmonary valve in the aortic position and inserting a right
regurgitation (MR). The FO flow is predominantly left to right. In ventricle (RV) to pulmonary artery conduit, and some will require
the LVOT view, the aortic valve appears thickened and does not aortic or mitral valve replacement later in childhood to maintain a
‘disappear’ during systole. Pulsed-wave Doppler reveals increased biventricular outcome.17 Univentricular palliation is performed in
310 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
LV
RV
Absent right
AV connection
(tricuspid valve)
• Fig. 29.4  Tricuspid atresia: Absence of a connection between the right
atrium and ventricle and right ventricular hypoplasia. LV, Left ventricle; RV,
right ventricle.
cases with significant LV hypoplasia or with a thin-walled poorly
functioning LV, provided the right heart continues to function well.  only one ventricular portion is well grown). With moderate to
Tricuspid atresia
Overview. Tricuspid atresia is characterised by the absence
of a connection between the right atrium (RA) and the RV. It is
typically associated with right ventricular hypoplasia and varying
degrees of PS or pulmonary atresia.  are usually better, and there is a better chance of following a biven-
Pathophysiology. In tricuspid atresia, all the systemic venous
return is shunted right to left across the (usually) large FO. Both
pulmonary and systemic venous return enter the dominant LV
through an enlarged mitral valve. A perimembranous VSD may
connect the rudimentary RV to the LV and supply the pulmonary
artery (80%) or aorta (20%). In the absence of a VSD the right
sided structures are miniature.  Associated anomalies
Associated anomalies
• Pulmonary hypoplasia and atresia are common, and CoA is pres-
ent in most fetuses with ventriculoarterial discordance (20%).44
• Chromosomal abnormalities are rare with tricuspid atresia.  there is no specific organ involvement.
Ultrasound findings. The four-chamber view has one dominant
and one rudimentary chamber. Only one inlet valve is identified,
and the other AV junction is represented by a band of echo-bright
tissue (Fig. 29.4). Colour Doppler confirms ventricular inflow into
the dominant chamber. Outflow tract hypoplasia and obstruc-
tion are confirmed with colour and pulsed-wave Doppler. Correct
identification of the dominant ventricle is essential for counselling.
Whereas a dominant LV will show a fish-mouthed mitral valve in
the posterior position, a dominant RV will show a trileaflet valve
with septal attachments lying anterior, in the short axis-view.  nary stenosis or atresia, known as the unipartite ventricle. This
Specific features to check at follow-up and perinatal
management
• Identify chamber morphology.
• Check ventriculoarterial connection.
• Check for outflow tract obstruction and coarctation.
• Check for reversal of flow in the arterial duct, confirming duct
dependency.
• Plan delivery in a tertiary care centre because PGE will be
required.  throughout pregnancy.
Treatment and outcome. The initial management of tricuspid
atresia depends on the degree of outflow tract obstruction and
associated anomalies. A systemic-to-pulmonary artery shunt is
required in case of severe PS or atresia. If the great arteries are
discordant, coarctation or extended arch repair may be necessary.
Ultimately, infants undergo a single-ventricle palliation. Survival
of tricuspid atresia was 83% at 1 year with no late deaths in a
large multicentre study.44 Long-term survival favours those with
a dominant LV.45 
Pulmonary atresia with intact septum and pulmonary stenosis
Overview. Pulmonary stenosis is characterised by a narrowing
of the RVOT. Valvular obstruction is most common in the fetus,
but mild PS is rarely diagnosed prenatally. Pulmonary atresia may
be membranous (80%) or a long segment and muscular obstruc-
tion (20%). This section discusses severe PS and pulmonary atre-
sia with intact ventricular septum (PAIVS). 
Pathophysiology. The RV is often characterised as tripartite
(with developed inlet, trabecular and outlet portions), bipartite
(where one of these is underdeveloped), or unipartite (where
severe PS or pulmonary atresia, the RV is exposed to significant
pressure-overload resulting in important fibrosis and subsequent
RV dysfunction. The TV is usually small, and the RV involutes
secondary to reduced filling. In hearts with severe tricuspid regur-
gitation (TR) through a dysplastic TV, growth of the TV and RV
tricular surgical pathway after birth. As in tricuspid atresia, all the
systemic venous return is shunted right to left through the large
FO. The pulmonary vasculature is supplied by reversed flow (left-
to-right shunting) through the arterial duct. Ventriculocoronary
fistulae are seen in the smallest, unipartite RVs and associated with
a poorer outcome.46,47 
• PS is a component of many other structural heart malforma-
tions. PAIVS is associated with ventriculocoronary fistulas in
up to one third of cases.30,46-48
• Extracardiac malformations may occasionally be found, but
• Chromosomal abnormalities are rare with PAIVS. PS may be
associated with syndromes such as Williams-Beuren, Alagille
or Noonan. 
Ultrasound findings. The four-chamber view is abnormal in
severe PS and PAIVS (Fig. 29.5A). The TV is often small and
dysplastic with bright chordae secondary to pressure overload and
may show moderate to severe TR.30 In severe PS or pulmonary
atresia, trabecular overgrowth leads to a bipartite ventricle. In the
severest case, the RV is hypoplastic with tricuspid and pulmo-
may be associated with ventriculocoronary fistulas, confirmed by
colour Doppler and bidirectional high velocity flow on pulsed-
wave Doppler. The branch pulmonary arteries are usually small
and confluent (Fig. 29.5B). Colour Doppler confirms flow across
the pulmonary valve, and the peak velocity can be estimated by
pulsed-wave or continuous-wave Doppler. In the 3VT, reversed
flow is seen in the arterial duct. Flow in the DV often shows
absent or reversed flow secondary to high right atrial pressure.30
This does not indicate fetal hypoxemia and may be recorded
 
 CHAPTER 29  The Heart 311

CEPH
Voluson
E8

Severe
TR

RA

LA
RV

LV

A
-69 cm/s
Voluson
E8

aAo
aAo
MPA

-69 cm/s
Duct with
reversed flow
PAs

LPA

B
• Fig. 29.5  Small right ventricle and severe tricuspid regurgitation in a fetus with pulmonary atresia and
intact ventricular septum (A). No forward flow is seen across the pulmonary valve and the small main pul-
monary artery is supplied by reversal of flow in the arterial duct (B). aAo, ascending aorta; LA, left atrium;
LPA, left pulmonary artery; LV, left ventricle; MPA, main pulmonary artery; PAs, branch pulmonary arteries;
RA, right atrium; RV, right ventricle; TR, tricuspid regurgitation.

Specific features to check at follow-up and perinatal
management
• Check right heart dimensions (TV, RV, pulmonary valve, branch
pulmonary arteries) and obtain gestational age–adjusted z-scores.39
• Check for significant TR and quantify it.
• Check direction of flow in the arterial duct.
• Check for ventriculocoronary fistulas.
• Consider referral for prenatal valvuloplasty in selected patients.
• Plan delivery in tertiary care centre to maintain ductal patency
with PGE. 
Treatment and outcome. As in most CHD, usually no hae-
modynamic compromise is seen before delivery unless hydrops
develops because of severe TR. There are theoretical reasons
why prenatal pulmonary valvuloplasty might lead to improved
RV function in childhood, but this is usually reserved for cases
with hydrops. A biventricular outcome may be feasible in early
childhood, but right heart failure may necessitate conversion to a
later Fontan circulation. Severe PS or PAIVS is a duct-dependent
lesion associated with a guarded prognosis, particularly in the
presence of ventriculocoronary fistulas, which can lead to coro-
nary artery occlusion and death.48,49 Cardiac catheterisation is
important when fistulas are detected to exclude a right ventricular
coronary-dependent circulation and determine the best postna-
tal approach. The pulmonary valve is not opened in such cases
because this will result in coronary steal and myocardial infarction. 
Double-inlet left ventricle
Overview. In double-inlet left ventricle (DILV), both atria are
connected to a dominant ventricle. The dominant ventricle is usu-
ally of left morphology (DILV). 
Pathophysiology. Any atrial arrangement is possible, but both
AV valves open into a single ventricle. These are usually balanced,
312 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

but one inlet valve may be dysplastic and become atretic during CEPH
fetal life, or there may be a common valve. The rudimentary ven- Voluson
E8
tricle is supplied by a VSD and may give rise to one or both great
arteries as in double outlet right ventricle (DORV). 

Associated anomalies LV RV
• DILV is associated with a wide variety of great arterial connec- Displaced
valve
tions and LVOT or RVOT obstruction is common. leaflets
• Heterotaxy syndrome should be excluded.
• Chromosomal abnormalities are rare.  Atrialised
RV
LA
Ultrasound findings. Abdominal situs should be carefully
assessed because right or left atrial isomerism may occur. The
four-chamber view shows one dominant and one rudimentary Dilated
RA
chamber. Correct identity of the dominant ventricle is essential
for counselling. 

Specific features to check at follow-up and perinatal

management
• Identify situs and chamber morphology. • Fig. 29.6  The displacement of the tricuspid valve results in atrialisation of
• Check AV and ventriculoarterial connections. the right ventricle (RV) in Ebstein anomaly. The valve is dysplastic, resulting
• Check for progressive outflow tract obstruction. in severe tricuspid regurgitation and right atrial dilatation. LA, Left atrium;
• Check for reversal of flow in the arterial duct or aortic arch, LV, left ventricle; RA, right atrium.
suggesting duct-dependent pulmonary or systemic circulations
respectively. Ultrasound findings. In the four-chamber view, the heart is large
• Plan for delivery in a tertiary care centre because this is a duct- and may be ‘wall to wall’. The RA is markedly enlarged and the FO
dependent circulation.  generous (Fig. 29.6). The hinge point (functional insertion) of the
TV is displaced apically, and the valve leaflets do not coapt, resulting
Treatment and outcome. The initial management of DILV in severe regurgitation that typically arises from below the valve and
depends on the presence of outflow tract obstruction and associ- reaches to the back of the atrial wall, demonstrated by colour Dop-
ated anomalies. A systemic-to-pulmonary arterial shunt is required pler. The pulmonary vasculature is underperfused and small, and the
in case of insufficient forward flow through the pulmonary valve, 3VT may show reversed ductal flow and pulmonary regurgitation. 
or aortic arch repair for CoA. Ultimately, infants will undergo
single ventricle palliation. Generally, postnatal intermediate-term Specific features to check at follow-up and perinatal
survival favours those with a dominant LV.45  management
• Check for prognostic indicators at diagnosis because they will
Ebstein Anomaly guide surveillance.
Overview. Ebstein anomaly is characterised by a displacement • Fetuses with severe TR should be monitored weekly for signs
of the TV annulus towards the cardiac apex. This results in ‘atri- of hydrops or supraventricular tachycardia by the local team.
alisation’ of the RV inlet. The presentation varies depending on These require urgent referral to a fetal centre for management
the degree of atrialisation and TR. Some may present with fetal of arrhythmia and monitoring for maternal mirror syndrome.
tachycardia or hydrops.  • Invasive genetic testing should be offered.
• Multidisciplinary team discussion should take place during the
Pathophysiology. The functional impairment of the RV and third trimester to decide whether a path of active treatment is
severity of TR cause right heart dilation, often resulting in a to be initiated after delivery. If so, plan for delivery in a ter-
wall-to-wall heart. Reduced forward flow contributes to hypo- tiary care centre to provide the appropriate postnatal support
plasia of the pulmonary vasculature and pulmonary atresia often or make arrangements for palliative care. 
develops prenatally. A pathophysiological ‘circle of death’ occurs
when there is reversal of flow through the arterial duct, severe pul- Treatment and outcome. The prognosis of Ebstein anomaly
monary regurgitation and severe TR. The high right atrial pressure diagnosed prenatally is dismal. In a large retrospective multicentre
wave results in reverse A waves in the DV and pulsations in the series, the fetal mortality rate was 17%, and the overall perina-
umbilical vein, and may result in fetal hydrops and demise.  tal mortality rate 45%.23 Tricuspid annular dilation, pulmonary
regurgitation, RVOT obstruction, pericardial effusion and diag-
Associated anomalies nosis before 32 weeks of gestation indicate a poor outcome.23,51
• Ebstein anomaly is commonly associated with RVOT obstruction. Only a minority of children will have surgery, and it is impor-
Atrial septal defects (ASDs) are usual.50 Tachycardia may occur. tant to offer expectant parents the option of palliative care.
• Severe Ebstein anomaly may result in hydrops from high right The surgical approach depends on the severity of the malfor-
atrial pressure and pulmonary hypoplasia. mation. A biventricular approach attempts to repair the TV and
• In a large multicentre study, genetic abnormalities have been reduce right atrial size. Univentricular surgery comprises patch
found in 20% of those fetuses that had an amniocentesis, most closure of the TV, atrial septectomy and systemic-to-pulmonary
commonly trisomy 21, CHARGE syndrome and chromosome arterial shunting followed by Glenn operation at 3 to 6 months
1p36 deletion.23 Therefore invasive testing should be offered.  and Fontan completion at around 3 years. 

BREECH Voluson
E8
RA
LA
RV
LV
• Fig. 29.7  Atrioventricular septal defect is characterised by a common
atrioventricular junction and valve. The atrial and ventricular compo-
nent of the septal defect are shown by the two arrows allowing mixing
of blood. LA, Left atrium; LV, left ventricle; RA, right atrium; RV, right
ventricle.
Atrioventricular septal defect
Overview. Atrioventricular septal defect accounts for about
4% of all CHDs.33 It is strongly associated with chromosomal
abnormalities, most commonly trisomy 21. AVSD is characterised
by a common AV junction. There is usually a common AV valve
but more rarely separate valves are identified. The septal defects
are of variable size and may even be absent, which makes prenatal
detection of this condition more difficult. Depending on the rela-
tive sizes of the RV and LV, AVSD may be considered balanced or
unbalanced.  with other malformations, particularly RAI with TAPVC.
Pathophysiology. Usually no haemodynamic effects are seen
before birth in isolated AVSD. When it occurs with tetralogy of
Fallot (ToF), varying degrees of RVOT obstruction may develop,
leading to ductal dependency. It is commonly seen in cases of
heterotaxy, and in right atrial isomerism (RAI), obstructed total
anomalous pulmonary venous connection (TAPVC) may occur
below the diaphragm and require early surgery. In uncomplicated
cases, the size of the atrial and ventricular component of the defect
will determine the potential for left-to-right shunting and for car-
diac failure in infancy.  similar in utero, VSDs have no haemodynamic effects until several
Associated anomalies
• Unbalanced AVSD is usually associated with hypoplasia of
the LV and aorta or pulmonary atresia and TAPVC. About
5% of AVSDs are associated with ToF, increasing the risk for
­trisomy 21.52
• Extracardiac malformations are commonly seen with no spe-
cific organ involvement with balanced AVSD.36 Unbalanced
AVSD is typically associated with ‘heterotaxy’ syndrome, usu-
ally RAI with asplenia.
• In 40% to 60%, a second trimester prenatal diagnosis of a bal-
anced AVSD is associated with trisomy 21 or less commonly
with trisomies 18 and 13.36,52,53 The prevalence is higher in the
first trimester.  formations should be offered karyotyping.
CHAPTER 29  The Heart 313
Ultrasound findings. The abdominal situs may be abnormal.
The heart may appear ‘rounded’ as the inlet septum is shorter
than the outlet septum. The atrial and ventricular component of
the septal defect vary in size (Fig. 29.7). During diastole, colour
Doppler shows the characteristic ‘butterfly’ H-shaped appear-
ance, and no offsetting of the inlet valves can be seen. Some-
times a dilated coronary sinus (from a persistent left superior
vena) can be mistaken for the atrial component of an AVSD.
The visualisation of a septum primum more anteriorly allows
their differentiation. A short-axis view at the level of the AV
valve should be obtained to confirm the diagnosis of AVSD
(Video 29.2). There is no fish-mouth mitral valve because even
if separate valves are identified, the left valve opens to the ven-
tricular septum. The outflow tract and 3VT are usually normal
in balanced AVSD. 
Specific features to check at follow-up and perinatal
management
• Check abdominal situs and position of cardiac apex.
• Check for ventricular disproportion.
• Check for regurgitation of the common valve.
• Check for LVOT or RVOT obstruction.
• Check pulmonary venous connection (space behind the heart
and diaphragm) for TAPVC and reassess later in pregnancy
• Offer invasive diagnosis.
• Plan for delivery in a tertiary care centre in cases of unbalanced
AVSD, heterotaxy and when noncardiac anomalies or chromo-
somal abnormalities are present. 
Treatment and outcome. Isolated, balanced AVSD is not duct
dependent. Most cases will undergo surgery on bypass at about 3
to 6 months of age or earlier if they are symptomatic. The opera-
tive mortality rate is less than 5%, and quality of life depends on
associated anomalies such as trisomy 21. The cardiac outcome is
good unless there is significant AV valve regurgitation.54 The out-
come for unbalanced AVSD is poorer because of its association
 
Ventricular septal defect
Overview. Ventricular septal defects are the most common form
of CHD seen in childhood, but isolated VSD is rarer in prenatal
series.33 Small isolated VSDs are likely to be benign and may close
spontaneously before delivery or in early childhood. Large or multi-
ple VSDs are likely to be symptomatic and require postnatal evalu-
ation, and they may be associated with syndromes or aneuploidy. 
Pathophysiology. Because the pressures in the RV and LV are
weeks after birth when the pressure in the pulmonary circulation has
decreased. This allows shunting of blood from left to right, resulting
in increased pulmonary circulation, which may present with poor
feeding, increased respiratory effort and reduction in weight gain. 
Associated anomalies
• VSDs form part of many major CHDs, including ToF, com-
plete or complex TGA, DORV, common arterial trunk (CAT),
interrupted aortic arch (IAA), HLHS and tricuspid atresia.
• VSDs may be associated with many types of extracardiac mal-
formations.
• The true association of isolated VSDs with aneuploidy remains
uncertain; large VSDs or those of any size with associated mal-
 
314 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Ultrasound findings. A VSD may be identified by a drop- Ultrasound findings. The diagnostic features are two LAs or
out in the interventricular septum where the borders typically RAs, but these are not always easy to assess. The first step is to
appear hyperechogenic. To reduce false-positive and false-nega- evaluate the position of the abdominal vessels and their relation-
tive diagnoses, the four-chamber view should be obtained with ship to the spine.
the ultrasound beam perpendicular to the septum. Flow across Most fetuses with LAI have an interrupted IVC with azygos con-
the VSD can be visualised best using bidirectional power Dop- tinuation characterised by a similarly sized vessel lying behind the aorta
pler with low pulse repetition frequency, with careful attention in the transverse and sagittal planes. In the coronal plane, the azygos
to gain settings.  and aorta lie side by side with flow in opposite directions. The azygos
usually enters the right SVC. A coronary sinus is usually identified.
Specific features to check at follow-up and perinatal In RAI, the IVC is typically on the same side as the aorta,
management which can be either on the left or more usually on the right side of
• Check for disproportion of ventricles and great arteries in the the spine. The stomach is often small and posterior and the liver
3VT. central. No coronary sinus is identified, and the posterior atrial
• Check for normal growth of the great arteries and their wall is symmetrical. An additional ascending or descending vein
flows. may be identified draining the pulmonary veins above the heart
• Consider invasive diagnosis with large VSDs and when there (supracardiac) or below the diaphragm (infracardiac). 
are extracardiac anomalies
• Delivery is possible in a local hospital for babies with isolated Specific features to check at follow-up and perinatal
VSD.  management
• Serial ultrasound scans should assess pulmonary venous course
Treatment and outcome. Postnatally, the diagnosis of isolated and signs of bowel obstruction.
VSD should be confirmed by clinical examination and echocar- • Consider fetal MRI for assessment of bronchial and gastroin-
diography. Provided the perinatal period is stable, the ideal time testinal system.
for this is at about 4 to 6 weeks of age when the pulmonary pres- • Prenatal consultation with paediatric and paediatric cardiac surgeons.
sures have fallen and the significance of the shunt can be assessed • Plan delivery in a tertiary care centre or discuss palliative care if
more reliably. Small VSDs usually close naturally. Large defects the lesion appears unbalanced, TAPVC appears obstructed or
may become symptomatic by about 4 to 6 weeks and can be man- there are important associated defects. 
aged medically by diuretics while surgery or, more rarely, inter-
ventional catheter closure is planned. There is usually no need for Treatment and outcome. The prognosis depends on the pres-
repeat surgery, and the outlook is good.  ence and severity of associated malformations. For children with
LAI without heart block and associated defects, the outlook is
Atrial isomerism or ‘heterotaxy syndrome’ generally good. However, there is increased mortality rate for all
Overview. Heterotaxy syndrome is characterised by a differ- surgical procedures in children with heterotaxy compared with
ent (Greek: heteros) arrangement (Greek: taxis) of major organs. those with normal situs. Sustainable repair of TAPVC is the most
In cardiology, it is defined based on the morphology of the atrial challenging aspect of the surgical management, but conduction
appendages rather than arrangement of the abdominal organs. abnormalities, poor ventricular function and valve regurgitation
Fetuses with atrial isomerism show bilateral left (left atrial isomer- make the Fontan circulation less successful. Large, long-term
ism (LAI)) or right atrial appendages (RAIs).  series report that fewer than 25% of affected children with RAI
reach 4 years of age because of the combination of complex cardiac
Pathophysiology. The pathophysiology in heterotaxy depends defects, gastrointestinal malformations and infection.55 A recur-
on associated cardiac and extracardiac malformations.  rence risk for up to 10% is reported in subsequent pregnancies.9 
Associated Anomalies Variations of systemic venous return
Left atrial isomerism (polysplenia) Overview. Variations of systemic venous return include inter-
• Most often there is an interrupted IVC with azygos continu- rupted IVC with azygos continuation and persistent left superior
ation to the right or persistent left superior vein cava draining vena cava (LSVC). Both can be considered as normal variants in
into a dilated coronary sinus. AVSD is common. In LAI, con- the absence of other cardiac and extracardiac anomalies.
genital heart block may occur because of absence of the sinus The diagnosis of absent DV is associated with growth restriction
node and is associated with poor prognosis. and aneuploidy. The umbilical vein reaches the systemic circulation in
• Associated malformations include biliary atresia, bowel malro- a variety of ways: directly to the IVC or iliac veins, via the portal sinus
tation, primary ciliary dyskinesia and pulmonary abnormalities to the right atrium or coronary sinus or through the hepatic sinusoids,
(bilobed lungs). or it may bypass the liver and enter the heart directly, leading to cardiac
• Chromosomal abnormalities are rare.  failure or postnatally leaving a defect in the diaphragm and potential
Right atrial isomerism (Ivemark syndrome, asplenia) for a right-sided hernia.56 The portal system may be absent, leading to
• The most common cardiac lesion is unbalanced AVSD with severe metabolic problems with high levels of morbidity and mortality. 
pulmonary stenosis or atresia. Because there are two morpho-
logic right atria, there is, by definition TAPVC. Bilateral caval Pathophysiology. During normal embryogenesis, the LSVC
veins are common. involutes. If it persists, it drains the blood from the left subcla-
• RAI is often associated with functional asplenia, two trilobed vian and jugular veins into the RA through the coronary sinus.
lungs and other gastrointestinal anomalies. A dilated coronary sinus, in the setting of the usual right SVC
• Chromosomal abnormalities are rare. Familial recurrence may may cause alteration of left ventricular inflow, resulting in a slim
be as high as 10%.  LV and arch hypoplasia. Interrupted IVC results from a failure
 CHAPTER 29  The Heart 315

COMP
Voluson
CEPH E8

RA LA
Duct
LSVC
aAo
CS RSVC

RV LV

A B
• Fig. 29.8  A dilated coronary sinus (A) and a ‘fourth’ vessel on the three-vessel view (B) are seen in this
fetus with persistent left superior vena cava. aAo, Ascending aorta; duct, arterial duct; LSVC, left superior
vena cava; RSVC, right superior vena cava.

of formation and anastomosis of segments of the IVC. Instead of
draining directly into the RA, the venous return from the lower
body drains via the azygous or hemiazygous vein into the SVC.  obtain gestational age–adjusted z-scores.39,40
Associated anomalies
• LSVC is commonly associated with many cardiac malforma-
tions. The dilated coronary sinus may alter left heart flows in
first trimester, leading to a degree of left heart hypoplasia, the
most important pathological effect being CoA. Absent DV
may lead to right atrial dilation and severe TR
• Both LSVC and interrupted IVC can be associated with het-
erotaxy syndromes.
• One series reported chromosomal anomalies in 9% of fetuses
with isolated LSVC57; however, this likely overestimates the
true incidence because of referral bias. Karyotyping should be
offered for absent DV.  Overview. Complete TGA accounts for about 5% of all
Ultrasound findings. In LSVC, a dilated coronary sinus can
be appreciated as a ‘circle’ in the standard four-chamber view(Fig.
29.8A). More posteriorly, it appears as two parallel lines with
dropout in the region of the atrial septum and may be confused
with primum ASD. In the RVOT and 3VT view, the LSVC is
identified on the left side of the arterial duct (Fig. 29.8B).
With interrupted IVC, no vessel is seen anterior and to the
right in the abdominal situs view in 90% of cases. Instead, a
dilated azygos or hemiazygos is observed lying behind the aorta.
In a longitudinal view, the azygos and aorta can be visualised side
by side with flow in opposite directions.
Both on transverse views of the abdomen and in the sagittal
view, a dilated portal sinus may be recognised and no DV iden-
tified. The portal veins should be sought and the connection of
umbilical and systemic veins identified.  not be administered before early expert assessment of the atrial
Specific features to check at follow-up and perinatal
management
• Check abdominal situs to detect an enlarged azygos vein and
the liver for portal venous abnormalities.
• Exclude heterotaxy syndrome by identifying atrial appendages
and coronary sinus.
• Check left heart dimensions in LSVC (mitral valve, LV, aor-
tic valve, ascending aorta, transverse aortic arch, isthmus) and
• Check the direction of flow through the FO and the mitral
inflow Doppler with LSVC.
• Delivery is possible in a local hospital in cases with isolated
sonographic findings. 
Treatment and Outcome. No treatment is required for
isolated LSVC and interrupted IVC, and the outlook is
excellent. 
Lesions Requiring Views of the Outflow Tracts
Complete transposition of the great arteries
CHDs.33 It is defined as AV concordance with ventriculoarterial
discordance, and the aorta arises from a morphologically RV and
the pulmonary trunk from the LV. 
Pathophysiology. In the fetal circulation, there is usually
no haemodynamic imbalance arising from TGA. Postnatally,
the RV pumps the oxygen-depleted systemic venous return
back into the body while the oxygen-rich blood from the pul-
monary veins is recirculated into the pulmonary vasculature.
Instead of a serial arrangement, the two circulations run in
parallel, which is fatal in the absence of sufficient communica-
tion through the FO and arterial duct. A restrictive FO occurs
in about 10% of fetuses with TGA and puts theses newborns
at increased risk for perinatal demise. Prostaglandin infusion
worsens oxygenation with this pathophysiology and should
communication after birth. 
Associated anomalies
• The most common associated heart malformation is a VSD.
Occasionally, LVOT obstruction may be found.
• TGA is rarely associated with extracardiac or chromosomal
abnormalities.36 
316 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
CEPH
Voluson
E8
RV
aAo
MPA HNV
LV
PAs
• Fig. 29.9  Transposition of the great arteries shows a parallel relationship
of the aorta and pulmonary arteries arising from the right and left ventricles,
respectively. aAo, Ascending aorta; HNV, head- and neck vessels; LV, left
ventricle; MPA, main pulmonary artery; PAs, branch pulmonary arteries;
RV, right ventricle.
Ultrasound findings. The four-chamber view and the LVOT
view are usually normal in isolated complete TGA. The diagnostic
clue is that the vessel arising from the LV divides shortly after its
origin. No great arterial cross-over is seen; instead the great arteries
run in a parallel course (Fig. 29.9). The normal 3VT view can-
not usually be obtained because of abnormal arterial relationship
and only two vessels can be visualised at the same time (the aorta
and SVC). Predicting perinatal well-being is difficult, and thus
FO and ductal flow are recorded serially. The hyperoxigenation
test after 34 gestational weeks may demonstrate responsiveness of
the pulmonary vascular bed and the ability for better postnatal
adaptation.58  side, and its inlet valve may be displaced apically and regurgitant
Specific features to check at follow-up and perinatal
management
• Check the size and patency of the FO until term.
• Check for dysplasia and obstruction of the semilunar valves
and outflow tracts that may preclude the arterial switch proce-
dure.
• Check patency and direction of flow in the arterial duct.
• Consider a late third trimester maternal hyperoxygenation test.
• Plan delivery in a tertiary care centre to assess atrial mixing
soon after delivery and maintain ductal patency with PGE
unless there is a restrictive FO.
• Prepare for urgent balloon atrial septostomy if restrictive FO is
suspected. 
Treatment and outcome. After birth, PGE is used to maintain
ductal patency unless there is a restrictive FO. If there is insuf-
ficient atrial mixing, a balloon atrial septostomy is indicated to
increase systemic oxygen saturation. The definitive surgical treat-
ment for TGA is the arterial switch procedure performed during
the first days of life in which the normal circulation is reestab-
lished. In cases in which corrective surgery is not feasible (e.g.,
significant pulmonary valve stenosis, hypoplastic RV, straddling
or overriding of the AV valves, some types of intramural coronary
artery course), palliative surgery may be considered (Rastelli or
Nikaido operation).
The postoperative survival rate with the arterial switch proce-
dure is excellent (>95%) and usually results in a normal quality
of life. Neurodevelopmental delay is a concern for some children
with CHD, including TGA.26 
Double discordancy or congenitally corrected transposition
Overview. Double discordancy (DD) or congenitally cor-
rected transposition (cTGA), accounts for fewer than 1% of all
CHDs. It is characterised both by AV and ventricular–arterial dis-
cordance. The RA is connected to a morphological LV and to the
pulmonary artery; the LA connects to the morphological RV and
into the aorta. 
Pathophysiology. There is usually no fetal haemodynamic
imbalance arising from DD, even with an associated VSD. How-
ever, the fetus may present with heart block or develop it later. If
there is associated severe pulmonary stenosis, there may be MR
(right-sided AV valve). TR (left-sided AV valve) may be found
because of associated Ebstein malformation. Isolated DD may
be unrecognised in childhood and may only present in adult life
when heart failure or heart block develops. Both the coronary
artery pattern and conduction tissue are not normal. 
Associated anomalies
• The most common associated heart malformations are VSD,
pulmonary stenosis and Ebstein malformation. Complete
heart block should be excluded at regular checkups.
• DD is rarely associated with extracardiac or chromosomal
abnormalities.36 
Ultrasound findings. Double discordancy may be suspected by
an abnormal cardiac apex; it is often located centrally or rightward
and the heart appears rounded. The inlet valves show differential
offsetting: the left-sided TV is inserted more apically than the
right-sided mitral valve. The trabeculated ventricle is on the left
if there is Ebstein malformation. The discordant VA connections
appear parallel without a cross-over, and the ventricles are often
side by side rather than in an anterior–posterior relationship. There
may be PS. The 3VT usually only shows the aortic arch and SVC. 
Specific features to check at follow-up and perinatal
management
• Check for valvular regurgitation (Ebstein anomaly).
• Exclude VSD and PS; ensure lesion is not ductal dependent at
term.
• Monitor for development of hydrops and heart block.
• Plan for local delivery unless ductal dependency, hydrops or
heart block develops. 
Treatment and outcome. After birth, no treatment is required
for uncomplicated DD. Postnatal echocardiography and electro-
cardiography (ECG) will provide confirmation of the diagnosis
and baseline information.
Later in childhood, a double switch operation may be consid-
ered to correct the circulation and establish the LV as the systemic
ventricle. This is a complex and relatively rare procedure that
should only be done in centres with experience. A large Japanese
series reports a 20-year survival rate of 80%.59 
 CHAPTER 29  The Heart 317

Voluson Voluson
E10 E10

LV RV
LV RV
VSD

aAo

Aortic
valve

• Fig. 29.10  A perimembranous ventricular septal defect with overriding aorta is seen in this fetus with
tetralogy of Fallot. Colour Doppler shows flow from both ventricles into the ascending aorta (‘Y-sign’). aAo,
Ascending aorta; LV, left ventricle ; RV, right ventricle; VSD, ventricular septal defect.

Tetralogy of fallot with pulmonary stenosis and atresia
Overview. Tetralogy of Fallot is a conotruncal malformation
in which the outlet septum is deviated anterocephalad, resulting
in the characteristic combination of (i) VSD, (ii) overriding aorta
and (iii) RVOT obstruction. The last feature (iv), right ventricu-
lar hypertrophy, is usually now only observed postnatally in cases
when children do not have access to timely surgical repair. The
degree of PS is variable, from very mild to complete obstruction
with no flow across the valve (pulmonary atresia). Another rare
variant is ToF with absent pulmonary valve. It is characterised by
massively dilated branch pulmonary arteries and severe pulmo-
nary regurgitation.  demonstrate the side of the aortic arch relative to the trachea. An
Pathophysiology. During fetal life, uncomplicated TOF
shows no important haemodynamic imbalance. However, this
may be severe when the pulmonary valve is ‘absent’, result-
ing in hydrops and fetal demise. In ToF or pulmonary atresia,
the central pulmonary blood vessels may be poorly developed,
but multiple sources of pulmonary blood supply may be pres-
ent arising from the aorta (major aortopulmonary collaterals
(MAPCAs)).
Postnatally, the VSD allows mixing of blood between the RV
and LV, but pulmonary overcirculation is usually prevented by
subvalvular and valvular PS. With severe PS or pulmonary atre-
sia, blood flow to the lungs is duct dependent. In the presence
of high pulmonary or low systemic vascular resistance, the blood
goes preferentially to the systemic circulation and can result in
episodes of hypoxia (‘spells’). Paradoxically, infants with MAPCAs
have pulmonary overcirculation and may be pink and breathless
after delivery.  • Offer invasive diagnosis.
Associated anomalies
• Associated CHDs are common with TOF60; right aortic arch
(RAA) is present in about 25% of cases and is associated with
an increased risk for 22q11 deletion.61
• Extracardiac malformations are seen in about a quarter of fetuses
with TOF36,60 with abdominal wall lesions such as omphalocele;
thymus hypoplasia increases the risk for 22q11 deletion.
• The risk for chromosomal abnormalities is about 30% with tri-
somies 21, 13 and 18 and 22q11 being the most prevalent.60,61  nary atresia or small pulmonary arteries, PGE maintains ductal
Ultrasound findings. Left-axis deviation may be the first clue
that a fetal conotruncal cardiac malformation is present. The four-
chamber view may appear normal, but reduced offsetting of the
TV may be a subtle sign. The VSD may be seen only in the LVOT
view, where septal continuity with the medial wall of the aorta
(ballerina’s foot) is lacking. The perimembranous outlet VSD
shows aortic override with a dilated ascending aorta. The RVOT
may appear small with an enlarged outlet septum contributing
to RVOT obstruction. Colour Doppler confirms that the aorta
receives blood from both ventricles, the Y sign (Fig. 29.10). The
3VT often shows just two vessels (the aorta and SVC) and will
RAA is seen in 25%. The direction of arterial duct flow should be
assessed using colour Doppler. 
Specific features to check at follow-up and perinatal
management
• Check for abnormalities of the inlet valves and additional
VSDs.
• Measurements of the pulmonary valve, main pulmonary artery
and branch pulmonary arteries should be obtained and trans-
formed into gestational age–adjusted z-scores.39
• Confirm that branch pulmonary arteries are confluent.
• If the branch pulmonary arteries appear dilated, use colour
Doppler across RVOT to confirm ‘absent’ PV with ‘to-and-fro’
flow.
• Check direction of flow in the arterial duct to confirm pulmo-
nary atresia.
• Check for MAPCAs in TOF with pulmonary atresia.
• Plan for delivery in a tertiary care centre. 
Treatment and outcome. Postnatal treatment depends on the
severity of the pulmonary obstruction. With adequately sized pul-
monary arteries, the infants are usually asymptomatic after birth.
Surgical repair is usually performed at around 4 to 6 months with
a survival of greater than 95% and a good outcome.61 However,
pulmonary regurgitation and right heart failure may occur in later
childhood, requiring pulmonary valve replacement. With pulmo-
318 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

patency, and a systemic-to-pulmonary artery shunt will be placed
early after delivery. The long-term problems are similar. Overall,
ToF is associated with a poorer prognosis than expected from the
cardiac features alone because of the frequent association with
other anomalies. ToF with ‘absent’ PV has a very poor outcome
due to tracheobronchial compression caused by the massive dila-
tion of the branch pulmonary arteries.  temic and pulmonary circulations. It accounts for about 1% of
Double outlet right ventricle
Overview. The term double outlet right ventricle is used when
both the aorta and the pulmonary artery arise from the RV in a
normal relationship (VSD type) or when the aorta arises anteriorly
from the RV and the pulmonary artery is central (TGA type).33  II is characterised by close but separate pulmonary branch origins,
Pathophysiology. The haemodynamics of a heart with DORV
are determined by the cardiac connections and presence of obstruc-
tion. The great arteries may have a normal relationship (when it shares
many of the features of TOF) or may be malposed with an anterior
aorta. The location of the VSD relative to the arterial outlets helps to
determine the surgical course. Obstruction to either the pulmonary
or systemic outflow tract will determine the need for PGE and early
surgery to allow adequate pulmonary or systemic flow.  is usually no normal arterial duct unless the aortic arch is inter-
Associated anomalies
• DORV is commonly associated with mitral atresia, pulmonary
stenosis or coarctation.
• Extracardiac malformations are common with DORV arrange-
ment, similar to TOF, and found in up to 70% of all fetuses.
• Chromosomal abnormalities are frequent and include triso-
mies and 22q11 deletion.62-64  sia and double aortic arch (DAA).
Ultrasound findings. Left-axis deviation may be the first clue
of a conotruncal cardiac malformation. The four-chamber view
may show any range of malformation, from normal to a single-
ventricle appearance with dominant large and small rudimentary
chamber. Colour Doppler will demonstrate flow from the RV into
both great arteries. In the 3VT, often only one great artery can
be visualised when the arteries are malposed, and reversed arch
flow may be seen with PS, pulmonary atresia or CoA. Pulsed-wave
Doppler will quantify pulmonary or aortic stenosis and diastolic
arch abnormalities in CoA.40  and branch pulmonary arteries arising directly from it. The trun-
Specific features to check at follow-up and perinatal
management
• Check for abnormalities of the AV connection (absent or
imperforate valves).
• Identify morphology of dominant ventricle.
• Check size and position of VSD relative to outflow tracts.
• Check relationship of great arteries (normal or malposed) and
relative sizes.
• Check size of branch pulmonary arteries and ensure conflu-
ence.
• Check direction of flow in the arterial duct
• Check size and direction of flow in the aortic arch and isthmus
• Offer invasive diagnosis.
• Plan for delivery in a tertiary care centre.  • Check for arch anomalies.
Treatment and outcome. Surgical treatment depends on the
precise cardiac anatomy and location of the VSD. Some combina-
tions of lesions will result in a biventricular outcome, some will
require conduit placement and others will follow a univentricu-
lar pathway from birth. Because of the common association with
cardiac and extracardiac association, generally, the prognosis is
poorer than anticipated from the cardiac findings alone. 
Common arterial trunk
Overview. Common arterial trunk is a conotruncal malfor-
mation characterised by a single outflow tract perfusing the sys-
all CHDs33 and is almost always associated with a large VSD.
Collett and Edwards classified CAT into four types, but usually
the fourth is now included within the spectrum of ToF with pul-
monary atresia: in type I CAT, a pulmonary trunk arises from the
CAT and branches into the left and right pulmonary arteries; type
and in type III there is wide separation of the origin of the branch
pulmonary arteries, with one sometimes arising from a small arte-
rial duct.65 
Pathophysiology. Common arterial trunk is not a duct-depen-
dent lesion. It behaves like a large VSD with increased pulmonary
blood flow after birth when the pulmonary vascular resistance
falls, and congestive heart failure may occur within weeks. There
rupted. In the rare subtype III, one branch pulmonary artery may
arise from a small arterial duct, creating a ductal-dependent pul-
monary circulation requiring PGE after delivery. 
Associated Anomalies
• CAT is frequently associated with arch anomalies. These
include right-sided aortic arch, interrupted arch, arch hypopla-
• Extracardiac malformations are seen in 40% of fetuses with no
specific organ involvement.66
• Chromosomal abnormalities are also frequent with 22q11
being the most prevalent, found in about 30% of fetuses.66,67 
Ultrasound Findings. Left-axis deviation may be the first
clue of a conotruncal cardiac malformation. The four-chamber
view can be unremarkable unless there is a large inlet VSD. Only
one semilunar valve is identified (the truncal valve). The LVOT
appears abnormal with a single great vessel overriding a VSD
cal valve is often dysplastic and thickened with regurgitation. If
severe, the LV may be dilated and bright. Colour Doppler dem-
onstrates blood flow from both ventricles into the common trunk
and confirms the presence of stenosis or regurgitation. The short-
axis view of the truncal valve may show an abnormal number of
valve leaflets. The thymus may be absent or hypoplastic.68 The
3VT shows a single arch (aortic arch) because the arterial duct is
usually absent. 
Specific features to check at follow-up and perinatal
management
• Check for truncal valve stenosis or insufficiency.
• Assess cardiac function and presence of endocardial fibroelasto-
sis.
• Check for hypoplastic or absent thymus.
• Offer invasive genetic testing.
• Plan for delivery in a tertiary care centre. Although types I and
II are not duct dependent, there is often confusion prenatally
with TOF with pulmonary atresia, which is duct dependent, so
caution is advised. 
 CHAPTER 29  The Heart 319

CEPH Voluson
E8
Voluson
E10

RV

LV
RA

LA

dAo

Increased
A B diastolic flow

• Fig. 29.11  Left–right disproportion on the four-chamber view in a midgestation fetus (A) suggests coarc-
tation of the aorta and warrants measurement of the aortic isthmus at the three-vessel and tracheal view.
Pulsed-wave Doppler shows increased flow at the aortic isthmus during diastole (B). dAo, Descending
aorta; LA, left atrium, LV, left ventricle; RA, right atrium; RV, right ventricle.

Treatment and outcome. Severe truncal valve insufficiency
may result in fetal heart failure and hydrops. Postnatal outcome
depends on the presence or absence of truncal valve stenosis or
insufficiency and IAA requiring neonatal arch repair. Otherwise,
surgery is usually performed during the first 4 weeks of life. It
consists of detachment of the pulmonary arteries from the CAT
and establishing continuity between the RV and the pulmonary
arteries through a conduit. The systemic outflow is reconstructed,
and the VSD is closed. Outcome after surgery is good provided
there is no 22q11 deletion or associated cardiac or extracardiac
complications. Conduit replacements are required during child-
hood and adulthood, which may have an important impact on
future morbidity.  pected, the diameter of the isthmus and the ductal arch should
Coarctation of the aorta and interrupted aortic arch
Overview. Coarctation of the aorta is common and accounts
for about 5% of all children born with CHD.33 It is defined as a
narrowing of the aortic arch and is typically located at the isthmus,
just proximal to the insertion of the arterial duct into the descend-
ing aorta.69 IAA describes the absence of an arch segment between
the left subclavian artery and duct (type A), the left carotid and
subclavian arteries (type B) and between the right and left carotids
(type C), respectively.  management
Pathophysiology. In utero CoA results in increased afterload
of the LV and reduces its filling, thus allowing more blood to be
shunted to the right heart. Consequently, there is often a marked
discrepancy in ventricular size.  • Trace head and neck vessels in IAA to confirm type.
Associated Anomalies
• CoA and IAA are associated with VSD and left heart obstruc-
tion. Bicuspid aortic valve is overrepresented and found in up
to 50% of infants with CoA.70
• Extracardiac abnormalities are common and affect all organ
systems.71 Vascular anomalies such as berry aneurysms are
found in up to 5% and represent a risk factor for stroke. Ele-
vated blood pressure occurs in one third of adults.
• CoA is associated with Turner syndrome; other associated
chromosomal abnormalities include trisomies 18 and 13.71
22q11 deletion is commonly associated with IAA type C.  severity. Surgery is performed off bypass via median sternotomy or
Ultrasound Findings. The suspicion of CoA is raised by four-
chamber disproportion (Fig. 29.11A). Unlike HLHS, the LV
remains apex forming with biphasic mitral valve flow. The gold
standard for detection of CoA is the 3VT,72 which shows dispro-
portion between the aortic and the ductal arches. In the sagittal
view, a ‘shelflike’ configuration may be seen. An increased distance
between the left common carotid and the left subclavian artery
may be a diagnostic clue. Colour Doppler shows flow continuing
into diastole at the aortic isthmus, and pulsed-wave Doppler typi-
cally demonstrates increased velocity of diastolic flow, often 30 to
40 cm/s (normally <15 cm/s) (Fig. 29.11B). Reversal of flow in
the prestenotic segment of the arch may be seen. If CoA is sus-
be measured from the 3VT and transformed into gestational age–
adjusted z-scores.40 Values below 2 standard deviations are indica-
tive of arch hypoplasia and warrant follow-up. The aortic valve
may be bileaflet. In IAA, the transverse arch cannot be traced past
the tracheal area. The sagittal views characteristically show con-
tinuation of the slim ascending aorta into the fetal neck as the
brachiocephalic. 
Specific features to check at follow-up and perinatal
• Measure the diameter of the aortic isthmus and the arterial
duct in the 3VT plane immediately proximal to the site of their
confluence and transform measurements into gestational age–
adjusted z-scores.40
• Obtain measurements of the mitral valve, LV, aortic valve,
ascending aorta and transverse arch.39
• Check mitral valve inflow Doppler, flow at the FO and aortic
valve to differentiate from other left heart anomalies.
• Check aortic valve leaflets to exclude bicuspid valve.
• Offer invasive diagnosis.
• Plan for delivery in a tertiary care centre to monitor arch anat-
omy and flow as the arterial duct closes. 
Treatment and outcome. Interrupted aortic arch requires early
surgery, but for CoA, the timing of surgical repair depends on its
320 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

a thoracotomy, depending on surgical preference. The prognosis

Double
after surgical repair is excellent; however, restenosis may occur. aortic arch
This is usually treated by balloon dilation, but repeat surgery is
sometimes required for recoarctation or aneurysm formation at
the site of operation. PA
One third of young adults will develop hypertension, underlin-
ing the importance for early detection and serial follow-up by a
cardiology team into adulthood.73,74 Bicuspid valve may require
surgery or replacement. 
Right aortic arch and double aortic arch
Overview. The reported prevalence of isolated RAA and DAA
is very low (<0.01%).75 The true prevalence, however, is likely to
be higher because both may be asymptomatic pre- and postnatally.  Duct

Pathophysiology. Arch anomalies are thought to result from dAo

abnormal development and regression of the DAA present dur-
ing embryologic development. Edwards suggested a hypothetical
model to explain abnormal development of the arches.76 DAA and
RAA with left duct encircle oesophagus and trachea77,78 therefore
have the potential to cause obstruction, which may cause polyhy-
dramnios, hyperechogenic lungs and flattened diaphragm before
birth and dyspnoea and dysphagia after birth. 
Associated anomalies
• RAA and DAA are sometimes seen with conotruncal CHDs.
• Extracardiac malformations are rare with isolated RAA and
DAA. Thymus hypoplasia is suggestive of 22q11 deletion. The
risk for chromosomal abnormalities is increased with RAA;
22q11 deletion is found in 5% to 10% of all fetuses with iso-
lated RAA.75,79  ventricular output is delivered to the lungs because the pul-
Ultrasound findings. The 3VT is the ‘gold standard’ to
detect arch anomalies. The trachea is used as a reference point
to evaluate the laterality of the great vessels. Normally, the aorta
and the duct are on the left side of the trachea. In RAA, the aor-
tic arch is on the right side of the trachea, and in 83%, the duct
is left sided so that the vessels surround the trachea, forming
a U-shape. In the remaining 17%, both arches are right sided.
In DAA, the ascending aorta bifurcates, and the left aortic arch
and RAA encircle the trachea and oesophagus completely80 (Fig.
29.12). An aberrant left subclavian artery (ALSCA) may com-
plete a vascular sling.  Specific features to check at follow-up and perinatal
Specific features to check at follow-up and perinatal
management
• Check for signs of tracheoesophageal compression: polyhy-
dramnios, hyperechogenic lungs, and a flattened diaphragm.
• Offer invasive genetic testing.
• Plan for delivery in a tertiary care centre. Consider an ex utero
intrapartum treatment (EXIT) procedure in fetuses with severe
tracheoesophageal obstruction.  PGE synthase inhibitors. Dietary manipulation may result in
Treatment and outcome. Treatment and outcome depend
on the presence or absence of tracheoesophageal obstruction and
associated anomalies. In cases of severe prenatal tracheal compres-
sion, an EXIT procedure may be indicated. In all other cases of
DAA, infants should be monitored for dyspnoea, tracheomalacia
and dysphagia, which direct surgical management. In RAA with
left duct, closure of the duct leaves a ligament that may cause tra-
cheal obstruction and require surgical resection. The ALSCA may
arise from the ductal pouch (Kommerell diverticulum), and PGE
may be required to maintain its patency, pending reimplantation.
• Fig. 29.12  Three-dimensional reconstruction of a fetus with double aortic
arch. dAo, Descending aorta; duct, arterial duct; PA, pulmonary atresia.
Paediatric surgeons usually electively clip the ductal remnant in
infancy to prevent tracheal wall damage or symptomatic oesopha-
geal obstruction. 
Constriction of the arterial duct
Pathophysiology. In utero, only 13% to 25% of the right
monary vasculature has a high impedance; the rest is shunted
through the arterial duct into the systemic circulation.83 Pre-
mature ductal constriction results in increased right ventricular
afterload and TR. 
Ultrasound findings. The right ventricle is dilated with reduced
function. Colour and pulsed-wave Doppler reveal significant TR.
The pulmonary valve may appear normal, but the pulmonary
artery is often dilated (Fig. 29.13A). Arterial duct Doppler shows
high systolic and diastolic velocities (Fig. 29.13B). 
management
• Stop administration of PGE synthase inhibitors such as non-
steroidal anti-inflammatory drugs.
• Consider dietary changes to avoid polyphenol-rich foods in
idiopathic ductal constriction.84
• Serial ultrasound to monitor for hydrops. 
Treatment and outcome. Discontinue drugs, particularly
reversal of ductal constriction. Children may require treatment
for pulmonary hypertension or reduced ventricular function after
birth.85 
Lesions Difficult to Detect Prenatally
Isolated anomalous pulmonary venous connection
Overview. In isolated total or partial anomalous pulmonary
venous connection (TAPVC or PAPVC), pulmonary venous
blood connects with the systemic circulation. Depending on the
location, it is classified into (i) supracardiac type, (ii) cardiac type,
 CHAPTER 29  The Heart 321

Turbulent
flow

Duct

Ao

60 60

45 45

30 30

15 15

cm/s cm/s
B
• Fig. 29.13  Significant left–right disproportion between aorta and pulmonary trunk and aliasing of flow is seen in this fetus with constriction of the ductus
arteriosus (A). Pulsed-wave Doppler shows increased flow velocities during diastole (B). Ao, Aorta; duct, ductus arteriosus.

(iii) infracardiac type and (iv) mixed type.86 TAPVC or PAPVC is mixed type and 19% with cardiac type.86 After successful surgery,
rarely detected during prenatal screening because the timing of the the prognosis is usually good; however, about 15% of patients have
screening examination is too early to identify the confluence and postoperative obstruction for which there is no good therapy.86
the low pulmonary blood flow in the four-chamber view.87  The recurrence risk is high (17%),9,86 and repeat fetal echocar-
diography is recommended in the third trimester in subsequent
Pathophysiology. Both PAPVC and TAPVC are asymptom- pregnancies. 
atic during fetal life.87 Postnatally, the neonates are profoundly
hypoxaemic and rapidly become acidotic, requiring urgent medi- Atrial septal defect
cal resuscitation and cardiovascular surgery.  Overview. Patency of the FO is essential for survival in utero;
therefore oval fossa ASDs are rarely diagnosed prenatally. The primum
Associated anomalies. ASD (defect located in the septum primum adjacent to the atrioven-
• TAPVC or PAPVC may occur in isolation or in combination tricular valves) is often part of an AVSD with different pathogenesis. 
with RAI.
• Extracardiac malformations include asplenia, malrotation and Associated anomalies
Scimitar syndrome. • ASD is seen in other CHDs with an obligate right-to-left shunt
• Chromosomal abnormalities are rare with TAPVC or PAPVC.  such as tricuspid atresia.
• ASD is commonly found with extracardiac anomalies and
Ultrasound findings. TAPVC is suspected when the flow in is associated with trisomy 21 or Holt-Oram, Noonan or
the pulmonary veins cannot be connected to the LA. A useful Treacher-Collins syndrome. 
clue is an increased distance between the LA and the descending
aorta.88,89 The presence of a dilated coronary sinus in the absence Specific features to check at follow-up and perinatal
of an LSVC should always raise suspicion for TAPVC.  management
• If primum ASD is suspected, check carefully for the presence
Specific features to check at follow-up and perinatal of dilated coronary sinus, which may mimic primum ASD.
management • Offer invasive diagnosis with primum ASD.
• Plan for delivery in a tertiary care centre. • Delivery is possible in a local hospital in cases with suspected
• Cardiac team and cardiovascular surgeons must be prepared isolated ASD.
for immediate intervention if obstructed pulmonary venous • Postnatal echocardiography is recommended at about 4 to
return is confirmed.  6 weeks of age when the pulmonary pressures have fallen to
determine its physiological significance. 
Treatment and outcome. Pulmonary venous obstruction is an
important risk factor for mortality and is present in 85% of infants Treatment and outcome. Generally, the outcome of isolated
with infracardiac type, 44% with supracardiac type, 41% with ASD is excellent. Infants are followed expectantly. Closure of
322 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Suspected abnormal
FHR

Obstetric
Fetal distress? assessment and intervention
(dependent on gestational age)

Irregular rhythm Bradyarrythmia Tachyarrythmia

FHR 110–180 beats/min FHR <110 beats/min FHR >180 beats/min

Assess AV conduction
Assess AV conduction Assess AV
conduction

Atrial contraction Ventricular

Atrial contraction
followed by beat without Atrial rate > 1:1 Ventricular
followed 1:1 conduction Atrial rate >
ventricular preceding atrial ventricular conduction: rate >
by ‘missed’ ventricular rate
contraction: contraction: rate atrial rate/
ventricular (2:1 or 3:1
conducted sinus complete
beat: conduction)
supraventricular ventricular ES bradycardia A-V-dissociation
nonconducted
ES
supraventricular
ES
lrregular AA Regular AA Sinus SVT with 1:1
Atrial flutter Ventricular
intervals: intervals: tachycardia conduction tachycardia
blocked atrial Is there any typically typically
bi-/trigeminy regularity in AV <180–200 220–280
conduction? beats/min beats/min

YES: NO: complete lnterval of lnterval of

increasing AV AV dissociation VA < AV VA > AV
intervals until CHB lll° AVRT AET
beat is skipped AVNRT PJRT
CHB ll° (type
Wenckebach)
Constant AV
interval, but
alternating non-
conducted beats:
CHB ll°
(type Mobitz)

Consider Treat with LQTS:

No treatment, No treatment; dexamethasone digoxin, Treat with Treat with magnesium,
monitor if monitor for to prevent flecainide sotalol sotalol lidocaine,
frequent development progression (or sotalol) or flecainide if >50% propanolol
or blocked of SVT to CHB Ill or if >50% if >50% of time AB mediated:
with hydrops of time of time dexamethasone

• Fig. 29.14  Flowchart for diagnosis of fetal arrhythmia. A suggested clinically oriented approach for the diagnosis of fetal abnormal fetal rhythm. AA
interval, Time interval between two consecutive atrial contractions; AB mediated, antibody-mediated; AET, atrial ectopic tachycardia; AV, atrioventricular;
AVNRT, atrioventricular nodal reentry tachycardia; AVRT, atrioventricular reentry tachycardia; CHB II°, second-degree congenital heart block; CHB III°,
third-degree congenital heart block; ES, extrasystole (i.e., premature contraction); FHR, fetal heart rate; LQTS, long-QT syndrome; PJRT, permanent junc-
tional reciprocating tachycardia; SVT, supraventricular tachycardia.

large defects is warranted either surgically or by a percutaneous on the mechanical interaction of the atria and ventricles using
approach in childhood.  ultrasound. M-mode tracings across atrium and ventricle and
simultaneous pulsed-wave Doppler of LV inflow and out-
flow,90,91 SVC and aorta91 or pulmonary artery and vein92 can
Arrhythmia be used interchangeably, but interpretation of recordings are
Overview. Abnormal cardiac rhythm is common during devel- sometimes difficult, and cases should be referred to an expert.
opment and is commonly detected by routine prenatal ultrasound, A suggested diagnostic flowchart for fetal arrhythmia is shown
cardiotocography or during auscultation of the fetal heart at a rou- in Fig. 29.14. 
tine antenatal appointment. Broadly, arrhythmia can be classified Perinatal management and treatment. Skin lotions contain-
into one of three categories: (i) irregular rhythm (110–180 beats/ ing cocoa butter and ingestion of cocoa products provoke ectopic
min), (ii) bradyarrhythmia (<110 beats/min) and (iii) tachyar- beats, and a reduction in their use is usually helpful therapeuti-
rhythmia (>180 beats/min). cally. No intervention is necessary in the majority of cases with
Ectopic beats (i.e., extrasystole) are the most common type ectopic beats; however, monitoring is warranted if they are fre-
of fetal arrhythmia and are usually benign. Sustained brady- or quent or blocked because there is an increased risk for the develop-
tachyarrhythmia warrants referral to an experienced fetal cardiolo- ment of supraventricular tachycardia.93
gist to assess cardiac morphology and perform rhythm analysis.  Prenatal treatment for supraventricular tachycardia is indicated
Ultrasound findings. Because fetal ECG is not readily avail- if it is sustained (>50% of the time) or in the presence of hydrops.
able, the prenatal classification and diagnosis rely primarily Digoxin, sotalol and flecainide have been widely used as first-line

therapy. To date, no randomised controlled trial has been per-
formed to demonstrate the superiority of any of these drugs.
No treatment is indicated for blocked atrial bigeminy, which
resolves spontaneously.
Anti-SSA or -SSB–positive pregnant women have a 3% risk
for developing congenital heart block (CHB) in their fetuses, with
15% in pregnancies following an affected case. Management of
these pregnancies by serial echocardiography in the office or hos-
pital has not been successful in preventing the development of
CHB. Studies are underway using home monitoring with a por-
table device to determine whether this will be more successful in
preventing CHB.94
The decision to treat bradycardia or tachycardia requires expert
evaluation, and there is great variety in treatment strategies because
the evidence base is poor.
Management of bradyarrhythmia depends on the underly-
ing cause: The use of transplacental steroids to treat and prevent
CHAPTER 29  The Heart 323
complete heart block has been extensively debated. It may reduce
hydrops or inflammation and prevent progression to CHB; con-
clusive evidence, however, is lacking, and side effects must be
weighed against possible benefits.95 
Conclusion
Prenatal detection of most CHD is feasible, and screening of the
entire pregnant population is recommended using the five trans-
verse view fetal heart examination protocol. Pregnancies compli-
cated by CHD should be referred to a tertiary care centre to make a
full diagnosis, provide interdisciplinary fetal and maternal monitor-
ing and planning of delivery and (immediate) postnatal interven-
tions depending on the type of CHD and any associated defects.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Arzt W, Wertaschnigg D, Veit I, et  al. Intra-
uterine aortic valvuloplasty in fetuses with
32. McElhinney DB, Marshall AC, Wilkins-
Haug LE, et  al. Predictors of technical suc-
critical aortic stenosis: experience and results cess and postnatal biventricular outcome
1. Hoffman JIE, Kaplan S. The incidence of
of 24 procedures. Ultrasound Obstet Gynecol. after in utero aortic valvuloplasty for aortic
congenital heart disease. J Am Coll Cardiol.
2011;37(6):689–695. stenosis with evolving hypoplastic left heart
2002;39(12):1890–1900.
18. Freud LR, McElhinney DB, Marshall AC, syndrome. Circulation. 2009;120(15):1482–
2.  Van Der Linde D, Konings EEM, Slager
et  al. Fetal aortic valvuloplasty for evolving 1490.
MA, et  al. Birth prevalence of congeni-
hypoplastic left heart syndrome: postnatal 33. Samanek M, Voriskova M. Congenital heart
tal heart disease worldwide: a systematic
outcomes of the first 100 patients. Circulation. disease among 815,569 children born between
review and meta-analysis. J Am Coll Cardiol.
2014;130(8):638–645. 1980 and 1990 and their 15-year survival: a
2011;58(21):2241–2247.
19. Peake LK, Draper ES, Budd JL, Field D. Out- prospective Bohemia survival study. Pediatr
3. Carvalho JS, Allan LD, Chaoui R, Copel JA,
comes when congenital heart disease is diag- Cardiol. 1999;20:411–417.
et  al. ISUOG Practice Guidelines (updated):
nosed antenatally versus postnatally in the UK: 34. Moerman PL, Van Dijck H, Lauweryns JM,
sonographic screening examination of
a retrospective population-based study. BMC et  al. Premature closure of the foramen ovale
the fetal heart. Ultrasound Obstet Gynecol.
Pediatr. 2015;15(1):1–8. and congenital pulmonary cystic lymphangiec-
2013;41(3):348–359.
20. Markkanen HK, Pihkala JI, Salminen JT, et al. tasis in aortic valve atresia or in severe aortic
4. The American Institute of Ultrasound in Medi-
Prenatal diagnosis improves the postnatal car- valve stenosis. Am J Cardiol. 1986;57(8):703–
cine. AIUM Practice Guideline for the Perfor-
diac function in a population-based cohort of 705.
mance of Fetal Echocardiography. J Ultrasound
infants with hypoplastic left heart syndrome. 35. Graziano JN, Heidelberger KP, Ensing GJ,
Med. 2013;32(6):1067–1082.
J Am Soc Echocardiogr. 2013;26(9):1073–1079. et al. The influence of a restrictive atrial septal
5. National Health Service. NHS Fetal Anomaly
21. Morris SA, Ethen MK, Penny DJ, et  al. Pre- defect on pulmonary vascular morphology in
Screening Programme: Standards [Internet].
natal diagnosis, birth location, surgical cen- patients with hypoplastic left heart syndrome.
https://www.gov.uk/topic/population-screen-
tre, and neonatal mortality in infants with Pediatr Cardiol. 2002;23(2):146–151.
ing-programmes/fetal-anomaly.
hypoplastic left heart syndrome. Circulation. 36. Song MS, Hu A, Dyamenahalli U, et al. Extra-
6. Yagel S, Cohen SM, Achiron R. Examination
2014;129(3):285–292. cardiac lesions and chromosomal abnormalities
of the fetal heart by five short-axis views: a
22. Peyvandi S, De Santiago V, Chakkarapani associated with major fetal heart defects: com-
proposed screening method for comprehensive
E, et  al. Association of prenatal diagnosis of parison of intrauterine, postnatal and post-
cardiac evaluation. Ultrasound Obstet Gynecol.
critical congenital heart disease with postnatal mortem diagnoses. Ultrasound Obstet Gynecol.
2001;17(5):367–369.
brain development and the risk of brain injury. 2009;33(5):552–559.
7. Mistry H, Gardiner HM. The cost-effective-
JAMA Pediatr. 2016;170(4):e154450. 37. Galindo A, Nieto O, Villagrá S, et  al. Hypo-
ness of prenatal detection for congenital heart
23. Freud LR, Escobar-Diaz MC, Kalish BT, et al. plastic left heart syndrome diagnosed in fetal
disease using telemedicine screening. J Telemed
Outcomes and predictors of perinatal mortal- life: associated findings, pregnancy outcome
Telecare. 2013;19(4):190–196.
ity in fetuses with Ebstein anomaly or tricuspid and results of palliative surgery. Ultrasound
8.  National Institute for Cardiovascular Out-
valve dysplasia in the current era: a multicentre Obstet Gynecol. 2009;33(5):560–566.
comes Research [Internet]. https://nicor4.nic
study. Circulation. 2015;132(6):481–489. 38. Glatz JA, Tabbutt S, Gaynor JW, et al. Hypo-
or.org.uk/chd/an_paeds.nsf/vwContent/Anten
24. Calderon J, Angeard N, Moutier S, et  al. plastic left heart syndrome with atrial level
atal Diagnosis?Opendocument.
Impact of prenatal diagnosis on neurocognitive restriction in the era of prenatal diagnosis. Ann
9. Allan LD, Crawford DC, Chita SK, et  al.
outcomes in children with transposition of the Thorac Surg. 2007;84(5):1633–1638.
Familial recurrence of congenital heart dis-
great arteries. J Pediatr. 2012;161(1):94–99. 39. Schneider C, McCrindle BW, Carvalho JS,
ease in a prospective series of mothers referred
25. Li Y, Yin S, Fang J, et al. Neurodevelopmental et al. Development of Z-scores for fetal cardiac
for fetal echocardiography. Am J Cardiol.
delay with critical congenital heart disease is dimensions from echocardiography. Ultrasound
1986;58(3):334–337.
mainly from prenatal injury not infant cardiac Obstet Gynecol. 2005;26(6):599–605.
10. Arya B, Glickstein JS, Levasseur SM, Wil-
surgery: current evidence based on a meta-anal- 40. Pasquini L, Mellander M, Seale A, et  al.
liams IA. Parents of children with congenital
ysis of functional magnetic resonance imaging. Z-scores of the fetal aortic isthmus and duct:
heart disease prefer more information than
Ultrasound Obstet Gynecol. 2015;45(6):639– an aid to assessing arch hypoplasia. Ultrasound
cardiologists provide. Congenit Heart Dis.
648. Obstet Gynecol. 2007;29(6):628–633.
2013;8(1):78–85.
26. Paladini D, Alfirevic Z, Carvalho JS, et  al. 41. Rychik J, Szwast A, Natarajan S, et al. Perinatal
11. Allan LD, Cook A, Sullivan I, Sharland GK.
ISUOG consensus statement on current and early surgical outcome for the fetus with
Hypoplastic left heart syndrome: effects of fetal
understanding of the association of neurode- hypoplastic left heart syndrome: a 5-year single
echocardiography on birth prevalence. Lancet.
velopmental delay and congenital heart disease: institutional experience. Ultrasound Obstet
1991;337(8747):959–961.
impact on prenatal counseling. Ultrasound Gynecol. 2010;36(4):465–470.
12. Ashburn DA, McCrindle BW, Tchervenkov
Obstet Gynecol. 2017;49(2):287–288. 42. Rychik J, Rome JJ, Collins MH, et  al. The
CI, et al. Outcomes after the Norwood opera-
27. Arzt W, Tulzer G. Fetal surgery for cardiac hypoplastic left heart syndrome with intact
tion in neonates with critical aortic stenosis or
lesions. Prenat Diagn. 2011;31(7):695–698. atrial septum: atrial morphology, pulmonary
aortic valve atresia. J Thorac Cardiovasc Surg.
28. Wohlmuth C, Wertaschnigg D, Wieser I, vascular histopathology and outcome. J Am
2003;125(5):1070–1082.
et  al. Tissue Doppler imaging in fetuses with Coll Cardiol. 1999;34(2):554–560.
13. Copel JA, Pilu G, Kleinman CS. Congeni-
aortic stenosis and evolving hypoplastic left 43. Donofrio MT, Moon-Grady AJ, Hornberger
tal heart disease and extracardiac anoma-
heart syndrome before and after fetal aor- LK, et  al. Diagnosis and treatment of fetal
lies: associations and indications for fetal
tic valvuloplasty. Ultrasound Obstet Gynecol. cardiac disease: a scientific statement from
echocardiography. Am J Obstet Gynecol.
2016;47(5):608–615. the American Heart Association. Circulation.
1986;154(5):1121–1132.
29. Arzt W, Tulzer G, Aigner M, et al. Invasive intra- 2014;129(21):2183–2242.
14. Tennstedt C, Chaoui R, Körner H, Dietel M.
uterine treatment of pulmonary atresia/intact 44. Wald RM, Tham EB, McCrindle BW, et  al.
Spectrum of congenital heart defects and extra-
ventricular septum with heart failure. Ultrasound Outcome after prenatal diagnosis of tricuspid
cardiac malformations associated with chro-
Obstet Gynecol. 2003;21(2):186–188. atresia: a multicentre experience. Am Heart J.
mosomal abnormalities: results of a seven year
30. Tworetzky W, McElhinney DB, Marx GR, 2007;153(5):772–778.
necropsy study. Heart. 1999;82(1):34–39.
et al. In utero valvuloplasty for pulmonary atre- 45. Beroukhim RS, Gauvreau K, Benavidez OJ,
15. Tulzer G, Arzt W, Franklin RCG, et al. Fetal
sia with hypoplastic right ventricle: techniques et al. Perinatal outcome after prenatal diagnosis
pulmonary valvuloplasty for critical pulmonary
and outcomes. Pediatrics. 2009;124(3):e510– of single-ventricle cardiac defects. Ultrasound
stenosis or atresia with intact septum. Lancet.
e518. Obstet Gynecol. 2015;45(6):657–663.
2002;360(9345):1567–1568.
31. Gardiner HM, Kovacevic A, Tulzer G, et  al. 46. Giglia TM, Mandell VS, Connor AR, et  al.
16. Chaturvedi RR, Ryan G, Seed M, et al. Fetal
Natural history of 107 cases of fetal aortic Diagnosis and management of right ventricle-
stenting of the atrial septum: technique and
stenosis from a European multicentre ret- dependent coronary circulation in pulmonary
initial results in cardiac lesions with left atrial
rospective study. Ultrasound Obstet Gynecol. atresia with intact ventricular septum. Circula-
hypertension. Int J Cardiol. 2013;168(3):2029–
2016;48(3):373–381. tion. 1992;86(5):1516–1528.
2036.

323.e1
323.e2 References
47. Daubeney PE, Sharland GK, Cook AC, et al.
Pulmonary atresia with intact ventricular sep-
tum: impact of fetal echocardiography on inci-
dence at birth and postnatal outcome. UK and
Eire Collaborative Study of Pulmonary Atresia
with Intact Ventricular Septum. Circulation.
1998;98(6):562–566.
48. Gardiner HM, Belmar C, Tulzer G, et al. Mor-
phologic and functional predictors of eventual
circulation in the fetus with pulmonary atresia or
critical pulmonary stenosis with intact septum. J
Am Coll Cardiol. 2008;51(13):1299–1308.
49. Daubeney PEF, Wang D, Delany DJ, et  al.
Pulmonary atresia with intact ventricular sep-
tum: predictors of early and medium-term
outcome in a population-based study. J Thorac
Cardiovasc Surg. 2005;130(4):1–9.
50. Watson H. Natural history of Ebstein’s anom-
aly of tricuspid valve in childhood and adoles-
cence. An international cooperative study of
505 cases. Br Heart J. 1974;36(5):417–427.
51. Baek JS, Yu JJ, Im YM, Yun T-J. Outcomes of
neonatal Ebstein’s anomaly without right ven-
tricular forward flow. J Thorac Cardiovasc Surg.
2016;152(2):516–521.
52. Fesslova V, Villa L, Nava S, et al. Spectrum and
outcome of atrioventricular septal defect in
fetal life. Cardiol Young. 2002;12(1):18–26.
53. Delisle MF, Sandor GG, Tessier F, Farquhar-
son DF. Outcome of fetuses diagnosed with
atrioventricular septal defect. Obstet Gynecol.
1999;94(5 Pt 1):763–767.
54. Craig B. Atrioventricular septal defect: from
fetus to adult. Heart. 2006;92(12):1879–1885.
55. Taketazu M, Lougheed J, Yoo S-J, et al. Spec-
trum of cardiovascular disease, accuracy of
diagnosis, and outcome in fetal heterotaxy syn-
drome. Am J Cardiol. 2006;97(5):720–724.
56. Jowett V, Paramasivam G, Seale A, et  al. Dia-
phragmatic hernia: a postnatal complication of
anomalous drainage of the umbilical vein. Ultra-
sound Obstet Gynecol. 2013;41(5):589–591.
57. Berg C, Knüppel M, Geipel A, et al. Prenatal
diagnosis of persistent left superior vena cava
and its associated congenital anomalies. Ultra-
sound Obstet Gynecol. 2006;27(3):274–280.
58. Szwast A, Tian Z, McCann M, et al. Vasoreac-
tive response to maternal hyperoxygenation in
the fetus with hypoplastic left heart syndrome.
Circ Cardiovasc Imaging. 2010;3(2):172–178.
59. Hiramatsu T, Matsumura G, Konuma T, et al.
Long-term prognosis of double-switch opera-
tion for congenitally corrected transposition of
the great arteries. Eur J Cardio-Thoracic Surg.
2012;42(6):1004–1008.
60. Poon LCY, Huggon IC, Zidere V, Allan
LD. Tetralogy of Fallot in the fetus in the
current era. Ultrasound Obstet Gynecol.
2007;29(6):625–627.
61. Zhao Y, Abuhamad A, Fleenor J, et al. Prena-
tal and postnatal survival of fetal Tetralogy of
Fallot: a meta-analysis of perinatal outcomes
and associated genetic disorders. J Ultrasound
Med. 2016;35(5):905–915.
62. Kim N, Friedberg MK, Silverman NH.
Diagnosis and prognosis of fetuses with
double outlet right ventricle. Prenat Diagn.
2006;26(8):740–745.
63. Hartge DR, Niemeyer L, Axt-Fliedner R, et al.
Prenatal detection and postnatal management
of double outlet right ventricle (DORV) in 21
singleton pregnancies. J Matern Fetal Neonatal
Med. 2012;25(1):58–63.
64. Smith RS, Comstock CH, Kirk JS, et  al.
Double-­ outlet right ventricle: an antenatal
diagnostic dilemma. Ultrasound Obstet Gynecol.
1999;14(5):315–319.
65. Collett RW, Edwards JE. Persistent trun-
cus arteriosus; a classification according
to anatomic types. Surg Clin North Am.
1949;29(4):1245–1270.
66. Volpe P, Paladini D, Marasini M, et al. Com-
mon arterial trunk in the fetus: characteristics,
associations, and outcome in a multicentre
series of 23 cases. Heart. 2003;89(12):1437–
1441.
67. Swanson TM, Selamet Tierney ES, Tworetzky
W, et  al. Truncus arteriosus: diagnostic accu-
racy, outcomes, and impact of prenatal diagno-
sis. Pediatr Cardiol. 2009;30(3):256–261.
68. Paladini D. How to identify the thymus in the
fetus: the thy-box. Ultrasound Obstet Gynecol.
2011;37(4):488–492.
69. Matsui H, Adachi I, Uemura H, et  al. Anat-
omy of coarctation, hypoplastic and inter-
rupted aortic arch: relevance to interventional/
surgical treatment. Expert Rev Cardiovasc Ther.
2007;5(5):871–880.
70. Becker AE, Becker MJ, Edwards JE. Anoma-
lies associated with coarctation of aorta:
particular reference to infancy. Circulation.
1970;41(6):1067–1075.
71. Paladini D, Volpe P, Russo MG, et al. Aortic
coarctation: prognostic indicators of survival in
the fetus. Heart. 2004;90(11):1348–1349.
72. Brown DL, Durfee SM, Hornberger LK. Ven-
tricular discrepancy as a sonographic sign of
coarctation of the fetal aorta: how reliable is it?
J Ultrasound Med. 1997;16(2):95–99.
73. Brown ML, Burkhart HM, Connolly HM,
et al. Coarctation of the aorta: lifelong surveil-
lance is mandatory following surgical repair. J
Am Coll Cardiol. 2013;62(11):1020–1025.
74. Canniffe C, Ou P, Walsh K, Bonnet D, Cel-
ermajer D. Hypertension after repair of aortic
coarctation—a systematic review. Int J Cardiol.
2012;167(6):2456–2461.
75. Mogra R, Saaid R, Kesby G, et al. Early fetal
echocardiography: experience of a tertiary
diagnostic service. Aust N Z J Obstet Gynaecol.
2015;55(6):552–558.
76. Edwards JE. Vascular rings related to anomalies
of the aortic arches. Mod Concepts Cardiovasc
Dis. 1948;17(8):1.
77. Yoo S-J, Min J-Y, Lee Y-H, et al. Fetal sono-
graphic diagnosis of aortic arch anomalies.
Ultrasound Obstet Gynecol. 2003;22(5):535–
546.
78. Naidu DP, Wohlmuth C, Gardiner HM. Pre-
natal diagnosis of double aortic arch: can we
predict airway obstruction (pseudo-CHAOS)
and need for airway EXIT? Ultrasound Obstet
Gynecol. 2017;49(5):660–661.
79. Perolo A, De Robertis V, Cataneo I, et al. Risk
of 22q11.2 deletion in fetuses with right aortic
arch and without intracardiac anomalies. Ultra-
sound Obstet Gynecol. 2016;48(2):200–203.
80. Wohlmuth C, Gardiner HM. The biplane
mode: it takes two to tango. Ultrasound Obstet
Gynecol. 2017;49(4):540.
81. Zielinsky P, Piccoli AL, Manica JLL, et  al.
Reversal of fetal ductal constriction after mater-
nal restriction of polyphenol-rich foods: an
open clinical trial. J Perinatol. 2012;32(8):574–
579.
82. Bubols GB, Zielinsky P, Piccoli AL, et  al.
Nitric oxide and reactive species are modulated
in the polyphenol-induced ductus arteriosus
constriction in pregnant sheep. Prenat Diagn.
2014;34(13):1268–1276.
83. Kiserud T. Physiology of the fetal circulation.
Semin Fetal Neonatal Med. 2005;10(6):493–
503.
84. Pérez-Jiménez J, Neveu V, Vos F, Scalbert
a. Identification of the 100 richest dietary
sources of polyphenols: an application of the
Phenol-Explorer database. Eur J Clin Nutr.
2010;64(suppl 3):S112–S120.
85. Gewillig M, Brown SC, De Catte L, et al. Pre-
mature foetal closure of the arterial duct: clini-
cal presentations and outcome. Eur Heart J.
2009;30(12):1530–1536.
86. Seale AN, Uemura H, Webber SA, et al. Total
anomalous pulmonary venous connection:
morphology and outcome from an interna-
tional population-based study. Circulation.
2010;122(25):2718–2726.
87. Seale AN, Carvalho JS, Gardiner HM, et  al.
Total anomalous pulmonary venous connec-
tion: impact of prenatal diagnosis. Ultrasound
Obstet Gynecol. 2012;40(3):310–318.
88. Berg C, Georgiadis M, Geipel A, Gembruch U.
The area behind the heart in the four-chamber
view and the quest for congenital heart defects.
Ultrasound Obstet Gynecol. 2007;30(5):721–
727.
89. Kawazu Y, Inamura N, Shiono N, et al. ‘Post-
LA space index’ as a potential novel marker for
the prenatal diagnosis of isolated total anoma-
lous pulmonary venous connection. Ultrasound
Obstet Gynecol. 2014;44(6):682–687.
90. Strasburger JF, Huhta JC, Carpenter RJ, et al.
Doppler echocardiography in the diagnosis and
management of persistent fetal arrhythmias.
J Am Coll Cardiol. 1986;7(6):1386–1391.
91. Nii M, Hamilton RM, Fenwick L, et al. Assess-
ment of fetal atrioventricular time intervals
by tissue Doppler and pulse Doppler echo-
cardiography: normal values and correla-
tion with fetal electrocardiography. Heart.
2006;92(12):1831–1837.
92. Carvalho JS, Prefumo F, Ciardelli V, et al. Eval-
uation of fetal arrhythmias from simultaneous
pulsed wave Doppler in pulmonary artery and
vein. Heart. 2007;93(11):1448–1453.
93. Donofrio MT, Moon-Grady AJ, Hornberger
LK, et  al. Diagnosis and treatment of fetal
cardiac disease: a scientific statement from
the American Heart Association. Circulation.
2014;129(21):2183–2242.
94. Prospective Maternal Surveillance of SSA
(Sjögren Syndrome A) Positive Pregnancies
Using a Hand-held Fetal Heart Rate Monitor.
NCT02920346. http://www.clinicaltrials.gov.
95. Eliasson H, Sonesson S-E, Sharland G, et  al.
Isolated atrioventricular block in the fetus: a ret-
rospective, multinational, multicentre study of
175 patients. Circulation. 2011;124(18):1919–
1926.
30
Fetal Lung Lesions
JAMES COOK, ANGELA YULIA, COLIN WALLIS AND PRANAV P. PANDYA
KEY POINTS
• A limited number of congenital malformations of the respira-
tory tract can be identified directly by prenatal sonography.
• These malformations should be described systematically
because definitive diagnosis requires histologic examination.
• The identification of subtle lesions that have no detrimental
effect on a fetus or postnatal respiratory function is increas-
ingly common.
• A lack of evidence surrounding the natural history of asymp-
tomatic cystic lung lesions has resulted in highly divergent
postnatal management strategies.
• A conservative approach to postnatal management of asymp-
tomatic cases is a reasonable option.
• Careful prenatal counselling is recommended.
Congenital Malformations of the Respiratory
Tract
Embryologic development of the respiratory tract requires the
appropriate growth of the upper airway and the six ‘trees’ that make
up the lower respiratory tract – bronchial, arterial (systemic and
pulmonary), venous (systemic and pulmonary) and lymphatic –
together with a normal thoracic volume, a normal thoracic skeletal
structure and normal neuromuscular function.
Defects in any of these elements (except the systemic venous
system because there are none known) can affect anatomical
organisation resulting in a variety of congenital abnormalities.
Whereas some lesions can be detected by direct prenatal sono-
graphic visualisation, other lesions are suspected because of the
presence of nonspecific findings (e.g., mediastinal shift) or form
part of a more generalised genetic syndrome, for example, the pul-
monary hypoplasia seen in skeletal dysplasias such asphyxiating
thoracic dystrophy. Other congenital malformations of the lungs
only become apparent in the postnatal period when they cause
symptoms.
Not all congenital malformations have a detrimental impact on
a fetus or postnatal respiratory function. Advances in sonographic
technology allow prenatal detection of subtle lesions which may
have no immediate clinical impact. A lack of evidence surround-
ing the natural history of asymptomatic cystic lung lesions has
resulted in divergent postnatal management strategies and diffi-
cult prenatal counseling.
In this chapter, we will identify thoracic malformations, exclud-
ing congenital diaphragmatic hernia (covered in Chapter 31), that
324
can be detected directly on prenatal sonography, describe a system
to classify these lesions sonographically, define their salient patho-
logical features and discuss the merits of prenatal and postnatal
management options. 
Thoracic Malformations Detected on
Prenatal Ultrasound
Thoracic malformations detectable on prenatal sonography are
detailed in Table 30.1; however, a definitive diagnosis of these lesions
(excluding congenital diaphragmatic hernia (CDH)) requires histo-
logic confirmation. A prenatal or postnatal diagnosis is not possible
by radiologic means alone. The imaging appearance of different
types of lesions can be identical, rendering specific pathological diag-
noses redundant and risking confusion in communication between
medical professionals and families. Moreover, it is now apparent
that there can be considerable overlap in histologic features within
lesions, further highlighting the increasing complexity of diagnosis
and the limitations of diagnosis based on imaging modalities alone.
In light of these difficulties, a system whereby malformations
detected by prenatal sonography are described meticulously in
simple language, based on their appearance, and without the
presumption of a single pathological diagnosis has been recom-
mended.1 Within this system, all thoracic malformations are
described under the umbrella term congenital thoracic malformation
(CTM). Malformations are then defined further using descriptive
terms, including the presence and size of cysts, the presence of a
feeding vessel, the degree of echogenicity, the presence or absence
of mediastinal shift, polyhydramnios and the presence of anoma-
lies in other systems. In practical terms, lesions have been most
usefully classified sonographically as either macrocystic or micro-
cystic (see Table 30.1).2
Detection of CTMs, often at the routine 20-week anomaly
scan, allows detailed planning of further prenatal management,
including serial sonographic monitoring, delineation of the lesion
and intrauterine therapeutic interventions if required. Planning
for appropriate neonatal support on delivery can be prepared in
advance. The exception to this pattern are pleural effusions, which
often present later when scanning is undertaken because of a sus-
picion of increased amniotic fluid or as an incidental finding on
ultrasound (USS) undertaken during the third trimester.
The appeal of enhanced prenatal radiologic definition of lung
CTMs has led to the exploration of magnetic resonance imag-
ing (MRI) as an additional modality. MRI has been used in the
delineation of fetal lung lesions3 and the identification of feeding
vessels,4 although this is readily done using Doppler ultrasound.
 CHAPTER 30  Fetal Lung Lesions 325

Whether this enhanced imaging provides any additional informa- mistaken for a cystic structure. The crucial importance of this dif-
tion of practical value over ultrasound alone has yet to be deter- ferentiation is the subsequent management strategy for CDH,
mined, and in most centres at present, MRI is not part of routine particularly the place of delivery, associations with chromosomal
practice.  abnormalities and genetic syndromes and general prognosis. Addi-
tional sonographic features that assist in the correct identification
Macrocystic Lung Lesions of a CDH include the absence of a stomach within the abdomen,
the visualisation of peristalsis within the thorax, the paradoxical
Macrocytic lung lesions include congenital pulmonary airway movement of abdominal viscera within the thorax during fetal
malformations (CPAMs), bronchogenic cysts, enteric cysts, bron- breathing movements and absence of a diaphragm. However, dif-
chial atresia and congenital lobar emphysema (see Table 30.1). ferentiation can remain difficult on occasion. In a case series of 110
The sonographic appearance includes a cystic lesion(s) of varying fetuses diagnosed with a CTM, two were later identified as having
size in the thorax with or without mediastinal shift (Fig. 30.1). a diaphragmatic hernia, both after serial prenatal scanning.2
An essential differential diagnosis to consider on identification
of a macrocystic lung lesion is a left-sided diaphragmatic hernia, Congenital Pulmonary Airway Malformations
in which the stomach or bowel herniated into the thorax can be
Congenital pulmonary airway malformations were previously
TABLE Differential Diagnosis of Congenital Thoracic known as congenital cystic adenomatoid malformations (CCAM).
30.1 Malformation In 2002, Stocker recommended the term CPAM as being pref-
erable to the term congenital cystic adenomatoid malformation
Macrocystic Lesions because not all types of CPAM are cystic and adenomatoid.5 The
Congenital pulmonary airway malformation new terminology enables a better description of the entity’s altera-
tions. For example, type 0 is not a cystic lesion, and types 0, 1 and
Bronchogenic cyst or enteric cyst
4 are not adenomatoid lesions.
Congenital diaphragmatic hernia Congenital pulmonary airway malformations represent the
most common cystic lesions diagnosed on prenatal ultrasound.
Bronchial atresia
Currently, the best estimate of incidence reported by the Euro-
Congenital lobar emphysema pean Surveillance of Congenital Anomalies (EUROCAT) is 0.94
Pleuropulmonary blastoma in 10 000 live births.6 Although CPAMs can be defined as mac-
rocystic or microcystic, both types are described in this section.
Microcystic Lesions Opinions vary as to the aetiology of these lesions. Genetic
Congenital pulmonary airway malformation abnormalities that may influence normal lung development or
external insults disrupting lung growth have been postulated.
Pulmonary sequestration
The subclassification of CPAM types remains contentious. Var-
Pleural effusion ious systems of classification have been proposed with the most
Tracheal or laryngeal atresia
widely accepted that of Stocker.5 Within this system, CPAMs are
classified into five types according to the level of the bronchial
Pulmonary hypoplasia or agenesis tree at which the defect is thought to have occurred. The strength
Mediastinal teratoma of this classification is that specific neoplasms are associated with
specific CPAM subgroups; however, these are not distinguishable
Rhabdomyoma with prenatal ultrasound, and postnatal histologic examination
Ectopia is required. There can be significant histologic overlap of lesions
previously considered distinct (e.g., hybrid forms of CPAM, pul-
   monary sequestration (PS) and bronchial atresia).

A B
• Fig. 30.1  A, Axial view through the chest of a fetus at 22 weeks with multiple cystic lesions in the chest. Note
the shift and compression of the heart. B, A single cyst is seen in the axial view with some mediastinal shift.
326 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Type 0 or acinar dysplasia. This is a rare type of CPAM and of cystic structures broadly termed foregut cysts.11 Subdivision of
is thought to develop at the level of the bronchus. On histologic these cysts can be made on the basis of their histology.
examination, bronchial airways are present, but the distal paren- Bronchogenic cysts have histologic features in keeping with the
chyma is highly unusual and consists mainly of mesenchymal primitive airway and are commonly identified as single cysts within
tissue. Macroscopically, the lungs are small, and the condition the mediastinum but may be situated anywhere along the bron-
is not compatible with life. The condition is also termed acinar chial tree or even in extrathoracic locations. They typically present
dysplasia.5  as a single cyst and are lined with respiratory-type epithelium and
Type 1. This is the most common type of CPAM, accounting contain cartilage in the wall on histologic examination. Clinical
for approximately 60% to 70% of cases.5 and thought to develop manifestations are most commonly attributable to airway com-
at the bronchial/bronchiolar level. The cysts range in diameter pression but cysts may also act as a nidus of infection and bleeding.
up to 10 cm, with at least one cyst more than 2 cm in diam- In contrast to bronchogenic cysts, enteric cysts have histologic
eter required for diagnosis. They are lined with pseudostratified features differentiated towards the gut rather than the bronchus.11
ciliated columnar epithelium, with mucous cell proliferation also They can be subdivided according to where they occur along the
present on occasion.5  gastrointestinal (GI) tract. Cysts arising from the oesophagus are
Type 2. These are less common than type 1 CPAMs, account- termed oesophageal cysts, and cysts arising at a distal point along
ing for 15% to 20% of cases,5 and are thought to arise at the the GI tract are termed gastroenteric. Malignant change has been
bronchiolar level. Lesions typically consist of multiple small cysts described in enteric cysts. 
which range in size but must be less than 2 cm in diameter for
diagnosis. The cysts are related to dilated bronchiole-like struc- Congenital Lobar Emphysema
tures and are surrounded by simplified alveolar tissue.5 These
lesions can be associated with other congenital abnormalities such This lesion is characterised by hyperinflation of a lobe or segment of
as renal agenesis or dysplasia7 as well as cardiovascular and neuro- the lung and ordinarily presents in a neonate or infant with respi-
logic abnormalities.  ratory distress. Occasionally, it is detected prenatally as an appar-
Type 3 or lung hyperplasia. These are a rare type of CPAM, ent macrocystic lesion on ultrasound or the cause of a profound
accounting for 5% to 10% of cases5 thought to arise at the bron- mediastinal shift. Partial airway obstruction caused by a mucosal
chiolar/alveolar duct level. Their inclusion as a CPAM is con- flap, twisting of the lobe on its pedicle or a defect in bronchial
troversial because the microscopic features of excess bronchiolar cartilage results in air trapping. Presumably, the prenatal features,
ducts and parenchyma typical of fetal lung is considered by some when present, are also a result of partial bronchial obstruction and
pathologists to represent lung hyperplasia.8 The lesions can be the accumulation of lung fluid. Histologically, a normal number
large and affect an entire lobe with consequent compression of of distended and sometimes ruptured alveoli are demonstrated.
surrounding lung tissue.  Rarely, there are increased alveolar counts, which has been termed
Type 4 or regressed pleuropulmonary blastoma. These are polyalveolar lobe.12 
very rare lesions and the most controversial of the CPAM diag-
noses. The cysts can be large and are impossible to distinguish
radiologically from type 1 CPAMs. On histologic examination, Microcystic Lung Lesions
however, the cysts are lined with alveolar or bronchiolar epithe- Congenital Pulmonary Airway Malformation
lial cells upon mesenchymal tissue.5 The only difference between
this lesion and a pleuropulmonary blastoma (PPB) is the absence Microcystic CPAMs appear as a uniformly hyperechogenic lesion
of blastema. Some argue that these lesions in fact represent a in the chest on prenatal USS (Fig. 30.2). As with macrocystic
regressed neoplasm rather than a form of CPAM.9  lesions, they can be associated with mediastinal shift and hydrops. 

Bronchial Atresia Pulmonary Sequestration

Bronchial atresia describes interruption of a lobar, segmental or
subsegmental bronchus either caused by discontinuity or membra-
nous interruption. It results in the cystic degeneration of the distal
lung parenchyma likely caused by accumulation of obstructed fetal
lung fluid. As previously stated, CPAMs are also thought to origi-
nate from a spectrum of bronchial defects, and bronchial atresia
may also lie along this spectrum. Indeed, evidence of bronchial
atresia is often identified in hybrid association with CPAMs and
PS.10 Despite complete interruption of the bronchus, the distal
lung can fill with air in the postnatal period and even become
hyperinflated, although the mechanism is not well understood.
Prenatally, there may be a cystic appearance or just mediastinal
shift.  bar lesions are identified beneath the left lower lobe or within the
Bronchogenic Cysts and Enteric Cysts
(Duplication Cysts)
The embryonic lung develops from an outgrowth of the primitive
foregut. Disruption of this division can result in the formation
Pulmonary sequestrations are defined as isolated areas of lung tis-
sue that do not communicate with the bronchial tree of the nor-
mal lung and receive a blood supply from a systemic vessel. They
are subdivided into two groups: intralobar, in which the lesion lies
within the visceral pleura, and extralobar, in which the seques-
tration is invested in its own pleura. There can be considerable
histologic overlap with features of type 2 CPAMs and bronchial
atresia,13 and in common with these other cystic lesions, the aeti-
ology of PS is not understood.
Many sequestrations present as an echogenic mass in the fetal
chest or abdomen at the 20-week anomaly scan. Intralobar seques-
trations are typically identified in the left lower lobe, and extralo-
abdomen. Doppler ultrasound can be used to identify the blood
supply (Fig. 30.3). Some sequestrations communicate directly
with the GI tract, most commonly the lower oesophagus and
stomach.14 Careful monitoring prenatally is required, particularly
if there are signs of hydrops as the anomalous blood supply can
result in cardiac failure. 

• Fig. 30.2  Axial view through the chest of a fetus at 21 weeks with micro-
cystic congenital pulmonary airway malformation.
• Fig. 30.3 Axial view through the chest of a fetus with a pulmonary
sequestration which is echogenic; the extra feeding vessel is seen.
A B
• Fig. 30.4  A, Axial view through the chest of a fetus at 23 weeks with laryngeal atresia. Note the appearance
of bilateral enlarged and hyperechogenic lungs. B, Parasagittal view of enlarged bilateral hyperechogenic
lungs with flattening of the diaphragm. C, Parasagittal view of laryngeal atresia showing a dilated trachea.
CHAPTER 30  Fetal Lung Lesions 327
Laryngeal or Tracheal Atresia
During embryogenesis, the epithelial lining of both the larynx and
trachea is derived from the endoderm. As the endoderm prolif-
erates, occlusion of the lumen of the larynx and trachea occurs
followed by recanalization at approximately 10 weeks’ gestation.
Failure of recanalization can be partial or complete, with complete
failure associated with laryngeal or tracheal atresia and partial fail-
ure associated with laryngeal webs.
Laryngeal and tracheal atresia are rare malformations but
should be considered if bilateral enlarged and hyperechogenic
lungs are present on ultrasound of the fetus. Careful ultrasound
examination may reveal a dilated trachea below the level of the
obstruction (Fig. 30.4). The mass effect of the distended lungs
results in the other typical radiologic features of mediastinal com-
pression and convexity of the diaphragms. Nonspecific features
such as polyhydramnios and fetal hydrops may also be evident.
Although these lesions can be isolated, they may also be part of
a genetic syndrome (e.g., Fraser syndrome), mandating detailed
fetal anomaly scanning and genetic investigations if deemed
appropriate. 
Pulmonary Hypoplasia and Agenesis
Pulmonary hypoplasia and pulmonary agenesis are related malfor-
mations characterised by a spectrum of lung parenchymal under-
development. Whereas pulmonary agenesis lies at the extreme end
of the spectrum with complete absence of lung parenchymal tis-
sue, pulmonary hypoplasia is defined by diminished numbers of
airways and alveoli resulting in a reduced lung size. The features
for both malformations may be unilateral or bilateral.
The lesions are thought to result from insults during lung
embryogenesis, the timing of which relates to the point on the
spectrum of the clinical features. Complete agenesis of one or
both lungs is exceptionally rare and is often associated with other
congenital abnormalities (e.g., scimitar syndrome associated with
anomalous pulmonary venous return). The remaining lung in uni-
lateral agenesis is hypertrophied and causes mediastinal shift.
Pulmonary hypoplasia is a more frequent finding and is char-
acterised by reduced lung volume on prenatal ultrasound with the
appearance of a small chest or increased heart:lung ratio. Associ-
ated congenital abnormalities are present in at least 50% of cases.
The pathogenesis might include:
• A deficiency in the thoracic volume inhibiting normal lung
growth, such as may be seen in skeletal dysplasias associated
with short ribs, in which the rib size restricts pulmonary growth
C
328 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A B
• Fig. 30.5  A, Parasagittal view of chest of a fetus at 21 weeks with fetal akinesia deformation sequence.
Note the appearance of the small chest and short ribs. B, Midsagittal view of the small chest.
• A deficiency in fetal breathing movements inhibiting normal
lung development, as in the fetal akinesia deformation sequence
(e.g., Pena Shokeir syndrome type 1) in which a general lack of
movement and minimal or no breathing or swallowing move-
ments inhibits normal lung development (Fig. 30.5)
• Severe oligohydramnios from early in pregnancy is the com-
monest cause of pulmonary hypoplasia, as may be seen in
bilateral renal agenesis, early-onset bilateral cystic renal disease
or severe preterm prelabour rupture of membranes (PPROM),
where a lack of amniotic fluid prevents the normal passage of
liquid in and out of the lungs which then cannot develop to
their full potential.
• A deficient vascular supply inhibiting normal lung growth,
which may result from congenital heart disease  of the diaphragm, do not add independent predictive value to the
Prenatal Management
General Considerations
The detection of a CTM on prenatal ultrasound should prompt
a detailed and expert scan to exclude CDH and detect other
congenital anomalies that may aid definition of the underlying
pathology. Identification of a systemic blood supply to the lesion
by Doppler USS is useful in defining a likely sequestration (see
Fig. 30.3).  • Maternal administration of betamethasone21,22
Cystic Lung Lesions
A definitive diagnosis of a congenital cystic lung lesion is not pos-
sible using ultrasound, and even histologic examination often
reveals hybrid features of different entities. As such, there is also
considerable overlap in the clinical management of these malfor-
mations. In general, the prognosis for a fetus with a cystic lung
lesion is good with the risk for intrauterine or perinatal death less
than 1% in the University College Hospital case series.2
Serial ultrasound scans should be undertaken at least every 4
weeks and sometimes more frequently if there is significant medi-
astinal shift because these lesions can change during the course of
gestation with most reducing in size over the course of the preg-
nancy.15,16 About half17 may increase in size until the middle of
the second trimester after which a reduction in size or even appar-
ent sonographic resolution is possible in up to 76% of cases.18
Features such as fetal hydrops or mediastinal shift, usually asso-
ciated with poor prognosis,19 may also resolve spontaneously.
Attempts have been made to identify prognostic features for use
within predictive outcome models such as various cyst volume
ratios.20 Crombleholme and colleagues reported a useful tool to
classify the risk for hydrops, the need for fetal intervention and
the perinatal survival rate.18 The CPAM volume ratio (CVR) is
calculated by dividing the volume of the CPAM (length × height ×
width × 0.52) by the head circumference. A CVR greater than 1.6
predicts an increased risk for fetal hydrops (≤75%), and a CVR
less than or equal to 1.6 is associated with a less than 3% risk
for hydrops.18 CVR has proven on retrospective and prospective
assessment to be the most useful predictor for the development of
fetal hydrops. In addition, CVR can also be very useful in coun-
selling parents, guiding the frequency of subsequent scans and
determining which patients to preemptively treat with antenatal
interventions described later. Other parameters such as a mass–
thorax ratio, cystic predominance of the lesion and eventration
CVR.20,21 However, the unpredictability of the natural history of
these lesions in utero means that regular sonographic assessment
remains the most powerful tool in the shaping of individual man-
agement strategies.
A variety of fetal interventions have been attempted to reduce
the mass effect of a lesion, prevent the progression of complica-
tions and improve the outcome for these fetuses. However, these
prenatal interventions are infrequently used and usually only in
cases of persistent preterm hydrops when the prognosis is poor.
Interventions include:
• Thoracocentesis23
• Shunt insertion23
• Resection of the cysts by open fetal surgery23
• Sclerotherapy by percutaneous injection24
• Radiofrequency ablation20
• Laser ablation25
Over the years, maternal betamethasone treatment has been sug-
gested to have beneficial effects on large microcystic CPAMs.26-28
In 1998, Higby and colleagues first described resolution of a large
CPAM when steroids therapy was initially given for fetal lung
maturation.29 Since then, several others have reported similar
effect with two standard doses of 12 mg of betamethasone intra-
muscularly, 24 hours apart, in which they showed a decrease in
CPAM growth lesions with CVR of 1.4 or greater at 19 to 26
weeks of gestation27,30 Betamethasone seems to be a good choice
because it does not cause decreased alveolarisation compared with
dexamethasone.31 The exact mechanism by which steroids induce
CPAM regression is not well understood. Curran and associates
hypothesised that steroids promote the maturation of the lung
cells,28 and San Feliciano and colleagues postulated that antena-
tal corticosteroids produce effects on vascular endothelial growth
factor, which plays a crucial role in pulmonary development.31

Others have speculated that steroids affect cell proliferation and
apoptosis and downregulate several genes related to abnormal
lung development, consequently reducing CPAM growth.22 Cur-
rent evidence suggests that in large CPAMs with hydrops, a course
of steroids appears to be a reasonable first-line therapy. However,
a variable response on maternal betamethasone treatment has
also been reported. In a study by Morris and colleagues 15 of 20
patients (13 were hydropic and 2 were nonhydropic) with high-
risk fetal CPAM (defined as a CPAM associated with hydrops
or a CVR >1.6) received at least one course of steroids.22 Seven
of the 13 hydropic fetuses (54%) showed an initial response to
steroid administration, but the 2 nonhydropic high-risk fetuses
progressed to birth without developing hydrops. Seven of the 15
patients, however, resulted in fetal demise or early postnatal death,
giving a survival rate of 53%.22 For a subset of high-risk CPAMs
with CVR greater than 1.6 that do not adequately respond to a
single course of steroids, multiple courses of antenatal betametha-
sone may facilitate the stabilisation or regression of CPAM and
result in favourable short-term outcomes without the need for
open fetal resection.32 Concerns regarding the long-term effects of
maternal betamethasone on fetal development have been raised,
but there are no studies so far to support deleterious effects occur-
ring after one or two courses of maternal betamethasone.33,34
Whether steroids should also be used in low risk CPAMs
without hydrops is more debatable, as the prognosis of low risk
CPAMs without intervention is generally good and spontaneous
regression may occur.35
One of the most commonly applied invasive intervention for the
management of cystic lung lesion is thoracocentesis. In the majority
of cystic lung lesions described by Cavoretto and colleagues, treat-
ment was aimed primarily at drainage of the effusion rather than sur-
gery of the lesions.36,37 However, treatment by thoracocentesis alone
lead to the subsequent reaccumulation of the effusion in most of the
cases, thus necessitating the placement of a thoracoamniotic shunt.
Schrey and colleagues studied 11 fetuses with macrocystic
CPAM who underwent thoracoamniotic shunting.37 Shunts were
inserted at a mean gestational age of 24.6 (range, 17–32) weeks.
Marked mediastinal shift was present in all cases. Six fetuses were
hydropic, and of the remaining five, one had severe polyhydram-
nios, three had lesions that were rapidly increasing in size and one
had a very large lesion at initial presentation. One hydropic fetus
that underwent the procedure at 17 weeks died 1 day later. The
mean gestational age of delivery of the 10 fetuses who survived
was 38.2 weeks. The group concluded that fetal thoracoamniotic
shunting for large macrocystic CPAM is associated with favour-
able outcome in most cases and should be considered in severe
cases even before hydrops develops.37 Open surgery to resect
lesions has serious maternal side effects and has only been prac-
ticed in one or two units in the United States.
Growing evidence supports a potential benefit of fetal laser abla-
tion of the systemic feeding artery (FLAFA) in improving survival
and avoiding the need for postnatal surgery in fetuses with bron-
chopulmonary sequestration (BPS).25,38,39 In one case report, a
hydropic fetus with a microcystic lung lesion and associated systemic
arterial supply underwent successful laser coagulation of the feeding
vessel and resolution of the hydrops.38 Cruz-Martinez and colleagues
assessed the effectiveness of FLAFA in five fetuses with hybrid lung
lesion associated with hydrops at risk for perinatal death.25,40 FLAFA
was performed successfully in all cases at a median gestational age of
24.9 (range, 24.4–31.7) weeks. After the intervention, the dimen-
sions of both lungs increased and fluid effusions resolved in all
cases. All cases were delivered liveborn at term, without respiratory
CHAPTER 30  Fetal Lung Lesions 329
morbidity, resulting in perinatal survival of 100%. During postna-
tal follow-up, three (60%) cases showed progressive regression of
the entire lung mass and did not require postnatal surgery; in two
(40%) cases, a progressive decrease in size of the mass was observed,
but a cystic portion of the lung mass persisted and postnatal lobec-
tomy was required. They concluded that in fetuses with large hybrid
lung lesions at risk for perinatal death, FLAFA is feasible and could
improve survival and decrease the need for postnatal surgery.25
However, it is important to be aware that these interventions are
not commonly applied techniques and are only reserved in cases
with a very poor prognosis, in which without any interventions, the
fetuses are unlikely to survive. Because of the rarity of these cases,
it is difficult to collect good quality controlled data to support the
use of these more aggressive interventions. Management should be
in a specialist centre with appropriate experience in fetal therapy.
The majority of lung lesions are small, not associated with
mediastinal shift, or the mediastinal shift resolves as pregnancy
progresses. In these cases, delivery can be planned without the
need for neonatal intensive care support. In contrast, if mediasti-
nal shift remains into the third trimester, neonatal intensive care
support with access to surgical backup is recommended. 
Laryngeal and Tracheal Atresia (see Fig. 30.4)
Upper airway atresia can occur in isolation but may also be associ-
ated with other congenital abnormalities (e.g., Fraser syndrome).41
In cases associated with multiple abnormalities termination of the
pregnancy may be discussed. When atresia is an isolated finding,
then a prenatal intervention involving the placement of a trache-
ostomy or tracheoplasty below the obstruction to assist in lung
development has been attempted with variable results.42,43
An intervention designed to secure a definitive airway during
delivery, before the onset of respiration, is termed ex utero intra-
partum treatment (‘EXIT procedure’). The technique requires the
positioning of a definitive airway by tracheostomy or intubation
during delivery, whilst fetal oxygenation is still maintained via the
placenta.44 When stable, delivery of the fetus is completed and sur-
gery to correct the obstruction can be planned (see Chapter 31).
Similar procedures may also be considered in cases of complete
high airway obstruction from other causes (e.g., cystic hygroma).
Malformations that cause complete high airway obstruction, includ-
ing laryngeal and tracheal atresia, have been classified into a group des-
ignated ‘congenital high airway obstruction syndrome’ (CHAOS).45 
Pulmonary Hypoplasia and Agenesis
There are multiple causes of lung hypoplasia (described previ-
ously). Correction of the underlying cause, if possible, may permit
further alveolar development, both prenatally and postnatally. In
many of the conditions associated with pulmonary hypoplasia or
pulmonary agenesis, such as those described earlier, the underly-
ing abnormality is so severe that survival would not be possible
even if the pulmonary hypoplasia could be corrected. Amnioinfu-
sion in women with early preterm rupture of membranes is not
proven to improve outcome and therefore is not recommended.46 
Postnatal Management
Cystic Lung Lesions
In common with prenatal management, there is considerable
overlap in management strategies for of all of these lesions. Only a
330 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
minority of neonates with a CTM exhibit respiratory distress due
to the mass effect of the lesion. In these cases, however, surgical
intervention is warranted, and the management is resection of the
lesion. In a case series of 119 neonates cared for at Great Ormond
Street Hospital (GOSH) diagnosed prenatally with CPAM or PS,
only 8 (6.7%) required emergency surgery during the neonatal
period.47 This is in keeping with another large case series from
Southampton of 72 neonates with a prenatal diagnosis of con-
genital lung malformation in which only one required emergency
surgery.
The vast majority of neonates remain asymptomatic. A CT scan
of the chest during the first 3 months of life48 should be under-
taken in all cases with a prenatally diagnosed cystic lung lesion
to enable an accurate assessment because chest radiographs alone
are unreliable and may falsely indicate resolution of the lesion.48
An echocardiogram can delineate the systemic feeding vessel in
cases of likely PS. After initial investigations, further management
remains the subject of intense debate because the need for sur-
gical intervention is less clear. This has resulted in highly diver-
gent management strategies largely because of a deficiency in our
knowledge of the natural history of these lesions. Supporters of a
conservative approach suggest that these lesions were largely clini-
cally silent in the population before routine anomaly scanning, the
risk for complications appears low49 and postnatal spontaneous
resolution may occur.50 However, advocates of a surgical approach
cite concerns regarding the potential for malignant transformation
and recurrent infection, resulting in approximately 70% of lesions
being resected worldwide.51
Divergent management strategies make prenatal counselling
of parents challenging and emphasises the need for a multidisci-
plinary approach involving the obstetrician, neonatologist, paedi-
atrician and paediatric surgeon, all providing a consistent message.
In the following section, we will summarise the arguments for and
against a surgical approach to asymptomatic cystic lung lesions.
There are four arguments used to justify a surgical approach9:
• The risk for malignancy
• The risk for complications such as infection
• The potential for compensatory lung regrowth after early
resection
• A low complication rate after elective surgery
The risk for malignancy. The two types of malignancy associ-
ated with cystic lung lesions are PPB and bronchioloalveolar car-
cinoma (BAC), both of which are rare in early life.49,50
Pleuropulmonary blastomas are a distinct pathological entity.
Crucially, a type 1 PPB cannot be distinguished radiologically
from a benign cystic lung lesion such as a type 1 CPAM. The risk
for a prenatally diagnosed benign cystic lesion in fact representing
a PPB is small because the majority of PBBs are identified post-
natally. A total of 350 pathology-confirmed PBB cases have been
identified to date by The International PPB Registry,52 of which
only 9 were identified on prenatal ultrasound scan. The probabil-
ity that a benign cystic lung lesion may be a PPB is increased if
there is a family history of PPB associated tumours (e.g., ovarian,
renal and thyroid) or mutations within the DICER1 gene (dem-
onstrated to be associated with 66% of cases) are detected.54,55 The
prognosis of these lesions depends on their histologic staging. Type
1 PBB has a better outcome (91% 5-year survival rate) than types
two and three (71% and 53% 5-year survival rates, respectively).52
Bronchioloalveolar carcinomas occur after the transformation of
mucinous cells, which are only identified within type 1 CPAMs.
These cells have the potential to develop into areas of atypical
adenomatous hyperplasia, transform into BAC (noninvasive) and
subsequently adenocarcinoma (AC) (invasive).54,55 The time course
of the progression described or if progression is inevitable is not
known. Both BAC and AC are exceptionally rare with only 24 cases
described in total, usually as an incidental finding in adult patients.
Importantly, there is evidence that prophylactic resection of
cystic lung lesions does not eliminate the risk for malignancy. Ade-
nocarcinoma has been described after previous resection of CPAM
in early life,56 and a PPB has been described after prior resection
of a cystic lesion from an anatomically distinct area of the lung.57 
The risk for other complications including recurrent infection.
The incidence of respiratory infection in children with CTM is
low in early life. Stanton and colleagues51 described 505 conser-
vatively managed infants, of whom 3.2% became symptomatic in
the first year. Ng and colleagues49 reported a series of 74 patients
of whom 5% of developed symptoms over a median follow-up
period of 5 years. The GOSH cohort (described earlier) has been
followed up for a median period of 9.9 years.58 We have estab-
lished that after the second year, the rate of resection for recurrent
respiratory infection diminishes, and there were no resections in
those over the age of 5 years.
In cases of PS, an additional complication is the possibility of
heart failure caused by high flow through the feeding vessel. In
these cases, embolization of the feeding vessel or resection of the
lesion has been performed. In addition, involution of these lesions
has been described after embolization.59
Bronchogenic and enteric cysts, if symptomatic, tend to cause
symptoms associated with airway compression, but other compli-
cations, including bleeding, have been described. 
The potential for compensatory lung growth after early
resection. Based on the traditional view that alveolar division
only continues for the first 2 years of life, it has been suggested
that improved compensatory lung growth may occur if resection
is completed during this period. However, new evidence suggests
that genuine new alveolar growth is likely to occur into adoles-
cence.60 Furthermore, the majority of lesions identified are small
and appear to continue to reduce in size as the lung grows in
early life. Indeed, in the GOSH cohort, we identified that 5.9%
of lesions had disappeared on CT imaging during the postnatal
period, which is a similar proportion to that reported previously.50 
The risk of surgery. If surgical intervention is being considered,
then the risks associated with surgery must be balanced against
the risks of complications and intended benefits described earlier.
In their meta-analysis, Stanton and colleagues51 reported a com-
plication rate of 5% in elective procedures carried out on asymp-
tomatic infants, including air leak, infection, effusion and death
in one case. More recently, Hall and colleagues61 have reported
a complication rate of 23% in a case series of 60 patients with
asymptomatic CPAM who underwent surgery. This included 3
cases of major complications: tension pneumothorax, aggressive
chest wall fibromatosis and near-fatal haemorrhage. 
Pulmonary Agenesis and Hypoplasia
The outcome of babies born with pulmonary hypoplasia or agen-
esis is generally poor and is associated with high neonatal mortality
and significant long-term morbidity rates.62,63 After delivery, these
infant need respiratory support, which can range from supplying
supplemental oxygen to mechanical ventilation, including high-
frequency ventilation and extracorporeal membrane oxygenation.
Infants with severe respiratory failure secondary to pulmonary
hypoplasia and documented persistent pulmonary hypertension
of the newborn may benefit from inhaled nitric oxide (NO), but

the data are limited.64 Aggressive ventilation in these infants can
cause overexpansion of lungs with compresses intraalveolar capil-
laries, which further exacerbates pulmonary hypertension. There
is a small population study which demonstrated that NO may be
beneficial as adjunct therapy in combination with sildenafil and
dopamine infusions to improve neonatal survival; however, larger
studies are needed to support the practice.65  A conservative approach to prenatally diagnosed asymptomatic
Conclusion
There are a wide range of congenital malformations of the respira-
tory tract, some of which are detected on prenatal sonography.
It is not possible to distinguish among these lesions sonographi-
cally, and prenatal reports should be based on a purely descrip-
tive system of reporting. There is also considerable overlap in the
pathological features of various cystic lung lesions, the aetiology of
which is poorly understood.
CHAPTER 30  Fetal Lung Lesions 331
Interventions are usually only undertaken in the prenatal and
postnatal period if significant symptoms are present. The vast
majority of lesions detected are asymptomatic, with many appear-
ing to resolve spontaneously in both the pre- and postnatal peri-
ods. Postnatal management remains controversial because of a lack
of knowledge of their natural history.
CTMs is a reasonable option in some asymptomatic cases. Careful
explanation to parents and prenatal counselling regarding these
lesions is recommended. Unfortunately, there remains no prospect
of a randomised trial to assist with decision making in these diffi-
cult cases, and clinical judgment is required. Continued long-term
follow-up of cases will assist in further definition of their natural
history.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 26. Loh KC, Jelin E, Hirose S, et al. Microcystic congenital pulmonary air-
way malformation with hydrops fetalis: steroids vs open fetal resection.
J Pediatr Surg. 2012;47:36–39.
1. Bush A. Congenital lung disease. A plea for clear thinking and clear
27. Tsao K, Hawgood S, Vu L, et al. Resolution of hydrops fetalis in con-
nomenclature. Pediatr Pulmonol. 2001;32:328–337.
genital cystic adenomatoid malformation after prenatal steroid therapy.
2. Bush A, Hogg J, Chitty LS. Cystic lung lesions—prenatal diagnosis and
J Pediatr Surg. 2003;38:508–510.
management. Prenat Diagn. 2008;28:604–611.
28. Curran PF, Jelin EB, Rand L, et  al. Prenatal steroids for microcystic
3. Liu YP, Chen CP, Shih SL, et al. Fetal cystic lung lesions: evaluation with
congenital cystic adenomatoid malformations. J Pediatr Surg. 2010;45:
magnetic resonance imaging. Pediatr Pulmonol. 2010;45:592–600.
145–150.
4. Zeidan S, Hery G, Lacroix F, et  al. Intralobar sequestration associated
29. Higby K, Melendez BA, Heiman HS. Spontaneous resolution of non-
with cystic adenomatoid malformation: diagnostic and thoracoscopic pit-
immune hydrops in a fetus with a cystic adenomatoid malformation.
falls. Surg Endosc. 2009;23:1750–1753.
J Perinatol. 1998;18:308–310.
5. Stocker JT. Congenital pulmonary airway malformation—a new name
30. Peranteau WH, Wilson RD, Liechty KW, et al. Effect of maternal beta-
and an expanded classification of congenital cystic adenomatoid malfor-
methasone administration on prenatal congenital cystic adenomatoid mal-
mations of the lung. Histopathology. 2002;41:424S–431S.
formation growth and fetal survival. Fetal Diagn Ther. 2007;22:365–371.
6. EUROCAT: European Surveillance of Congenital Anomalies. EURO-
31. San Feliciano L, Remesal A, Isidoro-García M, Ludeña D. Dexametha-
CAT Guide 1.4 and Reference Documents 2013. www.eurocat‐network.
sone and betamethasone for prenatal lung maturation: differences in
eu/.
vascular endothelial growth factor expression and alveolarization in rats.
7. Stocker J, Madewell J, Drake R. Congenital cystic adenomatoid mal-
Neonatology. 2011;100:105–110.
formation of the lung. Classification and morphologic spectrum. Hum
32. Derderian S, Coleman A, Jeanty C, et al. Favorable outcomes in high–
Pathol. 1977;8:155–171.
risk congenital pulmonary airway malformations treated with multiple
8. Langston C. New concepts in the pathology of congenital lung malfor-
courses of maternal betamethasone. J Pediatr Surg. 2015;50:515–518.
mations. Semin Pediatr Surg. 2003;12:17–37.
33. Stutchfield PR, Whitaker R, Gliddon AE, et al. Behavioural, educational
9. Kotecha S, Barbato A, Bush A, et al. Antenatal and postnatal manage-
and respiratory outcomes of antenatal betamethasone for term cesarean
ment of congenital cystic adenomatoid malformation. Paediatr Respir
section (ASTECS trial). Arch Dis Child Fetal Neonatal Ed. 2013;98:
Rev. 2012;13:162–170.
F195–F200.
10. Kunisaki SM, Fauza DO, Nemes LP, et al. Bronchial atresia: the hidden
34. Wapner RJ, Sorokin Y, Thom EA, et al. Single versus weekly courses of ante-
pathology within a spectrum of prenatally diagnosed lung masses. J Pedi-
natal corticosteroids: evaluation of safety and efficacy. Am J Obstet Gynecol.
atr Surg. 2006;41(1):61–65.
2006;195:633–642.
11. Nobuhara KK, Gorski YC, La Quaglia MP, et al. Bronchogenic cysts and
35. Witlox RS, Lopriore E, Oepkes D. Prenatal interventions for fetal lung
esophageal duplications. Common origins and treatment. J Pediatr Surg.
lesions. Prenat Diagn. 2011;31:628–636.
1997;32:1408–1413.
36. Cavoretto P, Molina F, Poggi S, et  al. Prenatal diagnosis and outcome
12. Mani H, Suarez E, Stocker JT. The morphologic spectrum of infantile
of echogenic fetal lung lesions. Ultrasound Obstet Gynecol. 2008;32:769–
lobar emphysema. A study of 33 cases. Paediatr Respir Rev. 2004;5(suppl
783.
1):S313–S320.
37. Schrey S, Kelly EN, Langer JC, et  al. Fetal thoracoamniotic shunting
13. Cass DL, Cromblehome TM, Howell LJ, et al. Cystic lung lesions with
for large macrocystic congenital cystic adenomatoid malformations of the
systemic blood supply: a hybrid of congenital cystic adenomatoid malfor-
lung. Ultrasound Obstet Gynecol. 2012;39(5):515–520.
mation and bronchopulmonary sequestration. J Pediatr Surg. 1997;32:
38. Oepkes D, Devlieger R, Lopriore E, et al. Successful ultrasound–guided
986–990.
laser treatment of fetal hydrops caused by pulmonary sequestration.
14. Newman B. Congenital bronchopulmonary foregut malformations: con-
Ultrasound Obstet Gynecol. 2007;29(4):457–459.
cepts and controversies [review]. Pediatr Radiol. 2006;36(8):773–791.
39. Mallmann MR, Geipel A, Bludau M, et al. Bronchopulmonary sequestra-
15. Cavoretto P, Molina F, Poggi S, et  al. Prenatal diagnosis and outcome
tion with massive pleural effusion: pleuroamniotic shunting vs intrafetal
of echogenic fetal lung lesions. Ultrasound Obstet Gynecol. 2008;32:
vascular laser ablation. Ultrasound Obstet Gynecol. 2014;44:441–446.
769–783.
40. Cruz-Martinez R, Martínez-Rodríguez M, Bermúdez-Rojas M, et  al.
16. Ierullo AM, Ganapathy R, Crowley S, et al. Neonatal outcome of antena-
Fetal laser ablation of feeding artery of cystic lung lesions with systemic
tally diagnosed congenital cystic adenomatoid malformations. Ultrasound
arterial blood supply. Ultrasound Obstet Gynecol. 2017;49(6):744–750.
Obstet Gynecol. 2005;26:150–153.
41. Lacy DE, Shaw NJ, Pilling DW, et  al. Outcome of congenital lung
17. Kunisaki SM, Barnewalt CE, Estroff JA, et al. Large fetal congenital cystic
abnormalities detected antenatally. Acta Paediatr. 1999;88:454–458.
adenomatoid malformations: growth trends and patient survival. J Pediatr
42. Paek BW, Callen PW, Kitterman J, et  al. Successful fetal intervention
Surg. 2007;42:404–410.
for congenital high airway obstruction syndrome. Fetal Diagn Ther.
18. Crombleholme TM, Coleman B, Hedrick H, et al. Cystic adenomatoid mal-
2002;17:272–276.
formation volume ratio predicts outcome in prenatally diagnosed cystic ade-
43. Saadai P, Jelin EB, Nijagal A, et  al. Long–term outcomes after fetal
nomatoid malformation of the lung. J Pediatr Surg. 2002;37(3):331–338.
therapy for congenital high airway obstructive syndrome. J Pediatr Surg.
19. Schott S, Mackensen-Haen S, Wallwiener M, et al. Cystic adenomatoid
2012;47(6):1095–1100.
malformation of the lung causing hydrops fetalis: case report and review
44. Wilson RD, Chitty LS. Anomalies of the fetal thorax and abdomen: diag-
of the literature. Arch Gynecol Obstet. 2009;280:293–296.
nosis, management and outcome. Prenat Diagn. 2008;28(7):567.
20. Vu L, Tsao K, Lee H, et al. Characteristics of congenital cystic adenoma-
45. Lim FY, Crombleholme TM, Hedrick HL, et  al. Congenital high air-
toid malformations associated with nonimmune hydrops and outcome.
way obstruction: natural history and management. J Pediatr Surg.
J Pediatr Surg. 2007;42:1351–1356.
2003;38:940–945.
21. Curran PF, Jelin EB, Rand L, et  al. Prenatal steroids for microcystic
46. Roberts D, Vause S, Martin W, et al. Amnioinfusion in very early preterm
congenital cystic adenomatoid malformations. J Pediatr Surg. 2010;45:
prelabor rupture of membranes (AMIPROM): pregnancy, neonatal and
145–150.
maternal outcomes in a randomized controlled pilot study. Ultrasound
22. Morris LM, Lim FY, Livingston JC, et  al. High–risk fetal congenital
Obstet Gynecol. 2014;43:490–499.
pulmonary airway malformations have a variable response to steroids.
47. Cook J, Chitty LS, De Coppi P, et al. The natural history of prenatally
J Pediatr Surg. 2009;44:60–65.
diagnosed congenital cystic lung lesions: long-term follow-up of 119
23. Adzick NS, Harrison MR, Crombleholme TM, et al. Fetal lung lesions.
cases. Arch Dis Child. 2017;102:789–790.
Management and outcome. Am J Obstet Gynecol. 1998;179:884–889.
48. Epelman M, Kreiger PA, Servaes S, et  al. Current imaging of prenatally
24. Bermúdez C, Pérez–Wulff J, Arcadipane M, et  al. Percutaneous fetal
diagnosed congenital lung lesions. Semin Ultrasound CT MR. 2010;31:
sclerotherapy for congenital cystic adenomatoid malformation of the
141–157.
lung. Fetal Diagn Ther. 2008;24:237–240.
49. Ng C, Stanwell J, Burge DM, et al. Conservative management of antenatally
25. Cruz–Martinez R, Mendez A, Duenas–Riano J, et al. Fetal laser surgery
diagnosed cystic lung malformations. Arch Dis Child. 2014;99:432–437.
prevents fetal death and avoids the need for neonatal sequestrectomy in
50. Butterworth SA, Blair GK. Postnatal spontaneous resolution of congeni-
cases with bronchopulmonary sequestration. Ultrasound Obstet Gynecol.
tal cystic adenomatoid malformations. J Pediatr Surg. 2005;40:832–834.
2015;46:627–628.

331.e1
331.e2 References
51. Stanton M, Njere I, Ade-Ajayi N, et  al. Systematic review and meta–
analysis of the postnatal management of congenital cystic lung lesions.
J Pediatr Surg. 2009;44:1027–1033.
52. Messinger YH, Stewart DR, Priest JR, et al. Pleuropulmonary blastoma:
a report on 350 central pathology–confirmed pleuropulmonary blastoma
cases by the International Pleuropulmonary Blastoma Registry. Cancer.
2015;121:276–285.
53. Schultz KA, Harris A, Williams GM, et al. Judicious DICER1 testing and
surveillance imaging facilitates early diagnosis and cure of pleuropulmo-
nary blastoma. Pediatr Blood Cancer. 2014;61:1695–1697.
54. Mani H, Shilo K, Galvin JR, et al. Spectrum of precursor and invasive
neoplastic lesions in type 1 congenital pulmonary airway malformation:
case report and review of the literature. Histopathology. 2007;51:552–580.
55. Travis WD, Garg K, Franklin WA, et al. Bronchioloalveolar carcinoma
and lung adenocarcinoma: the clinical importance and research relevance
of the 2004 world health organization pathologic criteria. J Thorac Oncol.
2006;1:S13–S19.
56. Ioachimescu O, Mehta A. From cystic pulmonary airway malformation,
to bronchioloalveolar carcinoma and adenocarcinoma of the lung. Eur
Respir J. 2005;26:1181–1187.
57. Papagiannnopoulos K, Sheppard M, Bush A, et  al. Pleuropulmonary
blastoma: is prophylactic resection of congenital lung cysts effective. Ann
Thorac Surg. 2001;72:604–605.
58. Cook J, Chitty LS, De Coppi P, et al. The natural history of prenatally
diagnosed congenital cystic lung lesions: long-term follow-up of 119
cases. Arch Dis Child. 2017;102(9):798–803.
59. Cho MJ, Kim DY, Kim SC, et al. Embolization versus surgical resection
of pulmonary sequestration: clinical experiences with a thoracoscopic
approach. J Pediatr Surg. 2012;47(12):2228–2233.
60. Narayanan M, Owers-Bradley J, Beardsmore CS, et  al. Alveolariza-
tion continues during childhood and adolescence: new evidence from
helium–3 magnetic resonance. Am J Respir Crit Care Med. 2012;185:186–
191.
61. Hall NJ, Chiu PP, Langer JC. Morbidity after elective resection of pre-
natally diagnosed asymptomatic congenital pulmonary airway malforma-
tions. Pediatr Pulmonol. 2016;51(5):525–530.
62. Williams O, Hutchings G, Debieve F, Debauche C. Contemporary
neonatal outcome following rupture of membranes prior to 25 weeks
with prolonged oligohydramnios. Early Hum Dev. 2009;85:273–
277.
63. Kilbride HW, Yeast J, Thibeault DW. Defining limits of survival: lethal
pulmonary hypoplasia after midtrimester premature rupture of mem-
branes. Am J Obstet Gynecol. 1996;175:675–681.
64. Keszler M. Guidelines for rational and cost–effective use of iNO therapy
in term and preterm infants. J Clin Neonatol. 2012;1(2):59–63.
65. Tiryaki S, Ozcan C, Erdener A. Initial oxygenation response to inhaled
nitric oxide predicts improved outcomes in congenital diaphragmatic her-
nia. Drugs D R. 2014;14(4):215–219.
31
Congenital Diaphragmatic Hernia
FRANCESCA MARIA RUSSO, LIESBETH LEWI, JUTE RICHTER AND JAN DEPREST
KEY POINTS
• Congenital diaphragmatic hernia occurs in 1 to 4 in 10,000
births. The condition is isolated in more than 50% of the
cases.
• The main causes of mortality and morbidity are respiratory
insufficiency and persistent pulmonary hypertension of the
newborn.
• Prenatal diagnosis should be made by screening ultrasound,
after which patients are referred to specialised centres.
• In isolated cases, the size of the lungs and the presence of
liver herniation are antenatal predictors of outcome.
• In cases with anticipated poor outcome, a potential option is
fetal treatment in the form of fetoscopic endoluminal tracheal
occlusion.
Epidemiology and Background
Congenital diaphragmatic hernia (CDH) is a developmental
anomaly with a prevalence ranging between 1 and 4 in 10,000
births, which means that in Europe, around 2000 children are
born with this condition every year.1 Despite being relatively
uncommon, CDH is a major clinical concern inside the realm of
neonatology, with important implications for diagnosis, manage-
ment and prognosis. Although medical and surgical management
have improved the outcome of this condition, CDH remains asso-
ciated with high mortality and significant morbidity.2
A primary characteristic of CDH is that the diaphragm fails to
form properly during embryogenesis. Normally, the diaphragm
develops to form a continuous sheet that completely separates the
thoracic and abdominal cavities before the major period of internal
organ growth. In the case of CDH, a significant proportion of the
diaphragm is absent.3 The defect is usually on the left side (85%)
but can also occur on the right (12%–15%) or bilaterally. In some
rare instances, a true agenesis of the hemidiaphragm is present,
but in most cases, the defect is limited to the posterolateral region
of the diaphragm (referred to as Bochdalek hernia). The anterior
(Morgagni hernia; 25%–30%) or central regions (2%–5%) can
also be affected.4 Less often, the diaphragm is present but thinned
and devoid of muscular fibres (diaphragmatic eventration).5
The diaphragmatic defect allows herniation of abdominal vis-
cera into the thorax, where they compete for the space normally
reserved to accommodate the growing lungs. When the defect is
located on the left side, the thorax may contain small and large
bowel, the spleen, the stomach, the left lobe of the liver and,
332
occasionally, the kidney. Right-sided CDHs virtually always con-
tain part of the right lobe of the liver and sometimes the bowel,
kidney, or both.6 The loss of a continuous diaphragmatic muscle
also impairs fetal breathing movements that are necessary for
proper stretch-induced lung maturation.7
Lungs of fetuses with CDH display variable degrees of lung
hypoplasia, with impairment of both airway and vascular matura-
tion. These changes become symptomatic immediately after birth,
when neonates have variable degrees of respiratory insufficiency
and persistent pulmonary hypertension (PPH), which is often
resistant to inhaled nitric oxide (iNO).8
In 50% to 60% of cases, the diaphragmatic defect and lung
hypoplasia are the only significant anomalies. In the remaining
cases, there are major nonpulmonary congenital anomalies.9 Car-
diovascular defects such as ventricular septal defects, cardiac out-
flow anomalies (tetralogy of Fallot, double outlet right ventricle,
transposition of the great vessels and others) and abnormal great
vessels (right aortic arch, double aortic arch, truncus arteriosus,
abnormal subclavian arteries and others) are the most common
associated anomalies, found in about one third of patients with
CDH.10 Left ventricular hypoplasia has also been described, yet
its occurrence and clinical relevance are debated.11 Musculoskel-
etal defects such as anomalies of the limbs or of the number and
shape of the vertebral bodies or ribs, neural tube defects,9 abdomi-
nal wall defects,12 craniofacial defects or urinary tract anomalies
have also been reported. Associated malformations are sometimes
components of Pallister-Killian and Fryns syndromes; Ghersoni-
Baruch syndrome; Wilms tumour, aniridia, genitourinary anoma-
lies, and mental retardation (WAGR); Denys-Drash; and other
syndromes. Some chromosomal anomalies, such as 9p tetrasomy,
have CDH as part of their spectrum. For further information we
refer to the excellent review by Slavotinek and colleagues.13
Finally, the presence of the intestine in the thorax during late
fetal development causes malrotation, malfixation, or both, which
can further complicate the disease. 
Aetiology and Pathogenesis
The causes of CDH are largely unknown, although exposure to
teratogens or pharmacologic agents has been suggested. In par-
ticular, phenmetrazine, thalidomide, quinine, nitrofen and vita-
min A deficiency have been linked to this disease.14 In the classical
view, the defect in CDH occurs first in the muscular part of the
diaphragm. However, studies in rats have suggested that CDH is a
primary lung pathology, even in humans.15 Keijzer and colleagues
proposed the ‘dual-hit hypothesis’, that is, that two independent
events cause the major features seen in CDH.16 These hits disturb

normal lung development (first hit) and diaphragm formation
(second hit). Data from animal studies confirm this hypothesis: in
the toxic nitrofen model in rats, abnormalities in the ipsilateral as
well as the contralateral lung are present already before the devel-
opment of the diaphragm.17
Another theory is based on the hypothesis that nonclosure of
the pleuroperitoneal canals would be caused by a defect in the
pleuroperitoneal folds (PPFs), the source of diaphragmatic cells.
Of interest, the diaphragmatic defect in the nitrofen model is
located more medial than could be expected from nonclosure of
these canals.18 Therefore it has been proposed that the origin of
the diaphragmatic defect lies in the amuscular mesenchymal pre-
cursor cells of the diaphragm, which are also derived from the
PPFs. This theory is based on the observation that although the
migration of muscular precursors is not disturbed, a defect occurs
in regions of the underlying mesenchymal substratum of the PPF.
This would subsequently contribute to the defective region in
CDH.
Finally, an involvement of the retinoid signalling pathway is
likely in CDH. Both animal and clinical studies have shown that
retinol and retinol-binding proteins are decreased in newborns
with this malformation.19,20 Moreover, some of the genes involved
in the pathogenesis of human CDH are tightly related to retinoid
signalling.21  Congenital Diaphragmatic Hernia–Related
Prognosis
Congenital diaphragmatic hernia was described many years ago,22
but survival after repair was not achieved until the 20th century.6
The symptoms of insufficient gas exchange are associated with
those of PPH, and unless invasive treatment is undertaken, the
respiratory condition deteriorates rapidly until death.
Survival rates vary widely amongst various neonatal manage-
ment centres. When only liveborn children undergoing surgery
are included, survival until discharge approaches 70% for isolated
CDH.23 If terminations of pregnancy, spontaneous abortions,
stillbirths, prehospital or preoperative deaths and surgical mortal-
ity are taken into account, the mortality rate is between 50% and
60%.12 The gap between these numbers is usually referred to as the
‘hidden mortality’. However, significant advances in the postnatal
management, with the introduction of ‘gentle ventilation and per-
missive hypercarbia’, have resulted in improved survival rates over
the past 2 decades.24 The improved survival of very sick babies
is, however, associated with a higher risk for severe morbidity24
persisting beyond the initial hospitalisation, especially in those
treated with extracorporeal membrane oxygenation (ECMO).
The severity of lung hypoplasia and PPH are the key determi-
nants of both mortality and morbidity and thus of quality of life.25
More than half of survivors are oxygen dependent at 28 days of
age,2 and 16% require oxygen at the time of discharge for a mean
duration of 14.5 months.26 Restrictive and obstructive lung dis-
eases have also been reported in CDH survivors many years after
operation.27 Diaphragmatic rigidity and thoracic deformities can
play a minor role in chronic lung disease.28 Bronchodilators may
be needed in 40% of patients in the first year of life.26 PPH may
persist in up to 30% of patients at 2 months of age and is associ-
ated with increased risk for early death and increased morbidity.29
PPH also deeply affects the quality of life in CDH survivors.30
Nonpulmonary diseases are also relatively frequent in CDH
survivors. Gastroesophageal reflux is caused by a distorted anatomy
of the diaphragmatic sling, both congenital and related to the sur-
gical CDH repair. Also, esophageal motility and gastroesophageal
CHAPTER 31  Congenital Diaphragmatic Hernia 333
sphincter function are disturbed.31 In addition, malrotation may
delay gastric emptying, and the abnormal balance of pressures in
the thorax and abdomen may facilitate retrograde passage of gas-
tric contents to the oesophagus. Reflux can complicate the preex-
isting respiratory disease, and for all these reasons, a considerable
proportion of these patients respond poorly to medical treatment
and ultimately require antireflux surgery.32 Reflux and the need
for antireflux surgery are dependent on the degree of pulmonary
hypoplasia.33
Neurodevelopmental deficits are possible in patients in whom
brain oxygenation was marginal for long periods of time and
particularly when ECMO required major vascular occlusion.34
Neurosensory deafness occurs in a small proportion of children
surviving CDH.34 This is progressive, and it is often tied to both
prolonged antibiotic treatment and a particular sensitivity or a
developmental defect of the inner ear.
These sequelae, and the frequent associated malformations,
require long-term follow-up for early diagnosis and proactive
management.6 Most babies eventually lead a life very close to
normal provided when managed in a multidisciplinary follow-up
program.35 
Aberrations in Lung Development
In CDH, the lung is hypoplastic not only on the side of the hernia,
but the contralateral side is affected as well but to a variable extent.
Peripheral airways are less developed, and there are markedly less
and smaller alveoli, thickened alveolar walls and an increased
amount of interstitial tissue36 so that there is less alveolar airspace
and gas exchange surface area. Parallel to airway changes, there
is a reduction in arteries, resulting in a hypoplastic vascular bed.
Morphologically, a thickening of the vascular wall is determined
by an increase in arterial media and adventitia and by neomus-
cularisation of the small pulmonary arteries, which are normally
partially or nonmuscularised.37 The structural remodelling of the
small pulmonary arteries reduces their ability to dilate to increase
the vascular bed capacity and reduce the pressure in the pulmo-
nary circulation after birth.38 After birth, further muscularisation
of this ‘immature’ pulmonary vasculature occurs, with migration
of adventitial fibroblasts into the media and smooth muscle cells
into the intima. These morphologically abnormal vessels respond
abnormally to mechanical and chemical stimuli, including the
shear stress accompanying raised blood flow through the nar-
rowed vessels. Increased contractility and impaired relaxation of
pulmonary arteries have been demonstrated in animal models39
and could be responsible for the low efficacy of conventional vaso-
dilatory therapy. 
Prenatal Diagnosis and Outcome Prediction
Today, high-resolution ultrasound and advances in prenatal diag-
nosis and genetic testing have made it possible to diagnose CDH
relatively early and to rule out a number of associated anomalies.
Ideally, antenatal ultrasound screening identifies cases in utero,
reportedly in more than 70% of cases, yet lower numbers have
been reported as well.40 Intrathoracic abdominal organs are the
hallmark of CDH. Left-sided CDH typically presents with a
mediastinal shift to the right caused by herniation of the stom-
ach, intestines and in some cases liver (Fig. 31.1A). In right-sided
CDH, part of the liver is visible in the chest, with mediastinal
334 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

L
L
B

A B

C D
• Fig. 31.1  Ultrasound appearance of congenital diaphragmatic hernia (CDH) on a cross-section at the
level of the four-chamber view. A, Fetus with left-sided CDH. The chest contains bowels (B), stomach
(S) and part of the liver (L), and there is a shift of the heart and mediastinum towards the right side. B,
Right-sided CDH: the liver (L) is visualised in the thorax, and there is a mediastinal shift towards the left.
C, Measurement of the lung area and D, measurement of the head circumference for calculation of the
observed to expected lung to head ratio. (Courtesy of UZ Leuven.)

shift to the left1 (Fig. 31.1B). Prenatal diagnosis allows in utero
referral to a tertiary care centre used to manage the condition for
expert assessment, counselling and perinatal management. Addi-
tional genetic and morphologic assessment using ultrasound or
magnetic resonance imaging (MRI) can be used to rule out associ-
ated malformations.
For isolated cases, clinicians should individualise prognosis to
counsel parents about prenatal options. Most prediction methods
are based on lung size, liver herniation and pulmonary circula-
tion, and more recently stomach position.41-46 Ultrasound mea-
surement of the lung-to-head ratio (LHR) is most widely used.
The LHR, first described by Metkus and colleagues,43 provides an
indirect estimate of the size of the lung contralateral to the hernia
normalised for the head circumference (Fig. 31.1C). It is a two-
dimensional measure and changes over gestation as the lung area
grows more rapidly than the head circumference. The observed to
expected (o/e) LHR has subsequently been proposed to eliminate
the effect of gestational age at assessment.47 The o/e LHR has been
shown to be an independent predictor of postnatal survival47 both
in left- and right-sided CDH48 and to some extent of short-term
morbidity.49 Other methods to assess lung size, such as the lung-
to-thorax ratio,50 the quantitative lung index51 and three-dimen-
sional measurements of lung volume,52 have also been proposed,
but the predictive value of these parameters has not been validated
to the same extent as the o/e LHR.
Liver herniation can be determined both for left- as well as
right-sided hernia, although in the latter case, the liver is nearly
always herniating through the defect. For left-sided CDH, her-
niation of the liver into the thorax has been recognised as a pre-
dictor of poor outcome by Harrison and colleagues53 and seems
to be independent from lung size.54 Although it is theoretically
possible to quantify the amount of liver in the thorax, liver posi-
tion on ultrasound is usually categorised binary as either ‘up’
(in the thorax), or ‘down’ (confined to the abdomen). Because
the echogenicity of the liver is very similar to that of the lung,
it can be difficult to assess liver position with ultrasound (Fig.
31.2A and Video 31.1). Additional indirect signs suggestive
of liver herniation are presence of hepatic vessels above the
diaphragmatic edge43 (Fig. 31.2B), abnormal position of the
ductus venosus or the gallbladder55 (Fig. 31.2C) or deviation
(bowing) of the umbilical vein on towards the left side56 (Fig.
31.2D). Finally, evaluation of stomach position has recently
been reintroduced as an indirect method to estimate severity
of the disease in left-sided CDH because it has been shown to
correlate with the proportion of intrathoracic liver determined
by MRI.57
The combination of liver herniation and o/e LHR is now a
widely accepted method to stratify fetuses with left- and right-
sided CDH into groups with an increasing degree of pulmonary
hypoplasia and corresponding mortality rates. It is also used to
select patients for fetal therapy trials (Fig. 31.3).
Severity assessment by MRI theoretically has several advantages
over ultrasound. Visualisation is not limited by maternal habitus,
amniotic fluid volume or fetal position. With MRI, the total
(left and right) lung volume can be measured, which may better
predict postnatal lung function. Volumetry may also accurately
quantify liver and stomach herniation.58,59 Although one study
has claimed that MRI better predicts outcome than ultrasound,60
the numbers do not allow such a claim nor has this been proven
in clinical practice.61
 CHAPTER 31  Congenital Diaphragmatic Hernia 335

A B

Right CDH Control

C Left D
• Fig. 31.2 Ultrasound evidence of liver herniation in left-sided congenital diaphragmatic hernia (CDH)
cases. A, Sagittal section of the fetal abdomen and thorax demonstrating liver herniation (L). B, Visualisa-
tion of the hepatic vessels above the diaphragmatic edge (arrow). C, As a consequence of the herniation
and rotation of the liver, the gallbladder (arrow) is visualised on the left side of the abdomen. D, Bowing of
the umbilical vein towards the left side; the same section from a fetus with CDH without liver herniation is
provided on the right for comparison. (Courtesy of UZ Leuven.)

Liver in thorax (‘up’) Liver in abdomen (‘down’)

Total trial for Total trial for Candidates for

severe lung hypoplasia moderate lung hypoplasia intrauterine therapy

100 100
90 90
Survival rate (%)

80 80
Survival rate (%)

70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
o/e LHR (%) <15 15–25 26–35 36–45 >45 o/e LHR (%) <30 30–45 >45
Extreme, Severe, Moderate, Mild, Severe, Mild,
1% of cases 14% of cases 45% of cases 40% of cases 53% of cases 47% of cases
A B
• Fig. 31.3 Patient stratification and selection of candidates for intrauterine therapy according to the
observed to expected (o/e) lung-to-head ratio (LHR) for left-sided (A) and right-sided (B) congenital dia-
phragmatic hernia. (Adapted from Jani et al.47 and DeKoninck et al.87)

Lung size and liver herniation also predict neonatal morbidity,
such as the duration of assisted ventilation, the need for supple-
mental oxygen, the need for patch repair and the time it takes
to full enteral feeding.46 The literature on prediction of PPH is
more limited (systematically reviewed by Russo and colleagues62).
Several candidate parameters have been suggested in single case
series, including lung size, presence of visceral herniation and
direct assessment of the pulmonary vasculature, which may pro-
vide additional information. However, to our knowledge, there is
currently no validated antenatal predictor for PPH. 
336 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Current Neonatal Management
In an attempt to improve outcome and permit comparison of
outcome data, the CDH EURO consortium has published a
standardised neonatal treatment protocol (revised in 201563).
Delivery is planned (either via induction or via caesarean section)
after 39 weeks in a high-volume tertiary centre, and the newborn
is immediately intubated.
Historically, high-pressure high oxygen conventional respira-
tory assistance was used, leading to iatrogenic pneumothorax and
acute death. Then it was realised that the hypoplastic and imma-
ture CDH lung was severely damaged by excessive oxygen deliv-
ery and by excessive airway pressure.64 Since Wung and colleagues
introduced the ‘gentle ventilation and permissive hypercarbia
strategy’, improved pulmonary outcomes and mortality rates have
been reported.65 This policy progressively gained support and it is
now the standard in most developed countries. High-frequency
oscillatory ventilation was also believed to improve survival and
reduce long-term morbidity. However, a recent randomised con-
trolled trial (RCT; the VICI trial: Ventilation in Infants with
Congenital diaphragmatic hernia: an International randomised
clinical trial.)66 has shown no superiority of this technique versus
conventional ventilation. The latter again shows that proper stud-
ies have to be done before implementing new technologies.
The management of PPH in newborns with CDH remains one
of the major concerns in neonatal intensive care units (NICUs).
Early cardiac ultrasound (within the first 24 hours of life) is rec-
ommended by the international consortia as a noninvasive tool
in the diagnosis of PPH. Serial cardiac ultrasonography is then
recommended to guide therapeutic choices and to monitor their
effect on PPH.25 The cornerstone of postnatal treatment of PPH
is the reversal of the vasoconstrictive component to prevent the
right ventricle overload and the development of irreversible vas-
cular remodelling. Currently, iNO is the first therapeutic choice
in CDH infants with PPH. However, in an RCT, although iNO
significantly decreased the need for ECMO in infants with PPH,
it did not reduce mortality rate, length of hospitalisation, chronic
lung injury or neurodevelopmental impairment.67 Furthermore,
although the response rate to iNO in all infants with PPH is
around 60%, in infants with CDH, it is estimated to be only
30%.68 Other vasodilatory drugs, such as phosphodiesterase type
5 (PDE5) inhibitors, endothelin antagonists or prostacyclins, have
been used alone or in association with nitric oxide in experimen-
tal series.69-71 However, their use is highly variable throughout
NICUs around the world, which makes it difficult to define their
exact role. Again, trials are being designed to clarify this.
Apart from pharmacologic therapy, ECMO is in some centres
an adjunct in the treatment of CDH-related PPH.25 It is used to
unload the right ventricle while putting the lungs at rest, thereby
reversing PPH, which would otherwise be lethal. It prevents addi-
tional lung injury induced by barotrauma and oxidative stress
in case of maximal ventilator support. As for iNO, experience
with ECMO achieved the worst results precisely in patients with
CDH.72 Therefore there is no evidence that the outcome of CDH
is better with ECMO. Together with the limitations of the tech-
nique (required weight above 2000 g, need for heparinisation), all
this has somewhat tempered the initial enthusiasm followed by its
diminished use in many centres.6
Surgical repair should be performed electively after the new-
born is stabilised. The optimal surgical technique remains under
debate. Minimal access surgery is gaining ground on the open
approach (thoracotomy or laparotomy). This approach has
aesthetic advantages but carries a higher risk for recurrence.63 It
may also not be applicable in severe cases. For defects that are too
large to be closed by primary repair, prosthetic patches may be
used to close the gap,73 ranging from first-generation nonabsorb-
able synthetic materials (Gore-Tex) to biomaterials (xenografts) as
well as composite materials. The ideal mesh remains elusive. 
Antenatal Therapeutic Strategies
The ability to prenatally identify a future nonsurvivor offers the
potential for prenatal interventions that could avoid that out-
come. The concept of tracheal occlusion (‘plug the lung until it
grows’) was first introduced by Wilson and associates74 and is
inspired by clinical observations in fetuses with congenital high
airway obstruction, who have a marked increased lung volume
and alveolar number.75 Airway obstruction prevents egress of pul-
monary fluid, which experimentally has been shown to prompt
lung growth by a mechanism of stretch of lung parenchymal
cells.76 This leads to increased airway branching morphogenesis,
an increase in both alveolar surface area and airspace volume and
stimulated alveolisation.77 This concept has been further explored
experimentally, demonstrating that tracheal occlusion improves
neonatal lung compliance and improves ventilation. The proce-
dure was first clinically attempted by open fetal surgery and clip-
ping of the trachea.78 Progress in minimally invasive fetal surgery
made percutaneous fetal endoscopic tracheal occlusion (FETO)
with a balloon possible.79 FETO is an investigational minimally
invasive, percutaneous procedure that can be done under mater-
nal local anaesthesia (Fig. 31.4A).80 The procedure is usually per-
formed at 27 + 0 to 29 + 6 weeks in severe cases and 30 + 0
to 31 + 6 weeks in moderate cases. The fetus is anesthetised and
immobilised with an intramuscular injection of a neuromuscu-
lar blocking agent, fentanyl and atropine. A flexible cannula is
inserted through the skin and myometrium and targeted to the
fetal nose tip under ultrasound guidance. Fetoscopic instruments
specifically designed for FETO are then introduced. These consist
in a 3.3-mm sheath loaded with a fiberoptic endoscope (1.3 mm;
Karl Storz) and a balloon occlusion system (catheter loaded with a
detachable inflatable latex balloon with integrated one-way valve
Goldbal 2, Balt Extrusion, France). A stylet or forceps can also be
inserted through the sheet to remove the balloon if wrongly posi-
tioned. Fetoscopic landmarks are the philtrum and upper lip, the
tongue and raphe of the palate, uvula, epiglottis, and eventually
the vocal cords. The endoscope is advanced into the trachea until
identification of the carina, above which the balloon is positioned
by inflation and detachment from the catheter (Video 31.2). The
median duration of FETO is 10 (range, 3–93) minutes, depen-
dent on both the experience of the operator and the position of
the fetus.81 A longer operation time is the main risk factor for
membrane rupture.
Experimental data suggest benefit of in utero reversal of tracheal
occlusion (‘plug-unplug’ sequence). It stimulates lung matura-
tion82 and has the logistic advantage of permitting vaginal delivery
and referral to the home institution of the patient. This prompted
clinicians to attempt timely in utero reversal as much as possible.
Meanwhile, clinical data suggest that prenatal balloon removal
increases neonatal survival83 and reduces neonatal morbidity.41
Elective intrauterine occlusion reversal is usually scheduled at 34
weeks in patients with an uneventful postoperative course. This
can be performed via ultrasound-guided puncture, fetoscopic
removal, tracheoscopic removal on placental circulation or post-
natal puncture. Ultrasound-guided in utero balloon puncture is
 CHAPTER 31  Congenital Diaphragmatic Hernia 337

B
• Fig. 31.4 Fetal therapy for congenital diaphragmatic hernia. A, Schematic drawing of percutaneous
fetoscopic endoluminal tracheal occlusion. Inset, A detachable balloon, normally used for endovascular
occlusion, is positioned in the trachea. B, Schematic drawing of balloon removal on placental circulation
by laryngeotracheoscopy. (Reproduced with permission from UZ Leuven, Leuven, Belgium. Drawing by
Myrthe Boymans.)

done after fetal immobilisation and analgesia. After puncture,
the balloon is pushed by the lung fluid into the pharynx, from
where it is either swallowed or falls into the amniotic cavity (Video
31.3). Fetoscopic removal is done with similar instruments as for
insertion. The balloon is first punctured with a stylet and then
grasped and retrieved with forceps.84
In 20% of cases, patients present earlier than planned with
threatening preterm delivery, with or without ruptured mem-
branes. Whenever clinically possible, in utero balloon removal is
still attempted using the same techniques. If in utero retrieval does
not seem safe or possible, the balloon is removed by laryngotra-
cheoscopy during a modified caesarean section under locoregional
338 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Main Potential Advantages and Risks of Fetal TABLE Main Inclusion and Exclusion Criteria for
31.1 Endoscopic Tracheal Occlusion 31.2 Participation in the TOTAL Trial, for Both the
Pros Cons Severe Arm and the Moderate Arm
Inclusion Criteria Exclusion Criteria
Stimulation of lung growth 15%–20% risk for pPROM
Minimally invasive procedure Mean gestational age at birth: 35 SEVERE ARM
weeks
Maternal age 18 years or older Patient not willing to undergo
Potential to increase survival Need for a second intervention
randomisation
(unplug)
Potential reduction in postnatal Potential risk for emergency unplug Singleton pregnancy Multiple pregnancy
morbidity
Effect on PPH not yet demonstrated Written informed consent Patient not able to consent in
full
PPH, Persistent pulmonary hypertension; pPROM, preterm premature rupture of membranes.
Left-sided diaphragmatic hernia Right-sided or bilateral
   diaphragmatic hernia
No associated structural anoma- Additional major structural or
anaesthesia, with the fetal head and shoulders delivered while the lies and normal karyotype genetic anomalies
fetus remains on placental circulation85 (Fig. 31.4B and Video
Gestational age at surgery, Balloon cannot be placed before
31.4). Postdelivery removal is a last resort, which is done by video 27.0–29.6 wk 29.6 wk
laryngotracheoscopy. Blind and ultrasound-guided ex utero punc-
ture have also been reported.86 In a recent study performed in Severe hypoplasia defined as o/e Moderate or mild hypoplasia
three FETO centres,84 balloon removal was elective in 72% of LHR <25% measured at the
cases and as an emergency in 28%. The primary method was by latest at 29 wk
fetoscopy in the majority of cases (67%), by ultrasound guidance Acceptance of responsibility to Maternal diseases or technical
in 21%, by tracheoscopy on placental circulation in 10% and by come to FETO centre for bal- limitations making prenatal sur-
postnatal tracheoscopy in 1%. The method of choice for primary loon removal gery hazardous or impossible
removals mainly depended on preference of the surgeon and, sur- Cervix >15 mm Cervix <15 mm at randomisation
prisingly, was not associated with a difference in the interval to
delivery. The main conclusion, however, was that delivery should MODERATE ARM
not take place outside FETO centres because the balloon could Maternal age 18 years or older Patient not willing to undergo
not be removed in three cases, leading to iatrogenic death. randomisation
Observational studies have shown that FETO leads to an appar- Singleton pregnancy Multiple pregnancy
ent increase in survival and reduced early neonatal respiratory
morbidity compared with historical controls of similar severity, Written informed consent Patient not able to consent in
for both severe left-sided CDH and severe right-sided cases.81,87,88 full
This potential benefit is now being investigated in two paral- Left-sided diaphragmatic hernia Right-sided or bilateral diaphrag-
lel RCTs called Tracheal Occlusion To Accelerate Lung growth matic hernia
(TOTAL; http://www.TOTALtrial.eu), in fetuses with left-sided
No associated structural anoma- Additional major structural or
CDH and severe or moderate lung hypoplasia (NCT01240057
lies and normal karyotype genetic anomalies
and NCT00763737).89 Right-sided CDH is too uncommon to
justify a RCT, and we use a cutoff of o/e LHR of less than45% Gestational age at surgery, Balloon cannot be placed before
based on a long single-centre series.  30.0–31.6 wk 31.6 wk
Moderate hypoplasia defined Severe or mild hypoplasia
Experimental Antenatal Treatments as o/e LHR 25%–45% in the
presence of liver herniation or
The main drawback of FETO is the increased risk for preterm o/e LHR 25%–35% without
delivery, partly offsetting the beneficial effects of fetal therapy. His- liver herniation
torical data on more than 200 cases demonstrated a 25% rate of Acceptance of responsibility to Maternal diseases or technical
preterm rupture of membranes at less than 34 weeks and a 30% rate come to FETO centre for bal- limitations making prenatal sur-
of delivery before 34 weeks, requiring urgent balloon retrieval.81 loon removal gery hazardous or impossible
Apart from being relatively invasive, FETO is also technically chal-
lenging and therefore difficult to widely implement. Moreover, the Cervix >15 mm Cervix <15 mm at randomisation
maximum post-FETO survival rate reported in severe cases so far FETO, Fetal endoscopic tracheal occlusion; LHR, lung-to-head ratio; o/e, observed to
is not higher than 50% to 60%, which in part is caused by insuf- expected.
ficient airway growth and, above all, limited vascular development.   
The problem of PPH seems, at least, to persist despite surgical fetal
treatment. The main advantages and disadvantages of FETO are
outlined in Table 31.1. The main inclusion and exclusion criteria PPH. It ideally should be a medical approach to overcome the risks
for participation in the TOTAL trial are outlined in Table 31.2. of fetal surgery. Translational research on alternative antenatal solu-
For the aforementioned reasons, complementary or alternative tions for CDH mainly focuses on three levels: (i) engineering tissue
therapeutic treatments would be welcomed. The ideal fetal therapy to substitute the affected lungs, (ii) regenerative therapy by stem cell
should address both the problem of ventilatory insufficiency and transplantation and (iii) medical therapy. We recently summarised the

progress in lung tissue engineering for congenital anomalies.90 Par-
ticularly attractive is the possibility of using decellularised matrices
and direct induced pluripotent stem cells and lung progenitor cells.
This could indeed be the ultimate alternative to lung transplanta-
tion. Even though one is still unable to engineer a fully functional
lung for transplantation, it is possible that, with the fast progress of
regenerative medicine, this technology will be adopted clinically in
the upcoming years. Stem cell–based strategies could also have a role
both during fetal development as well as in the postnatal period.91
The major mechanism of lung tissue regeneration, with either mesen-
chymal stem cells or amniotic fluid–derived stem cells, probably takes
advantage of the presence of endogenous epithelial progenitor cells.
The strategy with the highest translational potential in the short term
is most likely transplacental pharmacotherapy, which could modulate
cellular response in the fetus and ameliorate outcome. Research in
such field has recently focused on the prevention of PPH by using
drugs currently marketed for the treatment of PPH in adults, such as
the PDE5 inhibitor sildenafil,92 the endothelin antagonist bosentan93
or the prostacyclin receptor agonist ONO-1301SR.94  Self-assessment questions available at ExpertConsult.com.
CHAPTER 31  Congenital Diaphragmatic Hernia 339
Conclusion
Congenital diaphragmatic hernia should be detected at the prena-
tal anomaly ultrasound examination. Additional anomalies should
be searched for and appropriate genetic testing offered.
The severity of the condition should be assessed using ultra-
sound and possibly prenatal MRI. In utero referral to a centre
experienced with the postnatal management of CDH is manda-
tory to optimise outcome.
Today, the prenatal option of fetoscopic endoluminal tra-
cheal occlusion is available, and patients should be recruited to
ongoing randomised clinical trials to fully evaluate its efficacy.
Interventions in the future are likely to use a combination tis-
sue engineering, regenerative therapy by stem cell transplantation
and medical therapy to improve lung function and reduce the
risks of PPH.
Access the complete reference list online at ExpertConsult.com.
References development? Birth Defects Res A Clin Mol
Teratol. 2005;73:523–531.
diaphragmatic hernia: a histological study of
29 cases. J Pathol. 1999;189:112–118.
22. Irish MS, Holm BA, Glick PL. Congenital 39. Schmidt AF, Rojas-Moscoso JA, Gon-
1. Kotecha S, Barbato A, Bush A, et  al. Con-
diaphragmatic hernia. A historical review. calves FL, et  al. Increased contractility
genital diaphragmatic hernia. Eur Respir J.
Clin Perinatol. 1996;23:625–653. and impaired relaxation of the left pulmo-
2012;39:820–829.
23. Downard CD, Jaksic T, Garza JJ, et  al. nary artery in a rabbit model of congeni-
2.  van den Hout L, Schaible T, Cohen-
Analysis of an improved survival rate for con- tal diaphragmatic hernia. Pediatr Surg Int.
Overbeek TE, et al. Actual outcome in infants
genital diaphragmatic hernia. J Pediatr Surg. 2013;29:489–494.
with congenital diaphragmatic hernia: the
2003;38:729–732. 40. Gallot D, Boda C, Ughetto S, et al. Prenatal
role of a standardized postnatal treatment
24. Chiu P, Hedrick HL. Postnatal manage- detection and outcome of congenital diaphrag-
protocol. Fetal Diagn Ther. 2011;29:55–63.
ment and long-term outcome for survivors matic hernia: a French registry-based study.
3. Greer JJ. Current concepts on the patho-
with congenital diaphragmatic hernia. Prenat Ultrasound Obstet Gynecol. 2007;29:276–283.
genesis and etiology of congenital dia-
Diagn. 2008;28:592–603. 41. Done E, Gratacos E, Nicolaides K, et al. Pre-
phragmatic hernia. Respir Physiol Neurobiol.
25. Vijfhuize S, Schaible T, Kraemer U, et  al. dictors of neonatal morbidity in fetuses with
2013;189:232–240.
Management of pulmonary hypertension in severe isolated congenital diaphragmatic her-
4. Keijzer R, Puri P. Congenital diaphragmatic
neonates with congenital diaphragmatic her- nia undergoing fetoscopic tracheal occlusion.
hernia. Semin Pediatr Surg. 2010;19:180–
nia. Eur J Pediatr Surg. 2012;22:374–383. Ultrasound Obstet Gynecol. 2013;42(1):77–
185.
26. Muratore CS, Kharasch V, Lund DP, et  al. 83.
5. Numanoglu A, Steiner Z, Millar A, Cywes S.
Pulmonary morbidity in 100 survivors of 42. Claus F, Sandaite I, Dekoninck P, et  al.
Delayed presentation of congenital diaphrag-
congenital diaphragmatic hernia monitored Prenatal anatomical imaging in fetuses with
matic hernia. S Afr J Surg. 1997;35:74–76.
in a multidisciplinary clinic. J Pediatr Surg. congenital diaphragmatic hernia. Fetal Diagn
6. Tovar JA. Congenital diaphragmatic hernia.
2001;36:133–140. Ther. 2011;29:88–100.
Orphanet J Rare Dis. 2012;7:1.
27. Vanamo K, Rintala R, Sovijarvi A, et  al. 43. Metkus AP, Filly RA, Stringer MD, et  al.
7. Harrison MR, Adzick NS, Estes JM, Howell
Long-term pulmonary sequelae in survivors Sonographic predictors of survival in
LJ. A prospective study of the outcome for
of congenital diaphragmatic defects. J Pediatr fetal diaphragmatic hernia. J Pediatr Surg.
fetuses with diaphragmatic hernia. JAMA.
Surg. 1996;31:1096–1099; discussion 1099- 1996;31:148–151; discussion 151–152.
1994;271:382–384.
1100. 44. Done E, Allegaert K, Lewi P, et al. Maternal
8. Inhaled nitric oxide in full-term and nearly
28. Vanamo K, Peltonen J, Rintala R, et al. Chest hyperoxygenation test in fetuses undergoing
full-term infants with hypoxic respiratory
wall and spinal deformities in adults with FETO for severe isolated congenital dia-
failure. N Engl J Med. 1997;336:597–604.
congenital diaphragmatic defects. J Pediatr phragmatic hernia. Ultrasound Obstet Gynecol.
9. Sweed Y, Puri P. Congenital diaphragmatic
Surg. 1996;31:851–854. 2011;37:264–271.
hernia: influence of associated malformations
29. Dillon PW, Cilley RE, Mauger D, et al. The 45. Kitano Y, Okuyama H, Saito M, et  al. Re-
on survival. Arch Dis Child. 1993;69:68–70.
relationship of pulmonary artery pressure and evaluation of stomach position as a simple
10. Lin AE, Pober BR, Adatia I. Congenital dia-
survival in congenital diaphragmatic hernia. prognostic factor in fetal left congenital
phragmatic hernia and associated cardiovas-
J Pediatr Surg. 2004;39:307–312; discussion diaphragmatic hernia: a multicentre sur-
cular malformations: type, frequency, and
312. vey in Japan. Ultrasound Obstet Gynecol.
impact on management. Am J Med Genet C
30. Behrsin J, Cheung M, Patel N. Sildenafil 2011;37:277–282.
Semin Med Genet. 2007;145c:201–216.
weaning after discharge in infants with con- 46. Jani JC, Benachi A, Nicolaides KH, et  al.
11. Siebert JR, Haas JE, Beckwith JB. Left ventric-
genital diaphragmatic hernia. Pediatr Cardiol. Prenatal prediction of neonatal morbidity in
ular hypoplasia in congenital diaphragmatic
2013;34:1844–1847. survivors with congenital diaphragmatic her-
hernia. J Pediatr Surg. 1984;19:567–571.
31. Rayyan M, Omari T, Allegaert K, et al. Pp- nia: a multicentre study. Ultrasound Obstet
12. Brownlee EM, Howatson AG, Davis CF, Sab-
10 characterization of esophageal motility in Gynecol. 2009;33:64–69.
harwal AJ. The hidden mortality of congeni-
infants born with congenital diaphragmatic 47. Jani J, Nicolaides KH, Keller RL, et  al.
tal diaphragmatic hernia: a 20-year review. J
hernia using high resolution manometry. J Observed to expected lung area to head cir-
Pediatr Surg. 2009;44:317–320.
Pediatr Gastroenterol Nutr. 2015;61:524. cumference ratio in the prediction of survival
13. Slavotinek AM. The genetics of common
32. Bagolan P, Morini F. Long-term follow up of in fetuses with isolated diaphragmatic hernia.
disorders—congenital diaphragmatic hernia.
infants with congenital diaphragmatic hernia. Ultrasound Obstet Gynecol. 2007;30:67–71.
Eur J Med Genet. 2014;57:418–423.
Semin Pediatr Surg. 2007;16:134–144. 48. DeKoninck P, Gomez O, Sandaite I, et  al.
14. Stolar CJ, Dillon PW. Extracorporeal mem-
33. Verbelen T, Lerut T, Coosemans W, et  al. Right-sided congenital diaphragmatic her-
brane oxygenation and congenital diaphrag-
Antireflux surgery after congenital dia- nia in a decade of fetal surgery. BJOG.
matic hernia. Surgery. 1990;108:121–122.
phragmatic hernia repair: a plea for a tai- 2015;122:940–946.
15. Allan DW, Greer JJ. Pathogenesis of nitrofen-
lored approach. Eur J Cardiothorac Surg. 49. Cruz-Martinez R, Castanon M, Moreno-
induced congenital diaphragmatic hernia in
2013;44(2):263–267. Alvarez O, et  al. Usefulness of lung-to-head
fetal rats. J Appl Physiol. 1997;83:338–347.
34. Rasheed A, Tindall S, Cueny DL, et al. Neu- ratio and intrapulmonary arterial Doppler
16. Keijzer R, Liu J, Deimling J, et  al. Dual-hit
rodevelopmental outcome after congenital in predicting neonatal morbidity in fetuses
hypothesis explains pulmonary hypoplasia in
diaphragmatic hernia: extracorporeal mem- with congenital diaphragmatic hernia treated
the nitrofen model of congenital diaphrag-
brane oxygenation before and after surgery. J with fetoscopic tracheal occlusion. Ultrasound
matic hernia. Am J Pathol. 2000;156:1299–
Pediatr Surg. 2001;36:539–544. Obstet Gynecol. 2013;41:59–65.
1306.
35. Delacourt C, Hadchouel A, Toelen J, et  al. 50. Hidaka N, Murata M, Sasahara J, et al. Cor-
17. Iritani I. Experimental study on embryogen-
Long term respiratory outcomes of congenital relation between lung to thorax transverse
esis of congenital diaphragmatic hernia. Anat
diaphragmatic hernia, esophageal atresia, and area ratio and observed/expected lung area to
Embryol (Berl). 1984;169:133–139.
cardiovascular anomalies. Semin Fetal Neonat head circumference ratio in fetuses with left-
18. Kluth D, Kangah R, Reich P, et al. Nitrofen-
Med. 2012;17(2):105–111. sided diaphragmatic hernia. Congenit Anomal.
induced diaphragmatic hernias in rats: an ani-
36. Kitagawa M, Hislop A, Boyden EA, Reid 2015;55:81–84.
mal model. J Pediatr Surg. 1990;25:850–854.
L. Lung hypoplasia in congenital diaphrag- 51. Quintero RA, Quintero LF, Chmait R, et al.
19. Major D, Cadenas M, Fournier L, et  al.
matic hernia. A quantitative study of airway, The quantitative lung index (QLI): a gesta-
Retinol status of newborn infants with con-
artery, and alveolar development. Br J Surg. tional age-independent sonographic predic-
genital diaphragmatic hernia. Pediatr Surg Int.
1971;58:342–346. tor of fetal lung growth. Am J Obstet Gynecol.
1998;13:547–549.
37. Shehata SM, Sharma HS, van der Staak FH, 2011;205:544.e1–e8.
20. Beurskens LW, Tibboel D, Lindemans J,
et  al. Remodeling of pulmonary arteries in 52. Ruano R, Takashi E, da Silva MM, et al. Pre-
et al. Retinol status of newborn infants is asso-
human congenital diaphragmatic hernia with diction and probability of neonatal outcome
ciated with congenital diaphragmatic hernia.
or without extracorporeal membrane oxygen- in isolated congenital diaphragmatic hernia
Pediatrics. 2010;126:712–720.
ation. J Pediatr Surg. 2000;35:208–215. using multiple ultrasound parameters. Ultra-
21. Gallot D, Marceau G, Coste K, et  al. Con-
38. Shehata SM, Tibboel D, Sharma HS, Mooi sound Obstet Gynecol. 2012;39:42–49.
genital diaphragmatic hernia: a retinoid-
WJ. Impaired structural remodelling of pul- 53. Harrison MR, Langer JC, Adzick NS, et  al.
signaling pathway disruption during lung
monary arteries in newborns with congenital Correction of congenital diaphragmatic

339.e1
339.e2 References
hernia in utero, V. Initial clinical experience.
J Pediatr Surg. 1990;25:47–55; discussion
56–57.
54. Cannie M, Jani J, Chaffiotte C, et al. Quan-
tification of intrathoracic liver herniation by
magnetic resonance imaging and prediction
of postnatal survival in fetuses with congeni-
tal diaphragmatic hernia. Ultrasound Obstet
Gynecol. 2008;32:627–632.
55. Bootstaylor BS, Filly RA, Harrison MR,
Adzick NS. Prenatal sonographic predictors of
liver herniation in congenital diaphragmatic
hernia. J Ultrasound Med. 1995;14:515–520.
56. Harrison MR, Mychaliska GB, Albanese
CT, et al. Correction of congenital diaphrag-
matic hernia in utero IX: fetuses with poor
prognosis (liver herniation and low lung-to-
head ratio) can be saved by fetoscopic tem-
porary tracheal occlusion. J Pediatr Surg.
1998;33:1017–1022; discussion 1022–1023.
57. Cordier AG, Cannie MM, Guilbaud L, et al.
Stomach position versus liver-to-thoracic vol-
ume ratio in left-sided congenital diaphrag-
matic hernia. J Matern Fetal Neonatal Med.
2015;28:190–195.
58. Nawapun K, Eastwood M, Sandaite I, et al.
Correlation of observed-to-expected total
fetal lung volume with intrathoracic organ
herniation on magnetic resonance imaging in
fetuses with isolated left-sided congenital dia-
phragmatic hernia. Ultrasound Obstet Gynecol.
2015;46:162–167.
59. Cannie MM, Cordier AG, De Laveaucoupet
J, et  al. Liver-to-thoracic volume ratio: use
at MR imaging to predict postnatal survival
in fetuses with isolated congenital diaphrag-
matic hernia with or without prenatal tracheal
occlusion. Eur Radiol. 2013;23:1299–1305.
60. Bebbington M, Victoria T, Danzer E, et  al.
Comparison of ultrasound and magnetic
resonance imaging parameters in predicting
survival in isolated left-sided congenital dia-
phragmatic hernia. Ultrasound Obstet Gynecol.
2014;43:670–674.
61. Schaible T, Busing KA, Felix JF, et  al. Pre-
diction of chronic lung disease, survival and
need for ECMO therapy in infants with con-
genital diaphragmatic hernia: additional value
of fetal MRI measurements? Eur J Radiol.
2012;81:1076–1082.
62. Russo FM, Eastwood MP, Keijzer R, et  al.
Lung size and liver herniation predict the
need for extra corporeal membrane oxygen-
ation but not pulmonary hypertension in
isolated congenital diaphragmatic hernia: a
systematic review and meta-analysis. Ultra-
sound Obstet Gynecol. 2017;49(6):704–713.
63. Snoek KG, Reiss IK, Greenough A, et  al.
Standardized postnatal management of
Infants with congenital diaphragmatic her-
nia in Europe: the CDH EURO consor-
tium consensus—2015 update. Neonatology.
2016;110:66–74.
64. Sakurai Y, Azarow K, Cutz E, et  al. Pulmo-
nary barotrauma in congenital diaphragmatic
hernia: a clinicopathological correlation. J
Pediatr Surg. 1999;34:1813–1817.
65. Boloker J, Bateman DA, Wung JT, Stolar
CJ. Congenital diaphragmatic hernia in 120
infants treated consecutively with permissive
hypercapnea/spontaneous respiration/elective
repair. J Pediatr Surg. 2002;37:357–366.
66. Snoek KG, Capolupo I, van Rosmalen J, et al.
Conventional mechanical ventilation versus
high-frequency oscillatory ventilation for
congenital diaphragmatic hernia: a random-
ized clinical trial (The VICI-trial). Ann Surg.
2016;263:867–874.
67. Steinhorn RH. Therapeutic approaches using
nitric oxide in infants and children. Free Radic
Biol Med. 2011;51:1027–1034.
68. Kinsella JP, Ivy DD, Abman SH. Pulmo-
nary vasodilator therapy in congenital dia-
phragmatic hernia: acute, late, and chronic
pulmonary hypertension. Semin Perinatol.
2005;29:123–128.
69. Barst RJ, Langleben D, Badesch D, et  al.
Treatment of pulmonary arterial hyperten-
sion with the selective endothelin-A recep-
tor antagonist sitaxsentan. J Am Coll Cardiol.
2006;47:2049–2056.
70. Goissen C, Ghyselen L, Tourneux P, et  al.
Persistent pulmonary hypertension of the
newborn with transposition of the great arter-
ies: successful treatment with bosentan. Eur J
Pediatr. 2008;167:437–440.
71. McNamara PJ, Murthy P, Kantores C,
et al. Acute vasodilator effects of Rho-kinase
inhibitors in neonatal rats with pulmo-
nary hypertension unresponsive to nitric
oxide. Am J Physiol Lung Cell Mol Physiol.
2008;294:L205–L213.
72. Payne NR, Wright G, Kriesmer PJ, et  al.
Recurrent pulmonary arterial hypertension
following neonatal treatment with extracor-
poreal membrane oxygenation. Crit Care
Med. 1991;19:1210–1212.
73. Zani A, Zani-Ruttenstock E, Pierro A.
Advances in the surgical approach to congeni-
tal diaphragmatic hernia. Semin Fetal Neona-
tal Med. 2014;19:364–369.
74. Wilson JM, DiFiore JW, Peters CA. Experi-
mental fetal tracheal ligation prevents the
pulmonary hypoplasia associated with fetal
nephrectomy: possible application for con-
genital diaphragmatic hernia. J Pediatr Surg.
1993;28:1433–1439; discussion 1439–1440.
75. Hedrick MH, Ferro MM, Filly RA, et  al.
Congenital high airway obstruction syndrome
(CHAOS): a potential for perinatal interven-
tion. J Pediatr Surg. 1994;29:271–274.
76. Khan PA, Cloutier M, Piedboeuf B. Tracheal
occlusion: a review of obstructing fetal lungs
to make them grow and mature. Am J Med
Genet C Semin Med Genet. 2007;145C:125–
138.
77. Benachi A, Chailley-Heu B, Delezoide AL,
et al. Lung growth and maturation after tra-
cheal occlusion in diaphragmatic hernia. Am J
Respir Crit Care Med. 1998;157:921–927.
78. Flake AW, Crombleholme TM, Johnson MP,
et al. Treatment of severe congenital diaphrag-
matic hernia by fetal tracheal occlusion: clini-
cal experience with fifteen cases. Am J Obstet
Gynecol. 2000;183:1059–1066.
79. Deprest JA, Lerut TE, Vandenberghe K.
Operative fetoscopy: new perspective in fetal
therapy? Prenat Diagn. 1997;17:1247–1260.
80. Van der Veeken L, Russo FM, De Catte
L, et  al. Fetoscopic endoluminal tracheal
occlusion and reestablishment of fetal airways
for congenital diaphragmatic hernia. Gynecol
Surg. 2018;15(1):9.
81. Jani JC, Nicolaides KH, Gratacos E, et  al.
Severe diaphragmatic hernia treated by fetal
endoscopic tracheal occlusion. Ultrasound
Obstet Gynecol. 2009;34:304–310.
82. Flageole H, Evrard VA, Piedboeuf B, et  al.
The plug-unplug sequence: an important
step to achieve type II pneumocyte matura-
tion in the fetal lamb model. J Pediatr Surg.
1998;33:299–303.
83. Deprest J, Nicolaides K, Done E, et al. Tech-
nical aspects of fetal endoscopic tracheal
occlusion for congenital diaphragmatic her-
nia. J Pediatr Surg. 2011;46:22–32.
84. Jimenez JA, Eixarch E, DeKoninck P, et  al.
Balloon removal after fetoscopic endolumi-
nal tracheal occlusion for congenital dia-
phragmatic hernia. Am J Obstet Gynecol.
2017;217(1):78.e1–78.e11.
85. Deprest J, Brady P, Nicolaides K, et al. Pre-
natal management of the fetus with isolated
congenital diaphragmatic hernia in the era of
the TOTAL trial. Semin Fetal Neonatal Med.
2014;19:338–348.
86. Deprest J, Gratacos E, Nicolaides KH, FETO
Task Group. Fetoscopic tracheal occlusion
(FETO) for severe congenital diaphragmatic
hernia: evolution of a technique and pre-
liminary results. Ultrasound Obstet Gynecol.
2004;24:121–126.
87. DeKoninck P, Gomez O, Sandaite I, et  al.
Right-sided congenital diaphragmatic her-
nia in a decade of fetal surgery. BJOG.
2015;122(7):940–946.
88. Done E, Debeer A, Gucciardo L, et al. Predic-
tion of neonatal respiratory function and pul-
monary hypertension in fetuses with isolated
congenital diaphragmatic hernia in the fetal
endoscopic tracheal occlusion era: a single-cen-
tre study. Fetal Diagn Ther. 2015;37:24–32.
89. Deprest JA, Hyett JA, Flake AW, et al. Cur-
rent controversies in prenatal diagnosis 4:
Should fetal surgery be done in all cases of
severe diaphragmatic hernia? Prenat Diagn.
2009;29:15–19.
90. De Coppi P, Deprest J. Regenerative medi-
cine solutions in congenital diaphragmatic
hernia. Semin Pediatr Surg. 2017;26:171–
177.
91. Jeanty C, Kunisaki SM, MacKenzie TC.
Novel non-surgical prenatal approaches to
treating congenital diaphragmatic hernia.
Semin Fetal Neonatal Med. 2014;19:349–356.
92. Russo FM, Toelen J, Eastwood MP, et  al.
Transplacental sildenafil rescues lung abnor-
malities in the rabbit model of diaphragmatic
hernia. Thorax. 2016;71(6):517–525.
93. Lemus-Varela Mde L, Soliz A, Gomez-Meda
BC, et  al. Antenatal use of bosentan and/or
sildenafil attenuates pulmonary features in
rats with congenital diaphragmatic hernia.
World J Pediatr. 2014;10:354–359.
94. Umeda S, Miyagawa S, Fukushima S, et  al.
Enhanced pulmonary vascular and alveolar
development via prenatal administration of a
slow-release synthetic prostacyclin agonist in
rat fetal lung hypoplasia. PLoS One. 2016;11:
e0161334.
32
Abdomen
JON HYETT
KEY POINTS
• The embryologic processes involved in development of the
abdominal wall and viscera are complex and most anomalies
can be defined through their developmental origin.
• The abdominal viscera are our metabolic powerhouse but
have little functional significance in a fetus. Some signs of
abnormality develop late in pregnancy after the abdominal
viscera become functional.
• Most major abdominal defects can be detected sono-
graphically from early gestations if fetal anatomy is assessed
sequentially. Third trimester scans provide a window for op-
portunistic detection of anomalies that cannot be easily seen
before 22 weeks.
• Ultrasound diagnosis and surveillance of anomalies allows
obstetricians to work with multidisciplinary teams to improve
outcomes for fetuses that are affected by structural anoma-
lies.
Introduction
The abdomen constitutes that part of the body between the tho-
rax and the pelvis. The abdominal cavity is bounded by the dia-
phragm above but is contiguous with the pelvis; the boundary is
defined by the bony landmarks of the pelvic bones and lumbar
spine. Anteriorly and laterally, the abdominal cavity is bounded
by the soft muscular and fascial tissues of the anterior abdomi-
nal wall; posteriorly, the wall is more rigid, being formed by the
parietal peritoneum that lies over the vertebral bodies with their
muscular attachments.
From a functional perspective, the abdominal cavity essentially
acts as a repository for a number of organ systems responsible for
metabolic processing. This includes the hollow tubular structure
of the bowel, which enters cranially at the gastro-oesophageal
sphincter and develops into the remaining parts of the digestive
system, carrying and processing nutrients and waste before, at the
caudal end, passing these products back to the external environ-
ment. Organ systems such as the liver and kidneys are developed
through a number of embryologic stages bringing a variety of dif-
ferent cell lines together for functional effect. Other structures that
pass through the diaphragm and run into the pelvis include the
great vessels, lymphatics and peripheral nerves. Although prenatal
assessment of the abdomen may not inspire clinicians as much as
some other structures, this is the powerhouse of metabolic well-
being and includes and is bounded by many complex structures
340
that need to be coordinated with surrounding tissues. Abnormali-
ties of these systems can be lethal or cause significant morbidity in
a neonate, and there is significant value in prenatal diagnosis that
allows timely and appropriate intervention after birth.
Data from the European Congenital Anomalies Surveillance
(Eurocat) Registry show that abdominal wall defects and gas-
trointestinal (GI) anomalies are the fifth most prevalent type of
congenital anomaly, affecting approximately 1 in 400 pregnan-
cies (Table 32.1).1 About 75% of affected fetuses were liveborn,
3.2% of affected infants died in utero and 22% of women chose to
interrupt the pregnancy. The range of GI pathologies is shown in
Table 32.2. Abdominal wall defects are commonest, with
similar numbers of gastroschisis and exomphalos being identi-
fied although a significantly higher proportion of pregnancies
affected by exomphalos were terminated. As a consequence, gas-
troschisis is the commonest abdominal surgical complication
affecting liveborn infants. Herniation through the diaphragm is
also common and is dealt with elsewhere. The remaining pathol-
ogies predominantly result from developmental errors leading
to atresia of the variety of tubular structures seen through the
alimentary canal. 
Embryologic Development
The commonest abdominal anomalies seen prenatally, including
gastroschisis and bladder extrophy, relate to failures in embryologic
development of the abdominal wall. Formation of the abdominal
wall involves a combination of lateral plate mesoderm and overly-
ing ectoderm cell lines.2 The vertebrae and ribs and hypaxial flank
muscles develop in the midline by 5 weeks’ gestation and then
expand ventrolaterally and caudally.3 The rectus muscles reach the
level of the umbilicus by 8 weeks’ gestation. Further rapid dif-
ferentiation enables the development of the infraumbilical body
wall. Between 4 and 10 weeks (when the extruded bowel returns
to the intraabdominal cavity), there is a 25-fold increase in volume
of the abdominal cavity.3 Differential rates of cell proliferation
account for changes in shape, with a fivefold increase in abdomi-
nal circumference compared with length through this period.
Processes of cell migration, reorganisation and cell-to-cell adhe-
sion can all be disrupted, giving rise to the anomalies seen pre-
natally. Although exomphalos is also identified as an abdomi­nal
wall defect, the aetiology differs as the defect results from failure of
gut loops to return to the body cavity after normal physiological
herniation into the base of the umbilical cord.2
The cloaca is the endodermal lined cavity that forms the
boundary between the allantois (ventrally) and primordial
 CHAPTER 32 Abdomen 341

TABLE
32.1 Prevalence of Abdominal and Pelvic Abnormalities in Comparison to Other Systems
Systems or Aetiology Total LB, n (%) IUFD, n (%) TOP, n (%) Rate (95 CI)
Congenital heart disease 22,709 19,889 (88%) 380 (1.7%) 2440 (11%) 76.46 (75.47–77.47)
Limb defects 12,817 11,110 (87%) 199 (1.6%) 1508 (12%) 43.16 (42.41–43.91)
Urinary tract anomalies 10,082 8522 (85%) 170 (1.7%) 1390 (14%) 33.95 (33.29–34.62)
Central nervous system 7712 3402 (44%) 270 (3.5%) 4040 (52%) 25.97 (25.39–26.55)
Gastrointestinal and abdominal 7083 5283 (75%) 232 (3.2%) 1568 (22%) 23.85 (23.40–24.30)
wall defects
Genital anomalies 6217 5962 (96%) 39 (6.3%) 216 (3.5%) 20.93 (20.42–21.46)
Orofacial clefts 4198 3711 (88%) 66 (1.6%) 421 (10%) 14.14 (13.71–14.57)
Respiratory anomalies 1259 1001 (80%) 45 (3.6%) 213 (17%) 4.24 (4.01–4.48)
Chromosomal abnormalities 12,595 4841 (38%) 486 (3.9%) 7268 (58%) 42.41 (41.67–43.16)
Genetic syndromes 1811 1450 (80%) 35 (1.9%) 326 (18%) 6.10 (5.82–6.39)
Total 75,231 59,179 (79%) 1433 (1.9%) 14619 (19%) 253.31 (251.51–
255.13)

CI, Confidence interval; IUFD, intrauterine fetal death; LB, live birth; TOP, termination of pregnancy.
Data from Eurocat Database. European surveillance of congenital anomalies (all full member organisations; 2011–2015). http://www.eurocat-network.eu/accessprevalencedata/prevalencetables.
  

TABLE Prevalence of Gastrointestinal Anomalies and Abdominal Wall Defects Reported by the UK to the Eurocat
32.2 Database
Anomaly Total LB, n (%) IUFD, n (%) TOP, n (%) Rate (95 CI)
Gastroschisis 1182 1058 (90%) 39 (3%) 85 (7%) 4.67 (4.40–4.94)
Exomphalos 1012 360 (36%) 67 (6%) 585 (58%) 3.99 (3.75–4.25)
Diaphragmatic hernia 851 606 (71%) 30 (4%) 215 (25%) 3.36 (3.14–3.59)
Anorectal atresia or stenosis 797 558 (70%) 20 (3%) 219 (27%) 3.15 (2.93–3.37)
Oesophageal atresia +/- tracheo- 643 549 (85%) 32 (5%) 62 (10%) 2.54 (2.35–2.74)
oesophageal fistula
Duodenal atresia or stenosis 448 403 (90%) 18 (3%) 27 (7%) 1.77 (1.61–1.94)
Hirschsprung disease 396 395 (100%) 1.56 (1.41–1.72)
Other small bowel atresia or 244 235 (96%) 0.96 (0.85–1.09)
stenosis
Atresia of bile ducts 70 70 (100%) 0.28 (0.22–0.35)
Annular pancreas 14 11 (79%) 0.06 (0.03–0.09)

CI, Confidence interval; IUFD, intrauterine fetal death; LB, live birth; TOP, termination of pregnancy.
Data from Eurocat Database. European surveillance of congenital anomalies (all full member organisations; 2011–2015). http://www.eurocat-network.eu/accessprevalencedata/prevalencetables.
  

hindgut (dorsally).4 This is divided, at 4 to 6 weeks, into anterior
and posterior compartments by the urogenital sinus. Failure of
development of this sinus will lead to a condition described as
persistence of the cloaca in which the bowel, vagina and urethra
remain confluent and drain into a common opening. The ventral
part of the urogenital sinus develops into the bladder and urethra,
and as the bladder ‘descends’ into the pelvis, the remaining por-
tion of the allantoic duct involutes. Failure of involution will leave
a patent urachal remnant. The ventral wall is normally reinforced
through medial migration of mesoderm to form the lower part
of the anterior abdominal wall. If this does not occur, the cloa-
cal membrane can rupture, resulting in cloacal extrophy, bladder
extrophy or epispadias depending on the timing of this event.
The gut and major intraabdominal viscera are formed from a
tubular structure running through the craniocaudal axis of the
early embryo.5 This tube is lined by endodermal tissue originating
342 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A C
CRL
B D
• Fig. 32.1  A composite of ultrasound images showing normal anatomy in axial and longitudinal section (A
and B) and demonstrating the presence of gastroschisis (C) and exomphalos (D) at 12 weeks’ gestation.
CRL, Crown–rump length.
from the yolk sac. This is surrounded by a layer of mesoderm
contributing to the gut tube wall as well as splanchnic (visceral)
and somatic (parietal) mesoderms that continue to differentiate
to form the supporting mesenteries. The vascular bundle that
runs within the mesentery includes neural crest tissue that dif-
ferentiates into the nerves and neurons found throughout the
gut and associated viscera. The gut is traditionally divided into
three parts (fore, mid and hind gut) that have different vascular
supplies. Abnormalities of the bowel and other intraabdominal
viscera can result from a range of failures in normal embryo-
logic development, including anomalous differentiation of local
cell populations, failure in tubal canalisation, failure to pull the
gut into the abdominal cavity (or of closure of the ventral wall),
failure in bowel rotation and anomalous vascular or neuronal
connection. A number of resulting anomalies can potentially be
detected in the prenatal period.
The liver develops from an embryologic structure found at
the boundary of the embryonic pole and yolk sac known as the
septum transversum.6 This brings ectodermal, mesenchymal and
endodermal cell lines together. Internally, this aligns with the
boundary between the foregut and midgut. In the early embryo,
the liver buds out from the ventral surface. The cell lines undergo
significant differentiation between 5 and 8 weeks of gestation to
develop the complex architecture found between the portal field
and central vein, including development of the biliary tree. The
liver’s main embryonic and fetal functions are cardiovascular
and haemopoietic, providing a vascular connection between the
umbilical vein and the right side of the heart and producing blood
stem cells before bone marrow development.
From an ultrasound perspective, embryonic development of
the intraabdominal viscera can be followed from approximately
7 weeks of gestation.7 The anterior abdominal wall is already
formed at this gestation, but the cord insertion can be visualised,
and there is evidence of physiological herniation of the bowel into
the cord at this stage. At 8 to 9 weeks, the abdominal cavity is
almost completely filled by the liver and the stomach. The urethra
becomes patent (through rupture of the cloacal membrane) at 9
weeks’ gestation, and the diaphragm develops at approximately 10
weeks. The bowel rotates and returns to the abdominal cavity by
11 weeks.
Sonographic Features at 12 Weeks’ Gestation
Although the routine second trimester (18- to 20-week) scan
is still considered to be the ‘gold standard’ point for anatomi-
cal assessment, it is possible to detect major structural abnor-
malities, including some abdominal defects, at 11 to 13+6 weeks
(Fig. 32.1). In a series of 44,859 pregnancies that had a structured
sequential anatomical survey completed at the 11- to 13+6-week
scan, 488 (1.1%) fetuses had a structural abnormality, and 213
(43.6%) of these were successfully identified.8 This included all
104 cases of exomphalos, gastroschisis, megacystis and body stalk
anomaly. In contrast, none of the three reported cases of bowel
atresia were detected at 11 to 13+6 weeks.

The fetus is traditionally assessed in midsagittal section in the
first trimester, which allows accurate assessment of crown rump
length for confirmation of gestational age and measurement of
nuchal translucency thickness, but this is not the most valuable
plane for assessment of the abdomen and anterior abdominal
wall. This is best achieved by rotating the probe so that the fetus is
imaged in an axial section. The probe can then be manipulated to
sweep through the abdomen, visualising structures of importance
within a few seconds.9 Moving caudally from the thorax through
the diaphragm, in the upper abdomen, the stomach should be visi-
ble to the left of the midline. The stomach is normally visible in all
cases from 11 weeks’ gestation. Care should be taken to ensure that
situs is appropriate. To the right of the midline at this anatomical
level, the parenchyma of the liver can be seen, which is typically
homogeneous with no echogenic foci. The hepatic portion of the
umbilical vein can be used as a second landmark to define the cor-
rect plane for measurement of the abdominal circumference.
After the upper abdomen has been assessed, the probe can
be swept down to the level of the umbilicus, and care should
be taken to define the integrity of the insertion of the umbilical
cord. In early pregnancy (i.e. <9 weeks’ gestation), space within
the abdominal cavity is limited, and the developing small bowel
extrudes through the umbilicus into the base of the cord. This
should have returned to the abdominal cavity by 11 weeks’ gesta-
tion, and continued herniation of bowel or other intraabdominal
viscus should be regarded as evidence of exomphalos.10 The fea-
tures of this anomaly are discussed in more detail later. Colour
Doppler can also be used to identify the umbilical arteries as they
enter the abdomen, dividing around the bladder. A two-vessel
cord can be detected if care is taken in this evaluation and has been
reported to be associated with an increased risk for aneuploidy as
well as with renal anomalies and is therefore a useful marker in the
assessment of these disease processes.11 In addition to focusing on
the umbilicus, it is important to check that there is no evidence of
bowel herniation to the right of the midline; gastroschisis is also
readily detected at 11 to 13+6 weeks when free loops of bowel,
which are not contained within a membrane, can be seen floating
freely in the amniotic fluid.12  advancing gestation (the urinary system and abdominal defects
Sonographic Features at 20 Weeks’ Gestation
The abdomen and pelvis are traditionally assessed using three axial
sections at 18 to 20 weeks (Fig. 32.2). Moving sequentially from
the thorax through the diaphragm, the upper abdomen is assessed
Left
A B
• Fig. 32.2 A composite of ultrasound images showing normal anatomy in axial sections through the
upper abdomen (A), at the level of the umbilicus (B) and in the pelvis (C) at 20 weeks’ gestation.
CHAPTER 32 Abdomen 343
in an axial section that demonstrates the echolucent stomach, the
liver and the mid third of the umbilical vein at the level of the
portal sinus.13 The upper poles of the kidneys should not be visible
in this view. An additional cystic structure extending to the right
may be visible, representing the fetal gallbladder. The abdominal
circumference is measured at this level by placing callipers on the
outer surface of the skin line and using either perpendicular lin-
ear measures or an ellipse to complete assessment. From an ana-
tomical perspective, this view is used to check that the stomach is
visible and to check situs and consistent echogenicity across the
liver. In combination with coronal or longitudinal sections, this
view is also used to demonstrate the integrity of the left and right
hemidiaphragms.
Moving caudally, the fetal kidneys can be demonstrated lying
either side of the spine. Anteriorly, the cord insertion is assessed
to ensure integrity of the anterior abdominal wall. Turning into
a longitudinal section, the distance between the insertion of the
umbilicus and the genital tubercle can also be assessed. The lumen
of the small and large bowel is not typically obvious at this gesta-
tion, and dilated loops can be detected if present. The small bowel
may be echogenic, which has been associated with a range of
pathologies described later. An axial section of the pelvis is used to
demonstrate the presence of the bladder, and colour Doppler can
be applied to show the bifurcation of the umbilical arteries and
therefore the presence of a three-vessel cord. Moving caudally, the
external genitalia can also be assessed. 
Sonographic Features after 28 Weeks’ Gestation
Although a third trimester scan is not routinely performed in
many jurisdictions, the majority of women seen in our prac-
tice are referred by their obstetrician for sonographic review on
at least one occasion beyond 28 weeks’ gestation. The third tri-
mester scan typically focuses on fetal growth and well-being but
presents an opportunity for a brief review of systems that are
either difficult to examine at early gestations (e.g. the heart) or
of systems in which pathologies may only become apparent with
such as small and large bowel obstruction). Best practice there-
fore includes assessment of fetal anatomy during a third trimes-
ter growth and well-being scan; in the case of the abdomen, it is
sensible to specifically assess the stomach and bowel and to look
for any other aberrant cystic or solid masses within the abdomen
or pelvis. 
C
344 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Abdominal Wall Defects
Gastroschisis
Gastroschisis is an anterior abdominal wall defect in which bowel
herniates into the amniotic fluid. In contrast to an exomphalos,
the abdominal wall defect lies to the right of the midline, and the
herniated bowel is not covered by a peritoneal membrane. If the
defect is large, other abdominal organs may be involved. The prev-
alence is approximately 2 to 4 per 10,000 births.14,15 Postnatally,
infants with gastroschisis are often described as having simple
(isolated) or complex (associated with other bowel and structural
anomalies and with a more complex postoperative course) dis-
ease.16 Outcomes for these cohorts can be very different; complex
cases are more likely to need a bowel resection and to have ongo-
ing GI complications and have a significantly longer length of stay
after birth (37 vs 108 days) and significantly longer requirement
for parenteral nutrition (26 vs 71 days).
A number of theories have been extended in an attempt to
describe the embryologic derivation of gastroschisis.17 Amongst
the most popular are theories related to vascular disruption dur-
ing the development of the anterior abdominal wall. It has, for
example, been suggested that premature atrophy (before 5 weeks’
gestation) of the right umbilical vein interferes with development
of the junction of the somatopleure and the body stalk, result-
ing in a weakness in the umbilical ring. During the subsequent
process of herniation of the midgut into the extraembryonic coe-
lom, the small bowel herniates through this paraumbilical defect
and fails to return to the abdominal cavity at the end of this pro-
cess. This is supported by the fact that gastroschisis is associated
with intestinal nonrotation. An alternative theory suggests that at
a slightly later gestation (8–9 weeks), by the time the herniated
midgut has returned to the peritoneal cavity, there is a vascular
accident leading to obliteration of the right omphalomesenteric
arteries (the arterial plexus that is the precursor of the superior
mesenteric artery), resulting in necrosis of the cord base and her-
niation of the small bowel.
The cause of such a vascular insult has not been clearly identi-
fied. It is recognised that the prevalence of gastroschisis is higher
amongst younger pregnant women, and it has therefore been
suggested that there may be a teratogenic insult.17 Epidemio-
logic studies, many of which are limited by potential population
recruitment and reporting bias, have suggested that drugs such as
aspirin, pseudoephedrine, organic solvents, alcohol and cocaine
A B
• Fig. 32.3  Axial section of the upper abdomen at 20 (A) and 34 (B) weeks. The panel on the left (A) shows
loops of bowel lying freely (with no membranous covering) within the amniotic cavity. In B, the bowel is
slightly more pronounced, although the degree of dilatation is within normal limits for the later gestation.
have significant associations, although the quality of data is insuf-
ficient to correlate levels of exposure and effect. A recent case-
control study examining hair samples to identify recreational drug
use in the 3 months preceding diagnosis of a fetal abnormality was
not able to confirm an association between use of these drugs and
gastroschisis.18
In clinical practices that include routine ultrasound assess-
ment at 12 and 20 weeks, more than 95% of cases of gastroschisis
are detected prenatally, predominantly before the 20-week scan
(Fig. 32.3).1 In one recent study that included 44 cases of gastros-
chisis detected throughout the Northern region of the Nether-
lands, 86% of defects were detected at the 12-week scan, and the
remainder were all detected at the 20-week morphology scan; the
only cases that were detected postnatally involved women who
had declined routine ultrasound screening.14 A total of 12% of
affected fetuses had other structural anomalies, although chromo-
somal abnormalities were uncommon; one fetus was affected by
trisomy 18 and another by a genetic mutation in the NF1 gene.
After identification of gastroschisis, 14% of parents chose to ter-
minate the pregnancy, and 16% of the remaining infants died in
the perinatal period.
The contemporary challenge facing clinicians involved in pre-
natal diagnosis lies in defining the risk that an infant with gastros-
chisis has complex rather than simple disease and in defining risks
of preterm delivery (associated with an increased risk for neona-
tal mortality and morbidity) or of stillbirth.15,16,19 A number of
groups have retrospectively reviewed prenatal sonographic find-
ings in an attempt to distinguish between simple and complex
cases and to define risk for perinatal complication and mortality.
These findings have been well reviewed and brought together in a
meta-analysis published by a European consortium that reviewed
26 studies including 2023 fetuses.20 They reported that intraab-
dominal bowel dilation and polyhydramnios were associated with
bowel atresia (odds ratio (OR), 5.48; 95% confidence interval
(CI), 3.1–9.8 and 3.76; 1.7–8.3, respectively) and that gastric
dilation was associated with neonatal death (OR, 5.58; 95% CI,
1.3–24.1). No other ultrasound features were found to be signifi-
cantly associated with pregnancy outcome.
At the time of antenatal diagnosis, it is important to ensure
that a sequential anatomical survey is completed because fetuses
that have other structural anomalies are more likely to have a poor
outcome and are more likely to have an underlying chromosomal
or genetic disorder. Although fetuses with gastroschisis were tra-
ditionally thought to be at relatively low risk for chromosomal

16
(per 1000 continuing pregnancies)
14
12
10
Stillbirths
8 500
6 400
4
2 100
0 0
24
• Fig. 32.4 The impact of gestation on stillbirth and infant death rates (per 1000 ongoing cases) for
fetuses affected by gastroschisis. Stillbirth data are shown in circles and infant deaths by squares.
abnormality, the risk for conventional forms of aneuploidy is
significantly higher than that seen in an anatomically normal
population and recent studies have shown that use of genomic
microarrays for karyotyping is likely to detect pathogenic copy
number variations (CNVs) in 5% to 10% of cases.21 Conse-
quently, karyotyping should be offered to these families.
The nature of the gastroschisis anomaly can be assessed by iden-
tifying all herniated structures, measuring diameters of bowel both
intra and extraabdominally, measuring the size of the ‘neck’ or
abdominal wall defect. Infants with gastroschisis are frequently small
for gestational age. Although it is difficult to assess growth using the
abdominal circumference, which will be naturally reduced because
of herniation of intraabdominal contents, it is possible to look at the
bigger picture and include assessment of the amniotic fluid index
and maternal, placental and fetal Doppler. Doppler assessment of
the superior mesenteric artery has also been advocated, but this
is often difficult to perform, and there are no data showing this
improves management decisions and pregnancy outcome.
One publication on a large series of 860 infants affected by
gastroschisis has reported in utero and postnatal death rates by
gestational age (Fig. 32.4).15 Fetuses affected by gastroschisis that
are born very preterm are frequently impacted by infection (either
related to prematurity or their surgical morbidity), which has a
significant effect on outcomes, and infant death rates fall dramati-
cally after 32 weeks. In contrast, stillbirth rates start to climb late
in the third trimester. Sparkes and colleagues used these data to
look at the relative risk for expectant management compared with
elective delivery after 32 weeks. They showed that by 39 weeks,
there was a significant increase in risk for stillbirth through expect-
ant management, and most clinicians now advocate delivery
around 38 weeks’ gestation. According to the US-based data set,
the number needed to treat involves 17 elective deliveries at 39
weeks to prevent one in utero death.
It has been suggested that those cases that have a narrow ‘neck’
and dilated loops of bowel within the abdomen are more likely to
be complicated, with areas of bowel stenosis requiring postnatal
resection. Other authors have suggested that dilation of external
loops of bowel is also a poor prognostic indicator, being associated
with an increased risk for complex disease and preterm delivery,
but this can be difficult to interpret from a management perspec-
tive. Robertson and coworkers recently reported the value of ante-
natal sonography for prediction of neonatal outcome in a series
of 101 fetuses with gastroschisis.22 The group were able to define
that extraabdominal loops (>20 mm diameter) of bowel were
CHAPTER 32 Abdomen 345
1000
900
800
(per 1000 live births)
700
Infant deaths
600
300
200
26 28 30 32 34 36 38 40
Gestational age (weeks)
associated with an increased risk for complex disease with sensitiv-
ity of 73%. However, only 11 of the 38 cases with extraabdominal
bowel dilation had complex disease – a positive predictive value of
29% – making it difficult to act with confidence on this finding.
Fetuses that have gastroschisis need surgical intervention
shortly after delivery. It is therefore inappropriate to deliver them
outside of centres with tertiary neonatal and paediatric surgical
facilities. Although diagnosis, counselling about the condition and
any invasive testing may be done locally, it is normally best to
transfer care in the third trimester so that the obstetrician or fetal
medicine specialist who is responsible for coordinating delivery
can conduct serial surveillance to assess fetal well-being. This typi-
cally involves combinations of ultrasound and cardiotocography,
aiming for planned early delivery at 38 weeks’ gestation. One
research group recently reported a 58% reduction in stillbirth by
formalising this process of assessment.23
There is no good evidence that elective preterm delivery by
caesarean section improves the outcome of infants with gastros-
chisis.24 Induction is always more complex from the perspective
of being able to define the precise point of delivery, and for this
reason, many clinicians advocate that caesarean section allows bet-
ter synchronisation with neonatal and paediatric surgical teams.
Mothers will, however, be more able to support their infants if
they deliver vaginally, and this therefore appears to be a sensible
approach in most circumstances.
Gastroschisis may be repaired through primary closure, or if
the amount of prolapsed bowel is large, a silo may be constructed
and used to reduce the mass over the course of several days or
weeks. Infants who have gastroschisis will have delay in normal
feeding and are typically given parenteral nutrition for the first
few weeks of life. Although many infants will be home after 3 or
4 weeks, more complex lesions may lead to prolonged neonatal
admission. 
Exomphalos
An exomphalos or omphalocele is an anterior abdominal wall
defect that originates centrally at the umbilicus. Another con-
trasting feature in comparison to gastroschisis is that the defect is
bonded by the peritoneal membrane, although this can rupture in
some circumstances.
During fetal development, the bowel becomes too large to be
contained within the abdominal cavity, and the midgut herni-
ates into the extraembryonic coelom, being externalised from
346 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A B
• Fig. 32.5  Axial section of the abdomen in two fetuses at 12 (A) and 13 (B) weeks’ gestation. There is a
small exomphalos, which subtle echogenicity of the root of the cord in A. In contrast, B shows a mega-
exomphalos with more than 50% of the intraabdominal contents within the herniated sac.
approximately 5 to 9 weeks’ gestation. This feature is clearly
visible on early first trimester ultrasound. Typically, before the
bowel returns to the abdomen, it undergoes a process of rota-
tion. This process is typically incomplete in fetuses that have
an exomphalos. An exomphalos may contain other abdominal
organs such as the stomach, large bowel and liver, and the size of
the anterior abdominal wall defect may also vary widely because
the anterior abdominal wall muscles are often hypoplastic and
displaced laterally.
In a recent study reporting accuracy of prenatal diagnosis
in a regional Dutch dataset, 141 fetuses with exomphalos were
reported over a 4-year period.14 A total of 136 (96%) were identi-
fied prenatally, including 86% of those presenting in pregnancies
where women had opted for first trimester (11–13+6 week’ gesta-
tion) screening (Fig. 32.5). All 5 cases that were not identified in
this series had been missed at either the first (n = 1) or second (n
= 4) trimester scan. A total of 85% of fetuses that were identi-
fied as having an exomphalos had other structural anomalies, and
overall, 47% of this cohort had a chromosomal abnormality. The
commonest associated aneuploidy was trisomy 18, but trisomy
13, trisomy 21, 45X, triploidy and a variety of microscopic chro-
mosomal abnormalities were also reported. The strong association
with lethal aneuploidy and with other major structural abnormali-
ties led to a high rate of termination of pregnancy.
Because an exomphalos is associated with lethal forms of aneu-
ploidy, it is associated with a high spontaneous intrauterine loss
rate, and consequently, data on the prevalence of the anomaly
depend in part on the gestational age at which screening is per-
formed. In one study, first trimester (11–13+6 week) prevalence
was reported to be 0.25%.25 The risks of aneuploidy will therefore
be higher in those cases identified at earlier gestations. The other
impact of the difference in prevalence seen through pregnancy
and neonatal cohorts is that different clinicians who interact with
these fetuses have very different opinions on the likely outcome
for the infant. The paediatric surgeon who operates on the stable
neonate that is euploid and has an isolated structural defect has
a different vision of the spectrum of disease to a fetal medicine
specialist, and it is important to make sure that these experiences
are appropriately reflected in counselling. The commonest associ-
ated structural anomalies that are seen are cardiac and neurologic
abnormalities. In some series, about one third of infants who had
an apparently isolated exomphalos were found to have other struc-
tural anomalies after birth.26
Although the prognosis for a fetus affected by exomphalos is pri-
marily affected by associated chromosomal or structural anomalies,
other features can be assessed to determine the risk for a complicated
postnatal outcome. In an axial section, the circumference of the
omphalocele and abdominal cavity can be measured and expressed
as a ratio (the OC/AC ratio).27 A higher ratio is associated with liver
herniation, elective caesarean delivery, respiratory compromise and
delayed surgical closure. Total lung volumes can be calculated by
magnetic resonance imaging (MRI). Giant exomphalos is associated
with a 50% reduction in lung volume when fetuses are compared
with established normal ranges.28 These fetuses are likely to need
more ventilatory support, have a delay in establishing feeding and
require longer hospitalisation compared with who that have better
observed-to-expected lung volume ratios.
There is less controversy about ongoing surveillance than
is reported in fetuses with gastroschisis because there does not
appear to be an association with unanticipated late preterm or
term stillbirth. Although there is no good evidence supporting
elective caesarean section, many obstetricians prefer this delivery
route, particularly for larger lesions, citing the risk for intrapar-
tum rupture of the surrounding membrane, although this is rarely
reported.27 Timed delivery does have an advantage for timing neo-
natal and surgical assessment and may reduce the likelihood of
postnatal complication, although this has not been proven. The
goal of surgical repair is to effect complete fascial and abdominal
wall closure without causing cardiovascular or respiratory embar-
rassment or placing the repair under excessive intraabdominal
pressure. Although small defects are amenable to primary repair,
surgical intervention for larger defects is often delayed.29 The
membranous sac is allowed to form an eschar and epithelialize, a
process that may take up to 10 weeks. Some surgical groups use
tissue expanders or mesh to facilitate repair. 
Bladder and Cloacal Extrophy
Failure of closure of the lower anterior abdominal wall is a rare
event, leading to a spectrum of anomalies ranging from epispadias
through bladder extrophy to cloacal extrophy.4 The liveborn prev-
alence of bladder and cloacal extrophy are 1 in 40,000 and 1 in
250,000, respectively. Most sonographers will only see one or two
these defects in their career. Consequently, recognition of this defect
is poor, with a 25% (10 of 40 cases) prenatal detection rate reported
in one series.30
 CHAPTER 32 Abdomen 347

A B
• Fig. 32.6  Hyperechogenic bowel shown in axial (A) and mid-sagittal (B) section at 20 weeks’ gesta-
tion. This feature appears to be of clinical significance when the bowel is as bright as neighbouring bony
structures and is usually best assessed by reducing the gain on the image.

Bladder extrophy involves disruption of the anterior abdomi-
nal and bladder wall with exposure of the posterior wall of the
bladder and urethra. The upper urinary tract, which has a different
embryologic derivation, is typically normal. The distance between
the umbilicus and anus is shortened, and the pubic rami are short-
ened (by about a third in length) so that the pubic symphysis is
not closed. This is then associated with epispadias and a shortened
penile corpus in boys and vaginal stenosis and a bifid clitoris in
girls. Cloacal extrophy is even more extensive and may include
herniation of the foregut or exomphalos cranially together with an
imperforate anus and spinal anomalies caudally.
The ultrasound features associated with prenatal diagnosis
include an inability to define a normal filling bladder within the
pelvis, the presence of an anterior lower abdominal mass, low inser-
tion of the umbilical cord, difficulty in defining fetal sex (or the
finding of diminutive external genitalia) and widening of the pubic
rami.30 Charts have been produced that define the normal distance
between the base of the cord insertion and the genital tubercle, and
these can potentially be used to assess umbilical cord insertion in
fetuses that appear to have an absent bladder at the time of the
routine scan.31 On occasion, a urachal cyst has been mistaken for
a normal bladder.32 MRI has also been used to assist prenatal diag-
nosis and potentially has the advantage of being able to assess the
fetus in all three orthogonal planes with a more panoramic view of
the anomaly.33,34 This is particularly useful in identifying shortening
(and low umbilical cord insertion) of the anterior abdominal wall or
the presence of a mass in this area—features that can be difficult to
demonstrate using traditional ultrasound views.
Repair of bladder extrophy requires input from a multidisciplinary
surgical team and is typically delayed to 6 months of age. The principle
aims of surgery are to close the abdominal wall, secure continence and
reconstruct the genitalia. The bladder is repaired for primary closure,
and an osteotomy of the pelvic rami is performed simultaneously.
Many children subsequently need further bladder augmentation or
procedures to secure continence and may have ongoing orthopaedic
problems. Counselling is complex, and the intricacies of this for families
facing surgical repair of cloacal anomalies have been well described.35  The main ultrasound finding associated with both small and
Intraabdominal Masses
Cystic or solid intraabdominal masses are uncommon and often
present late rather than at the time of the routine 20-week
anomaly scan. The list of differential diagnoses is potentially
long and is dependent on the nature and site of the lesion as
well as the gender of the fetus. In most circumstances, the com-
bination of late presentation and uncertainty of diagnosis limit
prenatal intervention, and most of these lesions require further
assessment to define a management pathway during the postna-
tal period.
Hyperechogenic Bowel
The commonest intraabdominal mass seen during the 20-week
scan is hyperechogenic bowel; defined as loops of bowel that are
as bright or brighter than fetal bony parts (Fig. 32.6).36 Increased
echogenicity is described as being related to decreased gut motility
and the presence of thickened meconium within the small bowel.37
Up to 50% of fetuses affected by cystic fibrosis have hyper-
echogenic bowel and, in a white population, approximately 3%
of fetuses that have hyperechogenic bowel have cystic fibrosis.38,39
Hyperechogenicity is attributed to inspissation of meconium in
the distal ileum just proximal to the junction with the caecum.
Obstruction may vary between 5 and 10 cm in length and is often
associated with dilatation of the proximal ileum, which, when
seen sonographically, increases the risk to 25%.39,40
Nyberg and associates reported that 8 of 55 (14.5%) second
trimester fetuses affected by trisomy 21 had hyperechogenic bowel
compared to a prevalence of 0.9% in chromosomally normal
fetuses.41 Subsequent publications have suggested that the find-
ing of isolated echogenic bowel at 18 to 20 weeks is associated
with a fivefold increase in maternal age–related risk for Down
syndrome.42
Hyperechogenic bowel can also be a feature of intrauterine
infection and may has been associated with intraamniotic bleed-
ing, fetal growth restriction and stillbirth.43-45 These associations
are weak, and other features of these diagnoses should be sought. 
Dilated Bowel
large bowel atresia is dilation of proximal loops of bowel. Dila-
tion typically occurs later in pregnancy, when bowel function and
throughput has increased. Bowel atresia is rare with a prevalence
of approximately 1 in 1000 live births; only 25% of cases are rec-
ognised prenatally, and the screening tool ‘dilated bowel’ has a
predictive value of less than 50%.46,47
348 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Fig. 32.7  An axial section of the upper fetal abdomen at 22 weeks’ ges-
tation showing evidence of the ‘double bubble’ associated with duodenal
atresia.
Working distally, duodenal atresia affects approximately 1 in
5000 pregnancies and has a distinctive ‘double bubble’ appear-
ance in the third trimester (Fig. 32.7) but is typically not visible
at the routine 20-week anomaly scan.48 It may be found during
assessment of polyhydramnios. The stomach, which lies laterally
to the distended proximal part of the duodenum, can be followed
to demonstrate continuity and exclude the diagnosis of other
cystic lesions that can be found in the upper abdomen such as a
choledochal or hepatic cyst. Duodenal atresia is associated with
trisomy 21 and other structural anomalies.49 When isolated, pre-
natal diagnosis allows earlier postnatal intervention and reduces
complications related to severe fluid and electrolyte imbalances,
improving neonatal outcomes.50
The lumen of the small bowel is not readily visible at 18 to
20 weeks and is typically smaller than 7mm in the third trimes-
ter. Other intraabdominal tubular structures that can be dilated,
such as the ureter, can be mistaken for small bowel and need to
be excluded. Meconium ileus, jejunoileal atresia, volvulus and
Hirschsprung disease can all present in this way. Atresias can
occur at single or multiple points along the whole of the small
bowel, and perforation resulting in meconium peritonitis can
occur.
Dilated loops of large bowel can be identified through the
presence of haustration with a luminal diameter greater than 18
mm (Fig. 32.8). The prevalence of large bowel atresia is approxi-
mately 1 in 2000 pregnancies, but because it presents late, very
few (<10% in one series) are identified prenatally.51 Anorectal
atresia is frequently found in association with other structural
malformations, particularly anomalies affecting the genitourinary
system.52 Bowel proximal to an obstruction may be hyperecho-
genic or hypermobile, and the presence of these signs improves
diagnostic efficacy. If a bowel obstruction is suspected, it is useful
for parents to meet a neonatologist or paediatric surgeon before
birth to discuss management after delivery.  liver is the major source of fetal erythropoiesis between 8 and 28
Intraabdominal Cysts
Although dilated loops of bowel normally have a tubular appear-
ance, intraabdominal cysts are typically circular and more discrete.
Starting with the bowel, enteric duplication cysts can be found at
any point along the GI tract. Their embryologic derivation is not
well understood, but they are more prevalent in males.53 They are
found on the mesenteric side of the bowel and are therefore dif-
ficult to differentiate from mesenteric cysts.54 Although enteric
Voluson
E8
• Fig. 32.8  An axial section of the lower fetal abdomen at 34 weeks’ ges-
tation showing dilated loops of large bowel (with haustration).
duplication cysts typically cause pain, bleeding or intussusception
in later life, requiring surgical resection, mesenteric cysts cause few
problems.55
Localisation of a cystic structure helps refine the differential
diagnosis. Cysts in the upper right quadrant can be localised in
relation to the liver to help define whether they are intrahepatic
(e.g. a biliary cyst) or subhepatic (e.g. a choledochal cyst). Again,
diagnostic accuracy appears to be low.56
A lateral cystic structure in the lower abdomen or pelvis in a
female is most likely ovarian (Fig. 32.9). The hypothalamic–pitu-
itary–ovarian endocrine axis is active from the end of the first
trimester and folliculogenesis normally starts around 20 weeks’ ges-
tation. It has been suggested that the relatively high prevalence of
ovarian cysts more than 2 cm in diameter (∼1 in 2500 liveborn
females) is related to the enhanced level of ovarian activity occurring
at this time.57 Ovarian cysts tend to become more complex through
the antenatal period, and a cyst more than 4 cm in diameter at birth
is recognised as having a high risk for torsion or haemorrhage.58
Management options include antenatal aspiration, postnatal resec-
tion (of all or just of complex cysts) or a conservative approach. It is
not clear whether one or another approach is advantageous. 
The Liver
Because the placenta is the major metabolic regulator before birth,
the liver’s major functional roles in the fetus are its contribution to
erythropoiesis and facilitation of venous return to the heart. The
weeks’ gestation. In later pregnancy, the liver can continue to con-
tribute to erythropoiesis, and this commonly occurs in situations
of chronic fetal anaemia. One consequence of this is the develop-
ment of hepatomegaly, and it has been suggested that liver length
can be used as a marker for fetal anaemia.59 Prospective evaluation
by other groups did not show the same sensitivity as initial reports,
and noninvasive monitoring of fetal anaemia is now based around
assessment of the middle cerebral artery peak systolic velocity.60,61
Approximately 30% of venous return through the umbilical
vein is streamed directly to the right atria through the ductus
 CHAPTER 32 Abdomen 349

E2
E2

A B
• Fig. 32.9  Axial section of the pelvis (note the bladder in the midline) showing cystic (A) and solid (B)
ovarian masses.

venosus. From a physiological perspective, the intent is to

ensure supply of oxygenated blood to the brain. In addition to
this, assessment of flow through the ductus venosus provides
valuable insight into the function of the right heart. In the
first trimester of pregnancy a high proportion of fetuses that
have common chromosomal abnormalities, such as trisomy
21, have reversed or absent end diastolic flow in the ductus
venosus, and this haemodynamic marker can be used as a tool
to screen for these conditions.62 In addition, ductal flows are
often abnormal in fetuses that have major cardiac abnormali-
ties.63 The vessel is also valuable in assessing cardiac function
in the growth restricted fetus in which changes are indicative
of cardiovascular decompensation and should trigger deliv-
ery.64,65 Absence of the ductus venosus is rare but has being
more commonly recognised since the vessel has been used to
assess fetal cardiac function. Fetuses that have an absent ductus
venosus have a high risk for having both cardiac and extracar-
diac malformations, may be come hydropic in later pregnancy
and have higher rates of stillbirth.66
Hepatic tumours are rare but can present as solid or cystic
masses within the liver. The liver is typically enlarged and may • Fig. 32.10  An axial section of the upper abdomen showing an area of
compress other intraabdominal organs and cause elevation (and calcification within the liver parenchyma. Other views demonstrated that
splinting) of the diaphragm. Ultrasound can be used to localise this lay within the parenchyma but just below the diaphragm. Note the
the mass, document and monitor its size and determine whether acoustic shadowing below the lesion.
it is a vascular lesion. The commonest fetal hepatic tumours are
haemangioma (60%).67 Although these are described as ‘benign’ cavity and may be free (within the peritoneum) or within the
vascular lesions, if large, they can be associated with the develop- gastric or bowel wall or within the liver, biliary tree or spleen.
ment of high-output cardiac failure and fetal hydrops, thrombo- Lesions associated with the bowel or seeded in the peritoneum
cytopenia, tumour rupture and consumptive coagulopathy. The are typically related to bowel obstruction or perforation.71 Per-
value of intrauterine interventions in this circumstance is uncer- foration causes a sterile chemical peritonitis, and calcification
tain; the diagnosis has not been confirmed (differentials include is a longer term feature of the inflammatory response. There is
hamartoma and hepatoblastoma), and there are no trials that a trend to managing meconium peritonitis expectantly in the
demonstrate whether therapies such as hydrocortisone given into postnatal period, so no fetal intervention is recommended in this
the fetal circulation improve outcome.68,69 Hepatic tumours may circumstance.72
be associated with tumours in other systems, including the central Isolated areas of calcification seen abutting the diaphragm or
nervous system and the placenta, so a detailed systematic survey upper abdominal organs are common and should not cause alarm.
should be undertaken when a tumour is recognised.70  Multiple echogenic foci within structures such as the liver have
been described in cases of fetal infection and with cocaine use,
Intraabdominal Calcification which likely causes vascular disruption, inflammation and scar-
ring.73,74 Isolated intrahepatic foci have previously been reported
Areas of calcification are defined on ultrasound as discrete areas as markers of aneuploidy, but current comprehensive screening
of echogenicity which exhibit acoustic shadowing (Fig. 32.10). programmes are of such poor positive predictive value that there is
These can be found at a number of sites within the abdominal no value in using them to determine risk for aneuploidy. 
350 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Conclusions
The abdomen is often overlooked prenatally because its viscera
appear to be functionally less important than other systems before
birth. The commonest anomalies affecting the abdomen involve
the abdominal wall. Defects in the anterior abdominal wall can be
identified through routine prenatal imaging at 12 and 20 weeks’
gestation. This then allows involvement of a multidisciplinary
team in controlled delivery and repair, which improves outcomes.
Abnormalities of the intraabdominal viscera are relatively
uncommon and tend to present late in pregnancy so that detection
is opportunistic rather than systematic. Many of the characteris-
tics of these anomalies (presenting on ultrasound as a cystic or
solid mass) are shared, and it is often difficult to reach a precise
diagnosis before delivery. There are few indications for fetal inter-
vention or to deliver preterm on the basis of the finding of an
intraabdominal mass.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 19. D’Antonio F, Virgone C, Rizzo G, et al. Pre-
natal risk factors and outcomes in gastroschisis:
36. Slotnick RN, Abuhamad AZ. Prognostic
implications of echogenic fetal bowel. Lancet.
a meta-analysis. Pediatrics. 2015;136(1):e159– 1996;347:85–87.
1.  Eurocat: European Surveillance of Con-
e169. 37. Hill LM, Fries J, Hecker J, Grzybek P. Second
genital Anomalies. Prevalence Tables. http://
20. Overton T, Piere M, Gao H, et al. Antenatal trimester echogenic small bowel: an increased
www.eurocat-network.eu/accessprevalencedata/
management and outcomes of gastroschisis in risk for adverse perinatal outcome. Prenat
prevalencetables.
the UK. Prenat Diagn. 2012;32:1256–1262. Diagn. 1994;14:845–850.
2. Mekonen HK, Hikspoors JP, Mommen G,
21. Committee on Genetics and the Society for 38. Muller F, Frot JC, Aubury J, Boue A. Meco-
et  al. Development of the ventral body wall
Maternal-Fetal Medicine. Committee opin- nium ileus in cystic fibrosis fetuses. Lancet.
in the human embryo. J Anat. 2015;227:673–
ion no.682: Microarrays and next-generation 1984;2:223.
685.
sequencing technology: the use of advanced 39. Muller F, Dommergues M, Aubry MC, et al.
3. Sadler TW. The embryologic origin of ven-
genetic diagnostic tools in obstetrics and gyne- Am J Obstet Gynecol. 1995;173:508–513.
tral body wall defects. Semin Pediatr Surg.
cology. Obstet Gynecol. 2016;128:e262–e268. 40. Papp Z, Toth Z, Szabo M, Szeifert GT. Early
2010;19:209–214.
22. Robertson JA, Kimble RM, Stockton K, Sekar prenatal diagnosis of cystic fibrosis by ultra-
4. Arlen AM, Smith EA. Disorders of the blad-
R. Antenatal ultrasound features in fetuses with sound. Clin Genet. 1985;28:356–358.
der and cloacal anomaly. Clin Perinatol.
gastroschisis and its prediction in neonatal out- 41. Nyberg DA, Resta RG, Mahony BS, et al. Fetal
2014;41:695–707.
come. Aust N Z J Obstet Gynaecol. 2017;57:52– hyperechogenic bowel and Down’s syndrome.
5. Embryology Learning Resources, Duke Univer-
56. Ultrasound Obstet Gynecol. 1993;3:330–333.
sity Medical School. Gut development; 2017.
23. Perry H, Healy C, Wellesley D, et  al. Intra- 42. Nicolaides KH. Screening for chromo-
https://web.duke.edu/anatomy/embryology/
uterine death rate in gastroschisis following somal defects. Ultrasound Obstet Gynecol.
gi/gi.html.
the introduction of an antenatal surveillance 2003;21:313–321.
6. Hill M. Embryology Education and Research:
program: retrospective observational study. J 43. Simon-Bouy B, Satre V, Ferec C, et al. Hyper-
Gastrointestinal Tract—Liver Development.
Obstet Gynaecol Res. 2017;43:492–497. echogenic fetal bowel: a large French collab-
https://embryology.med.unsw.edu.au/embryol
24. Al-Kaff A, MacDonald SC, Kent N, et  al. orative study of 682 cases. Am J Med Genet.
ogy/index.php/Gastrointestinal_Tract_-_Liver
Canadian pediatric surgery network. Delivery 2003;121A:209–213.
_Development.
planning for pregnancies with gastroschisis: 44. Ghose I, Mason GC, Martinez D, et al. Hyper-
7. Blaas HG, Eik-Nes SH. Sonographic develop-
findings from a prospective national registry. echogenic fetal bowel: a prospective analysis of
ment of the normal foetal thorax and abdomen
Am J Obstet Gynecol. 2015;213:557.e1–e8. sixty consecutive cases. BJOG. 2000;107:426–
across gestation. Prenat Diagn. 2008;28:568–
25. Khalil A, Arnaotuglou C, Pacilli M, et al. Out- 429.
580.
come of fetal exomphalos diagnosed at 11–14 45. Whitten SM, Clayton S, Norbury G, Chitty
8. Syngelaki A, Chelemen T, Dagklis T, et  al.
weeks of gestation. Ultrasound Obstet Gynecol. LS. Etiology of hyperechogenic bowel within a
Challenges in the diagnosis of fetal non-chro-
2012;39:401–406. tertiary fetal medicine unit. Ultrasound Obstet
mosomal abnormalities at 11-13 weeks. Prenat
26. Cohen-Overbeek TE, Tong WH, Hatzman Gynecol. 2007;30:398.
Diagn. 2011;31:90–102.
TR, et  al. Omphalocele: comparison of out- 46. Stoll C, Alembik Y, Dott B, Roth MP. Evalu-
9. ISUOG Practice Guidelines. Performance of
come following prenatal or postnatal diagnosis. ation of prenatal diagnosis of congenital
first-trimester fetal ultrasound scan. Ultrasound
Ultrasound Obstet Gynecol. 2010;36:687–692. gastro-intestinal atresias. Eur J Epidemiol.
Obstet Gynecol. 2013;41:102–113.
27. Kleinrouweler CE, Kuijper CF, van Zalen- 1996;12:611–616.
10. Tassin M, Descriaud C, Elie C, et al. Omphalo-
Sprock MM, et  al. Characteristics and out- 47. Borsellino A, Zaccara A, Nahom A, et al. False-
cele in the first trimester: prediction of perina-
come and the omphalocele circumference / positive rate in prenatal diagnosis of surgical
tal outcome. Prenat Diagn. 2013;33:497–501.
abdominal circumference ratio in prenatally anomalies. J Pediatr Surg. 2006;41:826–829.
11. Martínez-Payo C, Cabezas E, Nieto Y, et  al.
diagnosed fetal omphalocele. Fetal Diagn Ther. 48. Lawrence MJ, Ford WDA, Furness ME, et al.
Detection of single umbilical artery in the
2011;30:60–69. Congenital duodenal obstruction: early ante-
first trimester ultrasound: its value as a marker
28. Danzer E, Victoria T, Bebbington MW, et al. natal ultrasound diagnosis. Pediatr Surg Int.
of fetal malformation. Biomed Res Int. 2014;
Fetal MRI-calculated total lung volumes in 2000;16:342–345.
2014: 548729.
the prediction of short-term outcome in giant 49. Grosfeld JL, Rescorla FJ. Duodenal atresia and
12. Tassin M, Benachi A. Diagnosis of abdominal
omphalocele: preliminary findings. Fetal Diagn stenosis: reassessment of treatment and out-
wall defects in the first trimester. Curr Opin
Ther. 2012;31:248–253. come based on antenatal diagnosis, pathologic
Obstet Gynecol. 2014;26:104–109.
29. Gamba P, Midrio P. Abdominal wall defects: variance and long term follow-up. World J Surg.
13. Salomon LJ, Alfirevic V, Berghella V, et al. on
prenatal diagnosis, newborn management, 1993;17:301–309.
behalf of the ISUOG Clinical Standards Com-
and long-term outcomes. Semin Pediatr Surg. 50. Romero R, Ghidini A, Costigan K, et al. Prena-
mittee. Practice guidelines for performance
2014;23:283–290. tal diagnosis of duodenal atresia: does it make
of the routine mid-trimester fetal ultrasound
30. Goyal A, Fishwick J, Hurrell R, et al. Antenatal any difference? Obstet Gynecol. 1988;71:739–
scan. ISUOG; 2010. http://www.isuog.org.
diagnosis of bladder/cloacal exstrophy: chal- 741.
14. Fleurke-Rozema H, van de Kamp K, Bakker M,
lenges and possible solutions. J Pediatr Urol. 51. Corteville JE, Gray DL, Langer JC. Bowel
et al. Prevalence, timing of diagnosis and preg-
2012;8:140–144. abnormalities in the fetus: correlation of pre-
nancy outcome of abdominal wall defects after
31. Fishel-Bartal M, Perlman S, Messing B, et  al. natal ultrasonographic findings with outcome.
the introduction of a national prenatal screen-
Early diagnosis of bladder exstrophy: quanti- Am J Obstet Gynecol. 1996;72:175.
ing program. Prenat Diagn. 2017;37:383–388.
tative assessment of a low-inserted umbilical 52. Keckler SJ, St Peter SD, Valusek PA, et  al.
15. Sparks TN, Shaffer BL, Page J, Caughey AB.
cord. J Ultrasound Med. 2017;36:1801–1805. VACTERL anomalies in patients with esopha-
Gastroschisis: mortality risks with each addi-
32. Riddell JV, Houle AM, Franc-Guimond J, geal atresia: an updated delineation of the spec-
tional week of expectant management. Am J
Barrieras D. Prenatal vesico-allantoic cyst trum and review of the literature. Pediatr Surg
Obstet Gynecol. 2017;216:66.e1–66.e7.
outcome—a spectrum from patent ura- Int. 2007;23:309–313.
16. Arnold MA, Chang DC, Nabaweesi R, et  al.
chus to bladder exstrophy. Prenat Diagn. 53. Gray SW, Skandalakis JE. The small intestines.
Risk stratification of 4344 patients with gas-
2015;35:1342–1346. In: Embryology for surgeons. Baltimore, MD:
troschisis into simple and complex categories.
33. Goldman S, Szejnfeld PO, Rondon A, et  al. Williams and Wilkins; 1994:63–100.
J Pediatr Surg. 2007;42:1520–1525.
Prenatal diagnosis of bladder exstrophy by fetal 54. Deshpande P, Twining P, O’Neill D. Prenatal
17. Curry JI, McKinney P, Thornton JG, Stringer
MRI. J Pediatr Urol. 2013;9:3–6. diagnosis of fetal abdominal lymphangioma by
MD. The aetiology of gastroschisis. BJOG.
34. Calvo-Garcia MA, Kline-Fath BM, Rubio EI, ultrasonography. Ultrasound Obstet Gynecol.
2000;107:1339–1346.
et  al. Fetal MRI of cloacal exstrophy. Pediatr 2001;17:445–448.
18. David AL, Holloway A, Thomasson L, et al. A
Radiol. 2013;43:593–604. 55. Foley PT, Sithasanan N, McEwing R, et  al.
case-control study of maternal periconceptual
35. Bischoff A, Calvo-Garcia MA, Baregamian N, Enteric duplications presenting as antenatally
and pregnancy recreational drug use and fetal
et al. Prenatal counseling for cloaca and cloa- detected abdominal cysts: is delayed resection
malformation using hair analysis. PLoS One.
cal exstrophy-challenges faced by pediatric sur- appropriate? J Pediatr Surg. 2003;38:1810–
2014;9:e111038.
geons. Pediatr Surg Int. 2012;28:781–788. 1813.

350.e1
350.e2 References
56. Casaccia G, Bilancioni E, Nahom A, et al. Cys-
tic anomalies of biliary tree in the fetus: is it
possible to make a more specific prenatal diag-
nosis. J Pediatr Surg. 2002;37:1191–1194.
57. Sakala EP, Leon ZA, Rouse GA. Management
of antenatally diagnosed fetal ovarian cysts.
Obstet Gynecol Surv. 1991;46:407–511.
58. Foley PT, Ford WDA, McEwing R, Furness
M. Is conservative management of prenatal
and neonatal cysts justifiable? Fetal Diagn Ther.
2005;20:454–458.
59. Roberts AB, Mitchell JM, Lake Y, Pattison
NS. Ultrasonographic surveillance in red blood
cell alloimmunization. Am J Obstet Gynecol.
2001;184:1251–1255.
60. Iskaros J, Kingdom J, Morrison JJ, Rodeck C.
Prospective non-invasive monitoring of preg-
nancies complicated by red cell alloimmuni-
zation. Ultrasound Obstet Gynecol. 1998;11:
432–437.
61. Dukler D, Oepkes D, Seaward G, et al. Non-
invasive tests to predict fetal anemia: a study
comparing Doppler and ultrasound param-
eters. Am J Obstet Gynecol. 2003;188:1310–
1314.
62. Maiz N, Wright D, Ferreira AF, et al. A mix-
ture model of ductus venosus pulsatility index
in screening for aneuploidies at 11-13 weeks’
gestation. Fetal Diagn Ther. 2012;31:221–229.
63. Maiz N, Plasencia W, Dagklis T, et  al.
Ductus venosus Doppler in fetuses with
cardiac defects and increased nuchal translu-
cency thickness. Ultrasound Obstet Gynecol.
2008;31:256–260.
64. Seravalli V, Miller JL, Block-Abraham D, Bas-
chat AA. Ductus venosus Doppler in the assess-
ment of fetal cardiovascular health: an updated
practical approach. Acta Obstet Gynecol Scand.
2016;95:635–644.
65. Lees CC, Marlow N, van Wassenaer-Leemhuis
A, et  al. TRUFFLE study group 2 year neu-
rodevelopmental and intermediate perinatal
outcomes in infants with very preterm fetal
growth restriction (TRUFFLE): a randomised
trial. Lancet. 2015;385:2162–2172.
66. Moaddab A, Tonni G, Grisolia G, et  al. Pre-
dicting outcome in 259 fetuses with agenesis of
ductus venosus—a multicenter experience and
systematic review of the literature. J Matern
Fetal Neonatal Med. 2016;29:3606–3614.
67. Isaacs H. Fetal and neonatal hepatic tumors. J
Pediatr Surg. 2007;42:1797–1803.
68. Mejides AA, Adra AM, O’Sullivan MJ, Nicho-
las MC. Prenatal diagnosis and therapy for a
fetal hepatic vascular malformation. Obstet
Gynecol. 1995;85:850–853.
69. Boull C, Maguiness SM. Congenital heman-
giomas. Semin Cutan Med Surg. 2016;35:124–
127.
70. Harris K, Carreon CK, Vohra N, et al. Placen-
tal mesenchymal dysplasia with hepatic mesen-
chymal hamartoma: a case report and literature
review. Fetal Pediatr Pathol. 2013;32:448–453.
71. Foster MA, Nyberg DA, Mahony BS, et  al.
Meconium peritonitis: prenatal sonographic
findings and clinical significance. Radiology.
1987;165:661–665.
72. Dirkes K, Crombleholme TM, Craigo SD,
et  al. The natural history of meconium peri-
tonitis diagnosed in utero. J Pediatr Surg.
1995;30:979–982.
73. Carroll SG, Maxwell DJ. The significance of
echogenic areas in the fetal abdomen. Ultra-
sound Obstet Gynecol. 1996;7:293–298.
74. Hume RF, Gringas JL, Martin LS, et al. Ultra-
sound diagnosis of fetal anomalies associated
with in utero cocaine exposure: further support
for cocaine-induced vascular disruption terato-
genesis. Fetal Diagn Ther. 1994;9:239–245.
33
Kidney and Urinary Tract Disorders
ROLAND DEVLIEGER AND AN HINDRYCKX
KEY POINTS
• When a fetal urinary tract anomaly is identified, careful
ultrasound examination is required to exclude coexistent
anomalies.
• In the presence of a coexistent anomaly, the risk for aneu-
ploidy and single-gene disorders as an underlying aetiology
should be considered and investigated.
• Sonographic features associated with long-term poor renal
function include hyperechogenic kidneys, renal cyst forma-
tion, oligohydramnios and the inability of the bladder to refill
after drainage.
• In cases of severe bilateral renal disease and associated severe
oligohydramnios in the midtrimester, lethal pulmonary
hypoplasia is probable and termination of pregnancy may be
considered.
• In ongoing pregnancies, multidisciplinary perinatal manage-
ment should be planned with involvement of paediatric
nephrologists and surgeons. Careful postdelivery assessment
of the baby should be performed.
• In selected cases of severe lower urinary tract obstruction,
prenatal procedures (shunting or cystoscopy) appear to
improve the short-term outcome and possibly also the long-
term outcome.
Embryology
The development of the urinary tract starts from the third week of
the embryonic period. Whereas the kidneys and ureters develop
out of the intermediate mesoderm, the bladder and urethra arise
from the urogenital sinus. Initially, the urogenital ridge is derived
from the intermediate mesoderm on both sides of the primitive
aorta. The intermediate mesoderm gives rise to a nephrogenic
cord, which forms the pronephros, mesonephros and metaneph-
ros. The pronephros is a primitive excretion system which invo-
lutes almost completely by the fourth week. The mesonephros is
composed of well-developed nephrons with vascularised glomer-
uli, draining into the mesonephric duct. It subsequently involutes
except for the most caudal part, which turns into the male gonads
and the vas deferens: the Wolffian duct. The permanent kidney
develops from the caudally migrating mesonephric duct, now
called the metanephric duct, from which a ureteric bud emerges
(Fig. 33.1). This ureteric bud outgrowth is a critical element in the
development of the kidney. Signals from the ureteric bud interact
with the adjacent metanephric mesenchyme. In response, these
mesenchymal cells differentiate into the different cell types of the
glomerulus and the proximal tubule, the loop of Henle and the
distal tubule. Consequently, mesenchymal cells excrete molecular
signals that induce the ureteric bud to branch and interact with
new mesenchymal zones to form a new set of glomeruli. This
branching of the ureteric bud is essential in the development of
the number of glomeruli or nephrons. The ureter, the pyelocaly-
ceal system and the most distal element of the nephron, the ductus
colligens, are all derived from the ureteric bud.
The lower urinary tract forms from the urogenital sinus which
is an ectodermal derivative, segregated from the cloaca by the
ingrowth of the urorectal septum (Fig. 33.2). The urogenital sinus
develops into bladder and proximal urethra and reaches out for
the caudal tail of the metanephric tube, which then connect as the
vas deferens.
At birth, each kidney contains about 1,000,000 functional
units, called nephrons. The branching process is completed by 22
weeks’ gestation, but the induction of the mesenchyme by the epi-
thelial ureteric structures is not completed before 34 to 36 weeks,
accounting for the progressive maturation of fetal renal func-
tion. The functional maturation continues further because of an
increase in size of the existing nephrons.
The metanephros is formed in the sacral region at the level
of S1 but in an adult the kidney lies at the upper lumbar level
(T12–L3). The ascent of the kidneys occurs between the 6th and
9th gestational weeks, probably as a result of differential growth
of the sacral and lumbar regions, which lead to an unfolding of
the lower pole of the embryonic body (Fig. 33.3). The transitory
vessels supporting the kidney during its ascent normally disap-
pear. During their ascent, the kidneys turn 90 degrees towards the
vertebral cords.
Fetal urine production begins by 10 to 11 weeks’ gestation.
As clearing waste products out of blood is done by the placenta,
the main function of the kidneys in the prenatal period is the
production of amniotic fluid. The biochemistry of fetal urine is
not well documented until 16 weeks. Later in gestation, analysis
of fetal urine suggests that the various functions of the kidney
do not develop at the same time. Whereas glomerular protein
resorption and tubular reabsorption of glucose and phosphate are
already mature at 20 weeks, the tubular reabsorption of sodium
and β-2-microglobulin and the tubular secretion of calcium is
more progressive during the second half of pregnancy.1
The estimated rate of urine production increases exponen-
tially with gestation from 5 mL/h at 20 weeks to 50 mL/h at 40
weeks.2,3 
351
352 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Mesonephric duct

Metanephrogenic blastema
B
Stalk of
Remnant of pronephros ureteric bud Ureteric bud

Renal pelvis
Mesonephros
C
Major calyx

Developing liver Ureter

Minor calyx

Renal pelvis
Nephrogenic cord
D

Mesonephric duct Mesenchymal

Cloaca cell cluster

Metanephric
Ureteric bud Metanephrogenic blastema blastema

Groove between lobes

Primordium of metanephros (permanent kidney)

Straight
Arched collecting collecting
tubule tubule
A E
• Fig. 33.1  Embryonic development of the fetal kidney and urinary tract. A, Development of the metaneph-
ros at the end of the fifth week. B, The ureteric bud emerges from the caudally migrating mesonephric
duct and interacts with the surrounding metanephrogenic blastema. C–E, Branching of the ureter and
development of the ureter, renal pelvis, calices and collecting tubules. (From Moore KL, Persaud TVN,
Torchia, MG. The urogenital system. In Before We Are Born: Essentials of Embryology and Birth Defects.
9th edn. Philadelphia: Saunders Elsevier, 2016.)

Allantois Urinary bladder

appearance in the first trimester. The visualisation of the renal
UG sinus arteries by colour Doppler can facilitate their identification (Fig.
Urogenital
33.4A). The sonographic corticomedullary differentiation starts at
membrane 15 weeks and becomes clearer with advancing gestational age (Fig.
Cloaca
33.5). Kidney echogenicity becomes less than that of the liver and
Perineal spleen from 17 weeks on.
body
The fetal bladder should always be visualised by 13 weeks (or
Anal
membrane
crown–rump length >67 mm), and megacystis can be identified
Anorectal canal
by ultrasound as early as 10 to 14 weeks at which time bladder
length should not exceed 7 mm5 (see Fig. 33.4A). Identification of
Cloacal Hindgut Urorectal
membrane septum the bladder later on in pregnancy is easy because of its location in
the pelvis, between the umbilical arteries (Fig. 33.4B). Fetal urine
• Fig. 33.2  Division of the cloaca into the urogenital (UG) sinus and the
production starts at 10 to 11 weeks of pregnancy and increases sig-
rectum by ingrowth of the urorectal septum. The UG sinus develops into
bladder and proximal urethra. (From T.W. Sadler PhD, Langman’s medical
nificantly beyond 16 weeks. At 20 weeks, about 90% of amniotic
embryology 12th ed. Philadelphia: Lippincott Williams & Wilkins; 2011.) fluid consists of fetal urine.
Ultrasound examination of the normal urinary tract consists
of the assessment of the presence, location and size of both
Normal Sonographic Development of the kidneys and the evaluation of their structure and echogenicity.
Fetal Kidneys and Urinary Tract In addition, the presence, size and shape of the fetal bladder
are examined, as well as development of the external genitalia
From about 10 to 12 weeks of pregnancy, the fetal kidneys and (Fig. 33.4C) and the amount of amniotic fluid. The amount
adrenal glands can be visualised at both sites of the lumbar spine.4 of amniotic fluid may be estimated subjectively or more objec-
The kidneys should be seen by ultrasound at 13 weeks, and they tively by measuring the deepest pocket or the amniotic fluid
are usually easy to identify because of their relatively hyperechoic index. 
 CHAPTER 33  Kidney and Urinary Tract Disorders 353

Superior mesenteric artery

Adrenal gland and artery Adrenal gland

Mesonephric duct

Mesonephros
Mesonephros
Inferior mesenteric artery

Genital ridge

Renal artery
Common iliac artery
Kidney
Renal artery

Metanephric blastema Mesonephric duct

Ureteric bud
(from mesonephric duct) Ureter
Allantois Bladder

Cloaca Urogenital sinus

5 weeks 6 weeks

Adrenal gland Adrenal gland

Renal artery
Kidney Kidney
Renal artery

Site of previous Sites of previous

renal artery renal artery

Gonad
Gonad

Mesonephric duct Mesonephric duct

Ureter Ureter
Bladder Bladder

Urogenital sinus Urogenital sinus

7 weeks 8 weeks

• Fig. 33.3  Renal ascent. (Case courtesy of Dr Matt Skalski, Radiopaedia.org, rID: 29856.)
354 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

C
• Fig. 33.4  Ultrasound appearance of the kidneys and fetal bladder in the first (A) and second (B) trimes-
ters of pregnancy. Normal female and male external genitalia at 20 weeks of pregnancy (C).

Classification and Pathology of Fetal Renal
and Urinary Tract Anomalies
The embryonic development of the urogenital system in humans
is a complex process; consequently, renal anomalies are common
and constitute about 20% of all congenital anomalies.6
Malformations of the urogenital system can be classified into:
1. Urinary tract anomalies
2. Renal malformations
3. Bladder malformations
4. Genital malformations  threshold value to diagnose urinary tract dilation is lacking, but
Urinary Tract Anomalies
Urinary Tract Dilation
Dilation of the urinary tract is a common finding occurring in
1% to 2% of pregnancies. Whereas pyelectasis is defined as a
dilation of the renal pelvis, hydronephrosis consists of a dila-
tion of both the renal pelvis and the calyces. The severity of uri-
nary tract dilation can be assessed by different grading systems:
descriptive (mild, moderate or severe hydronephrosis), quanti-
tative (value of the anteroposterior diameter of the renal pel-
vis (APRPD)) and semiquantitative (e.g., grading system of the
Society of Fetal Urology).
Measuring the APRPD is the generally accepted method. The
APRPD is measured in a cross-sectional plane through both kid-
neys at the level of the hilus (Fig. 33.6). A consensus on the ideal
the most accepted cutoff values are 4 mm in the second trimester
and 7 mm in the third.
The semiquantitative grading system, as proposed by the Soci-
ety of Fetal Urology (SFU), classifies antenatal hydronephrosis in
5 grades, taking into account the evaluation of the renal pelvic
dilation, the presence of minor or major calyceal dilation and

15w
24w
32w
• Fig. 33.5  The normal corticomedullary differentiation throughout pregnancy. With advancing gestational
age, the differentiation between the renal cortex and medulla becomes clearer. The medulla becomes
hypoechogenic because of the appearance of the medullary pyramids (arrows).
1 2
• Fig. 33.6  Measurement of the anteroposterior diameter of the renal pelvis.
the evaluation of parenchymal thickness7 (Table 33.1). Recently,
Nguyen and coworkers proposed the Urinary Tract Dilatation
(UTD) Classification System which is a consensus consolida-
tion of a number of previous studies including the SFU system.
CHAPTER 33  Kidney and Urinary Tract Disorders 355
20w 22w
26w 30w
34w 39w
This grading system is based on the APRPD, calyceal dilation,
parenchymal thickness and appearance, dilation of the ureter and
assessment of the bladder and provides a description of urinary
tract dilation that can be applied both prenatally and postna-
tally as well as a standardised approach for perinatal evaluation8
(Table 33.2 and Fig. 33.7).
The underlying aetiology of fetal urinary tract dilation is diverse
and may result either from overdistension secondary to a distal
obstructive lesion or from retrograde urine flow as a consequence of
reflux. It can occasionally be difficult to distinguish between these
two broad pathologies and significant overlap exists.6 Transient or
physiologic dilation is the most common aetiology, accounting for
50% to 70% of mostly mild urinary tract dilations. With increas-
ing dilation or progression to hydronephrosis, the likelihood of a
significant uropathy on postnatal evaluation increases.9
Fetal obstructive uropathies are usually classified by ultrasound
according to the level of the dilation (Table 33.3). For high- and
midlevel obstruction, the lesions can be either uni- or bilateral.
This is important from a prognostic point of view because post-
natal renal function is likely to be normal in unilateral cases with
preserved amniotic fluid volume. 
Mild Isolated Pyelectasis
Mild pyelectasis is a common and usually benign finding (see
Fig. 33.6). In some cases, however, it may be secondary to
356 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Hydronephrosis Grading System of the Society TABLE Ultrasonography Parameters Included in the
33.1 of Fetal Urology (SFU) 33.2 Urinary Tract Dilatation (UTD) Classification
Hydronephrosis Grade Pattern of Renal Sinus Splitting System and Normal Values
>28 Postnatal
0 No splitting
US Findings 16–27 wk wk (>48 hr)
1 Urine in pelvis barely splits sinus
APRPDa <4 mm <7 mm <10 mm
2 Urine fills intrarenal pelvis
Urine fills extrarenal pelvis; major Calyceal dilationb No No No
calyces dilated • Central No No No
• Peripheral
3 SFU grade 2+
minor calyces uniformly dilated and Parenchymal thicknessc Normal Normal Normal
parenchyma preserved Parenchymal appearanced Normal Normal Normal
4 SFU grade 3 with Ureter(s)e Normal Normal Normal
parenchymal thinning
Bladderf Normal Normal Normal
SFU, Society of Fetal Urology.
Adapted from Nguyen HT, Herndon CD, Cooper C, et al. The Society of Fetal Urology consen- Unexplained oligohydramnios No No No
sus statement on the evaluation and management of antenatal hydronephrosis. J Pediatr Urol
aAntero-posterior
renal pelvic diameter (AP RPD) (mm): measured on transverse image at the
6:212–231, 2010.
maximal diameter of infrarenal pelvis.
   bCalyceal dilation: yes or no.
cParenchymal thickness: normal or abnormal (subjective assessment).
dParenchymal appearance: normal/abnormal (evaluate echogenicity, corticomedullary differ-
entiation and for cortical cysts).
vesicoureteral reflux (VUR) or obstruction. Prenatal ultrasound eUreter:normal or abnormal (dilation = abnormal; however, transient visualisation is consid-
follow-up should be performed to ensure that the dilation does ered normal postnatally).
not increase with gestation. Postnatal ultrasound is advocated 6 fBladder: normal or abnormal (evaluate wall thickness, presence of ureterocele, dilated pos-
weeks after birth unless symptomatology suggestive of urinary terior urethra).
tract involvement occurs (e.g., fever). Adapted from Nguyen HT, Benson CB, Stein DR, et  al. Multidisciplinary consensus on the
The prevalence of mild pyelectasis is somewhat greater in classification of prenatal and postnatal urinary tract dilation (UTD classification system). J
Pediatr Urol 10(6):982–998, 2014.
fetuses with trisomy 21 than in euploid fetuses, and this finding
has been used for screening. In the presence of midtrimester pyel-   
ectasis, other soft markers for fetal aneuploidy should be sought
and the risk for aneuploidy, especially trisomy 21, calculated.10
Male fetuses tend to have a larger renal pelvis. Therefore the likeli- Ureterocele
hood ratio for aneuploidy associated with this finding is smaller in A ureterocele is a cystic dilation of the distal intravesical ureter.
male than in female fetuses.  Most ureteroceles arise from an abnormal location of the ureteral
meatus in the bladder and are therefore termed ectopic. Uretero-
Ureteropelvic Junction Stenosis celes are often associated with a double ureter and kidney (duplex
Ureteric obstruction at the ureteropelvic junction (UPJ) is the system), the ureterocele at the lower ureteric orifice draining the
most common lesion of the fetal urinary tract, occurring in 1 in upper pole of the duplex kidney. The lesion may be associated
2000 newborns. The cause can be intrinsic (e.g., abnormal muscle with obstruction accounting for the dilation of the correspond-
arrangement at the UPJ, anomalous collagen collar or urothelial ing ureter or renal pelvis. On ultrasound, the ureterocele is visible
fold) or extrinsic (e.g., compression of the ureter by a crossing as a ‘bubble’ in the fetal bladder (Fig. 33.11). A large ureterocele
vessel). UPJ stenosis can be bilateral in approximately 30% of may obstruct the bladder neck, the disease then resembling an
cases. The typical ultrasound presentation is severe hydronephrosis infravesical obstruction (see under lower urinary tract obstruction
without ureteral dilation and with a normal bladder (Fig. 33.8). A (LUTO)). In these cases, successful prenatal drainage has been
severely dilated renal pelvis can rupture and evolve to a perineph- described.11 
ric urinoma (Fig. 33.9). 
Obstruction of the Bladder Outlet (Lower Urinary Tract
Obstruction at the Vesicoureteral Junction (VUJ) (Vesicoure- Obstruction)
teral Junction Stenosis) and Megaureter Obstruction of the bladder outlet usually occurs in the urethra and
Obstruction at the VUJ can be uni- or bilateral. On prenatal can lead to bladder dilation (megacystis or megabladder) with muscle
ultrasound, the ureter (hydroureter, Fig. 33.10A) and renal hypertrophy and secondary hydroureteronephrosis (Fig. 33.12). The
pelvicalyceal system are dilated. The cause can be an ana- kidneys may ultimately become dysplastic, resulting in a variable asso-
tomical abnormality of the vesicoureteral junction (stricture ciation with chronic renal failure. Longstanding oligo- or anhydram-
or valve), or the obstruction can be functional. A primary nios will ultimately result in lethal fetal pulmonary hypoplasia.
megaureter (Fig. 33.10B) is most frequently the result of an Sonographically, a persistently dilated bladder is visualised
aperistaltic distal ureteral segment, but megaureters can also between the two umbilical arteries using colour-flow Dop-
be seen in high grade VUR or in cases of ectopic insertion in pler. The dilation is usually round in shape but can be larger
the bladder.  above the umbilical vessels, evoking the shape of a cork or
 CHAPTER 33  Kidney and Urinary Tract Disorders 357

Prenatal presentation

16–27 wk ≥28 wk 16–27 wk ≥28 wk

AP RPD AP RPD AP RPD AP RPD
4 to <7 mm 7 to <10 mm ≥7 mm ≥10 mm

Central or no Peripheral
calyceal dilationa calyceal dilationa

Parenchymal Parenchymal
thickness normal thickness abnormal

Parenchymal Parenchymal
appearance normal appearance abnormal

Ureters Ureters
normal abnormal

Bladder Bladder
normal abnormal

No unexplained Unexplained
oligohydramnios oligohydramniosb

UTD class A1: UTD class A2–3:

low risk increased risk

aCentral and peripheral calyceal dilation may be difficult to evaluate early in gestation.
bOligohydramnios is suspected to result from a GU cause.

• Fig. 33.7  Risk stratification based on UTD Classification System by Nguyen and coworkers.8 AP,
anterio-posterior; GU, genitourinary; RPD, renal pelvic diameter; UTD, urinary tract dilatation. (Nguyen HT,
Benson CB, Stein DR, et al. Multidisciplinary consensus on the classification of prenatal and postnatal
urinary tract dilation (UTD classification system). J Pediatr Urol 10(6):982–998, 2014.)

champagne bottle. The image of a dilated proximal urethra (key-

TABLE Level of Dilation, Sonographic Characteristics
33.3
hole) is suggestive of posterior urethral valves (PUVs) in a male fetus
and Possible Underlying Causes (Fig. 33.12C). PUVs are the most common cause of bladder
Most Distal obstruction, occurring exclusively in male fetuses. The ‘valves’
Level of Ultrasound Possible Underlying consist of folds of mucosa along the posterior wall of the urethra,
Uropathy Finding Causes most often extending from the veru montanum and resulting in a
very narrow urethral lumen. Urethral atresia can be seen in both
High Pyelectasis PUJ stenosis, polyuria, duplication male and female fetuses. This condition is less common than PUV
Hydronephrosis
and most frequently results in fetal lethality with anuria in the first
Mid Hydroureter VUJ stenosis, VUR, congenital mega- and early second trimesters. 
ureter, ureterocele
Low Megacystis Urethral atresia, PUV, obstructive
Vesicoureteral Reflux
ureterocele, polyuria, VUR, complex Vesicoureteral reflux is a common disorder that is associated with
cloacal malformations dilation of the ureters and calyceal collecting systems and is usu-
ally confirmed postnatally (Fig. 33.13). Direct imaging of the
PUJ, Pyeloureteral junction; PUV, posterior urethral valves; VUJ, vesicourethral junction; VUR, reflux is sometimes possible during real-time two-dimensional
vesicoureteral reflux. ultrasonography. In the most severe cases, the bladder and ureters
  
358 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A B
• Fig. 33.8  Severe hydronephrosis in uni- and bilateral ureteropelvic junction stenosis.
Voluson
E8
Urinoma
Kidney
• Fig. 33.9 Severe ureteropelvic junction stenosis with leakage of urine
into the retroperitoneal space: perirenal urinoma. Arrow indicates anteriorly
displaced, hydronephrotic kidney.
appear dilated because of a functional increase of the urinary out-
flow. The potential benefit of prenatal diagnosis of such cases is
to avoid postnatal urinary infection and subsequent kidney dam-
age. Postnatal assessment is essential, and postnatal surgery may be
required in the severe forms of VUR.  parenchyma. Amniotic fluid volume and associated anomalies
Complex Cloacal Anomalies
These anomalies result from the failure of the urogenital sinus
to divide properly. Therefore fistulae form in vesicovaginal or
urethrovaginal locations in females or urethrorectal locations in
males. The association with anorectal atresia is frequent.
Sonographically, the diagnosis can be quite difficult, and sono-
graphic signs may only appear late in pregnancy. In case of a uro-
enteric fistula, there may be bowel dilation containing echogenic
material. Direct visualisation of a duplicated and enlarged vagina
is suggestive for a cloacal malformation (Fig. 33.14). Although the
anal sphincter can be visualised on ultrasound, this does not rule
out anal atresia.  cloacal plate anomalies)
Voluson
E8
Microcolon-Megacystis-Intestinal Hypoperistalsis Syndrome
(MMIHS)
This syndrome is caused by an anomaly in the acetylcholine recep-
tor secondary to a variant of the ACTG2 gene. The disorder is
inherited as an autosomal dominant disorder but in many cases
occurs as a de novo mutation. It should be suspected when non-
obstructive megacystis is visualised in combination with ileal or
large bowel dilation and normal amniotic fluid. The condition has
a poor prognosis in the postnatal period because of the poor intes-
tinal function. 
Prenatal Management of Fetal Obstructive
Uropathies
In first trimester megacystis, further detailed fetal anatomy scan-
ning and karyotyping are warranted because the risk for an under-
lying chromosomal syndrome is increased. With a longitudinal
bladder diameter of 7 to 15 mm, there is a risk for about 25%
of a chromosomal defect. In the chromosomally normal group,
there is spontaneous resolution of the megacystis in about 90%
of cases. If the bladder diameter is greater than 15 mm, the risk
for chromosomal defects is about 10%, and in this chromosom-
ally normal group, the condition is almost invariably associated
with progressive obstructive uropathy (Table 33.4).12 If the fetal
bladder is not visualised during the first trimester, renal agenesis
should be actively excluded. This may require repeated (transvagi-
nal) scanning.
When a fetal obstructive uropathy is suspected during the
second trimester, a detailed assessment of the genitourinary tract
should be performed, including bladder size, renal size, shape and
should be evaluated. If there is severe oligohydramnios limiting
transabdominal ultrasound imaging, transvaginal ultrasound,
magnetic resonance imaging (MRI) and amnioinfusion can be
used to better visualise the fetus and urinary tract. When the diag-
nosis of obstructive uropathy is clear and associated malforma-
tions have been excluded, the prognosis should be established.
Factors associated with a poor prognosis include:
• Diagnosis before 24 weeks
• Severe oligohydramnios
• Renal dysplasia (hyperechogenic parenchyma, cortical cysts)
• Associated structural or chromosomal malformations
• Female gender (indicating urethral atresia or more complex
 
 CHAPTER 33  Kidney and Urinary Tract Disorders 359

A B
• Fig. 33.10  Ultrasound appearance of hydroureter (A) and megaureter (B).

Bladder
Bladder

A B
• Fig. 33.11  Typical ultrasound appearance of a ureterocele: a cystic structure (star) in the bladder, which
is the dilated, intravesical part of the ureter (arrow).

Bladder

A B C

D
• Fig. 33.12  Typical ultrasound appearance of severe lower urinary tract obstruction: megacystis (star) (A),
bilateral hydronephrosis and signs of renal dysplasia (arrows) (B), dilated proximal urethra or ‘keyhole’ sign
(C). Signs of bladder wall hypertrophy and trabeculation are shown in D (arrows).
360 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

B C D E
• Fig. 33.13  Ultrasound appearance of severe vesicoureteral reflux (VUR): a variable degree of hydrone-
phrosis with increase of hydronephrosis after voiding (A), hydroureter (B) and a persistent full bladder (C).
Postnatal confirmation of severe VUR on RX cystography (D and E).

Evaluation of the Fetus With an Obstructive Uropathy
Genetic Testing for Obstructive Uropathies
Overall, the incidence of fetal chromosomal anomalies is as
high as 10% to 15% in fetal renal defects, with the combina-
tion of renal and extrarenal malformations carrying the highest
risk.13,14 In isolated uropathies, the risk is lower, but most cli-
nicians offer invasive genetic testing including karyotyping or
preferably microarrays.6 No studies have specifically explored
the added value of microarrays compared with conventional
karyotyping in cases with LUTO. However, because chromo-
somal abnormalities are frequent in LUTO cases and genomic
imbalances are frequent in children with chronic kidney dis-
ease, it seems appropriate to screen fetuses with the highest
available scrutiny before invasive maternal procedures are con-
sidered.13  The presence of hyperechogenic renal parenchyma, thin-
Prediction of Fetal Renal Function
After a diagnosis of a uropathy is confirmed by ultrasound,
an attempt should be made to access the short- and long-term
outcomes. In unilateral disease, renal function is generally
preserved, and a conservative approach is warranted during
pregnancy, provided that the contralateral kidney is normal.
Postnatal management is dependent on the remaining func-
tion of the diseased kidney. In bilateral uropathies, the progno-
sis and management depend on the predicted damage to renal
function, which is usually predicted based on detailed renal
imaging supplemented by sampling of fetal urine, serum or
renal tissue, when imaging alone is uncertain. In some cases,
such as LUTO, the ultrasound appearance of the fetal kid-
neys and the urinary biochemistry are not strongly correlated,
so both ultrasound and biochemistry need to be taken into
account.15
Ultrasonography.  Ultrasonography is a good predictor of
renal function and includes evaluation of amniotic fluid volume
and renal parenchyma. In the second and third trimesters, the
amniotic fluid volume, measured using the amniotic fluid index
(AFI) or deepest vertical pocket (DVP), is an indirect reflection
of urinary output.16 Progressive development of severe oligohy-
dramnios in a fetus with a uropathy is suggestive of terminal
renal failure.
ning of the cortex, loss of corticomedullary differentiation
and cortical cysts are signs of severe renal damage.17 The
worst outcomes, defined as ‘intrauterine fetal renal failure’,
are found when there is no bladder refill for 2 days after
vesicocentesis.18 
Magnetic Resonance Imaging.  In cases of inconclusive
ultrasound diagnosis, MRI can be considered as a comple-
mentary diagnostic method, especially with oligohydam-
nios and in fetuses with genitourinary pathology involving
the pelvic and perineum region.19 Although MRI has been
attempted to predict long-term renal function, this approach

A
B tion of glomerular rather than tubular function.21 The most
• Fig. 33.14  A and B, Cloacal malformations. Dilated uterus with uterine
septum (large arrow), urinary ascites (stars) and hydronephrosis (arrow) in
a fetus with a persistent cloaca. (Courtesy of Luc De Catte.)
has been not sufficiently validated to contribute significantly
to management.  tational ages.
Analysis of Fetal Urine.  In a fetus, urine production is exclu-
sively reflective of renal function because the mother supplies
balanced nutrients and ensures fetal homeostasis without a contri-
bution from the fetal kidneys. Accordingly, because the composi-
tion of fetal urine depends only on fetal renal function (filtration,
excretion, reabsorption), fetal urinalysis can assess the ability of
the renal tubules to reabsorb a variety of components (sodium,
calcium, phosphorus, B2 microglobulin, glucose).
Clinically, the use of fetal urinalysis to predict renal func-
tion is a subject of controversy in fetal medicine. A systematic
review concluded that studies are extremely heterogenous and
globally of poor quality.20 From this systematic review, calcium
CHAPTER 33  Kidney and Urinary Tract Disorders 361
TABLE 
33.4 Outcome of First Trimester Megacystis
LONGITUDINAL BLADDER
LENGTH (MM)
7–15 >15
Total 110 35
Abnormal karyotype 26 4
Normal karyotype with follow up 79 30
• Spontaneous resolution 71 0
• LUTO 8 30
LUTO, Lower urinary tract obstruction.
Adapted from Liao AW, Sebire NJ, Nicolaides KH, et al. Megacystis at 10-14 weeks of ges-
tation: chromosomal defects and outcome according to bladder length. Ultrasound Obstet
Gynecol 21(4):338–341, 2003.
above the 95th percentile for gestational age and sodium above
the 95th percentile for gestational age appear most predictive
of poor renal outcome. The advantage of serial urinalysis as
opposed to evaluation at a single time point also remains a
matter of debate. Several authors have suggested that serial
sampling increased the predictive potential of urinary analytes,
but this observation has not been confirmed by others.21-23
Ruano and associates suggest repeating urinary biochemistry
48 hours after the first examination if the first is abnormal and
there is no ultrasound evidence of severe renal dysplasia. In
case of improvement, the renal prognosis is better, and prenatal
therapy can be considered.24 
Fetal Serum.  In contrast to the data of fetal urine, most
compounds in fetal blood do not reflect fetal renal function
because they cross the placenta and are cleared by the mater-
nal kidney. Serum microglobulins, however, do not cross the
placenta because of their high molecular weight (>40,000
Da), are filtered by the glomerulus and can provide an estima-
studied are β2 and A1 microglobulin. Advantages of serum
β2 microglobulin include levels that do not to vary with ges-
tational age, reflect glomerular function and can be sampled
serially, even after shunting. A β2-microglobulin level below
5 mg/dL is suggestive of preserved renal function. The dis-
advantage is the need for fetal blood sampling, carrying a
slightly higher risk than vesicocentesis, especially at early ges-
 
Fetal Renal Biopsy.  Few studies have used fetal renal biopsy
to predict renal function. Bunduki and associates assessed the
feasibility of renal biopsy in detecting renal dysplasia. They
performed ultrasound-guided renal biopsy, as well as urine
sampling, on 10 fetuses with severe urinary tract obstruction.
The sampling was successful in only 50% of the cases and was
clinically helpful in only one third.25 Although this approach
may have some value in predicting renal histology, it needs to
be remembered that a focal needle aspiration is not represen-
tative of the whole kidney parenchyma and can be mislead-
ing because renal dysplasia is patchily distributed in the renal
parenchyma. 
362 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Colour version available online

Colour version available online

d
b
c a

d b

A B
• Fig. 33.15  Endoscopic visualisation of the fetal bladder during fetoscopy. A, Anatomical landmarks: a,
veru montanum; b, plicae colliculi; c, urethral opening; and d, urethral valves. B, Laser fulguration of the
urethral valves. (Reproduced with permission from Martinez JM, Masoller N, Gratacos E, et al. Laser abla-
tion of posterior urethral valves by fetal cystoscopy. Fetal Diagn Ther 37:267–273, 2015.)

Management of Fetal Uropathies
After full assessment of the fetus, a plan of management should be
made based on the prognosis and the counselling of the parents by
a multidisciplinary team, allowing them to make informed deci-
sions. The options include termination of pregnancy, conservative
management with postnatal evaluation and treatment, or prenatal
treatment.
Termination of Pregnancy
In the most severe cases (multiple congenital anomalies, termi-
nal renal failure), the prognosis is unequivocally poor, and many
couples elect to terminate the pregnancy if legally possible. Post-
mortem testing (additional genetic testing including whole-exome
sequencing for identification of single-gene disorders, necropsy or
virtual necropsy) should be arranged and medical and psychoso-
cial follow-up offered to the parents.  (Fig. 33.15). In cases in which it is technically not feasible, vesi-
Conservative Management
Conservative management is often possible and requires follow-
up scans through the third trimester to reassess the situation and
determine postnatal care requirements.
In cases potentially requiring postnatal follow up and sur-
gery, the paediatric subspecialists (nephrologist, urologist) are
best involved in the prenatal counselling of the couples. In
many uropathies, no specific therapy is needed in the neonatal
period, allowing the mother to deliver in the maternity hospi-
tal of her choice. In situations requiring urgent surgical inter-
vention during the immediate neonatal period, the parents are
encouraged to give birth in a centre providing specialised pae-
diatric care.
Some parents also opt for conservative management in cases
with a lethal postnatal prognosis. In these cases, the parents need
to deliver in specialised units as the neonate will require adapted
palliative assistance.  collateral damage at the level of the bladder neck and adjacent
Fetal Therapy in Obstructive Uropathy
The rationale for in utero therapy is that restoration of the amni-
otic fluid volume prevents lung hypoplasia, and decompression
of the obstruction may relieve the pressure on the fetal kidneys
before irreversible renal damage occurs. The value of this approach
has been demonstrated in numerous animal models.26 The most
studied indication is LUTO caused by PUVs, but obstructive ure-
teroceles and occasional other malformations have been the sub-
ject of successful prenatal therapy reports. From a technical point
of view, restoring normal urinary outflow can be achieved by dif-
ferent approaches: 
Fetal Cystoscopy for Lower Urinary Tract Obstruction.  Direct
destruction of the urethral valves by laser or electrocoagulation
offers the advantage of restoring the urine flow through the ure-
thra and therefore the cycle of filling and emptying the bladder
in a single procedure. Several centres have reported successful
cases using fetal cystoscopy, but most studies do not include
long-term follow up.27 Identification of the vesical landmarks is
usually feasible, but successful treatment is not always possible
coamniotic shunting (VAS) is often offered. In a recent multi-
centre series, Martinez and coworkers reported on the results of
the centres in Barcelona and Leuven.28 In 20 cases operated at a
mean gestational age of 18.1 weeks (range, 15.0–25.6), and with
a median operation time of 24 min (range, 15–40), there were 9
(45%) terminations of pregnancy (6 persistent oligohydramnios
or evolution to oligohydramnios in the third trimester, 1 preterm
premature rupture of membranes (PPROM), 1 failed surgery, 1
aneuploidy), and 11 women (55%) delivered a live-born baby
at a mean gestational age of 37.3 (29.1–40.2) weeks. No infants
developed pulmonary hypoplasia, and all were alive at 15 to 110
months. Eight (40% of all fetuses, 72.7% of liveborn neonates)
had normal renal function, and 3 (27.3%) had renal failure
awaiting renal transplantation. Counselling for these cases should
include discussion that there is still a significant risk for progres-
sion to renal failure pre- or postnatally. Additionally, the direct
fulguration of the valves involves the risk for creating important
fetal anatomical structures. 
Vesicoamniotic Shunting (VAS).  The first reports on the use of
a shunt from the fetal bladder into the amniotic cavity to bypass the
obstruction are more than 30 years old.29 Technically, a double pig-
tail shunt is introduced percutaneously under ultrasound guidance
 CHAPTER 33  Kidney and Urinary Tract Disorders 363

Fetal abdominal wall

Amniotic fluid
Amniotic sac
Uterine wall
Maternal Bladder
abdominal wall

Shunt

B
• Fig. 33.16  A, Schematic representation of a vesicoamniotic shunt inserted in the bladder of the fetus
with lower urinary tract obstruction. (Reproduced with permission from the Children’s Hospital of Phila-
delphia. http://www.chop.edu/conditions-diseases/lower-urinary-tract-obstruction-luto.)  B, i, Schematic
of percutaneous vesicoamniotic shunting. ii, Postinsertion ultrasonography demonstrating correct place-
ment of vesicoamniotic pigtail catheter with the proximal end in the bladder and the distal end in the
amniotic cavity (arrows). (Reproduced with permission from Kilby MD, Morris RK. Fetal therapy for the
treatment of congenital bladder neck obstruction. Nat Rev Urol 11(7):412–419, 2014.)

to relieve the urinary obstruction by providing bladder drainage and
restoring amniotic fluid volume (Fig. 33.16).30 The use of VAS remains
controversial despite a large number of observational trials and one
attempted randomised controlled trial (the Percutaneous shunting
in Lower Urinary Tract Obstruction [PLUTO] trial)31 because case
selection is difficult, complications frequent and the effect on long-
term renal function uncertain.
A retrospective review from the University of California, San
Francisco, of fetuses with PUV that underwent fetal therapy
(mainly VAS) with favourable urine analysis results confirmed a
high fetal mortality rate of 42%. Chronic renal disease was diag-
nosed in 62% of the survivors, 25% of whom underwent renal
transplantation. This study, like that of Biard and associates,32 sug-
gests that fetal therapy does not cure fetuses with PUV but may
improve bladder function and decrease the morbidity of incon-
tinence and recurrent infections. Unfortunately, the PLUTO
Randomized Controlled Trial (RCT) initiated by the university
of Birmingham was stopped prematurely because of difficulty in
recruitment.31
A recent updated systematic review and meta-analysis on the
use of VAS in LUTO reported on 112 fetuses treated with VAS and
134 control participants.33 They found substantial heterogeneity
in the included studies. Perinatal survival was improved (odds
ratio (OR), 2.54; 95% confidence interval (CI), 1.14–5.67), but
there was no difference in survival at 1 or 2 years. There was no
difference in postnatal renal function between fetuses that under-
went intervention and those that did not (OR, 2.09; 95% CI,
0.74–5.94). Based on these observations, the authors proposed
a standardised grading system for LUTO that could help in the
selection of appropriate patients for antenatal therapy (Table
33.5). In addition, a standardised classification of patients based
on severity would facilitate comparison of treatments both retro-
spectively and prospectively.
This classification is hampered by the lack of uniform criteria
for renal dysplasia and normal or abnormal urinary biochemistry
and therefore needs further refinement and prospective validation.
Shunt complications include dislocation, blockage, fetal infec-
tion, fetal trauma, PPROM and preterm labour and occur in up to
40% of cases (Fig. 33.17). Also, it is important to recover the shunts
at delivery because they may remain in the uterine cavity and act as
a contraceptive device.34 
Vesicoamniotic Shunting Versus Cystoscopy.  No randomised
comparison of techniques is available. Cohort and observational
studies suggest that both fetal cystoscopy and VAS improve the
364 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
33.5 Proposed Staging for Lower Urinary Tract Obstruction According to Severity After 18 Weeks of Gestation
Stage II (Severe, Preserved Renal Stage III (Severe, Abnormal Fetal Renal
Stage I (Mild) Function) Function)
Amniotic fluid volume Normal Oligohydramnios or anhydramnios Oligohydramnios but usually anhydramnios
Renal cortical cysts Absent Absent Can be present
Renal dysplasia Absent Absent Can be present
Fetal urinary biochemistry Favourable Favourable within three consecutive Not favourable after three consecutive
evaluations evaluations
Fetal intervention Not indicated Potential prevention of pulmonary hypopla- Potential prevention of pulmonary hypoplasia but
sia and severe renal impairment not postnatal renal function

Adapted from Ruano R, Sananes N, Belfort MA, et al. Fetal lower urinary tract obstruction: proposal for standardised multidisciplinary prenatal management based on disease severity. Ultrasound Obstet
Gynecol 48(4):476–482, 2016.
  

Male fetus with megacystis

Exclude associated anomalies

Exclude renal dysplasia
Perform genetic testing (array CGH)
Perform vesicocentesis (consider cordocentesis)
for biochemical assessment

• Fig. 33.17  Latrogenic gastroschisis after delivery in a fetus with lower uri-
nary tract obstruction and treated with vesicoamniotic shunting. One part
of the shunt is wrapped around the thigh of the newborn (small arrow).
The bowels protrude through the abdominal insertion site of the shunt
(large arrow).
survival rate in cases of severe LUTO. However, based on limited
information, there is a suggestion that only fetal cystoscopy may
prevent impairment of renal function in fetuses with posterior
urethral valves.35 Current data support the idea of a randomised
controlled trial to compare the effectiveness of fetal cystoscopy
versus VAS for severe fetal LUTO.  (mainly VUR) but also with extrarenal malformations (mainly
Selection of Cases for Prenatal Therapy.  Based on the above,
we propose the following algorithm when a LUTO is visualised
(Fig. 33.18).  or chromosomal anomalies.37,38
Renal Abnormalities
Anomalies of Number
Renal agenesis
Renal agenesis is the result of either failure of the ureteric bud to
arise or failure of the bud to engage with the renal mesenchyme.
Differentiation from renal aplasia or atrophy cannot be made by
ultrasound imaging. The incidence of unilateral renal agenesis
(URA) is 1 in 2000 births and is more frequent in males and on
the left side.36 Bilateral renal agenesis is less common, with an
incidence of 1 in 4000.
Consider cystoscopy or VAS
• Fig. 33.18  Proposed flow-diagram for selection of fetuses for prenatal
therapy in cases of lower urinary tract obstruction. CGH, Comparative
genomic hybridization; VAS, vesicoamniotic shunting.
Unilateral renal agenesis is associated with anomalies of the
contralateral kidney and urinary tract in one third of patients
genital, gastrointestinal, cardiac and musculoskeletal).36 Both uni-
and bilateral agenesis can be a feature of many genetic syndromes
Compensatory hypertrophy of the solitary kidney as a positive
adaptive response to reduced nephron number can be identified
in utero in 90% of cases.39 Children with a solitary functioning
kidney have an increased risk for proteinuria, hypertension and
chronic kidney disease, especially in the presence of ipsilateral con-
genital anomalies of the kidney or urinary tract.40-43 Untreated,
bilateral renal agenesis is a lethal condition.
Ultrasound features are an empty lumbar fossa with an elon-
gated adrenal gland (Fig. 33.19). The amount of amniotic fluid os
normal in unilateral agenesis. Bilateral renal agenesis results in the
oligohydramnios sequence (pulmonary hypoplasia, dysmorphic
face and limb deformities).
 CHAPTER 33  Kidney and Urinary Tract Disorders 365

A B C
• Fig. 33.19  Fetus with unilateral renal agenesis (A): absence of the kidney and renal artery can be dem-
onstrated by using (colour) Doppler (B). (C) Elongated adrenals (arrows) on postmortem magnetic reso-
nance image in a fetus with bilateral renal agenesis. (Courtesy of P. Claus.)

A B
• Fig. 33.20  Duplex kidneys: uncomplicated normal variant (A) and complicated by hydronephrosis of
upper (large arrow) and lower (arrow) pole (B).

The recurrence risk to parents of a baby with isolated URA is Supernumerary Kidney
about 1%. If a parent has URA, the risk for renal anomalies in the A very early branching of the ureteric bud before invasion of the
offspring is around 7%.44 A higher sibling recurrence risk has been metanephric blastema can result in an extra kidney with its own
described for bilateral renal agenesis (8%).45  capsule, vascular supply and urinary tract. 

Duplex Kidney Anomalies of Position

A duplex kidney is characterised by the presence of two separate pel-
vicalyceal systems with complete or partial duplication of the ureters
(Fig. 33.20). It is a common renal anomaly occurring with an
incidence of 1 in 125 in the general population. Duplex kid-
neys can be uni- or bilateral and are more frequent in females. If
uncomplicated, this condition remains asymptomatic and is con-
sidered a normal variant. However, duplex kidneys are frequently
complicated by hydronephrosis, recurrent urinary tract infections
and need for surgical treatment. The ureter of the upper pole
ends ectopically (more caudally and medially) in the bladder or
truly ectopic (in the vagina, urethra, seminal vesicle or rectum),
which leads to obstructive hydronephrosis and renal dysplasia.
The lower pole ureter drains more laterally into the bladder tri-
gone than usual and this can result in VUR. Duplex kidneys
are frequently associated with a ureterocele, which is the dilated
intravesical part of the (upper pole) ureter. A ureterocele presents
as a cystic structure in the bladder on prenatal ultrasound, and its
detection is strongly associated with a confirmed duplex kidney
postnatally.46  syndromes (e.g., Turner syndrome).48
Ectopic kidneys
Ectopic kidneys occur in about 1 in 1000 pregnancies. They
are usually smaller and may be malrotated. The pelvic loca-
tion is the most common (Fig. 33.21A), but horseshoe kidneys,
crossed fused ectopia and even intrathoracic kidneys have been
described.
Horseshoe kidney
Horseshoe kidney is the most common type of fusion anom-
aly with an incidence of 0.15% in the general population (Fig.
33.21B).47 Most horseshoe kidneys result from fusion of the lower
poles and are located around the origin of the inferior mesenteric
artery. Half of patients with a horseshoe kidney have renal compli-
cations or associated extrarenal malformations. Horseshoe kidney
can be part of complex multiple malformation syndromes (e.g.,
VACTERL (vertebral anomalies, anorectal malformations, cardio-
vascular anomalies, tracheoesophageal fistula, esophageal atresia,
renal (kidney) or radial anomalies, and limb defects)) and genetic
 
366 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Voluson
E8

1
A B C
• Fig. 33.21  Ectopic kidneys: pelvic kidney (A), horseshoe kidney (B) and crossed fused ectopy; large
arrow points to the fusion of both kidneys (C).

A B
• Fig. 33.22  Echogenic kidneys.

Crossed fused ectopy
In crossed fused ectopy, one kidney migrates to the other side
(Fig. 33.21C). The ureter crosses the midline and inserts normally
into the bladder. Most patients with crossed fused ectopy remain
asymptomatic.  hyperechogenic kidneys without a family history or renal cysts can
Abnormalities in Renal Size, Structure and Echogenicity
Abnormal renal size
Small kidneys are defined as a kidney length or volume below
the 5th percentile for body length or weight, respectively. Hypo-
and or dysplastic kidneys can have various aetiologies. The
prognosis depends on the remaining renal function. The kidneys
are enlarged in cystic kidney disease, urinary tract dilation, renal
tumours and overgrowth syndromes as Beckwith-Wiedemann
syndrome.  underlying genetic syndromes, frequently showing an autosomal
Echogenic kidneys
Kidneys appear hyperechogenic if they look brighter
than the liver and spleen beyond 17 weeks of pregnancy
(Fig. 33.22).49,50 Fetal hyperechogenic kidneys have an aetiologic
diversity with each condition having a variable outcome. They
can be part of a systemic disease (aneuploidy, infection, meta-
bolic diseases or genetic syndromes), or they can be a manifes-
tation of intrinsic renal disease (polycystic kidney disease, renal
dysplasia, obstructive uropathy, nephrotic syndrome, renal vein
thrombosis). In some cases, increased renal echogenicity repre-
sents a normal variant.
The diagnosis of the underlying aetiology and the counseling of
the parents on the long-term prognosis in cases of bilateral, isolated
be challenging. Isolated hyperechogenic kidneys are most frequently
associated with polycystic kidney disease (both autosomal reces-
sive and dominant) and hepatocyte nuclear factor -1b (HNF1b)
mutations.51,52 In general, fetuses with very large kidneys or severe
oligohydramnios are likely to have a poor outcome. With normal
amniotic fluid volumes and moderately enlarged kidneys (<4 stan-
dard deviations (SDs)), prognosis seems to be better with a high
probability of survival without significant morbidity in infancy.53
The presence of associated malformations, however, is suspicious of
recessive inheritance pattern (e.g., Bardet-Biedl, Meckel-Gruber
or Beemer syndromes). Last, enlarged hyperechogenic or cystic
kidneys can be a manifestation of an underlying metabolic disease
(peroxisomal disorders, mitochondrial fatty acid synthesis defects or
congenital disorders of N-glycosylation) (Fig. 33.23).
Detailed fetal ultrasound examination, fetal karyotyping and
chromosomal microarray, family history and ultrasound exami-
nation of the parents ( and eventually the grandparents) are all
important in the workup of hyperechogenic enlarged kidneys.
Most recently, whole-exome sequencing or exome sequencing
 CHAPTER 33  Kidney and Urinary Tract Disorders 367

A B

C D E
• Fig. 33.23  Cystic kidney disease in a fetus with multiple acetyl-CoA dehydrogenase deficiencies. A, Pre-
natal ultrasound. B, Postmortem magnetic resonance image. C–E, Macro- and microscopic appearance
at fetal autopsy. (B courtesy of P. Claus. C to E courtesy of P. Moerman.)

panels including genetic disorders of the kidney have become
available to further differentiate the aetiology. If parents opt for
termination of pregnancy, histopathologic postmortem examina-
tion is crucial in determining the final diagnosis and recurrence
risk in a subsequent pregnancy. Collecting and storing a fetal
DNA sample will also be valuable as new genetic aetiologies of
renal disorders are rapidly being identified.  have fetal renal findings. Fetuses with ADPKD frequently present
Cystic Kidney Disease
There is a wide variety of renal cystic diseases that can be inherited
or acquired. A detailed ultrasound examination of the kidneys and
the urinary tract, a search for associated extrarenal malformations,
array comparative genomic hybridisation, molecular testing and
insight into the family history are all helpful to come to a diagnosis.  less frequent. Cysts may be visible in the third trimester, but usu-
Hereditary Kidney Disease
Polycystic Kidneys
Autosomal dominant polycystic kidney disease (ADPKD) is
the most common hereditary kidney disease, with an incidence of
1 in 800 live births. Two responsible genes have been identified,
PKD-1 and PKD-2, and the majority of patients (85%) carry a
mutation in PKD-1. De novo mutations are reported in 2% to
5%. The disease is characterised by large inter- and intrafamilial
phenotype variability.54,55 Patients usually remain asymptomatic
until 30 to 40 years of age. The kidneys enlarge progressively and
their surface is distorted by macrocysts. In addition, patients may
have cysts in the liver and pancreas and a variety of extrarenal
complications.
Early-onset ADPKD is described in 2% of cases and may
with moderately enlarged kidneys (+1-2 SD), hyperechoic cortex
and hypoechoic medulla (persisting corticomedullary differentia-
tion)56 (Fig. 33.24). Other patterns (normal kidney appearance,
hyperechoic kidneys with decreased cortico-medullary differentia-
tion (CMD) and even a form mimicking severe autosomal reces-
sive polycystic kidney disease (ARPKD)) are described but they are
ally they do not appear until after birth. Children with early-onset
ADPKD frequently have early manifestations, including hyperten-
sion, albuminuria and chronic kidney disease, although the pro-
gression to renal insufficiency seems to be slow.57,58 The risk for
recurrence with a similar manifestation in a subsequent pregnancy is
high. Preimplantation genetic diagnosis can be offered to ADPKD
patients with known mutations.
Autosomal recessive polycystic kidney disease (ARPKD) is
rarer than ADPKD and occurs with an incidence of 1 in 20,000
368 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A B
• Fig. 33.24  The most typical prenatal presentation of the kidneys in autosomal dominant polycystic kid-
ney disease: moderately enlarged kidneys with increased corticomedullary differentiation.
live births. Causative mutations occur in the PKHD-1 gene, but
there is evidence for locus heterogeneity and phenocopies. Disease
expression may vary widely within affected families.55 The disease
is characterised by cystic dilation of the tubules, predominantly
in the medulla. The outer cortex is spared. Additionally, patients
have biliary dysgenesis and hepatic fibrosis. ARPKD is typically
an infantile disease, identified late in gestation or at birth. Ultra-
sound scans can be normal up to 20 weeks of pregnancy. Kidneys
will be markedly enlarged (+4–15 SD) and hyperechoic with-
out (or with reversed) corticomedullary differentiation and with
a hypoechoic outer cortical rim (Fig. 33.25).59 In the perinatal
form, oligohydramnios as a consequence of renal failure results
in lethal pulmonary hypoplasia. Children with the infantile and
juvenile types develop chronic renal failure (with need for trans-
plantation in their teens), hepatic fibrosis and portal hypertension.
The recurrence rate is 25%, and if the mutation is known, prenatal
diagnosis can be offered.  occipital encephalocele (84%) and postaxial polydactyly (87%)
Renal Cysts and Maturity-Onset Diabetes of the Young Type 5
Deletions on the long arm of chromosome 17 (17q12) affect-
ing the hepatocyte nuclear factor 1b (HNF-1b) (TCF-2 gene)
cause a multitude of phenotypes, known as the renal cyst and
diabetes syndrome.60 HNF-1b deletions are an important cause
of bilateral hyperechogenic kidneys in fetuses.52 However, a
wide variety of renal phenotypes, including multicystic dys-
plastic kidney, renal agenesis, hypoplastic or dysplastic kidneys
and renal cysts, either isolated or in combination with extrare-
nal manifestations, have been described61,62 (Fig. 33.26). On
histopathologic examination, there is glomerulocystic kidney
disease.
The disorder is autosomal dominant with approximately 70%
of cases secondary to a de novo event.62,63 There is variable expres-
sivity, even in individuals with the same inherited mutation.
Prenatal identification of the typical 1.4-Mb deletion by chromo-
somal microarray confirms the diagnosis. The renal prognosis will
mainly be determined by ultrasound characteristics. Neurodevel-
opmental disability is seen in approximately 50% of cases.
Nephronophthisis comprises a heterogeneous group of auto-
somal recessive, tubulointerstitial cystic kidney disorders leading
to terminal renal failure in children and young adults. Kidneys are
normal to small and hyperechogenic with loss of corticomedullary
differentiation. About 20 involved genes have been identified. The
disorder is inherited as an autosomal recessive and is believed to
be secondary to alteration of ciliary function. Prenatal diagnosis is
possible by molecular testing.
Medullary cystic kidney disease is a similar tubulointerstitial
nephropathy but with an autosomal dominant inheritance pattern
and later onset of renal failure (fourth decade of life). 
Cystic Kidneys as a Part of Genetic Syndromes
Cystic kidneys can be the hallmark of a number of genetic
syndromes.
Ciliopathies. Ciliopathies comprise a group of disorders associ-
ated with genetic mutations encoding defective proteins, which
result in either abnormal formation or function of cellular cilia.
Meckel-Gruber syndrome is a rare autosomal recessive
lethal ciliopathy characterised by cystic kidney dysplasia (99%),
(Fig. 33.27). Other structural anomalies include oral cleft-
ing; genital anomalies; central nervous system malformations,
including Dandy-Walker and Arnold-Chiari malformation and
liver fibrosis.
Severe oligohydramnios leading to lung hypoplasia occurs in
the second trimester of pregnancy. Mutations in 14 genes have
been described in association with Meckel-Gruber syndrome.
Meckel-Gruber syndrome can be confirmed by molecular test-
ing in about 75% of cases. The aetiology of the other cases is
unknown.
Bardet-Biedl syndrome is an autosomal recessive ciliopathy
characterised by obesity, hypogonadism, mental retardation,
retinal degeneration, polydactyly and renal malformations. On
prenatal ultrasound, enlarged and hyperechogenic kidneys in
association with postaxial polydactyly can be detected. In 80%
of cases, 1 of 19 genes is associated.
Joubert syndrome is characterised by the absence or under-
development of the cerebellar vermis and a malformed brain-
stem. Together, these cause the characteristic appearance of a
molar tooth sign on MRI. Signs and symptoms can vary but
commonly include hypotonia, abnormal breathing, ataxia, dis-
tinctive facial features and intellectual disability. Joubert syn-
drome can be associated with other abnormalities, including
cystic kidney disease, retinal dystrophy, hepatic fibrosis and less
 1

A 2
B

C D E
• Fig. 33.25 Prenatal presentation of autosomal recessive polycystic kidney disease: grossly enlarged
hyperechogenic kidneys without corticomedullary differentiation, empty bladder and oligohydramnios (A
and B). Postmortem macroscopic image (C and D). Postmortem magnetic resonance image (E). (C and
D courtesy of P. Moerman. E courtesy of P. Claus.)

B
• Fig. 33.26 HNF-1β mutations: two fetuses (A and B) with unilateral multicystic kidney disease and con-
tralateral hyperechogenic kidney with multiple cortical cysts.
370 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C
• Fig. 33.27  Cystic kidney dysplasia (A), occipital encephalocele (B and C) (and postaxial polydactyly; not
shown) in a fetus with Meckel-Gruber syndrome. (Courtesy of L. De Catte.)

frequently postaxial polydactyly. More than 30 genes involved

in the formation and function of cilia have been described
as causing Joubert syndrome. Most commonly, Joubert syn-
drome is inherited in an autosomal recessive manner, but rarely
X-linked inheritance has been described. 

Other Inherited Causes of Cystic Kidneys

Other autosomal recessive inherited syndromes with cystic kid-
neys include renal-hepatic-pancreatic dysplasia (Ivemark syn-
drome) (Fig. 33.28), the short rib–polydactyly syndromes and
asphyxiating thoracic dysplasia (Jeune syndrome). On the other
hand, tuberous sclerosis, Von Hippel-Lindau syndrome and the
branchio-oto-renal syndrome have an autosomal dominant inher-
itance pattern. 

Nonhereditary Cystic Kidneys Disease

Multicystic dysplastic kidney (MCKD) is a developmental
anomaly of the kidney in which the normal renal parenchyma
is replaced by multiple, noncommunicating cysts of varying size.
The incidence ranges from 1 in 2400 to 1 in 4300 live births.
There is a male predominance, and the left side is most commonly
affected.
Multicystic kidneys are enlarged and have an irregular shape
(Fig. 33.29). The prenatal detection rate is around 91%.64 Extra-
renal malformations are described in 15%.64,65 Anomalies in the
contralateral kidney are present in one third of cases, mainly VUR
(20%) and UPJ stenosis (4%–5%). Recently, pathogenic copy
number variations were reported even in isolated MCKD.66 Bilat-
eral multicystic kidney disease is more frequently associated with
other congenital anomalies and syndromes.67
Because renal function in the multicystic kidney is minimal
or absent, bilateral MCKD is a lethal condition. In unilateral
MCKD, the prognosis is determined by the contralateral kid-
ney, which is compensatorally enlarged in up to 77% of cases.64
Multicystic kidneys involute completely in 5% before birth, in
49% after 1 year and in 95% at 15 years.68 The baseline kid-
ney size (length <62 mm) is the only significant predictor of
involution.69
Obstructive cystic dysplasia is the most common cause of
nonhereditary fetal renal cystic disease and hyperechogenic kid-
neys. Ultrasound features are those of a lower or upper urinary
tract obstruction in association with a hyperechogenic appearance
of the renal cortex and eventually the presence of cysts variable
in size (Fig. 33.30). Renal size can also vary. Obstructive cystic
dysplasia can be unilateral, bilateral or segmental, but it is usually
a progressive lesion. The amount of amniotic fluid is variable, and
renal function is usually impaired.
• Fig. 33.28  Cystic kidney dysplasia in a fetus with Ivemark II syndrome
(renal-hepatic-pancreatic dysplasia), due to a mutation in the NPHP3-
gene.
Simple renal cysts are rather uncommon in fetuses. They are
usually solitary and localised in the upper pole of a normal kidney
(Fig. 33.31). There is no association with other malformations,
and the prognosis is good. A follow up ultrasound examination
is usually recommended to exclude a more diffuse distribution or
other cystic renal diseases.
Renal tumours are uncommon in fetal life. Mesoblastic
nephroma, the most frequent one, is a benign, mostly large mes-
enchymal tumour, which appears as a solid or partially cystic mass,
commonly associated with polyhydramnios (Fig. 33.32). It has to
be differentiated from Wilms tumours, which are primary renal
cancers having an excellent postnatal prognosis. Nephroblasto-
matosis is characterised by multiple benign nodular lesions and
bilateral involvement.
Renal Anomalies in Association with Polyhydramnios
Congenital nephrotic syndrome of the Finnish type is an
autosomal recessive disorder characterised by massive protein-
uria and nephrotic syndrome from birth. Prenatal diagnosis can

A B
• Fig. 33.29  A–C, Typical ultrasound appearance of multicystic kidney dysplasia.
• Fig. 33.30  Obstructive cystic renal dysplasia (cortical cysts: arrows).
• Fig. 33.31  Simple renal cyst.
be suspected by the association of polyhydramnios, an enlarged
placenta and moderate growth restriction in the fetus. Analysis
of amniotic fluid or maternal serum shows a 10-fold increase in
α-fetoprotein levels, which can, however, also be found in fetuses
with a heterozygous mutation.
Finnish nephrosis is caused by mutations in the NPHS1 or
NPHS2 gene. NPHS1 gene mutations cause all cases of con-
genital nephrotic syndrome of the Finnish type. This form of the
condition is found in people of Finnish ancestry. NPHS1 gene
CHAPTER 33  Kidney and Urinary Tract Disorders 371
C
• Fig. 33.32  Typical ultrasound aspect of a fetal mesoblastic nephroma.
(Courtesy of L. De Catte.)
mutations can cause congenital nephrotic syndrome in non-Finn-
ish individuals, but they are a less common cause than NPHS2
gene mutations, which appear to be the most frequent cause of all
cases. Mutations in other genes cause a small number of cases of
congenital nephrotic syndrome. About 15% to 20% of individu-
als with congenital nephrotic syndrome do not have an identi-
fied mutation in one of the genes associated with this condition.
Prenatal diagnosis is possible in families with an index case and
known mutation.
Bartter syndrome, results from mutations in numerous
genes that affect the function of ion channels and transporters
that normally mediate transepithelial salt reabsorption in the
distal nephron segments, resulting in salt-losing polyuria lead-
ing to polyhydramnios. The antenatal or neonatal form of the
disease is most frequently caused by mutations in SLC12A1, the
sodium-chloride-potassium co-transporter gene, or mutations
in the ROMK gene. The disorder is inherited in an autosomal
recessive pattern. Prenatal genetic diagnosis requires molecular
testing, but based on an elevated amniotic fluid chloride level,
the syndrome can be prenatally suspected.70 Fetal treatment
with indomethacin and if necessary therapeutic amniocentesis
has been described.71 
372 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE helpful in establishing a diagnosis when no bladder is visualised

33.6 Differential Diagnosis of Abnormal Bladder (e.g., bladder extrophy should be suspected when no bladder is
visualised with normal amniotic fluid volume).
Bladder Anomaly Possible Causes The urachus connects the bladder to the anterior abdominal
Enlarged bladder Lower urinary tract obstruction wall in utero. The intrafunicular part of this canal can remain open
Severe VUR in utero and result in a hypoechogenic cystic mass located in the
Prune belly syndrome cord, next to its fetal insertion. Although the cyst itself may disap-
Megacystis microcolon hypoperi- pear in utero, it may result in a vesicocutaneous fistula that should
stalsis syndrome be managed appropriately in the neonatal period. 
Pseudo-enlarged bladder in females
Absent bladder Absent or nonfunctioning kidneys Summary
Severe IUGR
TTTS in multiple pregnancies Renal anomalies are often diagnosed prenatally because they often
Bladder extrophy appear as hypoechogenic fluid collections or affect the amniotic
Bladder rupture fluid volume.
Cloacal exstrophy This chapter has given a complete overview of the prenatal
Abnormal bladder Cloacal malformation sequence diagnosis and management of fetal renal conditions. In addition,
the embryology and pathophysiology of these conditions have
IUGR, Intrauterine growth restriction; TTTS, twin-twin transfusion syndrome; VUR, vesico- been summarised to provide readers with a comprehensive under-
ureteral reflux. standing of the origin of these conditions and their evolution.
   Detailed attention is given to the diagnosis, workup and pre-
natal therapeutic options in obstructive uropathies, especially
LUTO, including the results of the most recent clinical studies in
the field and alternatives to VAS.
Bladder Malformations Finally, the different types of cystic uropathies have been detailed
Bladder malformations are rare and mostly part of complex cloa- using the state-of-the-art classification and numerous pathologic
cal malformation syndromes or part of abdominal wall defect pictures to provide readers with an easy-to-read overview.
syndromes such as bladder extrophy. The main differential
diagnoses of enlarged, absent and abnormal bladder are rep- Access the complete reference list online at ExpertConsult.com.
resented in Table 33.6. The amount of amniotic fluid can be Self-assessment questions available at ExpertConsult.com.
References 18. Ruano R, Safdar A, Braun MC. Defining and
predicting ‘intrauterine fetal renal failure’ in
34. Kamphuis MM, Lim F, Oepkes D, et al. Sec-
ondary infertility as a late complication of
congenital lower urinary tract obstruction. vesico-amniotic shunt therapy. Prenat Diagn.
1. Muller F, Dommergues M, Bussieres L, et al.
Pediatr Nephrol. 2016;31(4):605–612. 2007;27(4):362–364.
Development of human renal function: refer-
19. Alamo L, Laswad T, Gudinchet F, et al. Fetal 35. Ruano R, Sananes N, Favre R, et  al. Fetal
ence intervals for 10 biochemical markers in
MRI as complement to US in the diagnosis and intervention for severe lower urinary tract
fetal urine. Clin Chem. 1996;42(11):1855–
characterization of anomalies of the genito- obstruction: a multicenter case-control study
1860.
urinary tract. Eur J Radiol. 2010;76(2):258– comparing fetal cystoscopy with vesicoam-
2. Rabinowitz R, Peters MT, Vyas S, et al. Mea-
264. niotic shunting. Ultrasound Obstet Gynecol.
surement of fetal urine production in normal
20. Morris RK, Quinlan-Jones E, Khan KS, et al. 2015;45(4):452–458.
pregnancy by real-time ultrasonography. Am J
Systematic review of accuracy of fetal urine 36. Westland R, Schreuder MF, van Wijk JA, et al.
Obstet Gynecol. 1989;161(5):1264–1266.
analysis to predict poor postnatal renal function Unilateral renal agenesis: a systematic review on
3. Lee SM, Park SK, Shim SS, et al. Measurement
in cases of congenital urinary tract obstruction. associated anomalies and renal injury. Nephrol
of fetal urine production by three-dimensional
Prenat Diagn. 2007;27(10):900–911. Dial Transplant. 2013;28(7):1844–1855.
ultrasonography in normal pregnancy. Ultra-
21. Nicolini U, Spelzini F. Invasive assessment 37. Woolf AS, Hillman KA. Unilateral renal agen-
sound Obstet Gynecol. 2007;30(3):281–286.
of fetal renal abnormalities: urinalysis, fetal esis and the congenital solitary functioning
4. Bronshtein M, Yoffe N, Brandes JM. First and
blood sampling and biopsy. Prenat Diagn. kidney: developmental, genetic and clinical
early second-trimester diagnosis of fetal urinary
2001;21(11):964–969. perspectives. BJU Int. 2007;99(1):17–21.
tract anomalies using transvaginal sonography.
22. Muller F, Dommergues M, Dumez Y, et  al. 38. Deshpande C, Hennekam RC. Genetic syn-
Prenat Diagn. 1990;10(10):653–666.
Fetal urinary biochemistry predicts post- dromes and prenatally detected renal anomalies.
5. Sebire NJ, Von Kaisenberg C, Nicolaides
natal renal function in children with bilat- Semin Fetal Neonatal Med. 2008;13(3):171–180.
KH, et  al. Fetal megacystis at 10-14 weeks
eral obstructive uropathies. Obstet Gynecol. 39. Van Vuuren SH, van der Doef R, de Jong TP,
of gestation. Ultrasound Obstet Gynecol.
1993;82(5):813–820. et  al. Compensatory enlargement of a solitary
1996;8(6):387–390.
23. Johnson MP, Corsi P, Evans MI. Sequential functioning kidney during fetal development.
6. Dias T, Sairam S, Kumarasiri S. Ultrasound
urinalysis improves evaluation of fetal renal Ultrasound Obstet Gynecol. 2012;40(6):665–668.
diagnosis of fetal renal abnormalities. Best Pract
function in obstructive uropathy. Am J Obstet 40. Sanna-Cherchi S, Ravani P, Corbani V, et al.
Res Clin Obstet Gynaecol. 2014;28(3):403–415.
Gynecol. 1995;173(1):59–65. Renal outcome in patients with congenital
7. Nguyen HT, Herndon CD, Cooper C, et  al.
24. Ruano R, Sananes N, Belfort MA, et al. Fetal anomalies of the kidney and urinary tract. Kid-
The society of fetal urology consensus state-
lower urinary tract obstruction: proposal for ney Int. 2009;76(5):528–533.
ment on the evaluation and management
standardized multidisciplinary prenatal man- 41. Corbani V, Ghiggeri GM, Sanna-Cherchi S.
of antenatal hydronephrosis. J Pediatr Urol.
agement based on disease severity. Ultrasound Congenital solitary functioning kidneys: which
2010;6:212–231.
Obstet Gynecol. 2016;48(4):476–482. ones warrant follow-up into adult life? Nephrol
8. Nguyen HT, Benson CB, Stein DR, et al. Mul-
25. Bunduki V, Saldanha LB, Zugaib M, et al. Fetal Dial Transplant. 2011;26(5):1458–1460.
tidisciplinary consensus on the classification of
renal biopsies in obstructive uropathy: feasi- 42. Westland R, Schreuder MF, van Wijk JA,
prenatal and postnatal urinary tract dilation
bility and clinical correlations—preliminary et  al. Renal injury in children with a soli-
(UTD classification system). J Pediatr Urol.
results. Prenat Diagn. 1998;18(2):101–109. tary functioning kidney—the KIMONO
2014;10(6):982–998.
26. Kitagawa H, Pringle KC, Nakada K, et  al. study. Nephrol Dial Transplant. 2011;26(5):
9. Lee RS, Cendron M, Nguyen HT, et al. Ante-
Optimal timing of prenatal treatment of 1533–1541.
natal hydronephrosis as a predictor of post-
obstructive uropathy in the fetal lamb. J Pediatr 43. Westland R, van Wijk JA, Schreuder MF.
natal outcome: a meta-analysis. Pediatrics.
Surg. 2003;38(12):1785–1789. The reason why mother nature provided us
2006;118:586–593.
27. Welsh S, Agarwal NM, Fisk NM, et  al. Fetal with two kidneys: the risks of a congenital
10. Sonek J, Croom C. Second trimester ultra-
cystoscopy in the management of fetal obstruc- solitary functioning kidney. Nephrol Dial.
sound markers of fetal aneuploidy. Clin Obstet
tive uropathy: experience in a single European 2012;27(6):2603–2604.
Gynecol. 2014;57(1):159–181.
centre. Prenat Diagn. 2003;23:1033–1041. 44. McPherson E. Renal anomalies in families of
11. Chalouhi GE, Morency AM, Ville Y. Prenatal
28. Martinez JM, Masoller N, Gratacos E, individuals with congenital solitary kidney.
incision of ureterocele causing bladder outlet
et  al. Laser ablation of posterior urethral Genet Med. 2007;9(5):298–302.
obstruction: a multicenter case series. Prenat
valves by fetal cystoscopy. Fetal Diagn Ther. 45. Bankier A, de Campo M, Danks DM. A pedi-
Diagn. 2017;37(10):968–974.
2015;37:267–273. gree study of perinatally lethal renal disease. J
12. Liao AW, Sebire NJ, Nicolaides KH, et  al.
29. Manning FA, Harrison MR, Rodeck C. Med Genet. 1985;22(2):104–111.
Megacystis at 10-14 weeks of gestation: chro-
Catheter shunts for fetal hydronephrosis 46. Whitten SM, McHoney M, Chitty LS, et  al.
mosomal defects and outcome according to
and hydrocephalus. Report of the interna- Accuracy of antenatal fetal ultrasound in the
bladder length. Ultrasound Obstet Gynecol.
tional fetal surgery registry. N Engl J Med. diagnosis of duplex kidneys. Ultrasound Obstet
2003;21(4):338–341.
1986;315(5):336–340. Gynecol. 2003;21(4). 342–246.
13. Verbitsky M, Sanna-Cherchi S, Gharavi AG,
30. Kilby MD, Morris RK. Fetal therapy for the 47. Weizer AZ, Silverstein AD, Auge BK, et  al.
et al. Genomic imbalances in pediatric patients
treatment of congenital bladder neck obstruc- Determining the incidence of horseshoe kid-
with chronic kidney disease. J Clin Invest.
tion. Nat Rev Urol. 2014;11(7):412–419. ney from radiographic data at a single institu-
2015;125(5):2171–2178.
31. Morris RK, Malin GL, Kilby MD, et  al. tion. J Urol. 2003;170(5):1722–1726.
14. Sanna-Cherchi S, Westland R, Gharavi AG,
Percutaneous vesicoamniotic shunting in 48. Je BK, Kim HK, Horn PS. Incidence and
et al. Genetic basis of human congenital anom-
Lower Urinary Tract Obstruction (PLUTO) spectrum of renal complications and extra-
alies of the kidney and the urinary tract. J Clin
Collaborative Group. Percutaneous vesi- renal diseases and syndromes in 380 children
Invest. 2018;128(1):4–15.
coamniotic shunting versus conservative and young adults with horseshoe kidney. Am J
15. Nassr AA, Koh CK, Ruano R, et al. Are ultra-
management for fetal lower urinary tract Roentgenol. 2015;205(6):1306–1314.
sound renal aspects associated with urinary
obstruction (PLUTO): a randomised trial. 49. Estroff JA, Mandell J, Benacerraf BR. Increased
biochemistry in fetuses with lower urinary tract
Lancet. 2013;382(9903):1496–1506. renal parenchymal echogenicity in the fetus:
obstruction? Prenat Diagn. 2016;36(13):1206–
32. Biard JM, Johnson MP, Adzick NS, et al. Long- importance and clinical outcome. Radiology.
1210.
term outcomes in children treated by prenatal 1991;181(1):135–139.
16. Magann EF, Sandlin AT, Ounpraseuth ST.
vesicoamniotic shunting for lower urinary tract 50. Carr MC, Benacerraf BR, Estroff JA, Mandell
Amniotic fluid and the clinical relevance of
obstruction. Obstet Gynecol. 2005;106(3):503– J. Prenatally diagnosed bilateral hyperechoic
the sonographically estimated amniotic fluid
508. kidneys with normal amniotic fluid: postnatal
volume: oligohydramnios. J Ultrasound Med.
33. Nassr AA, Shazly SAM, Ruano R. Effective- outcome. J Urol. 1995;153(2):442–444.
2011;30(11):1573–1585.
ness of vesicoamniotic shunt in fetuses with 51. Chaumoitre K, Brun M, Cassart M, et al. Dif-
17. Morris RK, Malin GL, Kilby MD, et al. Ante-
congenital lower urinary tract obstruction: an ferential diagnosis of fetal hyperechogenic cys-
natal ultrasound to predict postnatal renal
updated systematic review and meta-analysis. tic kidneys unrelated to renal tract anomalies:
function in congenital lower urinary tract
Ultrasound Obstet Gynecol. 2017;49(6):696– a multicenter study. Ultrasound Obstet Gynecol.
obstruction: systematic review of test accuracy.
703. 2006;28(7):911–917.
BJOG. 2009;116(10):1290–1299.

372.e1
372.e2 References
52. Decramer S, Parant O, Kessler S, et al. Anoma-
lies of the TCF2 gene are the main cause of
fetal bilateral hyperechogenic kidneys. J Am
Soc Nephrol. 2007;18(3):923–933.
53. Tsatsaris V, Gagnadoux MF, Dommergues
M, et  al. Prenatal diagnosis of bilateral iso-
lated fetal hyperechogenic kidneys. is it pos-
sible to predict long term outcome? BJOG.
2002;109(12):1388–1393.
54. Torres VE, Harris PC, Pirson Y. Autosomal
dominant polycystic kidney disease. Lancet.
2007;369(9569): 1287–12301.
55. Bergmann C. ARPKD and early manifesta-
tions of ADPKD: the original polycystic kid-
ney disease and phenocopies. Pediatr Nephrol.
2015;30(1):15–30.
56. Brun M, Maugey-Laulom B, Avni EF, et  al.
Prenatal sonographic patterns in autosomal
dominant polycystic kidney disease: a mul-
ticenter study. Ultrasound Obstet Gynecol.
2004;24(1):55–61.
57. Boyer O, Gagnadoux MF, Salomon R, et  al.
Prognosis of autosomal dominant polycystic
kidney disease diagnosed in utero or at birth.
Pediat Nephrol. 2007;22(3):380–388.
58. Selistre L, de Souza V, Dubourg L, et al. Early
renal abnormalities in children with postnatally
diagnosed autosomal dominant polycystic kid-
ney disease. Pediatr Nephrol. 2012;27(9):1589–
1593.
59. Avni FE, Garel L, Cassart M, et  al. Perinatal
assessment of hereditary cystic renal diseases:
the contribution of sonography. Pediatr Radiol.
2006;36(5):405–414.
60. Edghill EL, Bingham C, Hattersley AT, et al.
Mutations in hepatocyte nuclear factor-1beta
and their related phenotypes. J Med Genet.
2006;43(1):84–90.
61. Ulinski T, Lescure S, Beaufils S, et  al. Renal
phenotypes related to hepatocyte nuclear
factor-1beta (TCF2) mutations in a pediatric
cohort. J Am Soc Nephrol. 2006;17(2):497–
503.
62. Heidet L, Decramer S, Pawtowski A, et  al.
Spectrum of HNF1B mutations in a large
cohort of patients who harbor renal diseases.
Clin J Am Soc Nephrol. 2010;5(6):1079–1090.
63. Rasmussen M, Ramsing M, Sunde L, et  al.
A description of a fetal syndrome associated
with HNF1B mutation and a wide intrafa-
milial disease variability. Am J Med Genet.
2013;161a(12):3191–3195.
64. Schreuder MF, Westland R, van Wijk JA.
Unilateral multicystic dysplastic kidney: a
meta-analysis of observational studies on the
incidence, associated urinary tract malforma-
tions and the contralateral kidney. Nephrol
Dial Transplant. 2009;24(6):1810–1818.
65. Damen-Elias HA, Stoutenbeek PH, de Jong
TP, et  al. Concomitant anomalies in 100
children with unilateral multicystic kidney.
Ultrasound Obstet Gynecol. 2005;25(4):384–
388.
66. Xi Q, Zhu X, Wang Y, et  al. Copy number
variations in multicystic dysplastic kidney:
update for prenatal diagnosis and genetic coun-
seling. Prenat Diagn. 2016;36(5):463–468.
67. Winding L, Loane M, Wellesley D, et al. Pre-
natal diagnosis and epidemiology of multicys-
tic kidney dysplasia in Europe. Prenat Diagn.
2014;34(11):1093–1098.
68. Eickmeyer AB, Casanova NF, He C, et al. The
natural history of the multicystic dysplastic
kidney–is limited follow-up warranted? J Pedi-
atr Urol. 2014;10(4):655–661.
69. Rabelo EA, Oliveira EA, Tatsuo ES. Predictive
factors of ultrasonographic involution of pre-
natally detected multicystic dysplastic kidney.
BJU Int. 2005;95(6):868–871.
70. Garnier A, Dreux S, Vargas-Poussou R, et  al.
Bartter syndrome prenatal diagnosis based on
amniotic fluid biochemical analysis. Pediatr
Res. 2010;67(3):300–303.
71. Konrad M, Leonhardt A, Kockerling A,
et  al. Prenatal and postnatal management
of Hyperprostaglandin E syndrome after
genetic diagnosis from amniocytes. Pediatrics.
1999;103:678–683.
34
Diagnosis and Management of Fetal
Skeletal Abnormalities
LYN S. CHITTY, LOUISE C. WILSON AND FREDERICK USHAKOV
KEY POINTS
• Diagnosis of skeletal anomalies is challenging and requires
time and a team approach, including clinical geneticists,
paediatricians and pathologists.
• This chapter deals with the prenatal diagnosis of skeletal
anomalies. It gives aids to diagnosis and categorises condi-
tions by sonographic findings to help sonographers narrow
the differential diagnoses.
• Increasingly, with advances in genomic medicine, the defini-
tive diagnosis can be achieved prenatally after targeted
molecular genetic or metabolic investigations, sometimes by
safe approaches using analysis of cell-free DNA in maternal
blood.
• In the absence of a definitive diagnosis prenatally, expert
postmortem examination, including radiology or genome se-
quencing, should be offered for a diagnosis, which is essential
to define recurrence risks (which can vary from 1% to 50%) and
appropriate prenatal diagnosis in subsequent pregnancies.
• Molecular genetic diagnosis facilitates early prenatal diagno-
sis in subsequent pregnancies, and storage of DNA should be
encouraged, particularly in cases with an unknown diagnosis
as the genetic aetiology for these conditions is increasingly
being defined.
• This is a rapidly moving field, and discussion with geneticists is
helpful to ensure the most up-to-date information is available.
Introduction
Congenital skeletal anomalies are not uncommon, occurring with
an incidence of around 1 in 500. Many are amenable to prenatal
detection using ultrasound. The underlying aetiology is varied and
includes:
Aneuploidy
Genetic syndromes
Skeletal dysplasias
Teratogens
Isolated anomalies secondary to disruption
  
The sonographic detection of a fetus with a skeletal anomaly
can present a challenging diagnostic dilemma. Management
options can be very varied, and diagnosis may require biochemi-
cal, genetic or haematologic investigation. Increasingly more
sophisticated imaging, such as magnetic resonance imaging (MRI)
or computed tomography (CT), may elucidate features more eas-
ily interpreted by postnatal radiologists. Clinical genetic input is
invariably useful, not only because the family history or parental
examination may yield valuable clues to the diagnosis but also
because this is a field that is evolving rapidly. The underlying
genetic aetiology of skeletal dysplasias are increasingly known, and
new, safer, approaches to prenatal diagnosis based on analysis of
cell-free DNA (cfDNA) are being used.
This chapter discusses the normal embryology and sono-
graphic appearances of fetal limb development and go on to
suggest a systematic approach to the diagnosis of fetal skeletal
anomalies, as well as describing some of the more common condi-
tions in greater detail. Generalised skeletal dysplasias are discussed
as well as those groups of conditions associated with more local-
ised limb anomalies, which may or may not be part of a wider
genetic syndrome. Accurate sonographic identification of skeletal
abnormalities becomes increasingly important as more genes for
skeletal conditions are identified, raising the potential for accurate
prenatal diagnosis using molecular methods. 
Terminology
Fetal ultrasound diagnosis relies on the identification and accu-
rate description of sonographic findings. Skeletal anomalies are
associated with a range of genetic syndromes and dysplasias, and
discussion with other specialists (in particular clinical geneticists,
radiologists and orthopaedic surgeons) is necessary to try to define
both the diagnosis and prognosis to inform accurate parental
counselling. To be able to do this efficiently, a good understanding
of terminology is required. Normal bone nomenclature is illus-
trated in Fig. 34.1. The terminology used in describing abnormali-
ties of the limbs is given in Table 34.1. 
Embryology and Sonographic Appearance of
the Normal Fetal Skeleton
In humans, the upper limbs develop a few days in advance of the
lower limbs, with the arm buds appearing at about 5.5 postmen-
strual weeks. The fetal skeleton then forms in two ways, membra-
nous ossification (clavicle and mandible) and intracartilaginous
373
374 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Epiphysis
Diaphysis
• Fig. 34.1  Bone nomenclature.
(endochondral) when ossification occurs by calcium deposition
in preexisting cartilage matrix. Fetal ossification begins in the
clavicle at around 8 weeks’ gestation followed by the mandible,
vertebral bodies and neural arches at around 9 weeks; the frontal
bones at 10 to 11 weeks; and the long bones at around 11 weeks.
Most skeletal structures can be identified sonographically by 14
to 15 weeks. The appearance of the ossification in the fetal skel-
eton has been studied both radiographically and sonographically
using transabdominal ultrasound. However, probably the most
useful indication of which structures should be identified when
scanning in early pregnancy comes from a recent radiologic
study of human fetuses (Fig. 34.2).1 Identification of anomalies
of skeletal development requires detailed scanning and aids such
as charts of normal skeletal size, including length of long bones,
clavicles, mandible, scapulae, chest size, orbital diameters, renal
size and so on.2-5  may wish to avoid the risks associated with invasive prenatal test-
Classification of Skeletal Dysplasias
The genetic and pathological aetiology of skeletal anomalies is
wide, and there have been several classifications used. These have
evolved as understanding of the genetics and pathophysiology
of these rare but complex disorders becomes clearer. Classifica-
tions can be based on clinical or radiological features (or both),6
molecular genetic aetiology or the biological structure and func-
tion of genes and proteins involved (e.g., defects in structural
proteins, metabolic pathways, transcription factors etc.)7 or a
hybrid of both.8 A classification based on sonographic findings
is the most useful classification for the prenatal diagnostician
(Table 34.2), but there can be considerable overlap in condi-
tions, so a table listing the common diagnoses with gene loca-
tion, when known; inheritance; and main sonographic findings
is also given (Table 34.3). 
Clues to the Diagnosis of Skeletal Anomalies
Risk factors for skeletal anomalies include:
  
Family history
Drugs in early pregnancy
Maternal disease
Abnormal findings on routine ultrasound
Family History
Clearly, diagnosis in families in which there has already been an
affected child or when one parent is affected with a dominantly
inherited condition can be more straightforward than interpreta-
tion of findings that arise de novo. For some dominantly inher-
ited conditions, one parent may manifest mildly or subclinically
because of somatic (alteration in the DNA that occurs after con-
ception) mosaicism but be at high risk for a more severely affected
offspring who has inherited the mutation constitutively (nonmo-
saic), examples including osteogenesis imperfecta (OI) and spon-
dyloepiphyseal dysplasia congenita (SEDC).
Knowledge of the sonographic features and natural history of
the condition can aid prenatal diagnosis, but parents do need to
be aware that some conditions (e.g., achondroplasia) may pres-
ent relatively late and not be amenable to sonographic diagno-
sis until well into the second trimester. Others are more variable
(e.g., hypochondroplasia) and are not obvious until after birth or
in early childhood. For these reasons, molecular diagnosis may
be preferable in families in which the gene has been identified
before pregnancy. In the past, this required an invasive test (chori-
onic villus sampling) with its small risk for iatrogenic miscarriage
to obtain fetal genetic material for testing. However, technical
advances have made noninvasive prenatal diagnosis (NIPD) based
on analysis of cell-free fetal DNA in maternal blood possible for
several skeletal dysplasias.9 If the precise mutation is known before
pregnancy, then bespoke NIPD may be possible.10
With rapid advances in molecular genetics, the underlying
genetic aetiology for many of these conditions is known, and it is
imperative that tissue is available from affected pregnancies if par-
ents are subsequently to be given the opportunity of early testing.
Many conditions are heterogeneous (meaning that the causative
mutation(s) may reside in any one of a number of different genes)
so that extensive genetic analysis before pregnancy may be required
before prenatal molecular testing can be offered. Many families
ing. The advent of noninvasive prenatal testing using free fetal
DNA in the maternal plasma means that parents are increasingly
able to get a diagnosis without risk as this technology progresses.
Nonetheless, genetic workup before pregnancy will be required for
this technology as well as for traditional invasive testing. 
Drugs in Early Pregnancy
Although drugs are now extensively tested before release onto the
market, there are still those used regularly that may result in skel-
etal malformations if taken in early pregnancy (Table 34.4). Fur-
thermore, there is good evidence that some recreational drugs, if
used in early pregnancy, can cause skeletal anomalies, a postulated
vascular effect being responsible for some drugs (e.g., cocaine). 
Maternal Disease
The most common maternal conditions that can result in fetal
musculoskeletal anomalies (Table 34.5) include:
  
Diabetes
Myasthenia gravis
Myotonic dystrophy
  
Other conditions such as systemic lupus erythaematosus
(SLE) and hypothyroidism can also cause skeletal changes but
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 375

TABLE
34.1 Terminology Used in Describing Skeletal Abnormalities

Acheiria Absent hand(s)

Acheiropodia Absent hand(s) and feet
Acromelia Shortening of the distal segments of limbs (i.e., hands and feet)
Adactyly Complete absence of fingers, toes or both
Amelia Complete absence of one or more limbs from the shoulder or
pelvic girdle
Apodia Absent foot (feet)
Arthrogryposis Congenital joint contractures
Brachydactyly Short digits
Camptomelia Bent limb
Camptodactyly Bent digit(s)
Clinodactyly Incurved fifth finger
Ectrodactyly Split (cleft) hand(s) or feet, missing central ray(s), lobster claw
deformity
Hemimelia Congenital longitudinal absence or deficiency of a forearm or
lower leg bone
Kyphosis Dorsal convex curvature of the spine
Kyphoscoliosis Combination of lateral and anteroposterior curvature of the spine
Meromelia Partial absence of a limb Transverse Defect extending across the
whole width of the limb
Longitudinal Defects affecting one bone
along an axis
Terminal No bony part distal to the
defect
Intercalary With recognisable parts distal
to the defect
Mesomelia Shortening of the middle segment of a limb (i.e., radius/ulna and
tibia/fibula)
Micromelia Shortening of all long bones
Oligodactyly Absent or partially absent digit(s)
Phocomelia Relatively normal hands or feet are attached to the trunk either
directly or by extremely shortened long bones
Platyspondyly Flattening of the vertebral bodies
Polydactyly Extra fingers or toes Preaxial Extra digit on the radial or tibial
side
Postaxial Extra digit on the ulna of fibular
side
Rhizomelia Shortening of the proximal long bones (i.e., femur and humerus)
Syndactyly Fused digital rays Skin Fused skin only
Osseous Bony fusion
Scoliosis Lateral curvature of the spine
Talipes Club-foot Equinovalgus Foot twisted outwards
Equinovarus Foot twisted inwards
Equinus Extended foot

  
376 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Postnatal
Mandible
Maxilla
Occiput
Clavicles
Scapula
Ribs
Vertebral bodies:
T1–L5
C6–C7, S1–S2
C3–C6,S3
C2, S4–S5
Iliac bones
Ischium
Pubis
Distal phalanges
Metacarpals, metatarsals and phalanges
Middle phalanges
Calcaneum
Talus
Long bone diaphysis
Lower femoral epiphysis
Proximal humeral epiphysis

• Fig. 34.2  Ossification of the fetal skeleton by gestational age. (Adapted from Calder AD, Offiah AC. Foe-
tal radiography for suspected skeletal dysplasia: technique, normal appearances, diagnostic approach.
Pediatr Radiol 45:536–548, 2015.)

TABLE
34.2 Classification of Skeletal Dysplasias According to Major Sonographic Finding

Sonographic finding Condition Other investigations to be considered

SKULL
Hypomineralised Osteogenesis imperfecta types IIA and IIC Parental fracture history for possible somatic mosaicisma
Achondrogenesis type I a

Hypophosphatasia (severe neonatal form) Parental ALP and urinary phosphoethanolaminea

Mild hypomineralisation Achondrogenesis type 2 a

Cleidocranial dysostosis Parental historya

Osteogenesis imperfecta IIB Parental history as above
Cloverleaf Thanatophoric dysplasia type II NIPDa
Occasionally in SRPSs a

Antley-Bixler syndrome a

Craniosynostosis syndromes (Pfeiffer, Crouzon, Saethre- a

Chotzen syndromes)

SPINE
Hypomineralised Achondrogenesis type I *
Disorganised Jarcho-Levin syndrome Consider metabolic screeninga
Spondylocostal dysplasia
Dyssegmental dysplasia
Some chondrodysplasia punctatas
VATER/VACTERL
Face

Frontal bossing Thanatophoric dysplasia NIPD

Achondroplasia
Acromesomelic dysplasia
Depressed nasal bridge Chondrodysplasia punctatas Drug history, karyotype, ARSE deletion screen (CDPX1)a
Warfarin embryopathy Metabolic investigations: very-long-chain fatty acids and ste-
rol profile, CVS for peroxisomal enzyme studies, maternal
history of autoimmune disease
Micrognathia SEDC Karyotypea
Stickler syndrome a

Campomelic dysplasia a

Cleft lip Majewski syndrome a

Oral facial defect IV a

Continued
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 377

TABLE
34.2 Classification of Skeletal Dysplasias According to Major Sonographic Finding—cont’d

LEGS
Isolated straight, short long bones IUGR Fetal and maternal Doppler, maternal Down syn-
Constitutional short stature drome screening biomarkers, obstetric history
Femoral bowing Campomelic dysplasia As abovea
Osteogenesis imperfecta a

Hypophosphatasia a

Talipes Campomelic dysplasia NIPD for sex determinationa

Diastrophic dysplasia a

Stippled epiphyses Rhizomelic chondrodysplasia punctata Drug history, metabolic investigations; very-
Conradi Hunermann syndrome long-chain fatty acids and sterol profile,
X-linked recessive chondrodysplasia punctata CVS peroxisomal enzyme studies,a maternal
Warfarin embryopathy history of autoimmune disease
Maternal SLE

LIMB GIRDLES
Short clavicles Campomelic dysplasia a

Cleidocranial dysostosis a

Small scapula Campomelic dysplasia *

HANDS
Polydactyly Jeune asphyxiating thoracic dystrophy a

Ellis-van Creveld syndrome a

Short rib polydactyly syndromes a

Short fingers or trident hand Achondroplasia NIPDa

Acromesomelic dysplasia NIPDa
Thanatophoric dysplasia

THORAX
Narrow with short ribs SRPSS a

Jeune asphyxiating thoracic dystrophy a

Thanatophoric dysplasia NIPDa

Osteogenesis imperfecta types IIA, C and B a

Campomelic dysplasia a

Achondrogenesis a

Hypochondrogenesis a

Paternal UPD14 a

Beaded ribs Osteogenesis imperfecta type IIA and C a

Polyhydramnios Achondroplasia NIPDa

Thanatophoric dysplasia NIPDa
Paternal UPD14 a

aConsider molecular genetic testing.

ALP, Alkaline phosphatase; CVS, chorionic villus sampling; IUGR, intrauterine growth restriction; NIPD, noninvasive prenatal diagnosis; SEDC, spondyloepiphyseal dysplasia congenita; SLE, systemic lupus
erythaematosus; SRPS, short rib–polydactyly syndrome; VACTERL, vertebral anomalies, anorectal malformations, cardiovascular anomalies, tracheoesophageal fistula, oesophageal atresia, renal (kidney)
or radial anomalies and limb defects; VATER, vertebral anomalies, anorectal malformations, oesophageal atresia, and renal (kidney) or radial anomalies.
  

less commonly. Poorly controlled, insulin-dependent diabetes is
the most common maternal condition that can result in signifi-
cant skeletal anomalies in fetuses, which include developmental
field defects of the spine, vertebral segmentation defects, caudal
regression syndrome and deficiencies of the limbs (particularly
femoral hypoplasia, tibial hemimelia, preaxial hallucal polydac-
tyly) as well as anomalies of other viscera, in particular the heart
and urogenital tract.
In maternal myasthenia gravis, transmission of acetylcholine
receptor antibodies to the fetus can result in generalised arthro-
gryposis and neonatal or infant death.11 In some instances, the
antibodies are specific to the fetal subunit of the acetylcholine
receptor, and the mother may be asymptomatic.
Myotonic dystrophy is an autosomal dominantly inherited
condition which, when transmitted maternally to the fetus, can
result in congenital myotonic dystrophy (CMD). Mothers with
 378
TABLE
34.3 Skeletal Dysplasias: Gene Location, Inheritance and Sonographic Findings
Gestational

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

age at
presentation
Diagnosis Gene or location Genetics (wk) Limbs Thorax Spine Skull Other Features
Short Bowed Fingers Joints Ribs
Achondrogenesis IA/IB TRIP11 (1A); AR 12 +++ Narrow Short, +/- Hypo Hypo Oedema
SLC26A2 (DTDST) beaded
(1B)
Achondrogenesis II COL2A1 AD 12 ++ Narrow Short
Achondroplasia FGFR3 AD >24 + +/- mild Short +/- small Frontal bossing
Acromesomelic dysplasia NPR2, GDF5, AR Around 22 + Short +/- small Frontal bossing
BMPR1B
Beemer-Langer Unknown AR Around 20 + Poly Small Short Cloverleaf
Campomelic dysplasia SOX9 AD 16–20, var Legs Legs Talipes +/- small Micrognathia,
cardiac
defects, sex
reversal in
males
Conradi Hunermann CDP EBP XLD Var + Stippled Stippled
(CDPX2)
Diastrophic dysplasia SLC26A2 (DTDST) AR >16 + Hitchhiker Talipes Micrognathia
thumbs
Ellis-van Creveld syndrome EVC, LBN (EVC2 AR From 16 + Poly Narrow Short Cardiac anomaly
Hypophosphatasia (severe TNSALP AR >12 ++ ++ Hypo
neonatal form)
Jeune asphyxiating thoracic IFT80, DYNCH2H1, AR From 16, vari- + +/- poly Narrow Short CNS anomaly
dystrophy TTC21B, WDR19 able
and > 6 others
Kniest syndrome COL2A1 AD Variable + Mild Short Micrognathia,
depressed
nasal bridge
Majewski syndrome (SRPS2A; NEK1 AR >12 ++ Ovoid tibia Poly Narrow Short ++ Renal, cardiac,
SRTD6) CNS, genital
Osteogenesis COL1A1 COL1A2 AD, gm >12 +++ +++ Narrow Short, Hypo
imperfecta types IIA/C beaded
and IIC
 Osteogenesis COL1A1 COL1A2 AD, gm >16 ++ + (Narrow) (Beaded)
imperfecta type IIB
Osteogenesis COL1A1 COL1A2 AD, gm 20 + Legs
imperfecta type III
Osteogenesis COL1A1 COL1A2 AD, gm >20 Mild,
imperfecta type IV femora
Rhizomelic CDP PEX7, GNPAT, AR 20 Rhizomelic Stippled Nasal hypopla-
(RCDP1,2,3,5) NB AGPS, PEX5 stippled sia, cataracts
Overlapping heterogeneous
peroxisomal disorders,
Zellweger syndrome
Saldino-Noonan syndrome DYNC2H1 AR >12 ++ Poly Narrow Short ++ Renal, cardiac,
(SRPS2B; SRTD3; ATD3) genital
SEDC COL2A1 AD >12 ++ Short Micrognathia
Thanatophoric dysplasia I FGFR3 AD <16 Severe (Mild) Short Small ++ Short ++ Normal Frontal bossing
micro- trident

CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities

melia
Thanatophoric dysplasia II FGFR3 AD <16 Severe (Mild) Short, Small++ Short ++ Cloverleaf Frontal bossing
micro- trident
melia
X-linked recessive ARSE XLR Variable + stippled Short Stippled Stippled larynx
CDP (CDPX1) and trachea

AR, Autosomal recessive; AD, autosomal dominant; CNS, central nervous system; CDP, chondrodysplasia punctate; gm, germline mosaicism; SEDC, spondyloepiphyseal dysplasia congenita; XLR, autosomal recessive; XLD, X-linked dominant.
  

379
380 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
34.4 Skeletal Anomalies Associated With Drug Use in Early Pregnancy
Drug or
substance Skeletal anomalies Other sonographic findings
Warfarin Rhizomelic shortening of limbs, stippled epiphyses, kyphoscoliosis Flat face; depressed nasal bridge; renal, cardiac and CNS
anomalies
Sodium valproate Reduction deformity of arms, polydactyly, oligodactyly, talipes Cardiac and CNS anomalies, spina bifida, orofacial clefting
Methotrexate Mesomelic shortening of long bones, hypomineralised skull, syndactyly, CNS anomalies, including neural tube defects, micrognathia
oligodactyly, talipes
Vitamin A Hypoplasia or aplasia of arm bones or digits CNS and cardiac anomalies, spina bifida, cleft lip and pal-
ate, diaphragmatic hernia, exomphalos
Phenytoin Stippled epiphyses Micrognathia, cleft lip, cardiac anomalies
Alcohol Short long bones, reduction deformity of arm bones, preaxial polydac- IUGR, cardiac anomalies
tyly of hands, oligodactyly, stippled epiphyses
Cocaine Reduction deformities of arms +/- legs, ectrodactyly, hemivertebrae, CNS, cardiac, renal anomalies, anterior abdominal wall
absent ribs defects, bowel atresias

CNS, Central nervous system; IUGR, intrauterine growth restriction.

  

TABLE Sonographic Clues in the Fetal Skeleton to

34.5 Maternal Disease
Abnormal Findings on Routine Ultrasound
Sonographic
The first clue that there may be a skeletal anomaly pres-
ent is often the identification of a short femur at the time of a
findings Maternal condition Maternal diagnosis
scan for another reason, either a routine fetal anomaly scan
Caudal regression Diabetes Glucose tolerance test at around 20 weeks’ gestation or a scan later in pregnancy
Femoral hypoplasia for other indications. Careful examination of the rest of the
Multiple joint Myasthenia gravis Anti–acetylcholine
fetal anatomy can reveal further signs of a skeletal dysplasia
contractures receptor antibodies (Table 34.6). If limb shortening appears to be isolated, then intra-
(arthrogryposis) uterine growth restriction (IUGR) must be considered as a pos-
sible aetiology. In these circumstances, review of maternal serum
Talipes and polyhy- Myotonic dystrophy Examine for signs of screening results for levels of pregnancy-associated plasma pro-
dramnios myotonia, facial tein A (PAPP-A), β-human chorionic gonadotrophin (β-hCG)
appearance, genetic
and maternal serum alphafetoprotein (MSAFP) and assessment
referral
of fetal and maternal Dopplers can be useful diagnostic aids. In
Short limbs, Systemic lupus Autoimmune screen, a 4-year period at University College London Hospital of 130
stippled epiphy- erythaematosus history fetuses referred with ‘abnormal’ femora (short, bowed, hypoplas-
ses, depressed tic), 42 fetuses were thought to have short, straight femurs or
nasal bridge limbs with no other sonographic abnormalities detected. Only 2
   of these had a skeletal dysplasia. Many were normal and had either
had an incorrect assignment of gestational age or familial short
stature, but 31% had IUGR.
clinically detectable neuromuscular manifestations of myotonic Skull. The skull should normally be rugby ball in shape and
dystrophy have between a 10% and 50% risk for having a baby cast a good acoustic shadow, indicating normal mineralisation.
with CMD.12 In pregnancy, characteristic sonographic findings Decreased mineralisation is indicated by an anechoic skull
include talipes, decreased fetal movements or breathing and vault, which casts little or no acoustic shadow (Fig. 34.3). Fur-
polyhydramnios. Affected neonates are very floppy and often thermore, the intracranial contents will be more clearly visual-
have respiratory problems requiring ventilatory support, and ised than normal, and because the cerebral hemispheres appear
the mortality rate is high (20%). Most survivors have significant relatively anechoic, the appearances are not infrequently mis-
developmental delay and a reduced life expectancy. The finding taken for cerebral ventriculomegaly. Careful examination will
of talipes and polyhydramnios in a euploid fetus should prompt reveal the characteristic and hyperechoic, normally located,
examination of the mother for signs of myotonic dystrophy. choroid plexus (see Fig. 34.3). In conditions associated with
Maternal SLE can cause a variety of problems in fetuses, includ- hypomineralisation in later pregnancy, the skull shape can
ing limb shortening with stippled epiphyses; facial anomalies, in readily be deformed by pressure from the transducer. Varia-
particular a depressed nasal bridge; and an abnormal appearance tion in skull shape can be seen rarely in some craniosynostosis
of the spine secondary to extra calcification. Other findings can syndromes, and a cloverleaf skull is also seen in thanatophoric
include bradycardias, growth retardation and hydrops.13  dysplasia type II. 
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 381

TABLE Sonographic Examination Required When

34.6 Suspecting a Skeletal Abnormality

Anatomical part Features

Long bones Length
Pattern of shortening
• Short trunk vs short limbs
predominantly
• Rhizomelic vs mesomelic vs
acromelic
• Symmetrical vs asymmetric-
Which bones
Bowing or evidence of fractures
Width
Ossification
Epiphyses for stippling • Fig. 34.3 Anomalies of the fetal skull. Ultrasound images of a fetus with
osteogenesis imperfecta type IIa showing profound hypomineralisation
Spine Length
and lack of an acoustic shadow, resulting in very clear visualisation of the
Mineralisation
intracranial anatomy.
Alignment (hemivertebrae)
Organisation (stippling)
Cranium Shape vertebrae) (Fig. 34.4A and B) can occur with or without associ-
Mineralisation (acoustic shadow) ated rib anomalies. The appearance of general disorganisation may
Face Profile: frontal bossing indicate ectopic calcification seen in some chondrodysplasia punc-
• Depressed nasal bridge tates (see Fig. 34.4B and C) or bony developmental abnormalities
• Micrognathia as in Jarcho-Levin syndrome and other spondylocostal dysplasias,
Cleft lip VACTERL association (vertebral anomalies, anorectal malfor-
Cleft palate mations, cardiovascular anomalies, tracheoesophageal fistula,
Orbital diameters for hypo- or oesophageal atresia, renal (kidney) or radial anomalies and limb
hypertelorism defects) and maternal diabetes (see Fig. 34.4E). 
Chest Size Face. Many skeletal dysplasias have associated facial anom-
Ribs: length alies. The face should be examined in the coronal view to
• Shape exclude a cleft lip, which in some conditions, such as oral facial
• Beading (fractures) digital syndrome type IV or Ellis-van Creveld syndrome, can
be very small and difficult to detect (Fig. 34.5). An axial view
Hands Short fingers (trident hand)
Camptodactyly of the palate should be visualised in order to detect significant
Ectrodactyly degrees of cleft palate, which can be associated with several
Polydactyly dysplasias. A sagittal view will reveal micrognathia, flattening
Oligodactyly of the facial profile, frontal bossing or a depressed nasal bridge.
Measurement of the mandible and orbital diameters can be
Feet Size
useful but may be more difficult to achieve in later pregnancy
Polydactyly
with increasing acoustic shadowing from surrounding bony
Joints Contractures structures.3 
Pterygia Long bones. Length of long bones should be checked against
Talipes appropriate charts of long bones length.4 The pattern of shorten-
Radial club hand ing is a useful diagnostic aid. There may be generalised shortening
Associated abnormalities Cardiac abnormalities of all long bones (micromelia). It may be more marked in the
Renal anomalies proximal long bones (rhizomelia) or the forearms and lower legs
Intracranial abnormalities (mesomelia). In some conditions, the changes may be confined
Genital anomalies to the legs (e.g., campomelic dysplasia) or arms (e.g., Holt Oram
Fetal and maternal Dopplers Screen for IUGR syndrome). Deformation of the bones is a very useful diagnostic
feature. The position and degree of bowing or fracturing should
IUGR, Intrauterine growth restriction. be noted. Bones may appear short, thick and crumpled, indicat-
   ing severe degrees of fracturing and undermodelling (see images of
fetuses with OI in Fig. 34.9). Deformity; hypoplasia; or absence of
tibia, fibula, radius or ulna may be present. The ends of the bones
Spine. The spine should be examined carefully in all three should be carefully examined to exclude epiphyseal stippling that
orthogonal planes. There may be absent or decreased mineralisa- might indicate a chondrodysplasia punctata. If stippling is identi-
tion, but it must be remembered that ossification of the cervi- fied, various metabolic and cytogenetic investigations can be done
cal and sacral vertebral bodies is a late event; the sacral vertebral to define the underlying aetiology (see section on chondrodyspla-
bodies are not ossified until 27 weeks’ gestation. Vertebral seg- sia punctata and Fig. 34.20 for examples). Expanded metaphyses
mentation anomalies (hemivertebrae, butterfly vertebrae, fused may be seen in Kniest syndrome. 
382 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B

C D E
• Fig. 34.4 Spinal anomalies. A, Coronal view of a fetal spine showing multiple hemivertebrae. B,
Three-dimensional image of hemivertebrae. C, Coronal view of a spine in a fetus with brachytelepha-
langic chondrodysplasia punctata. D, Radiograph showing lateral view of the spine in this neonate with
brachytelephalangic chondrodysplasia punctata. E, Radiograph of a fetus with Jarcho-Levin syndrome.

A B
• Fig. 34.5  Small midline cleft lip as may be found in Majewski, oral facial digital (OFD) syndrome type
IV or Ellis-van Creveld syndrome. A, Three-dimensional ultrasound image of the same fetus. B, Coronal
view after birth.

Joints. The joints may be abnormal in a wide range of skel- more difficult to examine. However, some skeletal dysplasias have
etal, neurologic and neuromuscular conditions as well as a het- hypoplastic clavicles (cleidocranial dysostosis, pycnodysostosis) or
erogeneous group of hereditary distal arthrogryposes. Talipes in scapulae (camptomelic dysplasia). 
particular can be a feature of several dysplasias. The presence of Thorax. Many lethal dysplasias are associated with thoracic
webbing or pterygia may be helpful diagnostically.  abnormalities. Nomograms of thoracic circumference are avail-
Hands and feet. Polydactyly, either pre- or postaxial, can be able, but a small chest can often be identified by observation
a feature of a number of conditions (Fig. 34.6A and B). Oligo- alone. In normal circumstances, the thorax and abdomen should
dactyly, ectrodactyly and syndactyly are less common features be approximately the same size viewed in the axial plane, and
(Fig. 34.6C and D) but are good clues to the diagnosis when pres- sonographic comparison can help indicate a small chest as, for
ent. Rocker bottom feet are seen in trisomy 18 and some of the example, in OI and thanatophoric dysplasia (see Figs. 34.10 and
contractural syndromes (Fig. 34.6E).  34.12 in these sections for examples).14 The heart should nor-
Limb girdles. The limb girdles, shoulder and pelvis can be mally occupy one third of the chest. If the ribs are short, the
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 383

A B C

D E
• Fig. 34.6 Clues to the diagnosis that may be found in the hands and feet. A, Ultrasound image of
preaxial polydactyly of the feet in Greig acrocephalopolysyndactyly. B, The view of this foot after birth. C,
Ultrasound image showing the syndactyly resulting in the mitten hand seen in Apert syndrome. D, The
same hand as in C but visualised using three-dimensional ultrasound. E, Rocker bottom foot.

heart will appear to occupy a greater proportion of the chest
when viewed in the axial plane and, when there is extreme
shortening of the ribs, as, for example, in thanatophoric dyspla-
sia, the heart may appear to lie outside the thoracic cavity (see
Fig. 34.12). In the sagittal section, the thorax will be narrow and
the abdomen protuberant, a configuration that has given rise
to the expression ‘champagne-cork appearance’. The chest can
also be small secondary to a short spine, as in some of the spon-
dylodysplasias. In these situations, the chest may appear small
in a sagittal plane but, when viewed in the axial plane, the ribs
appear of normal length and the heart occupies the appropri-
ate proportion of the thoracic cavity (see Fig. 34.14). The ribs
themselves should be examined carefully as they may be short,
thick, thin, beaded or irregular in organisation or number. Short
ribs (extending less than halfway around the chest in transverse
section) can be viewed in the axial plane (see Figs. 34.7, 34.10
and 34.12 for examples), and they should be examined in a lon-
gitudinal plane to exclude beading, which is indicative of frac-
turing (see Figs. 34.9 and 34.10). Disorganisation of the ribs
(and spine) may be features of conditions such as Jarcho-Levin
syndrome (see Fig. 34.4E).  alies, including skeletal dysplasias.15,17 Improved technology
Increased nuchal translucency and oedema. One of the earli-
est signs of a musculoskeletal problem is an increased nuchal
translucency (NT), which has been reported in many skel-
etal dysplasias.15 Later in pregnancy, this is represented by an
increased nuchal fold and generalised skin thickening, as the
skin appears to outgrow the bones. In some conditions frank
hydrops can also occur.  a good time to examine the fetal skeleton. These factors result
Other structures. The rest of the fetal anatomy should be
examined carefully because many skeletal dysplasias and
genetic syndromes characterised by skeletal anomalies can
have abnormalities of the urogenital tract, heart and brain (see
Table 34.3). 
Timing of Diagnosis
Most reports of the diagnosis of skeletal dysplasias come
from incidental findings at the time of the routine second
trimester fetal anomaly scan or when women are scanned
later in pregnancy because of findings such as polyhydram-
nios or suspected IUGR. Indeed, the clinical presentation of
some dysplasias is such that sonographic findings may not be
evident until later in pregnancy.16 However, with the effec-
tiveness of first trimester combined screening for Down syn-
drome, there is an increasing use of early ultrasound as part
of routine obstetric care. This test includes measurement of
the NT which, when increased, is associated not only with an
increased risk for aneuploidy but also other congenital anom-
and ready availability of transvaginal scanning allows detailed
examination of the fetal anatomy in early pregnancy with the
subsequent detection of many structural abnormalities.17 Fur-
thermore, because long bones and many other fetal bony parts
are formed by 11 weeks’ gestation1 and charts of fetal limb
length are available from this gestation,4 the first trimester is
384 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
in significant potential for first trimester diagnosis of many
serious skeletal dysplasias, particularly those that are lethal or
carry a high chance of early mortality, as well as those with
significant degrees of limb reduction or serious orthopaedic
problems.15 As we go through the approach to diagnosis of
the various syndromes, we highlight those with which early
pregnancy diagnosis is possible. Furthermore, with increasing
knowledge of the underlying molecular pathology, identifica-
tion of markers of skeletal dysplasias can allow for targeted
invasive or increasingly noninvasive9 diagnosis for definitive
assessment of prognosis to inform parental counselling and
subsequent pregnancy management.  the spine are seen in type 1A cases.
Description of Individual Conditions:
Genetics and Sonographic Findings
There are increasing reports of the prenatal detection of a variety
of conditions associated with skeletal abnormalities in the litera-
ture. A few of the more common generalised skeletal dysplasias,
albeit all rare, will be described in detail here. In some cases, a
definitive diagnosis can be made antenatally after genetic muta-
tion or metabolic analysis, but, for the majority, detailed postna-
tal radiology and sometimes histopathology is required to make a
definitive diagnosis. This is needed to counsel parents with regard
to recurrence risks and to inform accurate prenatal diagnosis in
future pregnancies. However, in a few families, postnatal exami-
nation, including radiology without postmortem examination, is
declined, and in these situations, detailed sonographic informa-
tion can be the only diagnostic aid available.
Dysplasias Associated With Hypomineralisation
Achondrogenesis type 1A (Houston-Harris type).
Gene location 14q32.12
TRIP11 (thyroid hormone receptor interactor 11)
Autosomal recessive  sis by direct mutational analysis of the TNSALP gene is possible
Achondrogenesis type 1B (Parenti-Fraccaro type).
Gene location 5q32
SLC26A2 Diastrophic dysplasia sulphate transporter gene
(DTDST)
Autosomal recessive  serum levels of alkaline phosphatase consistent with being carri-
Achondrogenesis type II (Langer-Saldino type).
Gene location 12q13.11
COL2A1 (type 2 collagen)
Autosomal dominant
  
Achondrogenesis types 1 and 2 are difficult to distinguish
prenatally, but postnatal differentiation radiologically after birth
is vital as both forms of type 1 are inherited in an autosomal
recessive fashion (1:4 recurrence risk), but type 2 is usually spo-
radic (de novo autosomal dominant) with a low recurrence risk.
Achondrogenesis type 2 (Fig. 34.7A) probably represents the
most severe end of the spectrum of the type 2 collagenopathies,
which includes hypochondrogenesis, Kniest dysplasia, SEDC
and Stickler syndrome. Defects of type II collagen synthesis have
been demonstrated in some cases, and most cases are caused by
new heterozygous mutations in the COL2A1 gene. Both achon-
drogenesis types 1 and 2 result in stillbirth (or neonatal death).
Sonographic features include micromelia with very short but
straight long bones (Fig. 34.7B), a relatively large hypominer-
alised skull (in type 1), a short neck, a short trunk with a pro-
tuberant abdomen, hypomineralisation of the vertebral bodies
(Fig. 34.7C) and very short ribs (Fig. 34.7D). Other sonographic
features include skin oedema and polyhydramnios. Both condi-
tions have a flat nasal bridge with a short nose and anteverted
nostrils. Sonographically and radiologically, ossification of the
skull, spine and pelvis is more deficient in type 1 than in type 2.
The long bones are shorter in type 1, which has been subdivided
into types 1A (Houston-Harris) and 1B (Parenti-Fraccaro).18
Multiple rib fractures and almost complete lack of ossification of
 
Hypophosphatasia.
Gene locus 1p36.12
Tissue-nonspecific alkaline phosphatase gene (TNSALP)
Autosomal recessive
  
This condition occurs in congenital or infantile, childhood or
adult forms correlating with the severity of the reduction in activ-
ity of tissue nonspecific alkaline phosphatase. All have reduced
chondro-osseous mineralisation with low levels of alkaline phos-
phatase in blood, cartilage and bone, together with increased levels
of plasma pyridoxal 5′-phosphate and urine phosphoethanol-
amine. Measurement of alkaline phosphatase levels in chorionic
villi can be diagnostic.19 The severe neonatal form is detectable
using fetal ultrasound. Affected pregnancies usually result in still-
birth or neonatal death secondary to pulmonary hypoplasia. The
severe neonatal form is caused by biallelic mutations in the tissue-
nonspecific alkaline phosphatase gene (TNSALP) and as such is
inherited in an autosomal recessive fashion with a 1:4 recurrence
risk for parents. Sonographic features include increased NT and
short and sharply angulated long bones with occasional unusual
bony spurs at the point of angulation (Fig. 34.8A) and a hypo-
mineralised skull (Fig. 34.8B), which can assume a globular shape.
Long bones may also be also poorly mineralised. Prenatal diagno-
in families at known increased risk where molecular analysis has
confirmed a mutation in the TNSALP gene. The concentration of
phosphoethanolamine can be elevated in the urine of mutation
carriers and this can be a useful aid to diagnosis in cases arising
de novo in a family. In the case illustrated, both parents had low
ers of recessive hypophosphatasia and whole-exome sequencing of
chorionic villi confirmed a homozygous mutation in the ALPL
gene.10
Milder forms of dominant hypophosphatasia can also be pres-
ent in utero, with severe bowing of long bones in early pregnancy,
which improves over time. Neonates can show low alkaline phos-
phatase levels at birth, with mildly affected long bones and den-
tinogenesis that develops in childhood.20 
Osteogenesis imperfecta types IIA, IIB and IIC.
Gene locus 17q21.31-q22, 7q22.1
COL1A2 collagen I, α-2 polypeptide
COL1A1 collagen I, α-1 polypeptide
Autosomal dominant, autosomal recessive, germline mosaicism
  
Osteogenesis imperfecta is a disorder of connective tissue that
may affect 1 to 2 in 10,000 individuals. There are several types,
which result from multiple different mutations in the type 1 col-
lagen genes,COL1A1 and COL1A2, as well as a variety of other
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 385

A B

C D
• Fig. 34.7  Sonographic findings in achondrogenesis. A, Radiograph of a fetus with achondrogenesis
type II. B, Very short, straight leg bones, showing mesomelic shortening. C, Coronal view of the spine
showing the ‘tram-line appearance’ caused by hypomineralisation of vertebral bodies. D, Transverse sec-
tion through the lower thorax showing very short ribs.

A B
• Fig. 34.8  Sonographic findings in hypophosphatasia. A, Image of an acutely angulated femur in a fetus
with hypophosphatasia at 14 weeks’ gestation. B, Image of the same fetus at 14 weeks’ gestation show-
ing the hypomineralisation of the skull.

genes.21 The severity of the clinical features is related to the type of
mutation and the relative level of expression. Most mutations asso-
ciated with OI are inherited in a dominant pattern, and an autoso-
mal recessive inheritance is very rare but has been described. Most
severe cases of OI are caused by new dominant mutations. The
recurrence risk after an isolated case is about 5% to 7%, which is
thought to be mainly caused by somatic or gonadal mosaicism in
one of the parents.22,23
The most commonly used classification is that described by Sil-
lence and coworkers.24 Type I is the mildest form and usually only
386 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C

D E F
• Fig. 34.9  Sonographic findings in the osteogenesis imperfecta types IIA and IIC. A, Hypomineralised
skull. Note the lack of acoustic shadow and clarity of intracranial contents. B, Hypomineralised facial
bones. C, Hypomineralisation is also clearly seen in the sagittal view of the head, which also shows the
relatively flat profile. D, Short, beaded ribs. E, Bent distal leg. Note the relatively normal length foot with
absent mineralisation. F, Radiograph of a fetus with osteogenesis imperfecta type IIC.

presents with fractures occurring after birth. Occasional cases of
prenatal detection in the late third trimester have been reported. OI
type II refers to a group of severely affected neonates, who present
with prenatal sonographic abnormalities and who usually die in the
perinatal period. Type II has been further subdivided into subtypes
IIA, IIB and IIC, according to radiological features. Subtype IIB is
characterised by minimal or no rib fractures. The relatively normal
chest configuration renders it to be the only form of type II with
potential postneonatal survival. Type III is the most severe form,
which is compatible with life and presents with prenatal shortening
and fracturing of the long bones. Type IV is the most diverse group
in the classic classification. The more severely affected patients with
type IV OI present with fractures at birth, which can occasionally
be detected using prenatal ultrasound. New forms of OI based on
analysis of bone architecture and genetic analysis have been recently
added to the classification.21 Inheritance depends on genetic aetiol-
ogy, but recessive types have been described.
Type II OI is the severe, usually lethal form, which is associ-
ated with varying degrees of hypomineralisation. Sonographi-
cally, types IIA and IIC are difficult to differentiate; although
both types IIA and IIC have hypomineralised skull and facial
bones and skull, this is more extreme in type IIC (Fig. 34.9A–
C). In both types, there is continuous beading of the ribs (Fig.
34.9D), indicating multiple fractures. The long bones are broad
and crumpled, and it can be difficult to identify individual bones
in the distal limbs (Fig. 34.9E). The vertebrae are flattened and
hypoplastic, and the pelvis is hypoplastic with flattening of the
acetabular roofs and iliac crests, but these are features seen more
commonly on postnatal radiology (Fig. 34.9F). As with other
lethal skeletal dysplasias, an increased NT can be an early pre-
senting feature.15
In type IIB, sonographic findings are less severe. The skull
can show mild degrees of hypomineralisation, but this can be
difficult to recognise sonographically. The chest is less obviously
affected (Fig. 34.10A), but in the axial plane, there is a typical
configuration with slightly short ribs, which flare outwards at
the ends (Fig. 34.10B) and rib fractures, whilst universally pres-
ent after birth, can be difficult to identify antenatally with bead-
ing not always obvious. The long bones are longer and better
modelled but show obvious angulation secondary to fracturing
(Fig. 34.10C–E).
Prenatal diagnosis using molecular and biochemical methods
in 129 cases with different types of OI has been described.25 How-
ever, because the genes responsible for this condition are large,
with many potential mutations, and the recurrence risk is gener-
ally small, until recently, ultrasound was the preferred method for
prenatal diagnosis in subsequent pregnancies. With the advent of
exome sequencing, it is possible to screen multiple genes with a
single test, either after pregnancy or by analysis of chorionic villi
or amniocytes10 as in the case shown in Fig. 34.11. This fetus
was suspected of having OI IIB on prenatal ultrasonography. but
sequencing showed a homozygous mutation in the LEPRE gene,
indicating OI type VIII, which is associated with recessive rather
than sporadic inheritance.26 
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 387

A B

C D

Femur length in fetuses with osteogenesis imperfecta

90
IV III II-c II-b
80

70
Femur length (mm)

60

50

40

30

20

10

0
12 16 20 24 28 32 36 40
E Gestational week
• Fig. 34.10  Sonographic findings in osteogenesis imperfecta (OI) type IIB. A, Axial view comparing the
size of the thorax and abdomen demonstrating the slightly small chest. B, Axial view through the thorax
shows flaring of the ends of the ribs. C, The femur is short and angulated. D, The tibia and fibula are short,
but foot length is preserved. E, Fetal femur size chart showing the normal range with measurements from
fetuses with OI IIA, IIB, III and IV plotted.

Dysplasias Associated With a Small Chest This is the most common lethal skeletal dysplasia with an
incidence of around approximately 1 in 20,000. There are two
Achondrogenesis types 1 and 2. See earlier discussion. 
types, 1 and 2. At least 10 mutations in the FGFR3 gene have
Osteogenesis imperfecta types IIA and IIC. See earlier been shown to cause type 1.27 Most of these are heterozygous
discussion.  missense mutations (substitute an incorrect amino acid in the
FGF3 protein). The most common mutation is a substitution of
Thanatophoric dysplasia. the amino acid cysteine for the amino acid arginine at position
Gene location 4p16 248 (p.(Arg248Cys)). Other mutations cause the protein to be
Fibroblast growth factor receptor 3 gene (FGFR3) longer than normal. The mutated receptor is active independent
Lethal autosomal dominant, germline mosaicism of ligand binding, causing the severe problems with bone growth
   seen in this condition. All patients with type 2 thanatophoric
388 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
A B
• Fig. 34.11  Sonographic findings in osteogenesis imperfecta type VIII. A, Short slightly bowed humerus.
B, Slightly hypomineralised skull. Note how clearly the intracranial anatomy is visualised.
dysplasia27 have had a specific heterozygous missense mutation
(p.Lys650Glu) which occurs in a different part of the FGFR3 gene
than the mutations that cause type 1. The mutations arise de novo
and recurrence risks are very low (<1%), incorporating a small
germline mosaicism risk.
Thanatophoric dysplasia is a disorder characterised by short
long bones (Fig. 34.12A),28 with or without bowing, very
short ribs, short fingers giving rise to the classical trident hand,
platyspondyly and relative macrocephaly with frontal bossing
(Fig. 34.12B–F). Features are present from early pregnancy, and
sonographic diagnosis can be suspected from the first trimester.15
There are two types. Type 1 is the more common subtype and is
distinguished by the presence of curved femora and flattened ver-
tebral bodies (platyspondyly). An unusual head shape (cloverleaf
skull) is occasionally seen with type 1 and in virtually all cases
of type 2. Definitive molecular prenatal diagnosis can be made
by analysis of cfDNA in maternal plasma.9,28 Molecular diagnosis
is important as the differential diagnosis includes the short rib–
polydactyly syndromes, which are inherited in a recessive fashion,
and SEDC, which is viable.14 Neonates with thanatophoric dys-
plasia usually, but not always, die from pulmonary hypoplasia in
the neonatal period.  The incidence of SEDC is around 1 in 100,000, and it is caused
Short rib–polydactyly syndromes.
SRPS1 (SRTD3): 11q22.3, DYNC2H1
SRPS2 (SRTD6): 4q33, NEK1
SRPS3 (SRTD3): 11q22.3, DYNC2H1
SRPS4 (SRTD12): not mapped
SRPS5 (SRTD7): 2p24.1, WDR35
SRPS6 (SRTD8): 7q36.3, WDR60
Autosomal recessive inheritance
  
The short rib–polydactyly syndromes (SRPSs) comprise a
group of lethal skeletal dysplasias, all of which have a small thorax
secondary to short ribs, short limbs, pre- and postaxial polydac-
tyly and a variety of other visceral anomalies. There are four main
categories:
  
SRPS I (Saldino-Noonan type; SRPS1)
SRPS II (Majewski type, SRPS2)
SRPS III (Verma-Naumoff type, SRPS3)
SRPS IV (Beemer-Langer type, SRPS4)
  
All are inherited in an autosomal recessive fashion, and
there is considerable phenotypic overlap among the conditions
(Table 34.7). There are occasional case reports describing their pre-
natal detection.15,29,30 Sonographic features include an increased
NT, varying degrees of long bone shortening, a small chest with
short ribs (Fig. 34.13A and B), polydactyly (Fig. 34.13E) and a
variety of visceral abnormalities, which predominantly include renal
dysplasia, bladder outflow obstruction and cardiac and intracerebral
anomalies (Fig. 34.13C and D). Facial clefting is a feature of some
of these conditions; for example, in Majewski syndrome, a small
midline cleft lip may be a distinguishing feature (see Fig.34.5).30
The nomenclature is confusing because these conditions have
been incorporated into a wider grouping of short-rib thoracic dys-
plasias with or without polydactyly, including Ellis van Creveld
syndrome, Jeune asphyxiating thoracic dystrophy and Mainzer-
Saldino syndrome. The numbering of the SRPS and SRTD differs
as outlined earlier. 
Spondyloepiphyseal dysplasia congenita.
Gene locus 12q13.11
COL2A1 gene (type 2 collagen)
Autosomal dominant
  
by heterozygous mutations in the COL2A1 gene.18 Affected indi-
viduals are very short with a short trunk and marked lordosis.
Other complications include myopia, micrognathia, cleft palate
and deafness. Prenatally, shortening of the limbs is evident from
around 12 weeks’ gestation. Other findings include an increased
NT, micrognathia, poorly mineralised cervical vertebrae in early
pregnancy and a small chest secondary to a short trunk but normal
ribs (Fig. 34.14A and B).14 In early pregnancy the main differential
diagnosis is thanatophoric dysplasia, which can be distinguished
by screening maternal plasma for mutations in the FGFR3 gene.9
Later in pregnancy, achondroplasia and acromesomelic dysplasia
would be the main differential diagnoses. These may be distin-
guished from SEDC because this condition does not have relative
macrocephaly, frontal bossing or short fingers (Fig. 34.14C), and
the pattern of growth is different (Fig. 34.14D) compared with
that of fetuses with achondroplasia (see Fig. 34.22A). 
Asphyxiating thoracic dystrophy.
SRTD1: 15q13, no gene to date
SRTD2: 3q25.33, IFT80
SRTD3: 11q22.3, DYNC2H1
SRTD4: 2q24.3, TTC21B
SRTD5: 4p14, WDR19
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 389

80

60
Femur length (mm)

40

20

15 20 25 30 35 40
C
A Gestational age (wk)

D E F
• Fig. 34.12  Sonographic findings in thanatophoric dysplasia. A, Chart of femur length showing the size of
the femurs in thanatophoric (closed black dots) dysplasia and achondroplasia (open black circles) plotted
on centile charts for normal fetuses. B, Sagittal view of the thorax showing the typical ‘champagne cork’
appearance. C, Axial view showing the comparison between the thorax and abdominal circumferences.
Note how short the ribs appear in this plane, finishing halfway around the thorax such that the heart has
no protection from the ribs. D, View of the short legs in the typical ‘froglike’ position. E, The ‘trident hand’
viewed with three-dimensional ultrasound. F, Profile at around 22 weeks’ gestation showing marked fron-
tal bossing. (Part A from Chitty LS, Khalil A, Barrett AN, et al. Safer, accurate prenatal diagnosis of thana-
tophoric dysplasia using ultrasound and cell free fetal DNA. Prenat Diagn 33:416–423, 2013.)

TABLE
34.7 Features That May Be Sonographically Detected in Short Rib–Polydactyly Syndromes
Saldino-Noonan syndrome Majewski syndrome Verma-Naumoff syndrome Beemer-Langer syndrome
Short ribs + + + +
Short limbs + + + +
Bowed limbs Radius, ulna
Other skeletal anomaly Hypoplastic or absent fibula, Hypoplastic or absent tibia, Hypoplastic ilia Talipes, small scapula,
hypoplastic ilia, metaphy- ovoid tibia hypoplastic ilia
seal dysplasia
Preaxial polydactyly Fingers Fingers, including bifid
thumb
Continued
390 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
34.7 Features That May Be Sonographically Detected in Short Rib–Polydactyly Syndromes—cont’d
Saldino-Noonan syndrome Majewski syndrome Verma-Naumoff syndrome Beemer-Langer syndrome
Postaxial polydactyly Fingers and toes Fingers and toes Fingers and toes Fingers
Craniosynostosis Cloverleaf skull
Macrocephaly + +
Hypertelorism +
Micrognathia +
Cleft palate + + +
Midline cleft lip Midline Midline Midline
CNS anomalies + +
Ambiguous/absent + + + +
genitalia
Vaginal atresia +/- + + +
hydrometrocolpos
Cardiac abnormalities + + + +
Bowel atresias Anal atresia + + +
Exomphalos +
Renal dysplasia Renal cysts + + +
Urinary tract obstruction +
Oedema or hydrops Ascites + Skin oedema, ascites

CNS, Central nervous system.

  

A B

C D E
• Fig. 34.13  Sonographic findings in fetus with a short ribbed polydactyly syndromes (SRPS) at 16 weeks’
gestation. A, Longitudinal view through the thorax of a fetus with SPRS demonstrating the extremely short
ribs. B, The thorax of a fetus with achondroplasia viewed in the axial plane with the heart appearing to
lie virtually outside the thorax. Note the comparison with the abdominal size. C, Axial view through the
abdomen in this fetus with SRPS and obstructive uropathy. D, Views of another fetus with SRPS and large
echogenic kidneys. The very short femora are also visible. E, Three-dimensional view of a fetus with SRPS
showing the short limbs and polydactyly of the feet.
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 391

A B C

90 80
95th 95th
80 50th 70 50th
5th 5th
70 60

Humerus length (mm)

Compound Compound
Femur length (mm)

60 SEDC SEDC
50
50
40
40
30
30
20
20
10 10

0 0
10 15 20 25 30 35 40 10 15 20 25 30 35 40
Gestation (wk) Gestation (wk)

70 80
95th 95th
50th 70 50th
60
5th 5th
Compound 60 Compound
Radius length (mm)

50
Tibia length (mm)

SEDC SEDC
50
40
40
30
30
20
20
10 10

0 0
10 15 20 25 30 35 40 10 15 20 25 30 35 40
D Gestation (wk) Gestation (wk)
• Fig. 34.14  Sonographic findings in spondylo-epiphyseal dysplasia congenita (SEDC). A, Sagittal view of
a fetus with SEDC at 18 weeks’ gestation, demonstrating micrognathia but no frontal bossing and a short
thorax. B, Axial view through the chest and abdomen at 18 weeks’ gestation. Note that the chest appears
slightly small, but the heart only occupies one third of the chest, and the ribs are of normal length. C, A
hand in SEDC with normal length fingers compared with those seen in thanatophoric dysplasia at around
the same gestation. D, Long bone growth in two fetuses with SEDC plotted on charts of normal size. Note
the difference in size compared with measurements in thanatophoric dysplasia in Fig. 34.12A. (Part D from
Chitty LS, Tan AW, Nesbit DL, et al. Sonographic diagnosis of SEDC and double heterozygote of SEDC
and achondroplasia-a report of six pregnancies. Prenat Diagn 26:861–865, 2006.)

SRTD6: 4q33, NEK1 SRTD13: 5q23.2 CEP120

SRTD7: 2p24.1, WDR35 SRTD14: 14q23.1 KIAA0586
SRTD8: 7q36.3, WDR60 SRTD15: 2p21 DYNC2LI1
SRTD9: 16p13.3, IFT140 SRTD16: 2q13.12 IFT52
SRTD10: 2p23.3, IFT172 SRTD17: 3q29 TCTEX1D2
SRTD11: 9q34.11, WDR34 SRTD18: 14q24.3 IFT43
SRTD12: not mapped Autosomal recessive
  
392 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C
• Fig. 34.15  Jeunes asphyxiating thoracic dystrophy. A, Sagittal view of the thorax demonstrating the
narrow chest. B, Axial view of the thorax demonstrating the short ribs with heart occupying more than
one third of the chest. C, Radiograph of a 20-week fetus with Jeunes asphyxiating thoracic dystrophy
demonstrating the features, including short but straight limbs.

A B C

D
• Fig. 34.16  Sonographic findings in Ellis van Creveld syndrome. A, Short, straight femur. B, Polydactyly
of the toes showing six toes and a large duplicated great toe. C, Narrow chest seen in the sagittal plane.
D, A fetus with Ellis-van Creveld syndrome scanned at 13 weeks’ gestation when the short ribs, short
femur and lower leg are seen and polydactyly is clear.

These rare autosomal recessive conditions are characterised by
variable degrees of rhizomelic shortening of the long bones and
a small chest with short ribs (Fig. 34.15). Up to 50% of affected
individuals have postaxial polydactyly. Intracerebral anomalies
can occasionally occur. Neonatal death from respiratory difficul-
ties secondary to pulmonary hypoplasia is common, occurring in
around 70% of cases. Survivors may develop chronic renal failure
caused by cystic changes and periglomerular fibrosis in childhood
and also have an increased risk for retinal degeneration and haepatic
and pancreatic fibrosis. The limb shortening is usually rhizomelic,
and the condition might be mistaken for achondroplasia, but the
facial contours are normal. Prenatal sonographic findings are vari-
able, but presentation can be from around 16 weeks’ gestation
with short limbs and a long, narrow chest. The main differential
diagnosis is Ellis-van Creveld syndrome and paternal uniparental
disomy for chromosome 14 (pUPD14).  ential diagnosis includes Jeune asphyxiating thoracic dystrophy
Ellis-van Creveld syndrome.
Gene locus 4p16.2
EVC and EVC2 (LBN; Limbin gene)
Autosomal recessive
  
Ellis-van Creveld syndrome is a rare, autosomal recessive skeletal dys-
plasia characterised by disproportionate short stature, short limbs,
short ribs, postaxial polydactyly, midline cleft or notched upper
lip and dysplastic nails and teeth. Around 60% of affected indi-
viduals have a congenital cardiac defect, often an atrial-ventricular
septal defect. Some neonates have respiratory difficulties secondary
to the small chest, but this is rarely serious. Sonographic findings
include short long bones (Fig. 34.16A), postaxial polydactyly in
the hands and feet (Fig. 34.16B), a narrow thorax and short ribs
with or without a cardiac abnormality (Fig. 34.16C). The differ-
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 393

A B C
• Fig. 34.17 Sonographic findings in campomelic dysplasia. A, Femur showing midshaft bowing. B,
Image showing slight bowing of the tibia. C, Genital ambiguity in a male fetus with campomelic dysplasia.

and other SRPSs, although many of these have more profound
manifestations. Diagnosis from early pregnancy is achievable
through detection of the polydactyly and associated limb shorten-
ing (Fig. 34.16D).15  eral. Ambiguous genitalia occur in the majority of cases with an
Paternal uniparental disomy for chromosome 14. This condi-
tion results in a distinctive phenotype, which is recognisable and
diagnosable antenatally. It typically results in increased NT with a
subsequent small chest size with coat hanger-shaped ribs, anterior
abdominal wall defects ranging from marked diastasis recti to exom-
phalos, polyhydramnios and mild limb contractures. It is most com-
monly misdiagnosed as Jeune asphyxiating thoracic dystrophy both
pre- and postnatally. The postnatal chest radiograph is diagnostic.
When suspected antenatally or postnatally, it can be confirmed by
DNA studies on fetal material and samples from both parents or by
14q methylation testing using DNA from the proband alone. The
karyotype should be checked as a proportion of cases are associated
with Robertsonian translocations involving chromosome 14.  Autosomal dominant (somatic and gonadal mosaicism)
Dysplasias Associated With Bowing of the Long
Bones
Some conditions, such as hypophosphatasia and OI types IIA, B
and C, are associated with severe bowing or deformation of the
long bones but others may have less severe degrees (OI types III and
IV) or be more variable (campomelic dysplasia) in presentation.
Hypophosphatasia. See earlier discussion.  and disrupted growth plates. Many must use a wheelchair by late
Osteogenesis imperfecta types IIA, IIB and IIC. See earlier
discussion.  may now be amenable to significant amelioration with the use of
Campomelic dysplasia.
Gene locus 17q24.3
SOX9 gene
Autosomal dominant
  
This condition has an incidence of around 1 in 20,000. Most
cases are caused by mutations in the SOX9 gene, an SRY-related
gene at 17q24.3, but some are associated with a cytogenetically vis-
ible rearrangements of chromosome 17q24 upstream of SOX9 and
a milder clinical phenotype.31 Perinatal death is common, second-
ary to respiratory problems related to a small chest. Complications
in survivors include recurrent apnoea, kyphoscoliosis, learning
difficulties (mild to moderate), short stature and dislocation of
the hip.31 The main sonographic feature is bowing of the femur
and tibia, the arms being unaffected (Fig. 34.17A and B). Other
features include a large head, micrognathia, a cleft palate and a
flat nasal bridge, with occasional talipes. The chest can be narrow
and respiratory distress is common. About one third of cases have
cardiac defects (ventricular septal defect, atrial septal defect, tetral-
ogy of Fallot), and one third have hydronephrosis, mostly unilat-
XY karyotype. Confirmation of genetic sex and demonstration of
genital ambiguity in male fetuses (Fig. 34.17C) can be diagnostic
in the presence of other features described earlier. This can be done
after a maternal blood sample and analysis of cell-free fetal DNA
in maternal plasma.32,33 Other features, less easily identified on
ultrasound, include hypoplastic scapulae, narrow iliac wings and a
poorly ossified pubis. There may be only 11 pairs of ribs.34 
Osteogenesis imperfecta type III.
Gene locus 7q21.3, 17q21.33
COL1A2, COL1A1
Type I collagen polypeptide chain genes
Type III OI is the most severe form of OI that is compatible
with life. It is a rare form characterised by progressive deforma-
tion, which begins in utero. Individuals may present with mul-
tiple fractures at birth and sustain frequent fractures thereafter
(Fig. 34.18A).21 The skull is normally mineralised but has mul-
tiple wormian bones, not detectable with prenatal ultrasound.
Patients with type III OI progress to have severely short stature
because of spinal compression fractures, deformities of the limbs
childhood. Death in early life is common, but the clinical course
bisphosphonates.35 Most recently, in utero treatment with mesen-
chymal stem cells with repeat treatment at a few months of age has
been shown to reduce postnatal fracturing.36 Limb shortening and
bowing, particularly in the legs, is usually evident by 20 weeks’
gestation and often earlier (Fig. 34.18B and C). Other long bones
tend to be on or below the fifth centile. Some bowing of the arm
bones can also be seen. The chest appears normal, and often the
ribs, although thin, do not show beading, although fresh fractures
often occur at delivery (see Fig 18A and D–F). The head appears
normal and casts a reasonable acoustic shadow. 
Osteogenesis imperfecta type IV.
Gene locus 7q21.3, 17q21.33
COL1A2, COL1A1
Type I collagen polypeptide chain genes
  
Type IV is the most diverse group in the classic classification.
The more severely affected patients with type IV OI present with
394 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B

C D

E F
• Fig. 34.18  Sonographic findings in osteogenesis imperfecta (OI) type III. A, Radiograph of a fetus with
OI type III. Note the fresh rib fractures rather than the beading seen in OI type II. B, Note the symmetrical
bowing of the femora. C, Short, bowed lower legs. D, Sagittal view of the thorax and abdomen show-
ing relatively normal proportions. E, Axial view through the thorax and abdomen demonstrating relatively
normal proportions, with the heart occupying an appropriate amount of the thorax. F, The ribs are thin and
slightly irregular, but beading less obvious.

fractures at birth, moderate skeletal deformity and a relatively and sternum. The pattern of stippling together with the pattern
short stature. New forms of OI (types V–VII), based on analysis of limb shortening, degree of symmetry, nasal configuration, fetal
of bone architecture, have been recently added to the classifica- sex and other features can give a clue to the underlying aetiol-
tion.37 These types are moderately deforming, and before the ogy and allow for targeted biochemical, cytogenetic or molecular
introduction of the new classification, they would have all fallen analysis.38
into type IV category. Prenatal detection is more difficult. In the
authors’ experience of two cases, long bone length has remained Rhizomelic chondrodysplasia punctata.
within the normal range throughout pregnancy (Fig. 34.19), Gene locus
and the other feature detectable with prenatal ultrasound was RCDP1 6q23.3 PEX7 encoding the peroxisomal type 2 targeting
mild symmetrical bowing of the femora and lower leg bones (see signal (PTS2) receptor
Fig. 34.19).  RCDP2 1q42.2, GNPATacyl-CoA:dihydroxyacetonephosphate
acyltransferase (DHAPAT)
RCDP3 2q31.2, AGPS alkyldihydroxyacetonephosphate synthase
Conditions Associated With Stippled Epiphyses (alkyl-DHAP synthase) gene
Several conditions are associated with ectopic calcification around RCDP5, 12p13.31, PEX5
the epiphyses and in other parts of the skeletal, spine, hands, feet Autosomal recessive
  
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 395

A B

C D
• Fig. 34.19  Sonographic findings in osteogenesis imperfecta type IV. A, Image of femur showing mild
bowing in the proximal one third. B, Image of lower leg demonstrating minimal bowing of the tibia. C,
Image of lower leg demonstrating minimal bowing of the tibia and fibula. D, Radiograph showing the bow-
ing of the femora in this neonate.

Rhizomelic chondrodysplasia punctata (RCDP) is a rare dis-

order, characterised by rhizomelic shortening of long bones
(particularly the humerus), joint contractures, nasal hypoplasia,
congenital cataracts in about 70% and ichthyosis in 30%. Postna-
tally, these individuals have progressive microcephaly and severe
mental retardation. Many affected individuals die in early child-
hood, but survivors have shown severe growth restriction and
developmental delay.
Rhizomelic chondrodysplasia punctata is a peroxisomal disorder
associated with impaired plasmalogen biosynthesis and phytanic
acid alpha-oxidation and accumulation of phytanic acid. It most
commonly results from mutations in PEX7, which result in mistar-
geting of several peroxisomal matrix proteins. However, it can also
result from mutations in genes for specific peroxisomal enzymes,
GNPAT or AGPS, or from mutations in PEX5 encoding another
receptor required for peroxisomal matrix protein targeting. • Fig. 34.20  Sonographic findings in a fetus with rhizomelic chondrodys-
Prenatal diagnosis can be achieved through biochemical and plasia punctata. Note the extra calcification at the epiphysis of the femur.
enzyme studies using amniotic fluid or chorionic villi39 or through
mutation testing when the specific family mutation is known.
With advances in molecular technology, exome sequencing of cho-
rionic villi or amniocytes may also yield a definitive diagnosis.10 This condition is associated with asymmetric shortening of bones;
Sonographic features include rhizomelic long bone short- ichthyosis and other patchy skin changes; areas of alopecia and
ening, ectopic calcification in the hyaline cartilage, epiphyses sparse, coarse hair; and occasional cataracts. Intelligence is usu-
(Fig. 34.20), pelvis and around the vertebral column, resulting ally normal. Prenatal sonographic detection has been reported
in the appearance of stippled epiphyses and a disorganised spine. but most cases arise de novo.41 Sonographic findings include short
Prenatal detection of the cataracts associated with this condition limbs, which may be asymmetrical, with epiphyseal stippling
has been reported.40  and occasional bowing, with an irregular appearance of the spine
resulting from stippling. Affected fetuses are generally, but not
Conradi Hunermann syndrome. exclusively, female. Examination of the mother for cataracts and
Gene locus Xp11.23 skin changes, which may indicate she is a carrier, might be helpful
Emopamil-binding protein (EBP) gene in defining the diagnosis in the affected fetus. In pregnancies at
X-linked dominant known high risk, in which molecular investigations done before
  
396 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C

D E F
• Fig. 34.21  Sonographic findings in X-linked recessive chondrodysplasia punctata. A, Radiograph of a
fetus with X-linked recessive chondropunctata dysplasia showing the extra calcification in the anteropos-
terior view. B, Lateral spine radiograph. C, Ultrasound image of the femur showing the extra calcification
at the epiphysis. D, Ultrasound image of the humerus showing the extra calcification at the epiphysis. E,
Profile demonstrating the depressed nasal bridge and flat profile. F, Sagittal view of the spine showing the
‘disorganised’ appearance caused by ectopic calcification.

pregnancy have demonstrated mutations in the EBP gene, inva-
sive prenatal diagnosis may be offered.  Straight Limbs
X-Linked recessive chondrodysplasia punctata.
Gene locus Xp22.33
Arylsulphatase E (ARSE) gene
X-linked recessive
In this condition, only males are affected. They have short
limbs with stippling of the epiphyses as well as the larynx
and trachea (Fig. 34.21A and B), which disappears in early
childhood. 42 There can be marked nasal hypoplasia, which
also improves with age, and cataracts can be present in some
cases. The condition may arise because of large deletions in
Xp22.33, which result in contiguous deletion encompassing
the ARSE gene when developmental problems can also ensue,
or as a result of smaller deletions or point mutations involving
the ARSE gene when additional problems, including learning
disability, are not associated. Female carriers, who are hetero-
zygous for the deletion, tend to have mild short stature but no
radiologic stippling. Prenatal sonographic findings include
short limbs with epiphyseal stippling (Fig. 34.21C and D);
a very depressed nasal bridge and a small nose (Fig. 34.21E);
and stippling of the larynx, trachea and spine (Fig. 34.21F).
Testing for contiguous deletions on the X chromosome is
routinely available, but for those without detectable dele-
tions, definitive diagnosis is only possible prenatally after
invasive testing and identification of mutations in the ARSE
gene in families in which the causative mutation has been
identified.  blood.9 This approach can also be used to exclude a recurrence
Other Skeletal Dysplasias Associated With Short,
Achondroplasia.
Gene locus 4p16.3
FGFR3
Autosomal dominant
Achondroplasia is the most common nonlethal skeletal dysplasia
with an incidence of around 5 to 15 per 100,000 births. Almost
all patients have a specific heterozygous missense mutation
p.(Gly380Arg) in the FGFR3 gene, although other disease caus-
ing mutations have also been reported. The majority of cases are
sporadic and result from a de novo paternal mutation.43 Prenatal
sonographic diagnosis is often not achieved because limb length
is preserved until around 22 weeks’ gestation when growth slows
and measurements fall below normal centiles (Fig. 34.22A).16
Presentation of de novo cases often occurs in the third trimester,
when the fetus is scanned for some other reason, and short limbs
and other features may be evident. These include relative mac-
rocephaly (Fig. 34.22B), frontal bossing (Fig. 34.22C and D)
and short fingers (trident hand) (Fig. 34.20E and F). Other fea-
tures that can occur are a small chest (Fig. 34.20G) and mild
ventriculomegaly. Increased liquor volume or polyhydramnios
(Fig. 22C) is an almost universal feature in the third trimester.16
Other complications, such as craniocervical junction compres-
sion and spinal cord stenosis, can occur in later life.44 The pre-
natal diagnosis can be confirmed in de novo cases by screening
for mutations in the FGFR3 gene in cfDNA in the maternal
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 397

80

350
Femur length (mm)

300
60

Head circumference
250
40

200
150
20

100
15 20 25 30 35 40 15 20 25 30 35 40
A Gestational age (wk) B Gestation

C D E

F G
• Fig. 34.22  Sonographic findings in achondroplasia. A, Femur length in a cohort of fetuses with achon-
droplasia compared with normal centiles. Note how length falls away from normal centiles in midpreg-
nancy. B, Head circumference in a cohort of fetuses with achondroplasia plotted on normal size centiles.
Note the relative macrocephaly with majority lie on or above the 97th centile. C, Two-dimensional (2D)
profile demonstrating the marked frontal bossing. Note the increased amniotic fluid. D, 3-D view of the
face showing the frontal bossing. E, 3-D view of the short figures. F, 2-D view of short ‘trident’ fingers
at 32 weeks’ gestation. G, View of the thorax demonstrating the narrowing of the chest compared with
the abdomen. (Charts with permission from Chitty LS, Griffin DR, Meaney C, et al. New aids for the non-
invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular
confirmation using cell free fetal DNA in maternal plasma. Ultrasound Obstet Gynecol 37:283–289, 2011.)

from 9 weeks’ gestation in future pregnancies. Although the risk Acromesomelic dysplasia type is a rare skeletal dysplasia that
for recurrence is low, parents very much welcome the reassurance disproportionately affects the forearms, lower legs, hands and feet.
this can give.45  Other characteristics include relative macrocephaly, frontal boss-
ing, bowing of the forearms and a narrow funnel chest. The condi-
Acromesomelic dysplasia. tion may be obvious at birth but more frequently becomes evident
Gene locus during the first year of life. Eventual height is below the third cen-
9p13.3: NPR2, Maroteaux type tile.18 The condition is heterogeneous, and mutations in NPR2,
20q11.22: GDF5, Hunter-Thompson type GDF5 and BMPR1B have been reported. The prenatal phenotype
4q22.3: BMPR1B, Demirhan type is not well described, which is unsurprising given the rarity of the
Autosomal recessive condition and the difficulty in making a diagnosis even in the
398 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C
• Fig. 34.23 Sonographic findings in acromesomelic dysplasia. A, Profile showing frontal bossing. B,
Short, stubby fingers. C, Sagittal view through the thorax demonstrating the relatively narrow chest.

A B C
• Fig. 34.24  Sonographic findings in diastrophic dysplasia at 14 weeks’ gestation. A, Hitchhiker thumbs.
B, Feet demonstrating the ‘hitchhiker’ great toes. C, Short, straight femur and lower leg.

postnatal period. In the authors’ experience of two cases, the sono-
graphic features are very similar to those seen in achondroplasia,
although the pattern of limb shortening in the latter is rhizomelic
compared with mesomelic in the former. Relative macrocephaly,
frontal bossing, short fingers and a small chest are seen in both
conditions (Fig. 34.23A–C), and the gestation at onset of limb
shortening appears to be similar (i.e., after 20 weeks). However, it
must be remembered that of the two, achondroplasia is by far the
most common, and thus molecular analysis by screening maternal
plasma cfDNA for mutations in the FGFR3 gene will aid defini-
tive diagnosis.9  diagnosis has to await postnatal examination. Even then, although
Diastrophic dysplasia.
Gene locus 5q32
SLC26A2 (DTDST) gene
Autosomal recessive
  
This is a severe skeletal dysplasia characterised by vari-
able degrees of rhizomelic shortening of the long bones. Bilat-
eral talipes, micrognathia and the typical ‘hitchhiker’ thumbs
(Fig. 34.24A) and toes (Fig. 34.24B) are all common findings.
Sonographic detection has been reported from 13 weeks15
(Fig. 34.24A–C) when the hitchhiker thumbs and toes with short
limbs may be very evident, but the classical features are more fre-
quently reported slightly later in pregnancy.46  prenatally, although definitive diagnosis is difficult to achieve.
Kniest dysplasia.
Gene map locus 12q13.11
COL2A1
Autosomal dominant
The inherited disorders resulting from mutations in COL2A1,
the gene for pro α2 collagen (a fibrillar collagen) form a spectrum
of phenotypes. These are collectively known as the type II col-
lagenopathies, the milder forms of which are Stickler syndrome
and familial osteoarthritis. The spectrum includes disorders
which range from the lethal or perinatal lethal disorders such as
achondrogenesis II and hypochondrogenesis through to condi-
tions with varying degrees of short stature, including SEDC and
Kniest dysplasia. Many of these conditions present prenatally with
nonspecific limb shortening of varying degrees, but the definitive
the affected individual can have features typical of a type II colla-
genopathy with platyspondyly, kyphosis, short limbs, a depressed
nasal bridge and a prominent forehead, definitive radiologic clas-
sification can be difficult.
Kniest dysplasia is an autosomal dominant condition caused by
the defective formation of type II collagen. The phenotype is vari-
able and characterised by a significant degree of disproportionate
short stature, a short trunk and small pelvis, kyphoscoliosis, short
limbs, prominent joints and premature osteoarthritis that often
restricts movement.
Characteristic craniofacial manifestations of Kniest dyspla-
sia include midface hypoplasia, cleft palate, early-onset myopia,
retinal detachment and hearing loss. This condition is manifest
Sonographic findings can be variable. Limb length around 20
weeks was on or just above the fifth centile in all four cases seen
by the authors. Two cases scanned later in pregnancy appeared
to have a small chest because of a short chest with platyspondyly
rather than short ribs, which is of interest as respiratory difficulty
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities 399

in the newborn period is a recognised feature of Kniest dyspla-
sia. Careful sonographic examination of the limbs can reveal the
metaphyseal flaring seen on postnatal radiographs. Other features
that may give a clue to the underlying condition include micro-
gnathia, a flat face and mild frontal bossing. The findings can be
relatively subtle, and definitive diagnosis usually has to await post-
natal radiology and COL2A1 analysis.  isolated, are much more likely to be associated with an underlying
Limb Deficiency or Congenital Amputations
Conditions associated with forearm defects. Forearm defects,
including radial club hand, transverse limb defects and hand
anomalies, may be associated with a variety of aetiologies,
including aneuploidy (particularly trisomy 18), teratogens
or many genetic syndromes, or they may occur as isolated
findings. It is out of the scope of this chapter to go into great
detail, and the more common of these, still rare, conditions are
given in Table 34.8. It is reasonable to suggest that if the lesion
is unilateral, no other abnormalities are seen on ultrasound and
fetal growth is normal, it is more likely than not that this is an
isolated abnormality. However, detailed fetal echocardiography is
to be recommended, and the spine should be carefully examined
to exclude hemivertebrae. Bilateral lesions, even when apparently
genetic syndrome or aneuploidy than unilateral lesions. In a
review of 52 fetuses with forearm defects, 21 had bilateral forearm
anomalies, 4 with complete absence of the forearms. Fetuses with
bilateral lesions or other abnormalities were more likely to have
an underlying genetic or chromosomal pathology (Table 34.9).47 
Conditions associated with isolated lower limb defects. Fem-
oral anomalies are rarely isolated, the majority being associated
with other limb or somatic anomalies, which may give clues to the
underlying diagnosis. If there appears to be isolated shortening
of the legs with no bowing and no other abnormalities present,

TABLE
34.8 Selected Genetic Syndromes Associated With Forearm Reduction Defects
Cardiac Other potential
Forearms Lower limbs abnormality Growth USS findings FH Other diagnostic aids
De Lange Asymmetrical Small feet +/- IUGR, micro- Diaphragmatic - Low PAPP-A
syndrome and variable cephaly hernia
forearm
reduction,
absent
fingers or
oligodactyly,
syndactyly
TAR Bilateral radial Absent or +/- Usually Absent/abnormal + (AR) Fetal platelet count
aplasia, abnormal normal humeri, flexion DNA for the common
thumbs tibia, fibula, deformities 1q12 microdeletion
always femur,
present and talipes
normal; ulna
hypoplasia
Trisomy 18 Radial club Talipes, rocker + IUGR Multiple - Fetal karyotype
Trisomy 13 hand, flexed bottom feet,
fingers, polydactyly
polydactyly,
absent
thumbs
Holt Oram Bilateral radial Normal + Normal + (AD) Examine parents
syndrome abnormalities +/- radiography
of variable of the wrists and
severity, echocardiography
absent digits
(especially
thumb)
Fanconi syn- Hypoplastic Normal +/- IUGR Cryptorchidism, + (AR) Chromosome breakage
drome thumbs, renal anomalies studies
radial
anomalies,
polydactyly
Baller-Gerold Bilateral radial Normal +/- Normal Intracerebral + (AR) Genetic consultation
syndrome defects, anomalies, renal
absent or anomalies, anal
hypoplastic atresia, cranio-
thumbs synostosis
Continued
400 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
34.8 Selected Genetic Syndromes Associated With Forearm Reduction Defects—cont’d
Cardiac Other potential
Forearms Lower limbs abnormality Growth USS findings FH Other diagnostic aids
Nager syn- Bilateral radial Normal, +/- Microceph- Micrognathia, cleft + (AD) Genetic consultation
drome defects, occasional aly lip, absent/
absent or syndactyly abnormal fibula,
hypoplastic of toes renal anomalies
thumbs,
preaxial
polydactyly
Roberts syn- Bilateral radial Hypoplastic +/- Normal Cleft lip, cystic + (AR) Fetal karyotype and
drome and ulna or absent kidneys look for chromo-
hypoplasia lower limb some puffing
bones
VATER (L) Asymmetrical Normal +/- Normal Hemivertebrae, - Polyhydramnios
radial defects renal anomalies (caused by tracheo-
esophageal atresia)
Femur fibu- Variable degrees Asymmetrical - Normal Ectrodactyly -
lar ulna of hypoplasia bowing or
of ulnae, radii hypoplasia
and humeri of lower
limbs

AD, Autosomal dominant; AR, autosomal recessive; FH, family history; IUGR, intrauterine growth restriction; PAPP-A, pregnancy-associated plasma protein A, TAR, thrombocytopenia absent radius syn-
drome; USS, ultrasound; VATER, vertebral anomalies, anorectal malformations, oesophageal atresia and renal (kidney) or radial anomalies.
Adapted from Pajkrt E, Cicero S, Griffin DR, et al. Fetal forearm anomalies: prenatal diagnosis, associations and management strategy. Prenat Diagn 32:1084–1093, 2012.
  

TABLE
34.9 Outcomes in Fetuses With Forearm Defects Seen in Two Tertiary Referral Units

Diagnosis Patients (n) Forearm defect Other skeletal anomaly IUGR Other structural anomaly
Uni Bi
Trisomy 18 10 4 6 7 6 16
Other chromosomal 1 - 1 1 - -
Cornelia de Lange syndrome 5 2 3 5 4 3
TAR 2 - 2 2 - -
Baller-Gerold syndrome 1 - 1 1 1 3
Holt Oram syndrome 1 - 1 1 - 1
Femur fibular ulna 2 1 1 2 - -
Robert syndrome 2 - 2 2 2 2
Gollop syndrome 1 - 1 1 - -
Goldenhar syndrome 1 1 - 1 - 1
VATER 2 2 - - - 5
Other or unknown syndrome 12 9 3 12 5 16
Isolated unilateral 7 7 - - 1 -
Vascular incident 1 1 - 1 - 1
Lost to follow-up 4 4 - - - -
Total 31 21 18 14

IUGR, Intrauterine growth restriction; TAR, thrombocytopenia absent radius syndrome; VATER, vertebral anomalies, anorectal malformations, oesophageal atresia and renal (kidney) or radial anomalies.
(Adapted from Pajkrt E, Cicero S, Griffin DR, et al. Fetal forearm anomalies: prenatal diagnosis, associations and management strategy. Prenat Diagn 32:1084–1093, 2012.)
  
 CHAPTER 34  Diagnosis and Management of Fetal Skeletal Abnormalities
then IUGR and constitutional short stature must be considered in
the differential diagnosis. When bowing is present and the lesion
is unilateral, the most likely diagnosis is one of focal hypoplasia,
which is at the mild end of the caudal regression spectrum. Of
note, in 10 cases with unilateral femoral reduction seen by the
authors, serial scanning showed that in most cases, the growth of
the affected limb continued at a normal velocity, albeit below the
normal centiles. This information is useful in prenatal counselling
because it allows the counsellor to predict the degree of shortening
and, in consultation with orthopaedic surgeons, advise the parents
as to the likely postnatal management. In the five fetuses seen with
bilateral, symmetrical bowing of the femora, all had skeletal dys-
plasias. In three cases, growth of other long bones was initially on
the fifth centile and then fell below this after 20 weeks. These all
had OI type III. The remaining two cases had OI type IV. Three
fetuses had bilateral asymmetrical femoral or lower leg anomalies,
and in all cases, this was an isolated finding on examination after
birth. 
General Counselling and Follow-Up Issues
The finding of a skeletal anomaly at the time of a routine scan
should stimulate a detailed examination of the rest of the fetus,
paying particular attention to the features listed in Table 34.7.
A careful and systematic approach can often lead to the identi-
fication of features that narrow the differential diagnosis and
suggest other investigations that may be useful (see Table 34.3).
Karyotyping should be considered, particularly in the presence
of other somatic anomalies or when there appears to be isolated
generalised limb shortening. In this latter case, fetal and maternal
Doppler examination should be performed because IUGR com-
monly presents with short limbs. In the majority of cases, consul-
tation with a geneticist and, in all cases with a viable condition,
401
a paediatric orthopaedic surgeon, can be of great assistance, not
just in achieving a diagnosis but also for informing the parents of
likely prognosis and postnatal management. Definitive diagnosis
may be possible in some cases prenatally after mutation screening,
karyotyping or metabolic testing. A clinical geneticist is usually
best placed to advise on the most appropriate tests.
After birth, confirmation of the diagnosis is usually made
by expert radiology. In cases ending in fetal or perinatal death,
radiographs should be obtained, and tissue should be stored for
DNA analysis, which may be placental if consent for fetal biopsy
or postmortem is declined. A skin biopsy should be taken in case
collagen studies are needed. Survivors are best seen in a multi-
disciplinary clinic with access to clinical geneticists, orthopaedic
surgeons, rheumatologists, ophthalmologists, physiotherapists,
occupational therapists and orthotics. Many children will require
input from other specialists such as cardiologists, nephrologists
and audiologists. 
Conclusions
The elucidation of the underlying pathology in a fetus with a skel-
etal anomaly is challenging but can be achieved in many cases if
a logical and systematic approach to diagnosis is used. It requires
careful attention to detail and a multidisciplinary team approach.
As more genes are identified and molecular technology improves
with availability of exome sequencing15 and NIPD based on anal-
ysis of cfDNA (see Chapter 22), it will become increasingly pos-
sible to come to a definitive diagnosis prenatally and thereby offer
accurate prognostic information to the parents.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Syngelaki A, Chelemen T, Dagklis T, et  al.
Challenges in the diagnosis of fetal non-
34. Mansour S, Hall CM, Pembrey ME, et  al. A
clinical and genetic study of campomelic dys-
chromosomal abnormalities at 11-13 weeks. plasia. J Med Genet. 1995;32:415–420.
1. Calder AD, Offiah AC. Foetal radiography for
Prenat Diagn. 2011;31:90–102. 35. Harrington J, Sochett E, Howard A. Update
suspected skeletal dysplasia: technique, nor-
18. Hall CM, Offiah AC, Forzano F, et  al. Fetal on the evaluation and treatment of osteo-
mal appearances, diagnostic approach. Pediatr
and Perinatal Skeletal Dysplasia, an Atlas of genesis imperfecta. Pediatr Clin North Am.
Radiol. 2015;45:536–548.
Multimodality Imaging. London: Radcliffe 2014;61:1243–1257.
2. Loughna P, Chitty L, Evans T, et al. Fetal size
Publishing; 2012. 36. Westgren M, Götherström C. Stem cell trans-
and dating: charts recommended for clini-
19. Mornet E, Muller F, Ngo S, et al. Correlation plantation before birth - a realistic option for
cal obstetric practice. Ultrasound. 2009;17:
of alkaline phosphatase (ALP) determination treatment of osteogenesis imperfecta? Prenat
161–167.
and analysis of the tissue non-specific ALP gene Diagn. 2015;35:827–832.
3. Chitty LS, Campbell S, Altman DG. Measure-
in prenatal diagnosis of severe hypophosphata- 37. Roughley PJ, Rauch F, Glorieux FH. Osteo-
ment of the fetal mandible—feasibility and
sia. Prenat Diagn. 1999;19:755–757. genesis imperfecta: clinical and molecular
construction of a centile chart. Prenat Diagn.
20. Comstock C, Bronsteen R, Lee W, et al. Mild diversity. Eur Cell Mater. 2003;5:41–47.
1993;13:749–756.
hypophosphatasia in utero: bent bones in a 38. Irving M, Chitty LS, Mansour S, Hall C.
4. Chitty LS, Altman DG. Charts of fetal size:
family with dental disease. J Ultrasound Med. Chondrodysplasia punctata: an aetiological
limb bones. Br J Obstet Gynaecol. 2002;109:
2005;24:707–709. classification and a clinical and radiological
919–929.
21. Valadares ER, Carneiro TB, Santos PM, et  al. review. Clin Dysmorphol. 2008;17:229–241.
5. Chitty LS, Altman DG. Charts of fetal size:
What is new in genetics and osteogenesis imper- 39. Steinberg SJ, Elçioglu N, Slade CM, et al. Per-
kidney and renal pelvis measurements. Prenat
fecta classification? J Pediatr. 2014;90:536–541. oxisomal disorders: clinical and biochemical
Diagn. 2003;23:891–897.
22. Thompson EM, Young ID, Hall CM, et  al. studies in 15 children and prenatal diagnosis in
6. Spranger J. International classification of osteo-
Recurrence risks and prognosis in severe spo- 7 families. Am J Med Genet. 1999;85:502–510.
chondrodysplasias. The international working
radic osteogenesis imperfecta. J Med Genet. 40. Basbug M, Serin IS, Ozcelik B, et al. Prenatal
group on constitutional diseases of bone. Eur J
1987;24:390–405. ultrasonographic diagnosis of rhizomelic chon-
Pediatr. 1992;151:407–415.
23. Cole WG, Dalgleish R. Syndrome of the drodysplasia punctata by detection of rhizo-
7. Superti-Furga A, Bonafe L, Rimoin D. Molec-
month. Perinatal lethal osteogenesis imper- melic shortening and bilateral cataracts. Fetal
ular-pathogenetic classification of genetic
fecta. J Med Genet. 1995;32:284–289. Diagn Ther. 2005;20:171–174.
disorders of the skeleton. Am J Med Genet.
24. Sillence DO, Senn A, Danks DM. Genetic 41. Lefebvre M, Dufernez F, Bruel AL, et al. Severe
2002;106:282–293.
heterogeneity in osteogenesis imperfecta. J Med X-linked chondrodysplasia punctata in nine new
8. Hall CM. International nosology and classifi-
Genet. 1979;16:101–116. female fetuses. Prenat Diagn. 2015;3:675–684.
cation of constitutional disorders of bone. Am J
25. Pepiin M, Atkinson M, Starman BJ, Byers PH. 42. Horikoshi T, Kikuchi A, Tamaru S, et al. Pre-
Med Genet. 2002;113:65–77.
Strategies and outcomes of prenatal diagnosis for natal findings in a fetus with contiguous gene
9. Chitty LS, Mason S, Barrett AN, et  al. Non-
osteogenesis imperfecta: a review of biochemi- Syndrome caused by deletion of Xp22.3 that
invasive prenatal diagnosis of achondroplasia
cal and molecular studies completed in 129 includes locus for X-linked recessive type of
and thanatophoric dysplasia: next-generation
pregnancies. Prenat Diagn. 1997;17:559–570. chondrodysplasia punctata (CDPX1). J Obstet
sequencing allows for a safer, more accurate,
26. Moul A, Alladin A, Navarrete C, et al. Osteo- Gynaecol Res. 2010;36:671–675.
and comprehensive approach. Prenat Diagn
genesis imperfecta due to compound hetero- 43. Wilkin DJ, Szabo JK, Cameron R, et al. Muta-
35:656–662.
zygosity for the LEPRE1 gene. Fetal Pediatr tions in fibroblast growth factor receptor 3 in
10. Drury S, Williams H, Trump N, et al. Exome
Pathol. 2013;32:319–325. sporadic cases of achondroplasia occur exclu-
sequencing for prenatal diagnosis of fetuses
27. Tavormina PL, Shiang R, Thompson LM, et al. sively on the paternally derived chromosome.
with sonographic abnormalities. Prenat Diagn.
Thanatophoric dysplasia (types I and II) caused Am J Hum Genet. 1998;63:711–716.
2015;35:1010–1017.
by distinct mutations in fibroblast growth fac- 44. Hunter AGW, Bankier A, Rogers JG, et  al.
11. Vincent A, McConville J, Farrugia ME, et al.
tor receptor 3. Nat Genet. 1995;9:321–328. Medical complications of achondroplasia:
Antibodies in myasthenia gravis and related
28. Chitty LS, Khalil A, Barrett AN, et  al. Safer, a multicentre patient review. J Med Genet.
disorders. Ann N Y Acad Sci. 2003;998:324–
accurate prenatal diagnosis of thanatophoric 1998;35:705–712.
335.
dysplasia using ultrasound and cell free fetal 45. Lewis C, Hill M, Chitty LS. Non-invasive pre-
12. Upadhyay K, Thomson A, Luckas MJ. Con-
DNA. Prenat Diagn. 2013;33:416–423. natal diagnosis for single gene disorders: experi-
genital myotonic dystrophy. Fetal Diagn Ther.
29. Elçioglu NH, Hall CM. Diagnostic dilemmas ence of patients. Clin Genet. 2014;85:336–342.
2005;20:512–514.
in the short rib-polydactyly Syndrome group. 46. Wax JR, Carpenter M, Smith W, et al. Cartin
13. Kozlowski K, Basel D, Beighton P. Chondro-
Am J Med Genet. 2002;111:392–400. A Second-trimester sonographic diagnosis of
dysplasia punctata in siblings and maternal
30. Chen CP, Chang TY, Chen CY, et  al. Short diastrophic dysplasia: report of 2 index cases.
lupus erythematosus. Clin Genet. 2004;66:
rib-polydactyly syndrome type II (Majewski): J Ultrasound Med 22:805–880, 20038.
545–549.
prenatal diagnosis, perinatal imaging findings 47. Pajkrt E, Cicero S, Griffin DR, et  al. Fetal
14. Chitty LS, Tan AW, Nesbit DL, et  al. Sono-
and molecular analysis of the NEK1 gene. forearm anomalies: prenatal diagnosis, associa-
graphic diagnosis of SEDC and double hetero-
Taiwan J Obstet Gynecol. 2012;51:100–105. tions and management strategy. Prenat Diagn.
zygote of SEDC and achondroplasia-a report
31. Mansour S, Offiah AC, McDowall S, et al. The 2012;32:1084–1093.
of six pregnancies. Prenat Diagn. 2006;26:
phenotype of survivors of campomelic dyspla-
861–865.
sia. J Med Genet. 2002;39:597–602.
15. Khalil A, Pajkrt E, Chitty LS. Early prenatal
32. Hill M, Finning K, Martin P, et  al. Non-
diagnosis of skeletal anomalies. Prenat Diagn.
invasive prenatal determination of fetal sex:
2011;31:115–124.
translating research into clinical practice. Clin
16. Chitty LS, Griffin DR, Meaney C, et al. New
Genet. 2011;80:68–75.
aids for the non-invasive prenatal diagnosis of
33. Everett T, Chitty LS. Cell-free fetal DNA: the
achondroplasia: dysmorphic features, charts of
new tool in fetal medicine. Ultrasound Obstet
fetal size and molecular confirmation using cell
Gynecol. 2015;45:499–507.
free fetal DNA in maternal plasma. Ultrasound
Obstet Gynecol. 2011;37:283–289.

401.e1
35
Diagnosis and Management of
Abnormalities of the Face
ELISABETH DE JONG-PLEIJ AND CATERINA M. BILARDO
KEY POINTS
• The fetal face can be visualised by ultrasound from 9 weeks’
gestation onwards.
• After 9 weeks’ gestation, only proportional changes occur in
the fetal face.
• Clefts and micrognathia are the most common facial anoma-
lies.
• In many genetic disorders, the face has a deviant appearance.
• At the routine anomaly scan the profile, the lip and the eyes
should be examined.
• A detailed ultrasound investigation in high-risk patients
requires complete assessment of the face in all three orthogo-
nal planes.
Introduction
Of all ultrasound views, the fetal face is highly appreciated by par-
ents and frequently imaged during ultrasound examinations.
The face is anatomically and functionally a complex structure
which poses several challenges for prenatal imaging. The complex-
ity is caused by its particular varied three-dimensional morphol-
ogy and curved nature. Furthermore, the face includes various
sensory organs, each with a function of vital importance.
A lot of information can be obtained by ultrasound exami-
nation of the fetal face. Besides obvious and clinically relevant
anomalies, such as clefts or microphthalmia, changes in shape,
subtle dysmorphic features or markers, can provide clues to spe-
cific genetic syndromes. Facial anomalies are frequently associated
with other anomalies or part of syndromes or sequences.1,2 The
finding of a facial anomaly therefore requires a thorough examina-
tion of the entire fetus. In high-risk patients or when anomalies
are found, a facial segment-specific analysis should be performed
in all three orthogonal planes.
The first characteristics of the fetal face become visible at 9
weeks’ gestation (postmenstrual age) when the jawbones become
ossified (Fig. 35.1). After 9 weeks’ gestation, growth and devel-
opment of the face are dominated by proportional changes and
changes in the relative positions of the facial elements until long
after birth. Imaging and diagnosis of some facial anomalies such as
bilateral clefts and severe variants of retrognathia are possible from
11 weeks’ gestation, but with a high-resolution equipment and
402
transvaginal approach, they may also become visible at an earlier
stage.
In each trimester, the face has specific characteristics (Fig. 35.2).
The first trimester face has a triangular shape. The orbital plane
surmounted by the relatively large neurocranium forms the base
and the two rami of the mandible, the sides, ending in the pointed
chin. Between 16 and 36 weeks’ gestation, the facial height grows
relatively more than the width, especially before 25 weeks’ ges-
tation.3 In the third trimester, the face looks round because of
subcutaneous fat accumulation, especially below the zygomatic
bones, and because of the widening of the mandible.4 As opposed
to the adult face, the fetal face is relatively small, which is the
result of the rudimentary upper and lower jaws, the small nasal
cavities and sinuses and the unerupted primary teeth.
When looking at the midsagittal view, the fetal profile (FP) is
concave throughout gestation as opposed to the relative flat profile
in postnatal life (Fig. 35.3).5 The concavity, mainly caused by the
rather retrognathic position of the small mandible, with respect
to the maxilla, is probably meant to facilitate for the newborn the
concomitance of sucking movements and breathing during breast
feeding. 
Ultrasound Investigation of the Fetal Face
The International Society of Ultrasound in Obstetrics and
Gynecology (ISUOG) has published guidelines for the routine
midtrimester ultrasound scan. The recommended minimum
requirements for basic midtrimester anatomical examination of
the fetal face should include an attempt to visualise the upper lip
for exclusion of cleft lip. If technically feasible, other facial fea-
tures that can be assessed include the median facial profile and
evaluation of the orbits, nose and nostrils.6 Many countries have
developed guidelines for the routine midtrimester scan, usually
recommending visualisation of the profile, eyes and lips. In a
screening setting, the midsagittal (profile) plane, the coronal plane
(orbits) and the anterior coronal (nose–mouth) plane are usually
sufficient to visualise these structures with two-dimensional (2D)
ultrasound (Fig. 35.4).
A median facial profile provides important diagnostic clues for
the diagnosis of cleft lip, bossing or sloping forehead, microgna-
thia and nasal (bone) anomalies. The coronal or axial views can
be used to assess the orbits that should appear symmetrical and
intact. The distance between the orbits is about the same as the
diameter of one orbit. The symmetrical nose and nostrils, mouth
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face
and lips are evaluated in an anterior oblique coronal view to detect
in particular cleft lip.
When anomalies are encountered at a routine scan, the preg-
nant woman is commonly referred to a fetal medicine unit for a
more detailed ultrasound examination. In this setting, examination
of the fetal face usually starts with a subjective evaluation with 2D
ultrasound. A more systematic examination of the fetal face should
include sagittal, axial and coronal planes. In the midsagittal plane
profile view, the forehead, nasal bones, prenasal thickness, soft tissue
of the nose, philtrum, tongue, palatal bone, vomer, lower lip and
chin can be evaluated, and clear anomalies or dysmorphic features
may be observed. The oropharynx with the uvula (equal sign) can
be informative and indicate for the existence of a cleft palate. The
equal sign can also be obtained in both the axial or coronal planes.7
In the paramedian sagittal planes the orbits, eyelids, lenses and the
ears can be visualised. Of the coronal planes, the slightly tilted nose-
mouth plane is the most used for evaluation of the nose (tip, alae nasi
and nostrils), upper lip and mouth. More tilted views, towards the
deeper structures of the face, allows for visualisation of the maxilla,
palate, both eyelids, orbits with lenses and frontal bones. Serial axial
images of the fetal face are particularly useful to analyse the maxilla
and mandible with the tooth buds but also to view the orbits and
• Fig. 35.1  Two-dimensional ultrasound image of an embryo of 9 weeks
and 4 days showing the jawbones as bright with spots.
A B
• Fig. 35.2  Three-dimensional rendered ultrasound images of a first (A), second (B) and third trimester
(C) fetus.
403
the lenses, cheekbones, choanae, lips and tongue. A detailed facial
segmental analysis is presented later in this chapter.
The use of three-dimensional (3D) ultrasound examination is
the next step. This technique has been a major breakthrough in
the study of the fetal face and has given rise to a new discipline
called ‘fetal dysmorphology’ (see later discussion).
Suspected anomalies can be validated by objective measures of the
craniofacial features at a single point in time as well serially at different
gestational ages. The combination of typical facial features combined
with additional findings such as associated structural anomalies affect-
ing all organs but especially the central nervous system (CNS), poly- or
oligohydramnios, fetal size, information obtained from family history
and a genetic workup can lead to recognition of a known syndrome. 
Three-Dimensional Ultrasound of the Fetal
Face
Three-dimensional ultrasound is especially useful in demonstrat-
ing curved structures (Fig. 35.5) and surface malformations (Fig.
35.6) and improves accurate topographic depiction of structures.
Therefore, this technique is a major addition in the assessment of
the fetal face.8,9
In 2D ultrasound, a single plane is imaged at a time, and the
sonographer has to construct in her or his mind the complex 3D
anatomy of the face. 3D ultrasound enables to collect a volume
of ultrasound information consisting of multiple 2D slices. The
A
M
M
B
A B
• Fig. 35.3  Prenatal two-dimensional ultrasound image of a fetus (A) with
the maxilla–nasion–mandible angle (MNM) and postnatal cephalogram of
a child (B) with point A–nasion–point B angle (ANB); the MNM angle is
larger than the ANB angle, indicating a more convex profile in prenatal life.
C
404 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
examination starts by placing a box (region of interest) of variable
size on the 2D image of the object that we want to investigate.
Then a sweep is produced by a motor within the probe, whereby all
the adjacent 2D slices within the box are stored. When the fetus is
quiet, a slow sweep can be used, which improves spatial resolution.
Ideally, there should only be amniotic fluid in front of the face and
no other structures such as limbs. All stored sectional planes are
integrated to form the volume. After the volume is digitally stored,
the volume can be manipulated by modifying the colours and the
image settings and by using different modes. These options can
extract different information from the same dataset. Volumes of
the fetal face are usually analysed by using cross-sectional images
through the volume or by rendered images. Both approaches
improve understanding of the complex anatomy of the face; cross-
sectional imaging by enhancing spatial awareness and rendering
imaging by facilitating a lifelike 3D view of the face (see Fig. 35.5).
Cross-Sectional Imaging
When cross-sectional imaging is used, usually the three orthogonal
planes are displayed simultaneously, named multiplanar imaging
(Fig. 35.7). The ultrasound volume of the fetal face can be rotated
and reviewed millimetre by millimetre by scrolling through the
volumes.
A B
• Fig. 35.4  Ultrasound images showing the median profile view (A), the orbits in an axial view (B) and an
anterior oblique coronal nose–mouth view (C).
• Fig. 35.5  Three-dimensional surface-rendered image with high definition
technique of a normal second trimester fetus, showing a symmetrical face;
clearly an intact upper lip; and a sharp, definable cupid’s bow.
It is advisable to turn the face in the standard position with the
coronal view in the upper left box, the profile view with the nose
pointing to the left in the upper right box and the axial view with
the nose pointing downwards in the lower left box (see Fig. 35.7).
In this way, it is clear what is the right and left side of the fetus,
and using standard positions improves subjective pattern recogni-
tion. The reference dot, which marks the intersection of the three
orthogonal planes, is very helpful in identifying structures.
Multiplanar imaging is extremely helpful in defining the exact
midsagittal plane. When examining the fetal profile (FP) subjec-
tively, the true midsagittal plane is usually assumed to be present.
However clear landmarks to define the exact midsagittal plane
are missing. It is shown that presumed 2D profile images are
significantly oblique in 30% of cases.10 The multiplanar imaging
of 3D ultrasound provides the ultrasonographer with a unique
tool: the possibility to visualise contemporarily the three orthog-
onal planes. A deviation from the exact midsagittal plane is easily
recognised and can be corrected to the true midsagittal plane.
Evaluation of the profile in an incorrect midsagittal plane can
lead to diagnostic inaccuracies. For example, in a view deviating
from the midsagittal plane, the most protruding part of the chin
will not be visualised, creating the impression of retrognathia.
Also, the forehead will suggest more bossing and the nose will
seem small (Fig. 35.8).
C
• Fig. 35.6  Three-dimensional surface-rendered image of fetus with naso-
frontal encephalocele (arrow).
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 405

R L

A B
C

R L

• Fig. 35.7  Example of multiplanar imaging with the fetal face turned in
the standard position. R is the right side, and L is the left side of the fetus.
A B
• Fig. 35.8  Ultrasound images of the fetal profile. A, A deviating midsagit-
tal profile view, creating an image of retrognathia, flat nose and slightly
bossing forehead. B, The exact midsagittal profile view obtained with mul-
tiplanar imaging of the same fetus, showing a normal profile.

• Fig. 35.9  Example of tomographic ultrasound imaging display of the fetal face.

Another way to display a volume cross-sectionally is by tomo-

graphic ultrasound imaging or the ‘multislice method’. Several
slices of a volume, which are parallel to each other, are simultane-
ously displayed with predefined number and spacing of the slices
(Fig. 35.9). This offers the examiner a more complete picture. The
mandible and maxilla, for example, can be viewed at the same
time. 

A B
• Fig. 35.10 Three-dimensional rendered frontal view of the fetal face
before (A) and after (B) use of the electronic scalpel to remove the hand in
front of the face.
Rendered Images
In rendering mode, the image includes information from the
entire volume. The images from the three orthogonal planes are
combined to obtain a realistic 3D picture (see Fig. 35.5). It is
possible to turn and rotate the volume and view the volume from
various positions. Technical options such as the electronic scalpel
used to remove unwanted structures can improve the image qual-
ity and the diagnostic value of the ultrasound examinations (Figs.
35.10 and 35.11).
406 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C
• Fig. 35.11  Three-dimensional rendered frontal view of the face with high definition technique (A). Half of
the picture is deleted with the electronic scalpel (B). After turning the head around the y-axis of the fetus,
the profile is visible against a clear black background (C).

A B C D E
• Fig. 35.12  A–E, Three-dimensional rendered ultrasound pictures in several settings showing various
facial expressions.

By choosing various threshold values, the rendered volume
can be studied in a variety of ways. In the surface view, the
outer surface of the fetus is highlighted. The fetus is displayed
as a 3D sculpture. There are various technical options for edit-
ing this sculpture, for example, adjusting the gain or the posi-
tion of the light or using high-definition techniques that provide
skin-like pictures with sharp contrasts (Fig. 35.12). By observing
the fetal face in rendering mode, we will have a general subjec-
tive visual impression comparable with the ‘gestalt’ approach of
clinical genetics, offering the possibility to suspect dysmorpholo-
gies, especially later in pregnancy (Fig. 35.13). Rendering mode
is also helpful in assessing tumours, clefts and ear anomalies. In
the maximum mode, the bones are emphasised. The strongest
echoes are kept, and the echoes from the soft tissue are eliminated
(Fig. 35.14). This especially allows visualisation of curved skeletal
structures such as sutures and fontanels of the skull, hard palate
and nasal bones.
Finally, the possibility to store 3D ultrasound volumes
and edit them offline facilitates communication and research.
Realistic 3D images improve communication with the parents
and health professionals involved in the management of the
pregnancy.
Four-dimensional (4D) ultrasound added the fourth dimen-
sion ‘time’ to the 3D picture. This dramatically improved
dynamic assessment of the fetal face. Movements of the mouth
(yawning), tongue, eyelids and lenses can be visualised with 2D,
but with 4D ultrasound, these movements can be visualised with
greater ease and more details. Complex movement as seen in
facial expression can be observed (see Fig. 35.12). 4D ultrasound
might be an important modality for future prenatal behaviour
studies. 
Facial Clefts
Of all congenital anomalies involving the face, clefts are the most
common, affecting about 1 or 2 per 1000 live births.11 The inci-
dence of facial cleft varies by gender, ethnicity and geographic
location, being more frequent in boys (twice as much as in girls),
in Asians and in central–north Europe compared to the Euro-
pean average.11 In contrast, isolated cleft palate occurs equally
often in all races (about 4 in 1000 births) and is more common
in females. The distribution of clefts is estimated to be 25% for
cleft lip, 50% for cleft lip and palate and 25% for isolated cleft
palate.12,13
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face
A B
• Fig. 35.13 Surface-rendered images of syndromic fetuses, trisomy 21 (A), Binder syndrome (B) and
Apert syndrome (C). (Courtesy Dr B. Benoit)
A B
• Fig. 35.14  Examples of three-dimensional rendered ultrasound pictures of a normal face using surface
(A) and maximum (B) mode.
Facial clefts may either be isolated or associated with other
anomalies. The incidence of associated structural anomalies, chro-
mosomal aberrations (mainly trisomy 13 and 18) or an underlying
genetic syndrome or sequence varies with the anatomical cleft type.
Therefore adequate characterisation of the cleft is important not
only to counsel parent appropriately on the severity of the defect
but also on its likely association with chromosomal or syndromal
anomalies. Isolated clefts are associated with low mortality and
morbidity rates and are primarily an aesthetic and functional prob-
lem. The percentage of isolated cases is the highest in the cleft lip
(without a defect in the alveolar ridge or palate) and close to 0%
in midline and atypical cleft groups. Bilateral clefts have lower per-
centages of isolated cases than unilateral clefts. The most frequent
associated defects are musculoskeletal anomalies (polydactyly and
limb reductions) followed by malformations of the CNS and mal-
formations of the cardiovascular system. For all clefts grouped
together, the reported incidence of isolated clefts varies between
31% and 71%.11,14 It should be noted that the incidence in reality
may be lower because of unidentified syndromes or late manifesta-
tion of developmental delays (e.g., learning difficulties). About 350
syndromes are associated with facial clefting.13,15
The most common syndromes and sequences identified after
birth in cleft patients (including isolated cleft palate) are Pierre
Robin sequence, Van der Woude syndrome, Stickler syndrome,
CHARGE (coloboma, heart defects, atresia choanae, retardation
of growth, genital abnormalities, and ear abnormalities) associa-
tion, ectrodactyly-ectodermal dysplasia-cleft syndrome, Goldenhar
syndrome, Apert syndrome, Treacher Collins syndrome, Wolf-
Hirschhorn syndrome, velocardiofacial syndrome (22q11deletion),
407
C
Smith-Lemli-Opitz syndrome, Fryns syndrome and oral-facial-dig-
ital syndrome.
In a small percentage of cases, facial clefts are atypical and occur in
different regions of the face. According to the well-established Tessier
classification, the defects are numbered from 0 to 14 and classified
based on the anatomical localisation of the cleft, with the orbit as refer-
ence structure.16 The most common atypical facial cleft found at birth
is Tessier 7, a lateral cleft between the corner of the mouth and the ear.
When there is suspicion of a fetal cleft with associated anom-
alies the parents should be referred for genetic counselling and
additional genetic investigations. Usually a multidisciplinary team
consisting of a medical specialist (fetal medicine), a plastic sur-
geon (ear, nose and throat) and a specialised nurse or social worker
will explain the surgical options, aesthetic outcomes and specific
feeding needs of a newborn with a cleft lip or palate and will take
care of the psychological impact of the anomaly for the family.
The recurrence risk for isolated cases is 5% if one sibling or par-
ent is affected and 10% if two siblings are affected. In syndromic
cases, all kinds of inheritance have been described (autosomal
dominant, autosomal recessive, X-linked).
Ultrasound Examination of Facial Clefts
In the first trimester, the following findings are clues to the pres-
ence of a facial cleft: the premaxillary protrusion (Fig. 35.15), an
abnormal maxilla–nasion–mandible (MNM) angle,17 an abnor-
mal retronasal triangle18 (Fig. 35.16A) and the maxillary gap19
(Fig. 35.15 and 35.16B). Also, an enlarged nuchal translucency
increases the risk for facial clefts.20
408 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B
• Fig. 35.15  Two- and three-dimensional images of a first trimester fetus with bilateral clefts, premaxillary
protrusion, maxillary gap and micrognathia. A, Sagittal 2D scan. B, 3D rendered image.

A B
• Fig. 35.16  A, Coronal view (posterior to the nose) of a normal first trimester fetus, showing a normal
retronasal triangle with intact palate (arrow) and a normal mandibular gap (asterisk). The retronasal triangle
is formed by three echogenic lines formed by the two frontal processes of the maxilla and the palate. B,
Sagittal view of a normal first trimester fetus, showing an intact palate and no maxillary gap (arrow).

A B C
• Fig. 35.17  Two-dimensional image of frontal nose–mouth view showing an intact upper lip (A), a unilat-
eral cleft lip (B) and a bilateral cleft lip (C).

The ultrasonographic assessment usually starts by 2D ultra-
sound examination. The slightly tilted coronal nose–mouth view
is an important and obligatory plane for the diagnosis of facial
clefts. In this plane, the nostrils, philtrum and upper lip can be
evaluated (Fig. 35.17).
In the axial plane, the alveolar ridge and the upper lip can also
be evaluated (Fig. 35.18).
In the midsagittal plane, the premaxillary protrusion can be
seen, especially in bilateral clefts, although all types of clefts show
some protrusion of the premaxilla except when there is an intact
alveolar ridge21 (Fig. 35.19). The protrusion is caused by the miss-
ing restraining effect of an intact musculus orbicularis oris, and its
severity can be quantified with the MNM angle (see Fig. 35.18).
In unilateral clefts, the profile may look flat because of missing soft
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 409

tissue (in front of the maxilla) and because the muscularis orbicu-
laris oris pulls the lip to the noncleft side. (Fig. 35.18A).
Prenatal diagnosis of a cleft palate is a challenge because of the
shadows produced by surrounding osseous structures interfering
with a good visualisation of the palate. When the fetal head is tilted
backwards and the mouth is slightly open, the whole palate can
be visualised from the hard palate to the uvula (soft palate). Care
has to be taken to scan in the exact midsagittal plane (Fig. 35.20).
If the fetus makes breathing movement, adding colour may
reveal a cleft in the palate by showing bidirectional flow of amni-
otic fluid over the palate.
The equal sign described by Wilhelm is an important marker
for an intact uvula7 (Fig. 35.21). It can be visualised in in all three
planes, and its visualisation proves an intact palate. If the uvula
cannot be visualised in its typical presentation (equal sign), there
should be a strong suspicion of a cleft palate.7
There were great expectations for the role of 3D ultrasound
in the diagnosis of facial clefts, especially in the challenging diag-
nosis of the palate. 3D ultrasound does not seem to improve the
detection rate of cleft lip and palate; however, a more precise and
reliable diagnosis can be achieved. For example, rendered images
of the face can show unequivocally that the upper lip is intact (see
Fig. 35.5) or may be useful in defining the extent of the cleft(s)
in the lip (complete or incomplete, uni- or bilateral) (Fig. 35.22).
Several techniques have been proposed to improve detection of
especially cleft palate.
First the ‘reverse-face’ view was introduced by Campbell.22
With this technique, a frontal 3D-rendered view of the face is
obtained and rotated 180 degrees around the y-axis of the fetus.
Then by looking from the oropharynx (from inside out), the
palate, tongue, nasal cavity and orbits can be seen by scrolling
through the volume (Fig. 35.23).

• Fig. 35.18 Two-dimensional image of axial plane through maxilla with • Fig. 35.20  Two-dimensional ultrasound picture of a fetus with a back-
unilateral cleft lip and palate. Arrow indicates the cleft. ward-tilted head and open mouth, showing an intact hard and soft palate.

A B C
• Fig. 35.19  Ultrasound pictures of a case with isolated unilateral cleft lip and palate (A), a case with tri-
somy 13 and bilateral cleft lip and palate (B) and a case with an isolated bilateral cleft lip and palate (C). In
all three cases, the maxilla–nasion–mandible angles were enlarged.
410 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Hereafter different strategies using 3D volumes have been
developed to visualise the palate; these techniques include the
‘flipped-face’ view23 (Fig. 35.24), the ‘flipped face’ view with
angled insonation,24 the anterior axial 3D view reconstruc-
tion25 and variants of these. They differ mainly in starting plane,
insonation angles, the way the volume is rotated and the position
of the view bar. The best images are obtained when the head of the
fetus is slightly extended, the mouth is open and amniotic fluid
is present in the oral cavity (see Fig. 35.20). In everyday practice,
fetuses have very different positions, and the image quality differs
in each patient. Therefore the best strategy must be considered on
a case-by-case basis.
Recently, new 3D tools such as the ‘polyline’ enable tracing a
line along the curved 2D sagittal view of the palate and to obtain
a rendered image of it (Fig. 35.25). Also in these cases, it is impor-
tant that the fetus has a slightly open mouth and that the palate
lies as much as possible perpendicular to the scanning plane.  nonchromosomal syndromic conditions (e.g. Meckel-Gruber
• Fig. 35.21 Coronal view through the pharynx showing the equal sign
(circle) cranial to the vocal cords (arrows).
A B
• Fig. 35.22 Three-dimensional frontal rendered views, showing an incomplete unilateral cleft lip
(A), a complete unilateral cleft lip (B) and a bilateral complete cleft lip and palate with protrusion of the
premaxilla (C).
Micrognathia
Micrognathia refers to a hypoplastic mandible. In retrognathia,
the anteroposterior axis is mostly affected. Both conditions usu-
ally coexist, but frequently only the term micrognathia is used to
refer to the combination micrognathia and retrognathia. After
facial clefts, micrognathia is the most common congenital facial
anomaly. Micrognathia can be isolated but is often associated.
Conditions presenting micrognathia can be categorised in syn-
dromic conditions primarily involving the mandible (e.g. Pierre
Robin sequence, Treacher Collins syndrome, acrofacial dysostosis,
orofaciodigital syndromes), skeletal and neuromuscular diseases
(e.g. Pena–Shokeir syndrome, multiple pterygium syndrome,
achondrogenesis, campomelic dysplasia), chromosomal aberra-
tions (e.g. trisomy 18, trisomy13, triploidy, cri du chat syndrome,
Wolf-Hirschhorn syndrome, Pallister Killian syndrome) and other
syndrome, Fryns syndrome, Goldenhar syndrome, Peters plus
syndrome). A search in the OMIM website (Online Mendelian
Inheritance in Man, a database of human genes and genetic dis-
orders)26 retrieved 564 hits for ‘micrognathia’ and 180 for ‘ret-
rognathia, (in 2017). Table 35.1 summarises publications on the
incidence of associated conditions. The very low percentage of iso-
lated cases illustrates the severity of this facial anomaly, although
it is likely that mild isolated cases escape prenatal identification.
The search for associated anomalies is therefore paramount
when micrognathia is diagnosed. The prognosis is usually dismal
when micrognathia is not isolated.32 The neonatal mortality rate
is more than 80%, usually because of associated anomalies. In
isolated Pierre Robin sequence the survival rate is generally high.
Ultrasound follow-up should be performed to detect polyhydram-
nios due to glossoptosis and swallowing problems. Genetic inves-
tigations should always be discussed with the parents. Delivery
should be planned in a tertiary centre because in some cases, airway
obstruction may create an emergency, and intubation of the new-
born or another intervention by an ear, nose and throat specialist
can be necessary.
The recurrence risk in isolated cases is close to 0%. In syn-
dromic cases, it depends on the genetic background.
Ultrasound Examination of Micrognathia
Severe forms of micrognathia may be recognised in the profile view
in the first trimester (Fig. 35.26). The absence of the mandibular
gab in the frontal retronasal triangle view (Fig. 35.16A) may also
C
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 411

• Fig. 35.23 Three-dimensional ultrasound image of reversed-face view

showing an intact palate (arrow) above the tongue (asterisk). • Fig. 35.25  Three-dimensional rendered image of intact hard palate and
alveolar ridge.

• Fig. 35.24  Three-dimensional multiplanar image with flipped-face technique of a fetus with an intact
palate. The profile image in the left upper quadrant is turned upside down. The render box is adjusted
to the size of the palate. In the box in the right lower quadrant, a rendered image of the palate is visible.
412 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Overview of Published Data on the Incidence of Associated Structural Anomalies, Chromosomal Anomalies,
35.1 Skeletal or Neuromuscular Diseases, Syndromes or Sequences and Isolated Cases in Fetuses With
Micrognathia
Skeletal or
Reference Structural anomalya Chromosomal aberration neuromuscular disease Syndrome or sequence Isolated
Nicolaides et al.27 10/56 18% 37/56 66% 6/56 11% 2/56 4% 1/56 2%
(1993)
Turner and Twining28 1/9 11% 3/9 33% 4/9 44% 1/9 11% 0/9 0%
(1993)
Bromley and 3/20 15% 5/20 25% 4/20 20% 7/20 35% 1/20 5%
Benacerraf29
(1994)
Vettraino et al.30 1/58 2%
(2003)
Basburg et al.31 2007 5/32 22% 7/32 22% 11/32 33% 7/32 22% 2/32 6%
Paladini32 (2010) b 22/50 44%
Summary and mean 19/117 16% 74/167 44% 25/117 21% 17/117 14% 5/175 3%
percentage
aFetuses with structural anomalies without chromosomal aberrations, syndromes, sequences or skeletal or neuromuscular disease.
bOnly chromosomal aberrations were stated.
  

NT
A B

• Fig. 35.26  Two-dimensional image of a fetus with retrognathia (arrow) at • Fig. 35.27 Two-dimensional images of fetuses with retrognathia. A, A
12 weeks’ gestation. fetus with Smith-Lemli-Opitz syndrome. B, A fetus with Nager syndrome.
(Note also the sloping forehead.)

be a hint to the diagnosis of retrognathia in the first trimester. In
case of retrognathia, the mandibular gap is absent and replaced by
a bony structure representing the receding mandible.33
The ultrasound diagnosis of micrognathia is usually made
in the profile view, with 2D ultrasound (Fig. 35.27) or 3D
ultrasound (Fig. 35.28), in which the retracted and small man-
dible is visible. In severe cases, a hanging upper lip may be visible.
In the axial plane, the angle between the two bodies of the man-
dible is enlarged, creating a flattened round shape of the mandible
(Figs. 35.29 and 35.30).
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 413

A B
• Fig. 35.28  Three-dimensional surface-rendered images of a fetuses with retrognathia. A, Cri du chat
syndrome. B, Pierre Robin sequence. (Courtesy Dr B. Benoit.)

A B
C

• Fig. 35.29  Three-dimensional multiplanar image of a fetus with a lethal skeletal dysplasia at 16 weeks’
gestation showing retrognathia in box B with an enlarged maxilla–nasion–mandible angle. Note also the
flattened round shape of the mandible in box C.

For the diagnosis or exclusion of micrognathia, it is of vital

• Fig. 35.30 Two-dimensional axial ultrasound image showing the flat-
tened round shape of the mandible in a fetus with retrognathia.
importance to define with certainty the exact midsagittal plane. 3D
multiplanar ultrasound is ideal for this purpose (see Fig. 35.29).
Biometrical tools can be helpful in supporting the diagnosis
or in ambiguous cases. The first who provided prenatally objec-
tive measurements of the mandible were Otto and Platt in 1991
followed by Chitty and coworkers in 1993.34,35 All these authors
measured one body of the mandible in an axial scan plane parallel
to the mandible from the symphysis mentis to the temporoman-
dibular joint. Three authors used the same plane to measure man-
dibular depth and width.36-38 Paladini and coworkers introduced
the jaw index: anteroposterior diameter/biparietal diameter (BPD) ×
100. Using a cutoff value of 23, the jaw index showed greater diag-
nostic accuracy than subjective evaluation.37 In the latest studies,
angular measurements in the midsagittal plane are used to define
the relative position of the mandible. The inferior facial angle, for
example, is constant at 65.5 degrees (standard deviation [SD],
8.13 degrees) between 18 and 28 weeks’ gestation39 (Fig. 35.31).
414 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
The only angle using only bony landmarks is the MNM angle,
defined as the angle between the lines maxilla–nasion and mandi-
ble–nasion in the exact median plane (see Figs. 35.3A and 35.29).
The nasion is defined as the most anterior point at the intersection
of the frontal and nasal bones. Jaw landmarks were defined as the
middle points of the anterior borders of the maxilla and mandible.
Calipers are placed on the outermost borders of the bone. The
IFA
• Fig. 35.31  Two-dimensional image showing the inferior facial angle (IFA;
31 degrees) in a fetus with retrognathia. The IFA is defined as the angle
formed by the line orthogonal to the vertical part of the forehead at the
level of the synostosis of the nasal bones and the line joining the tip of the
mentum and the anterior border of the more protruding lip.
N
M
A B
• Fig. 35.32  Maxilla–nasion–mandible angle (MNM) angle in a normal fetus (A) and an enlarged MNM
angle in a fetus with Pierre Robin sequence (B).
mean MNM angle is 13.5 degrees and does not change signifi-
cantly during pregnancy. The 5th and 95th centiles are 10.39 and
16.91 degrees, respectively.5 In retrognathia, the MNM angle is
enlarged. (Fig. 35.32). 
Facial Segmental Analysis
Forehead
Although the forehead is part of the neurocranium and not of the
viscerocranium (facial bones), the forehead is discussed here because
the two are anatomically closely interwoven. The shape of the fore-
head is an important component in the evaluation of the FP. In
addition, anomalies of the neurocranium such as craniosynostosis,
macrocephaly and microcephaly influence the shape of the face.
Faro and colleagues described the morphology of the frontal
bones and metopic suture between 9 and 34 weeks of gestation
using 3D ultrasound.40 At 9 weeks’ gestation, small ossification
centres are visible, and by 11 weeks’ gestation, the frontal bones
appear as ‘thick eyebrows’. In the second trimester, the metopic
suture becomes delineated, and closure starts at 32 weeks’ ges-
tation moving upwards from the glabella (Fig. 35.33). From
16 to 18 weeks onwards, the frontal bone is visible in the pro-
file view, indicating that the ultrasound beam is wider than the
metopic suture. If the bony forehead is not depicted in the profile
view, a pathologically wide metopic suture should be suspected
(Fig. 35.34). Chaoui and associates reported on four patterns of
abnormality in the metopic suture in association with fetal mal-
formations during the second and third trimesters of pregnancy:
delayed closure, premature closure, a U-shaped open suture and
the presence of additional bone between the frontal bones, the
so-called wormian bones.41
Faro and coworkers investigated the development of the
frontal bones and metopic suture in normal fetuses and fetuses
with holoprosencephaly at 11 + 0 to 13 + 6 weeks of gestation.
N
M
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 415

9 wks, CRL 28.0 mm 11 wks, CRL 45.1 mm 12 wks, CRL 56.1 mm 13 wks, CRL 70.2 mm

14 wks, BPD 30.4 mm 15 wks, BPD 33.8 mm 16 wks, BPD 35.4 mm 18 wks, BPD 40.6 mm

20 wks, BPD 48.6 mm 22 wks, BPD 54.4 mm 24 wks, BPD 59.9 mm 26 wks, BPD 66.4 mm

28 wks, BPD 73.8 mm 30 wks, BPD 81.6 mm 32 wks, BPD 85.1 mm 34 wks, BPD 89.4 mm
• Fig. 35.33  Fetal face in coronal view rendered with a combination of transparent maximum and sur-
face texture modes of display of 16 normal fetuses from 9 to 34 weeks of gestation. In each case, the
crown–rump length (CRL) or biparietal diameter (BPD) is given. (From Faro C, Benoit B, Wegrzijn P, et al.
Three-dimensional sonographic description of the frontal bones and metopic suture. Ultrasound Obstet
Gynecol 26:618–621, 2005.)

Holoprosencephaly is associated with an accelerated development
of the frontal bones and premature closure of the metopic suture.42
Bossing and sloping foreheads. Bossing and sloping foreheads
are markers for severe underlying conditions. Unfortunately,
both markers are subtle in the first half of pregnancy but become
increasingly evident during pregnancy. Frontal bossing occurs in
more than 50 syndromes, such as craniosynostosis syndromes and
skeletal dysplasias.1 A sloping forehead is usually a sign of a devel-
oping microcephaly and is frequently accompanied by neurode-
velopmental delay.
The FP line and the nasofrontal (NF) angle, both measured
in the exact midsagittal profile view, can support the diagnosis of
bossing or sloping forehead in the second and third trimesters of
pregnancy.43,44 The FP line passes through the middle point of
the anterior border of the mandible and the nasion. In normal
fetuses the line passes through the frontal bone (zero position of
FP line) (Fig. 35.35A) but also may pass just behind the frontal
bone (positive position of the FP line), especially later in preg-
nancy when the frontal bone may acquire a rounded shape. In
frontal bossing, the FP line is exaggeratedly positive (distance
416 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B
• Fig. 35.34  Fetus of 25 weeks’ gestation with severe hydrocephalus. In the profile view (A), no frontal
bone is visible because of the wide metopic suture, visible in the coronal view (B).

A B C
• Fig. 35.35  Two-dimensional profile views of a normal fetus (A), a fetus with a bossing forehead (B) and
a fetus with a sloping forehead (C). The profile line (with line) is zero in A, exaggerated positive in B (arrow)
indicates enlarged distance between fetal profile line and frontal bone) and negative in C. The nasofrontal
angle (black angle) is normal in A, small in B and large in C.

between FP line and the frontal bone is >4 mm) (Fig. 35.35B). If
the FP line passes in front of the frontal bone (negative position
of FP line), this is always pathological (Fig. 35.35C) and may
indicate a sloping forehead or retrognathia.
The NF angle is defined as the angle between the nasal and the
frontal bones. The NF angle does not change significantly with a
mean of 117 degrees (10th and 90th centiles are 105 and 129 degrees,
respectively). In frontal bossing, the angle is small (Fig. 35.35B) and
the angle becomes large in sloping forehead (Fig. 35.35C) but also
in cases with flat noses (Figs. 35.36 to 35.38).  tumours. Nose anomalies, such as single nostril (Fig. 35.40B) or
Nose
The nose with its central position probably plays an important
role in the visual impression of the face. The nose can easily be
identified from 11 weeks’ gestation. In many syndromes, the nose
has specific characteristic features (Fig. 35.39).
Most publications describing nose anomalies are case reports
or series, reporting frontonasal dysplasia, maxilla-nasal dysplasia,
oculo-auriculo-fronto-nasal syndrome, total arhinia, split nose or
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face
A B
• Fig. 35.36  Ultrasound images of a case with Binder syndrome: second trimester two-dimensional (A),
second trimester three-dimensional rendered (B) and third trimester three-dimensional rendered images
(C). Note the large nasofrontal angle.
• Fig. 35.37 Two-dimensional image of a case with chondrodysplasia
punctata. The nose is flat with a large nasofrontal angle.
proboscis (Fig. 35.41), are frequently encountered in association
with holoprosencephaly and often with trisomy 13.
The nasal bones have received a lot of attention, and there is
overwhelming evidence that absent or hypoplastic nasal bones
are strong markers for trisomy 21. Excellent reviews have been
published by Sonek45 in 2003 and Shank and Odibo46 in 2009.
Prenasal thickness, defined as the skin thickness above the nose,
tends to be larger in trisomy 21 but also in some other condi-
tions as trisomy 1847 and other syndromes (Fig. 35.42B). In nor-
mal fetuses, the ratio of prenasal thickness to nasal bone length
is stable throughout gestation with a mean of 0.6 (5th and 95th
centiles are 0.5 and 0.8, respectively) and therefore an easy-to-use
tool when a suspicion of a chromosomal anomaly arises.48
417
C
The NF angle can be helpful in identifying flat noses as seen in
Binder syndrome (see Fig. 35.36), chondrodysplasia punctate (see
Fig. 35.37) and frontonasal dysplasia (see Fig. 35.42).
Small noses can be seen in several syndromes (see Fig. 35.42) in
contrast to large noses, which are extremely rarely seen prenatally
unless there is a tumour or an encephalocele. Care has to be taken
to image the exact midsagittal plane when assessing the nose.
In the axial plane–oblique nose–mouth view, the nostril and
the choanae can be assessed. The nostrils should be symmetrical
and are usually round or wide oval. A single nostril can be seen in
holoprosencephaly and narrow nostrils in several syndromes (see
Fig. 36.40). Normal ranges of nasal width and nostril distance are
available.49
Patency of the nasal choanae to exclude choanal atresia is best
assessed in the axial plane. Especially during breathing move-
ment, the choanae can be clearly visible with colour Doppler
(Fig. 35.43). 
Philtrum
Two studies that generated nomograms of philtrum length are
available.50,51 In normal fetuses, the philtrum is, subjectively,
about as long as the nasal protrusion (Fig. 35.44)
Abnormal philtrum lengths are part of many syndromes,
although long philtra are more common than short philtra.1
When the philtrum is long, it is also often bulging (Fig. 35.45). 
Jaws
The two main anomalies affecting the jaws are clefts and micro-
gnathia (see later), both generating a convex profile reflected by a
large MNM angle (see Figs. 35.19, 35.29 and 35.32). In several
syndromes, such as Binder syndrome, craniosynostosis syndromes
and skeletal dysplasias and in about 17% of trisomy 21 cases,52 the
maxilla is hypoplastic, producing a flat profile with a small MNM
angle instead (Fig. 35.46).
418 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B
C

• Fig. 35.38  Three-dimensional multiplanar image of a case with mild fronto-nasal dysplasia. Note the
dimple in the nose (arrow, box A), the flat nose (box B) and hypertelorism (box C). There was also septo-
optic dysplasia in this case.

A B

C D
• Fig. 35.40  Two-dimensional images of normal symmetrical round nos-
trils (A), a single nostril in holoprosencephaly (B), flat nostrils in frontonasal
dysplasia (C) and narrow nostril in Stickler syndrome (D).

• Fig. 35.39  Two-dimensional image of case with thanatophoric dysplasia,
showing typical saddle nose.
Rare anomalies affecting the mandible are agnathia, otocephaly
and atypical clefts. Otocephaly is a sporadic lethal condition with
severe micrognathia or even agnathia; midline defects, including
holoprosencephaly, anterior encephalocele, cyclopia, aglossia; and
midfacial location of the ears (Fig. 35.47). Tessier cleft nr 0, is a
midline cleft, which can affect the mandible.  genital ichthyosis.
Mouth
The mouth can be evaluated in the standard coronal nose–mouth
view. A nomogram of mouth length is available.53 The typical
clefts of the lip but also atypical Tessier clefts can affect the nor-
mal appearance of the mouth. Thick lips are part of Noonan
(Fig. 35.48A) and Costello syndromes. Macrostomia or micro-
stomia are part of some conditions. Macrostomia can be seen
in ablepharon-macrostomia, Pena Shokier syndrome or Costello
syndrome. Microstomia is described in agnathia, otocephaly, tri-
somy 21(Fig. 35.48B), Freeman-Sheldon syndrome and cerebro-
costo-mandibular syndrome.
A continuous open, tent-shaped or fish-like mouth can be a
marker of several neurologic or dermatologic conditions such as
congenital myotonic dystrophy, a restrictive dermopathy or con-
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face
• Fig. 35.41 Two-dimensional image of a fetus with holoprosencephaly
showing a proboscis.
A B
• Fig. 35.42  Two-dimensional images of fetuses with small noses: Stickler syndrome (A), Pallister Killian
syndrome (B) and campomelic dysplasia (C).
A B
• Fig. 35.43  Axial view of clearly normal patent choanae, without (A) and with (B,C) colour Doppler during
breathing movements.
419
Tumours. Tumours arising from the oral cavity can also give
the impression of a constantly open mouth. Examples are epigna-
thus (oral teratoma), epulis (gingival hyperplasia), ranula (muco-
cele occurring in the floor of the mouth), hamartoma or a foregut
duplication cyst (Fig. 35.49). These tumours arise usually later in
pregnancy and are often isolated. A large teratoma may distort
the orofacial anatomy and through the vascular steel phenomenon
cause cardiac failure, polyhydramnios, hydrops, preterm delivery
or IUD. Oral tumours carry a risk for development of polyhy-
dramnios because of swallowing problems and postpartum emer-
gency because of airway obstruction. An elective caesarean section
with EXIT (ex utero intrapartum treatment) procedure may be
necessary. 
Tongue. Anomalies of the tongue are macroglossia and micro-
glossia. A bifid tongue and tumours arising from the tongue are rare
anomalies (see Fig. 35.49). Whereas macroglossia can be an isolated
sonographic feature (trisomy 21), microglossia is rarely isolated
and likely combined with other more striking facial abnormalities.
Achiron and coworkers established nomograms for tongue cir-
cumference and identified cases with microglossia (partial trisomy
1) and macroglossia (trisomy 21).54 Bronshtein and coworkers
constructed nomograms for lingual width and identified a small
C
C
420 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

• Fig. 35.44  Two-dimensional images of normal second trimester fetuses showing that subjectively, nasal
protrusion is about equal to philtrum length.

A B C
• Fig. 35.45  Examples of a long philtrum in Pallister Killian syndrome (A) and Cornelia de Lange syndrome
(B) and a short philtrum in cri du chat syndrome (C). (B courtesy Dr B. Benoit.)

1
1 1

A B C D E

• Fig. 35.46  Two-dimensional images of flat profiles with small maxilla–nasion–mandible angle angles in
fetuses with Binder syndrome (A), Apert syndrome (B), thanatophoric dysplasia (C), achondrodysplasia
(D) and trisomy 21 (E).
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face
tongue in three cases with micrognathia.55 Subjectively, macro-
glossia is an abnormal protuberance of the tongue beyond the
alveolar ridge. A resting tongue that is protruding beyond the lips
or a protruding tongue during swallowing is especially abnormal.
The tongue may protrude because of increased size as in Beckwith-
Wiedemann syndrome but also because of hypotonia as seen in tri-
somy 21. A large oral tumour may resemble macroglossia. A resting
tongue between the alveolar ridges is a normal feature in prenatal
life and should not be mistaken for macroglossia (Fig. 35.50).  sound. Many studies published normal values of interocular,
Eyes
The hypoechoic eyeballs surrounded by the bony orbital ridges are
very easy to recognise with ultrasound examination; therefore, it
is not surprising that normal values of ocular diameter, interocular
• Fig. 35.47  Three-dimensional rendered image of a fetus with a severe
form of otocephaly. Note the cyclopia, agnathia and midline location of the
ears. (Courtesy Dr R. Chaoui.)
A B
• Fig. 35.48  Three-dimensional rendered image of a fetus with Noonan syndrome, showing a small mouth
and thick lips (A) and a fetus with trisomy 21 showing a small mouth (B). (B courtesy Dr B. Benoit.)
421
distance and external or binocular distances have already been
published in the early eighties with the aim to diagnose hyper-
telorism, hypotelorism or microphthalmia (Fig. 35.51).
Thanks to technical improvements, more details such as the
eyelids, eyelashes, hyaloid artery, lenses and pupils are visible (Fig.
35.52).
Fetal orbital measurements are reported both in early and
late gestation using both transvaginal and transabdominal ultra-
binocular and ocular distances followed by studies publishing
normal values of orbital circumference, orbital area and the ante-
rior and posterior chamber length.56-58 The axial ocular growth is
reported by Achiron and coworkers, which may be important for
families at risk for infantile glaucoma or persistent hyperplastic
primary vitreous.58 Values of fetal eyeball volume with 3D ultra-
sound were determined by Odeh and coworkers.59 This may be
helpful in the diagnosis of microphthalmia when the diagnosis is
not clear and obvious using 2D ultrasound. However, one must
beware of false negatives because microphthalmia may sometimes
be secondary to degenerative processes that occur in middle or
late gestation.
It is generally known by ultrasonographers that the distance
between the orbits is about the same as the diameter of one
orbit, a facial proportion anecdotally even described by Leon-
ardo da Vinci as being ideal.60 Hypertelorism and microphthal-
mia are associated with a wide range of different syndromes
and conditions (Figs. 35.53 and 35.54), and hypotelorism is
often associated with holoprosencephaly (Fig. 35.55); in severe
cases, even only one orbit (cyclopia) is present. Assessment of
the orbits is part of the routine midtrimester ultrasound scan.
Ocular anomalies are often associated with other malforma-
tions, especially of the CNS. Asymmetry is common for eye
anomalies, such in Goldenhar syndrome (oculo-auriculo-vertebral
spectrum).
Anophthalmia. The clinical distinction between real anoph-
thalmia and severe microphthalmia is difficult, and pathological
examination is usually necessary to make the correct diagnosis.
Real anophthalmia is probably a lethal condition as severe malfor-
mations of the forebrain accompany this anomaly. The antenatal
diagnosis of anophthalmia is made using 2D and 3D ultrasound,
and first trimester diagnosis of anophthalmia is also possible
because orbits are visible from 11 weeks’ gestation. 
422 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B

C D E
• Fig. 35.49  Image of a fetus with a foregut duplication cyst of the tongue. The first (A) and second (B)
trimester ultrasound scans were normal. In the third trimester, a thick-walled cyst protruding from the
mouth was seen. C, Axial plane. D, Nose–mouth view. E, Magnetic resonance image. Asterisk indicates
duplication cyst.

Hyaloid artery. The hyaloid artery is a transient fetal vessel Ears

visible as a continuous thin echogenic line between the posterior
wall of the orbit and the posterior border of the lens. In the first
trimester, low peak systolic flow can be detected in this artery.
The conversion of the primary vitreous to the secondary vitreous
begins in the second month and is completed near term, which
is accompanied by degeneration of the hyaloid artery. Achiron
and Birnholz describe ultrasonographic regression of the hya-
loid artery between 18 and 29 weeks’ gestation.58,61 Insufficient
regression of the primary vitreous and hyaloid artery results in
persistent hyperplastic primary vitreous (PHPV) and may be seen
in genetic disorders such as Walker-Warburg syndrome. The pre-
natal diagnosis of PHPV is possible: an irregular hyperechogenic
line extended from the posterior surface of the lens to the poste-
rior wall of the eye, often accompanied by cataract and microph-
thalmos (Fig. 35.56). PHPV has usually severe consequences for
vision.  shape. Other anomalies that could be recognised are pre-auricular
Dacryocystocele. As a result of delayed canalisation of the dis-
tal end of the nasolacrimal duct and an uncommon valve mecha-
nism at the cranial end, the lacrimal sac expands and is visible as a
round hypoechogenic mass. A dacryocystocele is rarely larger than
10 mm, is located near the nasal canthus and is only described
in the second half of pregnancy (Fig. 35.57). The differential
diagnosis includes encephalocele, haemangioma, lymphangioma,
teratoma, glioma, rhabdomyosarcoma and neurofibromatosis.
These conditions can easily be differentiated by echotexture, size,
localisation, colour Doppler and time of appearance. Canalisation
of the nasolacrimal pathway solves the problem, which even may
occur spontaneously before birth. A dacryocystocele may be part
of some syndromes with facial anomalies but often is an isolated
finding. Bilateral dacryocystoceles have been described.  18 weeks on with a mean of 65.4% (SD, 8.43%).66
Ear anomalies are frequently encountered in syndromes. Yet the
fetal ears have received little attention in prenatal ultrasound,
although 3D ultrasound has renewed interest in the external ear.
A retrospective analysis of 16,698 fetuses between 2000 and 2005
in Sweden revealed that no ear malformations were detected on
routine ultrasound, although the prevalence of minor ear anoma-
lies was 2.4 per 1000 and of major ear malformations 0.3 per
1000.62 The best time for fetal external ear observation is 17 to 25
weeks’ gestation, when ears are surrounded by amniotic fluid and
quite easy to identify. Hence case reports describing ear anomalies
are published since the 1980s, first with 2D ultrasound followed
by reports using 3D ultrasound. It must be noted that the ear
anomalies were not isolated in most case reports. Anomalies of
the ear can be categorised as aberrant size, location, rotation and
ear tags, preauricular pits, earlobe deformities and asymmetry (not
uncommon). Detailed descriptions for routine use with objective
measures or markers for location, rotation and shape of the exter-
nal ear are lacking. Location, rotation and shape of the external ear
can be examined with 2D ultrasound; however, 3D rendered views
offer a much clearer image (Fig. 35.58). Mostly, these diagnoses
are made subjectively, although proposals to analyse quantitative
fetal ear rotation with 3D ultrasound have been published.63,64 In
contrast, auricular biometry (length and width) during the second
and third trimesters using 2D or 3D ultrasound is published in
many studies (Table 35.2). A first trimester study was published in
2003.74 A linear growth of ear length and width with gestational
age is mostly described. The width-to-length ratio is stable from
 
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 423

C
• Fig. 35.50  A–C, Three-dimensional multiplanar representation of normal fetuses. The marker dot is
positioned on the tip of the tongue. On the left the profile views, the resting tongue is seen between the
alveolar ridges. On the right, there is clear identification of the tongue in the axial view.

OD IOD

BOD

• Fig. 35.51  Example of ocular distance (OD), interocular distance (IOD) and binocular distance (BOD),
also named external ocular distance measurements on a two-dimensional image in the axial plane.
424 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C

D E
• Fig. 35.52  Two-dimensional images of eyelids (A), eyelashes (B), hyaloid artery (C), lens (D) and pupil (E).

A B C
• Fig. 35.53  Hypertelorism in cases with Wolf Hirschhorn syndrome (A), thanatophoric dysplasia (B) and
frontonasal dysplasia (C).

A B C
• Fig. 35.54  Two- and three-dimensional images of microphthalmia at 21 weeks’ gestation in a fetus with
Spear syndrome. A and B axial planes showing smaller orbits and absence of the eye bulb. C, Rendered
3D imaged.
 CHAPTER 35  Diagnosis and Management of Abnormalities of the Face 425

Voluson
E10

• Fig. 35.55  Three-dimensional rendered image of a fetus with trisomy 13

and holoprosencephaly showing hypotelorism. (Courtesy Dr B. Benoit.)

• Fig. 35.57  Two-dimensional image showing a dacryocystocele between

the nose and the orbit (arrow). The orbits were normal but look asymmetri-
cal in this image because of the slightly oblique plane.

• Fig. 35.56  Three-dimensional multiplanar representation of a third trimester fetus with persistent hyper-
plastic primary vitreous. The long arrows point at the persistent hyaloid artery and the short arrows at the
cataract.

Conclusions segment–specific analysis should be performed, including

• When a facial anomaly is diagnosed, a thorough examination
of the fetus should be performed because the risk for associated
anomalies, chromosomal abnormalities and genetic syndromes
is increased.
• For evaluation of fetuses with clefts, 2D and 3D ultrasound are
both helpful in characterisation of the cleft.
• For evaluation of fetuses with micrognathia, it is important to
use 3D ultrasound for defining the exact midsagittal plane and
the use objective biometric tools.
• The face consists of several segments that can all show path-
ological variants. Therefore, in high-risk patients, a facial
assessment of the forehead, nose, philtrum, mouth and lips,
maxilla, mandible, eyes and ears.
• 3D ultrasound is a valuable tool in the assessment of the fetal
face, especially in defining the exact midsagittal profile plane
(multiplanar view), assessing the palate, recognising dysmor-
phic faces and visualising the external ear.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
426 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B C

D E F
• Fig. 35.58  Images of the fetal ear. A, A normal ear. B, Reduced ear length (22 mm at 31 weeks’ gestational
age) in a fetus with Pallister Killian syndrome. C, Anteverted and low implanted ear in a fetus with Noonan
syndrome. D, Ear tags. E and F, Malformed ear. (D–F courtesy Dr B. Benoit.)

TABLE
35.2 Summary of Publications on Objective Measurements Related to the Ear
Reference Methoda Measure GA (wk) Patients (n) Relation with GA (if not available another parameter is set out)
Birnholz and Farell65 2D sagittal EL 15–42 180 Linear
(1988) EL = 1.1011 × GA - 9.5089, r2= 0.962
Shimizu et al.66 2D sagittal EL 18–42 124 EL linear, r = 0.956, P = .0001
(1992) EW EW linear, r = 0.898, P = .001
ER ER stable at 65.39 ± 8.43%, r = 0.046, P = .605
Scatterplots with percentile lines are provided
Lettieri et al.67 2D coronal EL 14–25 452 Linear
(1993) EL = 1.161 ×GA - 9.731, r = 0.84, P = .001
Awwad et al.68 20–28 418 Linear
(1994) EL = -6.000 + 1.075 × GA
Chitkara et al.69 2D EL 15–40 2583 Linear
(2000) EL =1.076 × GA – 7.308 r = 0.96, P = .0001
Yeo et al.70 (2003) 2D sagittal or EL 14–41 447 Quadratic
coronal EL = -9.458 + 0.964 × GA + 0.024 × GA2 + 0.0005 × GA³,
r = 0.96, P < .001
Chang et al.71 (2000) 3D EL 17–41 122 EL = 1.752 ×GA – 0.016 ×GA2 – 10.765, r = 0.881
EW EW = 0.398 × GA – 0.989, r = 0.848
EA EA = 0.171 × GA – 2.239, r = 0.890
Roelofsema72 (2007) 3D multiplanar EL 18–34 494 Quadratic
mode sagittal EL = 14.40 + 1.310 × –(GA-20) - 0.0158 × (GA - 20)2
Hatanaka et al.73 3D rendering EL 19–24 114 EL = exp(1.215 × GA - 8.692) r2 = 0.423
(2010)(2011) mode sagittal
Sacchini et al.74 2D coronal EL 11–14 450 Linear
(2003) EL = 0.095 + 0.081 × CRL (mm), r = 0.76, P < .0001 r = 0.76,
P < .0001
aMethod included dimensionality and plane used for measurement when stated.
2D, Measurement performed with two-dimensional ultrasound imaging; 3D, measurement performed with three-dimensional ultrasound imaging; CRL, crown–rump length; EA, ear area; EL, ear length;
ER, ear ratio; EW, ear width; GA, gestational age.
  
References 26. Online Mendelian Inheritance in Man. https://www.omim.org/.
27. Nicolaides KH, Salvesen DR, Snijders RJM, et  al. Micrognathia fetal
facial defects: Associated malformations and chromosomal abnormalities.
1. Jones KL. Smith’s recognizable patterns of human malformation. Philadel-
Fetal Diagn Ther. 1993;8:1–9.
phia: WB Saunders; 1997.
28. Turner GM, Twining P. The facial profile in the diagnosis of fetal abnor-
2. Gorlin RJ, Cohen AM, Michael MJ, et al. Syndromes of the head and neck.
malities. Clin Radiol. 1993;47:389–395.
4th ed. New York: Oxford University Press; 2001.
29. Bromley B, Benacerraf BR. Fetal micrognathia: associated anomalies and
3. den Boogert AG, de Jong-Pleij EA, Ribbert LS, et al. Facial shape; height
outcome. J Ultrasound Med. 1994;13:529–533.
and width in the second and third trimester of pregnancy. J Matern Fetal
30. Vettraino IM, Lee W, Bronsteen RA, et al. Clinical outcome of fetuses
Neonatal Med. 2017:1–7. https://doi.org/10.1080/14767058.2017.1384
with sonographic diagnosis of isolated micrognathia. Obstet Gynecol.
807.
2003;102:801–805.
4. Zalel Y, Gindes L, Archiron R. The fetal mandible: in utero sono-
31. Basburg M, Ozgun MT, Ozcelik B, et al. Outcome of fetuses with sono-
graphic evaluation between 11 and 13 weeks’ gestation. Prenat Diagn.
graphic diagnosis of micrognathia OC 241. Ultrasound Obstet Gynecol.
2006;26:163–167.
2007;30:441.
5. de Jong-Pleij EA, Ribbert LS, Manten GT, et al. Maxilla-nasion-mandible
32. Paladini D. Fetal micrognathia: almost always an ominous finding. Ultra-
angle: a new method to assess profile anomalies in pregnancy. Ultrasound
sound Obstet Gynecol. 2010;35:377–384.
Obstet Gynecol. 2011;37:562–569.
33. Sepulveda W, Wong AE, Viñasl F, et al. Absent mandibular gap in the
6. Salomon LJ, Alfirevic Z, Berghella V, et  al. On behalf of the ISUOG
retronasal triangle view: a clue to the diagnosis of micrognathia in the first
Clinical Standards Committee. Practice guidelines for performance of the
trimester. Ultrasound Obstet Gynecol. 2012;39:152–156.
routine mid-trimester fetal ultrasound scan. Ultrasound Obstet Gynecol.
34. Otto C, Platt LD. The fetal mandible measurement: an objective determi-
2011;37:116–126.
nation of fetal jaw size. Ultrasound Obstet Gynecol. 1991;1:12–17.
7. Wilhelm L, Borgers H. The ‘equals sign’: a novel marker in the diagnosis
35. Chitty LS, Campbell S, Altman DG. Measurement of the fetal man-
of fetal isolated cleft palate. Ultrasound Obstet Gynecol. 2010;36:439–444.
dible-feasibility and construction of a centile chart. Prenat Diagn.
8. de Jong-Pleij EAP, Ribbert LSM, Bilardo CM. Three-dimensional multipla-
1993;13:749–756.
nar ultrasound is a valuable tool in the study of the fetal profile in the second
36. Watson WJ, Katz VL. Sonographic measurement of the fetal mandible:
trimester of pregnancy. Ultrasound Obstet Gynecol. 2010;35:195–200.
standards for normal pregnancy. Am J Obstet Gynecol. 1993;10:226–228.
9. Tutschek B, Blaas H-GK, Abramowicz J, for the ISUOG 3D Special
37. Paladini D, Morra T, Teodoro A, et al. Objective diagnosis of microgna-
Interest Group, et al. Three-dimensional ultrasound imaging of the fetal
thia in the fetus: the jaw index. Obstet Gynecol. 1999;93:382–386.
skull and face. Ultrasound Obstet Gynecol. 2017;50:7–16.
38. Zalel Y, Gindes L, Achiron R. The fetal mandible: an in utero sono-
10. Merz E, Weber G, Bahlman F, et  al. Application of transvaginal and
graphic evaluation between 11 and 31 weeks’ gestation. Prenat Diagn.
abdominal three-dimensional ultrasound for the detection or exclusion of
2006;26:163–167.
malformations of the fetal face. Ultrasound Obstet Gynecol. 1997;9:237–
39. Rotten D, Levaillant JM, Martinez H, et al. The fetal mandible: a 2D and
243.
3D approach to the diagnosis retrognathia and micrognathia. Ultrasound
11. Calzolari E, Pierini A, Astolfi G, et  al. Associated anomalies in multi-
Obstet Gynecol. 2002;19:122–130.
malformed infants with cleft lip and palate: an epidemiologic study of
40. Faro C, Benoit B, Wegrzijn P, et  al. Three-dimensional sonographic
nearly 6 million births in 23 EUROCAT registries. Am J Med Genet.
description of the frontal bones and metopic suture. Ultrasound Obstet
2007;143:528–537.
Gynecol. 2005;26:618–621.
12. Mulliken JB. The changing faces of children with cleft lip and palate.
41. Chaoui R, Levaillant JM, Benoit B, et al. Three-dimensional sonographic
N Engl J Med. 2004;351:745–747.
description of abnormal metopic suture in second- and third-trimester
13. Gorlin RJ, Cervenka J, Pruzansky S. Facial clefting and its syndromes.
fetuses. Ultrasound Obstet Gynecol. 2005;26:761–764.
Birth Defects Orig Artic Ser. 1971;7:3–49.
42. Faro C, Wegrzyn P, Benoit B, et al. Metopic suture in fetuses with holo-
14. Bergé SJ, Plath H, Van de Vondel PT, et  al. Fetal cleft lip and palate:
prosencephaly at 11 + 0 to 13 + 6 weeks of gestation. Ultrasound Obstet
sonographic diagnosis, chromosomal abnormalities, associated anoma-
Gynecol. 2006;27:162–166.
lies and postnatal outcome in 70 fetuses. Ultrasound Obstet Gynecol.
43. de Jong-Pleij EAP, Ribbert LSM, Pistorius LR, et  al. The fetal profile
2001;18:422–431.
line: a proposal for a sonographic reference line to classify forehead and
15. Cohen Jr MM, Bankier A. Syndrome delineating involving orofacial
mandible anomalies in the second and third trimester. Prenat Diagn.
clefting. Cleft Palate Craniofac. 1991;28:119–120.
2012;32:797–802.
16. Tessier P. Anatomic classification of facial, cranio-facial and latero-facial
44. de Jong-Pleij EAP, Bilardo CM, Manten GTR. Look at the fetal nose 1;
clefts. J Maxillo-Facial Surg. 1976;4:69.
the nasofrontal angle. Ultrasound Obstet Gynaecol. 2017;50:297–298.
17. Bakker M, Pace M, de Jong-Pleij EAP, et al. Prenasal thickness, prefron-
45. Sonek JD. Nasal bone evaluation with ultrasonography: a marker for fetal
tal space ratio and other facial profile markers in first-trimester fetuses
aneuploidy. Ultrasound Obstet Gynecol. 2003;22:11–15.
with aneuploidies, cleft palate, and micrognathia. Fetal Diagn Ther.
46. Shanks A, Odibo A. Nasal bone in prenatal trisomy 21 screening. Obstet
2018;43(3):231–240.
Gynecol Surv. 2009;65:46–52.
18. Sepulveda W, Wong AE, Martinez-Ten P, et  al. Retronasal triangle: a
47. Vos FJ, de Jong-Pleij EA, Tromp E, et al. Fetal facial profile markers in
sonographic landmark for the screening of cleft palate in the first trimes-
second and third trimester fetuses with Edwards syndrome. Ultrasound
ter. Ultrasound Obstet Gynecol. 2010;35:7–13.
Obstet Gynecol. 2015;46:66–72.
19. Chaoui R, Orosz G, Heling KS, et  al. Maxillary gap at 11–13 weeks’
48. de Jong-Pleij EAP, Vos FJ, Ribbert LSM, et al. Prenasal thickness-to-nasal
gestation: marker of cleft lip and palate. Ultrasound Obstet Gynecol.
bone length ratio: a strong and simple second- and third-trimester marker
2015;46:665–669.
for trisomy 21. Ultrasound Obstet Gynecol. 2011;39:109–185.
20. Timmerman E, Pajkrt E, Maas SM, et al. Enlarged nuchal translucency
49. Goldstein I, Tamir A, Itskovitz-Eldor J, et al. Growth of the fetal nose
in chromosomally normal fetuses: strong association with orofacial cleft.
width and nostril distance in normal pregnancies. Ultrasound Obstet
Ultrasound Obstet Gynecol. 2010;27:427–432.
Gynecol. 1997;9:35–38.
21. de Jong-Pleij EAP, Pistorius LR, Ribbert LSM, et al. Premaxillary pro-
50. Sivan E, Chan L, Mallozzi-EberleA, et  al. Sonographic imaging of the
trusion assessment by the maxilla-nasion-mandible angle in fetuses with
fetal face and the establishment of normative dimensions for chin length
facial clefts. Prenat Diagn. 2013;33:354–359.
and upper lip width. Am J Perinat. 1997;14:191–194.
22. Campbell S, Lees C, Moscoso G, et al. Ultrasound antenatal diagnosis of
51. Gull I, Wolman I, Merlob P, et  al. Nomograms for the sonographic
cleft palate by a new technique: the 3D ‘reversed face’ view. Ultrasound
measurement of the fetal philtrum and chin. Fetal Diagn Ther. 2005;20:
Obstet Gynecol. 2005;25:12–18.
127–131.
23. Platt LD, Devore GR, Pretorius DH. Improving cleft palate/cleft lip
52. Vos FI, de Jong-Pleij EA, Bakker M, et al. The facial profile of Down syn-
antenatal diagnosis by 3-dimensional sonography: the ‘flipped face’ view.
drome fetuses in the second and third trimester of pregnancy. Ultrasound
J Ultrasound Med. 2006;25:1423–1430.
Obstet Gynecol. 2015;46:168–173.
24. Pilu G, Segata M. A novel technique for visualization of the normal and
53. Vimercati A, Scioscia M, Burattini MG, et al. Prenatal diagnosis of Free-
cleft fetal secondary palate: angled insonation and three-dimensional
man-Sheldon syndrome and usefulness of an ultrasound fetal lip width
ultrasound. Ultrasound Obstet Gynecol. 2007;29:166–169.
nomogram. Prenat Diagn. 2006;26:679–683.
25. Faure JM, Captier G, Baumler M, et al. Sonographic assessment of nor-
mal fetal palate using three-dimensional imaging: a new technique. Ultra-
sound Obstet Gynecol. 2007;29:159–165.

426.e1
426.e2 References
54. Achiron R, Ben Arie A, Gabbay U, et  al. S. Development of the fetal
tongue between 14 and 26 weeks of gestation: in utero ultrasonographic
measurements. Ultrasound Obstet Gynecol. 1997;9:39–41.
55. Bronshtein M, Zimmer EZ, Tzidony D, et al. Transvaginal sonographic
measurement of fetal lingual width in early pregnancy. Prenat Diagn.
1998;18:577–580.
56. Jeanty P, Dramaix-Wilmet M, Van Gansbeke D, et al. Fetal ocular biom-
etry by ultrasound. Radiology. 1982;143:513–516.
57. Goldstein I, Tamir A, Zimmer E, et al. Growth of the fetal orbit and lens
in normal pregnancies. Ultrasound Obstet Gynecol. 1998;12:175–179.
58. Achiron R, Kreiser D, Achiron A. Axial growth of the fetal eye and
evaluation of the hyaloid artery: in utero Ultrasonographic study. Prenat
Diagn. 2000;20:894–899.
59. Odeh M, Feldman Y, Degani S, et al. Fetal eyeball volume relationship to
gestational age and biparietal diameter. Prenat Diagn. 2009;29:749–752.
60. Farkas LG, Kolar JC. Anthropometrics and art in the aesthetics of wom-
en’s face. Clin Plast Surg. 1987;14:599–616.
61. Birnholz JC, Farrell EE. Fetal hyaloid artery: timing of regression with
US. Radiology. 1988;166:781–783.
62. Romosan G, Henriksson E, Rylander A, et al. Diagnostic performance
of routine ultrasound screening for fetal abnormalities in an unselected
Swedish population in 2000–2005. Ultrasound Obstet Gynecol.
2009;34:526–533.
63. Hatanaka AR, Rolo LC, Araujo Junior E, et al. Analysis of fetal ear rota-
tion by three-dimensional rendering mode using a novel method: the
ear angle—preliminary results OC10.04. Ultrasound Obstet Gynecol.
2011;38:1–55.
64. Ginsberg NA, Cohen L, Dungan JS, et al. 3-D ultrasound of the fetal ear
and fetal autosomal trisomies: a pilot study of a new screening protocol.
Prenat Diagn. 2011;31:311–314.
65. Birnholz JC, Farell EE. Fetal ear length. Pediatrics. 1988;81:555–558.
66. ShimizuT Salvador L, Allanson J, et al. Ultrasonographic measurements
of the fetal ear. Obstet Gynecol. 1992;80:381–384.
67. Lettieri L, Rodis JF, Vintzileos AM, et al. Ear length in second trimester
aneuploidy fetuses. Obstet Gynecol. 1993;81:57–60.
68. Awwad JT, Azar GB, Karam KS, et al. Ear length: a potential sonographic
marker for Down syndrome. Int J Gynaecol Obstet. 1994;44:233–238.
69. Chitkara U, Lee L, El-Sayed YY, et al. Ultrasonographic ear length mea-
surement in normal second- and third-trimester fetuses. Am J Obstet
Gynecol. 2000;183:230–234.
70. Yeo L, Guzman ER, Ananth CV, et al. Prenatal depiction of fetal aneu-
ploidy by sonographic ear length. J Ultrasound Med. 2003;22:565–576.
71. Chang CH, Chang FM, Yu CH, et al. Fetal ear assessment and prenatal
detection of aneuploidy by the quantitative three-dimensional ultraso-
nography. Ultrasound Med Biol. 2000;26:743–749.
72. Roelofsema NM. Three-Dimensional Ultrasound Study of Fetal Craniofa-
cial Anatomy [thesis]. Rotterdam, Netherlands: Erasmus University; 2007.
73. Hatanaka AR, Lobo GR, Rolo LC, et al. A. Comparison of second tri-
mester ear length measured by two dimensional and three dimensional
ultrasound. Preliminary results P18.09. Ultrasound Obstet Gynecol.
2010;36:240.
74. Sacchini C, El-Sheikhah A, Cicero S, et  al. Ear length in trisomy 21
fetuses at 11–14 weeks of gestation. Ultrasound Obstet Gynecol. 2003;22:
460–463.
36
Fetal Hydrops
SYLVIE LANGLOIS AND R. DOUGLAS WILSON
KEY POINTS
• Hydrops fetalis is a pathological condition of excessive fluid
accumulation in at least two extravascular compartments,
including fetal soft tissues and body cavities. Hydrops fetalis
is a clinical finding and not a final diagnosis.
• There are two main pathophysiologies for hydrops fetalis,
immune and nonimmune. For nonimmune, the diagnostic
­categories are placental, cardiovascular, chromosomal,
­haematologic, lymphatic dysphasia, infection, thoracic
malformations, genetic syndromes, inborn errors of
­metabolism, extrathoracic tumours and genitourinary
tract and gastrointestinal malformations. Undiagnosed
causes are still common.
• Hydrops fetalis should be considered as an urgent finding
with timely evaluation, which requires a directed ‘stepwise
approach’.
• Fetal hydrops can cause a maternal physiological distur-
bance, mirror syndrome, with possible severe maternal
consequences.
• Fetal therapy is an option for certain causes of hydrops but
requires complete evaluation and informed consent before
its use.
• Prognosis and recurrence risk counselling is dependent on
the underlying cause, but overall the mortality rate remains
high. If no specific diagnosis is made prenatally, autopsy
should be offered for all perinatal losses because a significant
proportion of cases have a genetic cause with risk of recur-
rence in future pregnancies.
Introduction
Hydrops fetalis (fetal hydrops) is a pathologic condition of
excessive accumulation of fluid in at least two extravascular
compartments, including fetal soft tissues and body cavities.
It is the physiological end-stage process in a number of fetal
conditions and placental pathologies. The underlying causes
are classified under two major categories, immune and non-
immune. In countries that have introduced immunoglobulin
prophylaxis for Rhesus-D alloimmunisation, the incidence of
immune fetal hydrops has significantly decreased, and in these
countries, represents only 10% to 15% of fetal hydrops cases.
Nonimmune fetal hydrops has a reported incidence of 1 in
2000 to 3000 pregnancies but accounts for a disproportionate
share (3%) of overall mortality in the perinatal period.1 There
is a broad spectrum of well-recognised causes, and with the use
of next-generation sequencing (NGS) technology, an increas-
ing number of single-gene disorders are being diagnosed in
cases of fetal hydrops. Determining the cause of the fetal
hydrops is important because some conditions are treatable.
Furthermore, knowing the underlying cause provides addi-
tional information that may be useful to predict the prognosis
and is a key element to determine recurrence risk in future
pregnancies. 
Diagnosis of Hydrops Fetalis by Ultrasound
The diagnosis of immune (IHF) or nonimmune hydrops fetalis
(NIHF) requires an abnormal fluid accumulation in two or
more fetal body compartments. Body compartments (and the
fluid collections) are designated as subcutaneous space (skin
oedema or cystic hygroma [CH]), pleural space (pleural effu-
sion), pericardial space (pericardial effusion) and abdomen
(ascites).
Subcutaneous skin or scalp oedema requires an ultrasound
measurement or thickness of at least 5 mm (Fig. 36.1). The fluid
collection over the scalp or forehead can be identified with the
use of both the transverse and sagittal planes. Skin thickening of
at least 5 mm is required because some macrosomic fetuses may
have thickened skin secondary to fat deposition measuring up
to 5 mm.
Cystic hygroma is a cystic lymphatic lesion. It can be a single
or multiple congenital cysts, most commonly found within the
soft tissues of the neck. Generally, the CH is bilateral, asym-
metric, with thin-walled, multiseptate cystic lesions, and often
located posterior and lateral to the high cervical vertebrae.
Nuchal CH is the clinical consequence of a delay in develop-
ment or absence of the communications that normally develop
between the jugular lymph sacs and the internal jugular veins at
approximately 40 days of gestation.
Pleural effusion is an accumulation of fluid in the pleural space.
Effusion composition can be either chylous or clear (hydrothorax)
with most primary congenital effusions being chylous and occur-
ring on the right. Ultrasound identifies an anechoic space periph-
erally around the compressed lung. If the effusion is unilateral and
large, there may be additional mediastinal shift; bilateral effusions
can be either symmetric with no shift or asymmetric with shift
(Fig. 36.2).
427
428 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A B
• Fig. 36.1  Axial sections of the head (A) and abdomen (B) of a first trimester fetus with significance skin
oedema. (Courtesy Fred Ushakov, Department of Fetal Medicine, University College London Hospitals,
United Kingdom.)

A B
• Fig. 36.2  A, Moderate pleural effusion that is essentially unilateral. B, Severe bilateral pleural effusion.
(Courtesy Fred Ushakov, Department of Fetal Medicine, University College London Hospitals, United
Kingdom.)

Pericardial effusion is identified by the appearance of an echo-

lucent rim greater than 2 mm around both cardiac ventricles
(Fig. 36.3). Pericardial effusions of 1 to 2 mm are considered a
physiological fluid collection.
Fetal ascites is identified by ultrasound by visualisation of an
echolucent fluid rim encompassing the entire fetal abdomen in
the transverse view, usually at the level of the umbilical cord inser-
tion or liver. The echolucent collection of fluid outlines the vis-
ceral contents (Fig. 36.4). 

Pathophysiology
Fetal hydrops is the result of abnormal fluid movement between
the plasma and tissues that leads to excess fluid in both the tissues
and serous cavities (skin oedema, ascites, pleural and pericardial
effusions). Four main mechanisms have been postulated to explain
this abnormal distribution of body fluids: (i) an increase in hydro-
static capillary pressure (resulting for primary or secondary heart
failure or from obstruction of venous return), (ii) a reduction in
plasma osmotic pressure (from decreased albumin production or
increased albumin loss), (iii) obstruction or reduction of lymphatic
flow and (iv) damage to peripheral capillary integrity.1 Table 36.1 • Fig. 36.3  Transverse four-chamber view of the heart showing an abnor-
provides examples of conditions linked to each mechanism. In mal pericardial effusion. (Courtesy Fred Ushakov, Department of Fetal
some conditions, more than one mechanism may be involved.  Medicine, University College London Hospitals, United Kingdom.)
 CHAPTER 36  Fetal Hydrops 429

A B
• Fig. 36.4  Axial sections of the fetal abdomen showing a subtle rim (A) and a moderate collection of fetal
ascites (B). (Courtesy Fred Ushakov, Department of Fetal Medicine, University College London Hospitals,
United Kingdom.)

TABLE Mechanisms of Abnormal Fluid Distribution in

36.1 Hydrops Fetalis Conditions hydrops will not be further discussed in this chapter. However, in
the approach to a fetus with fetal hydrops, immune fetal hydrops
Mechanism Hydrops fetalis conditions is an important condition to consider because it is one of the treat-
able causes with strong evidence-based approaches. 
Increased hydrostatic Fetal congestive heart failure caused by fetal
capillary pressure arrhythmias, congenital heart disease,
obstructive intracardiac tumour, fetal anaemia Nonimmune Hydrops Fetalis
Elevated central venous pressure secondary
to high intrathoracic pressure caused by
Nonimmune hydrops fetalis has a variety of aetiologies. A systematic
intrathoracic masses or heart failure review of the aetiology of NIHF divided the affected cases into 14
classification groups (Table 36.2).2 This section reviews the different
Reduced intravas- Fetal hypoalbuminaemia secondary to haepatic NIHF aetiologies according to the same classification. Some of the
cular osmotic failure seen in metabolic conditions or aetiologies of NIHF are the focus of other chapters in this book.
pressure increased albumin loss seen in chylothorax These aetiologies will only be mentioned briefly in this chapter. 
or nephrotic syndrome
Obstructed or Disorders associated with abnormal lymphatic Twin-to-Twin Transfusion Syndrome and
reduced lymphatic development (e.g., Down syndrome, Noonan
flow syndrome, primary lymphangiectasia) or Placental Abnormalities
with decreased lymph flow associated with Twin-to-twin transfusion syndrome (TTTS) is a complication
reduced fetal movement (e.g., fetal akinesia associated with monochorionic twins caused by an imbalance
sequence, multiple pterygium syndrome)
of the vascular communications in the placenta between the
Damaged peripheral Conditions of fetal hypoxemia caused by fetal two fetuses and represents approximately 5% of cases of NIHF.
capillary integrity anaemia or placental insufficiency Haemodynamic and osmotic changes in the fetuses can lead to
Conditions of fetal inflammation caused by fetal hydrops in the recipient twin most commonly, but donor
fetal infection fetal hydrops has been identified as well.3 (See Chapter 44 for
Adapted from Randenberg AL. Nonimmune hydrops fetalis part I: etiology and pathophysiol-
more details on TTTS.)
ogy. Neonatal Netw 29(5):281–295, 2010. Placental chorioangiomas are rare benign placental tumours.
   Large chorioangiomas act as peripheral arteriovenous shunts, lead-
ing to increased cardiac output, cardiomegaly and finally heart
failure and hydrops. Some cases are complicated by fetal anaemia,
which further contributes to the development of the heart failure and
Aetiology hydrops.4 Umbilical cord haemangiomas have also been reported
with NIHF. (See Chapter 9 for more on placental pathology.) 
Immune Fetal Hydrops
Maternal sensitisation to a fetal red blood cell (RBC) antigen (usu- Cardiovascular Conditions
ally from a previous pregnancy) is followed in the next pregnancy
by transplacental passage of the circulating maternal IgG antibody Cardiovascular conditions are the primary aetiology in 21% of
against that red blood cell antigen resulting in fetal haemolytic NIHF cases and are identified as the most common group of disor-
anaemia, and if severe, fetal hydrops. The severity of the hae- der leading to NIHF. The cardiovascular aetiology can be divided
molytic process is dependent on the antigen–antibody involved into structural malformations, arrhythmias, cardiac tumours and
and the maternal antibody load. Chapter 40 details the diagnosis cardiomyopathy5 (Table 36.3). (See Chapter 29 for more details
and management of RBC alloimmunisation. This aetiology for on cardiovascular conditions.)
430 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Aetiologic Classification of Nonimmune Hydrops Fetalis With Percentage of Total Cases Each Category
36.2 Represents, Most Frequent Pathologies and Fetal Therapy Specific to Each Aetiology
Category Organ or System Most Frequent Pathologies Fetal Therapy
Placental or Placenta or umbilical cord (5.3%) Vascular shunting, TTTS, TRAP, chorioan- Endoscopic laser, RFA
umbilical cord gioma
FETAL

Cardiovascular (21.4%) Abnormal structural Balloon dilatation for stenotic valves

Arrhythmias Systemic or fetal medication
Tumour No prenatal option
Chromosomal (12.5%) Trisomy 21, trisomy 18, Turner syndrome No option
Haematologic (10.1%) α-Thalassemia other rare inherited forms IUT in selected cases
of anaemia
Lymphatic dysplasia (7.5%) Chylothorax Percutaneous shunt or pleurodesis
Congenital lymphatic dysplasia
Noonan syndrome
Infection (6.8%) Parvovirus B19, CMV, toxoplasmosis, IUT for anaemia seen with parvovirus B19 only
syphilis, varicella, rubella
Thoracic malformations (5.3%) Diaphragmatic hernia Experimental balloon tracheal occlusion
CPAM Steroids, RFA, percutaneous shunt, pleurodesis
Syndromic (4.6%) Multiple pterygium, fetal akinesia, skeletal No option
dysplasias
Genitourinary tract (2.0%) Finnish nephrosis No option
Bladder outlet obstruction Shunt, percutaneous cystoscopy
Inborn errors of metabolism (1.1%) Lysosomal storage disorders No option
Extrathoracic tumours (0.7%) SCTs Open fetal surgery
Gastrointestinal (0.7%) Meconium peritonitis, intestinal atresias No option
Miscellaneous (3.7%)
Idiopathic (18.2%) Case by case

CPAM, Congenital pulmonary airway malformation; CMV, cytomegalovirus; IUT, intrauterine transfusion; RFA, radiofrequency ablation; SCT, sacrococcygeal teratoma; TRAP, twin-reversed arterial perfusion;
TTTS, twin-to-twin transfusion syndrome.
  

For structural cardiac malformations, fetal hydrops can

result from lesions that place a burden on the right-sided car-
diac chambers, resulting in increased right atrial pressure such
as left-sided obstructive lesions, hypoplastic left heart syn-
drome or premature closure or restriction of the ductus veno-
sus and right-sided obstructive lesions such as pulmonary and
tricuspid atresia. Nonobstructive lesions that result in right
atrial volume overload such atrioventricular canal defect are
reported in fetal hydrops.
Fetal tachyarrhythmias are the second most common cardio-
vascular abnormality associated with fetal hydrops. This aetiology
has evidenced-based treatment protocols with secondary reversal
of the fetal hydrops. Fetal bradyarrhythmias are less commonly
associated with fetal hydrops and are not easily treatable.5
Intracardiac tumours represent a rare cause of fetal hydrops.
The most common fetal cardiac tumour is a rhabdomyoma with
fetal hydrops caused by tumour obstruction to cardiac flow or an
associated arrhythmia.6 Cardiac rhabdomyomas are frequently
a fetal manifestation of tuberous sclerosis (autosomal dominant
condition) and when suspected prenatally should result in a care-
ful family history and assessment of the couple. (Details on fetal
tumours are provided in Chapter 37.)  have been included or excluded from the study. Cohort studies of
Chromosomal Disorders
Chromosomal abnormalities are a frequent cause of fetal hydrops,
accounting for 13% of cases according to two systematic reviews
(selected publications from 1979 to 2013).2,7 However, amongst
the publications included in these two reviews, the frequency of
chromosomal abnormalities ranged from 0% to 77%. Other pub-
lications, which were excluded from the systematic reviews because
they did not address all causes of NIHF but focused only on the
incidence of chromosomal abnormalities, indicate an incidence of
chromosomal abnormalities in fetal hydrops ranging from 20% to
80%.8-10 The lower frequency reported by Bellini and colleagues
may in part be due, as discussed in their publication, to the dif-
ficulty in correctly classifying patients with chromosomal abnor-
malities and fetal hydrops. Such patients may be classified in the
chromosomal category or in the cardiovascular category because
the hydrops in a proportion of these patients may be secondary to
the cardiac malformation present. Structural cardiac abnormalities
are a common finding in trisomies 21, 18 and 13.
Another factor that may influence the frequency of chromo-
somal abnormality is whether or not cases of CH with fetal hydrops
TABLE
36.3 Cardiovascular Conditions Associated With Nonimmune Fetal Hydrops

Cardiovascular Condition Specific Diagnosis Key Points

Congenital heart defect (CHD) Hypoplastic left heart syndrome (HLHS) • Spectrum of disorders involving aortic atresia with or without mitral atresia or
stenosis
Atrioventricular (AV) canal defect • Involves both lower atrial and upper ventricular septal defects and a common
AV valve orifice; prenatal prognosis is variable
• 70% are associated with other cardiac malformations
• Associated with trisomy 21
Atrial septal defect • If large, may result in RV overload
Ventricular septal defect • Most common congenital cardiac lesion
• Can be associated with tetralogy of Fallot or transposition of the great vessels
Tetralogy of Fallot • Complex congenital cardiac malformation consisting of varying degrees of RV
outflow tract obstruction, overriding aorta and RV hypertrophy
Hypoplastic right heart syndrome • Less common than hypoplastic left heart syndrome
Ebstein anomaly • Malformation of the tricuspid valve causing insufficiency and atrialisation of a
significant portion of the RV
• Progression creates RV outflow tract obstruction and arrhythmias
• Prenatal diagnosis of Ebstein anomaly increases the obstructive or arrhythmia
risk because of the possible severity, thereby allowing prenatal identification
Truncus arteriosis • Fetal hydrops is rare
• Single cardiac outflow tract gives rise to the pulmonary, coronary and sys-
temic circulations
• 40% associated with 22q11 deletion;
Transposition of the great arteries • Complete TGA results in separation of the systemic and pulmonary circula-
(TGA) tions and with the AV discordance results in severe hypoxia postnatally
Aortic stenosis or atresia • Rare cases of critical aortic stenosis associated with hydrops
Cardiomyopathy • Hypertrophic: most common causes are maternal diabetes, TTTS, Noonan
syndrome and inborn errors of metabolism
• Dilated: most common causes are infection, endocardial fibroelastosis,
dysrhythmia and carnitine deficiency
Endocardial fibroelastosis • Thickening of the endocardium caused by a proliferation of cellular and
elastic tissue; it can be associated with obstruction of the great vessels and
familial inheritance
• Ultrasound imaging shows bright echogenic areas within the ventricular walls
Premature closure of ductus arteriosus • Inhibitors of prostaglandin synthesis (e.g., indomethacin) can constrict the
fetal ductus arteriosus both in vitro and in vivo
• This constriction effect is most pronounced after 30 weeks of gestation
Premature closure of foramen ovale • Rare cardiac abnormality
Cardiac arrhythmia Supraventricular tachycardia (SVT) • Prolonged duration with rate >200 beats/min is associated with hydrops
• Congenital heart disease is associated with SVT in 5%–10%
Atrial flutter • Rate of 300–500 beats/min
Bradyarrhythmia or congenital heart • Immune-mediated CHB (maternal SS-A and SS-B antibodies) can lead to
block (CHB) permanent heart damage
• Congenital heart defects can be associated with CHB
• Associated cardiac malformations with CHB: LA isomerism, TGA, ASD, pul-
monic atresia, anomalous pulmonary venous connection, double outlet right
ventricle, AV discordance, absent right AV connection,double inlet ventricle,
right atrial isomerism, pulmonic stenosis
Cardiac tumours Rhabdomyoma • 60%–80% fetal intracardiac tumours are caused by rhabdomyomas
• 60%–95% of rhabdomyomas are secondary to tuberous sclerosis (AD inheri-
tance with a high rate of new mutations)
• Fetuses with intramural cardiac tumours are at an increased risk for cardiac
arrhythmias and WPW syndrome
Intrapericardial teratoma, fibroma, • Fetuses with intramural cardiac tumours are at an increased risk for cardiac
myoma arrhythmias and WPW syndrome

AD, Autosomal dominant inheritance; ASD, atrial septal defect; AV, atrioventricular; LA, left atrial; RV, right ventricle; SVT, supraventricular tachycardia; TTTS, twin to twin transfusion syndrome; WPW,
Wolf-Parkinson-White.
  
432 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
36.4 Inherited Haematological Disorders Associated With Nonimmune Fetal Hydrops
Condition Pathophysiology Gene Inheritance Reference
Haemoglobinopathies: Haemolysis caused by absent α- or HBA1 Autosomal recessive Arcasoy and Gallagher16
α-thalassemia syn- abnormal α-globin chains
dromes
Glucose 6-phosphate Haemolysis associated with G6PD X-linked recessive Arcasoy and Gallagher16
dehydrogenase enzyme deficiency and maternal
deficiency fava bean ingestion or certain
medications
Pyruvate kinase defi- Haemolysis caused by enzyme PKLR AR Arcasoy and Gallagher16
ciency deficiency, resulting in
decreased ATP
Glucosephosphate isom- Haemolysis caused by enzyme GPI AR Arcasoy and Gallagher16
erase deficiency deficiency, resulting in
decreased ATP
Hereditary spherocytosis Haemolysis caused by abnormal SPTB Autosomal recessive for the fetal form Gallagher et al17
membrane skeleton leading to hydrops
Heterozygous carriers present with post-
natal hereditary spherocytosis (AD)
Congenital dyserythro- Haemolysis due to KLF1’s key role KLF1 Autosomal recessive Magor et al18
poietic anaemia in haemoglobin switching
Haemophagocytic lym- Erythrocyte under production sec- PRF1 AR Balta et al19
phohistiocytosis ondary to benign abnormal pro- UNC13D Iwatani et al20
liferation of histiocytes, which Bechara et al21
leads to multiorgan failure
Diamond-Blackfan Congenital erythroblastopenia RPS19 AD; de novo or inherited. Da Costa et al22
anaemia

AD, Autosomal dominant inheritance; AR, autosomal recessive inheritance; ATP, adenosine triphosphate.
  

fetal hydrops that have reported on the incidence of chromosomal
abnormalities and have separated the cases of fetal hydrops with
CH from the cases of fetal hydrops alone, have shown a higher fre-
quency of chromosomal abnormality in the former group (∼55%
compared with 20%–25% in NIHF without CH).8,9 The most
common chromosomal abnormalities were Turner syndrome and
Down syndrome. Other chromosomal abnormalities seen in both
groups included trisomy 18, trisomy 13 and structural chromo-
somal abnormalities.
Gestational age at presentation and ethnicity are other factors
to consider in assessing the risk of chromosomal abnormality. A
review of 100 cases of fetal hydrops in a southeast Asian popula-
tion found a relatively low incidence of chromosomal abnormality
(8%) compared with 22% of the cases diagnosed with haemoglo-
bin Bart’s disease (α-thalassemia hydrops fetalis).11 In the same
study, cases with and without chromosomal abnormalities were
compared, and the mean gestational age at diagnosis in the cases
with fetal hydrops and a chromosomal abnormality was 18 weeks’
gestation, 10 weeks earlier than in the group without a chromo-
somal abnormality.
Other cohort studies have supported the concept that aetiol-
ogy varies with gestational age at presentation, and in fetuses with
NIHF before 24 weeks, a chromosomal abnormality is seen in a
higher percentage of cases (32%12 vs 45%13). However, even if
fetal hydrops is detected in the third trimester, chromosome anal-
ysis is justified even if second trimester ultrasound was normal.
In a series of 214 cases of fetal hydrops, 4 fetuses were diagnosed
with transient abnormal myelopoiesis (1.9%) presenting between
29 and 33 weeks of gestation, and all cases had trisomy 21.14 This
is further supported by a retrospective case series of 79 cases of
trisomy 21 prenatally diagnosed. Eleven of these cases had fetal
hydrops, and in three, it was associated with haepatomegaly and
a myeloproliferative disorder presenting in the second and third
trimesters.15 A diagnosis of transient myeloproliferative disorder
can be suspected prenatally by the finding of blast cells and an
elevated white blood cell count in fetal blood. 
Haematologic Conditions
Fetal anaemia is a common finding in fetal hydrops and has mul-
tiple aetiologies, including blood group alloimmunisation (IHF),
infection (discussed later), fetal haemorrhage (fetomaternal haem-
orrhage), donor twin in TTTS and fetal inherited hematologic
disorders, most commonly α-thalassemia. Other less common
inherited disorders resulting in fetal anaemia are summarised in
Table 36.4. As discussed earlier, anaemia can also result from a
myeloproliferative disorder as is seen with trisomy 21 and lead to
NIHF.
The percent contribution of α-thalassemia to NIHF var-
ies greatly and is dependent on the ethnicity or geographic
location of the cohort. Typically, the -thalassemia four-gene
deletion (--(SEA)/--(SEA) genotype at the α globin locus, hae-
moglobin Bart’s hydrops fetalis) is the cause of the fetal anaemia
which results in high-output cardiac failure and fetal hypoxia.

Less commonly, haemoglobin H (HbH) disease caused by a
double-α gene deletion (--(SEA)) inherited from one parent
and Hb Constant Spring, an unstable haemoglobin variant
(Hb CS; HBA2:c.427T>C), inherited from the other parent
causes hydrops fetalis. α-Thalassemia carrier status is common
in Southeast Asian populations, and this autosomal recessive
disorder accounts for 28% to 55% of their NIHF. Most other
population cohorts report approximately 10% of their cases
caused by hematologic disorders.  with hydrothorax, and all five had associated polyhydram-
Lymphatic Dysplasia
Over the past decade, an increased proportion of cases with NIHF
have been diagnosed with conditions associated with abnormal
lymphatic development (CH and NIHF, chylothorax, or chyloas-
cites and NIHF).2
As discussed in the previous section, CH and NIHF is asso-
ciated with a high risk of chromosomal abnormalities. When
a chromosomal cause has been excluded, a syndromic cause
must be considered, with Noonan syndrome being the most
common syndrome associated with CH. Noonan syndrome is
an autosomal dominant disorder caused by mutations in genes
that participate in RAS–mitogen-activated protein kinase
signal transduction (RAS-MAPK pathway genes: PTPN11,
KRAS, SOS1, RAF1, MAP2K1, BRAF, SHOC2, NRAS, CBL
and RIT1) with PTPN11 mutations found in approximately
50% of patients. In 2006, a report of PTPN11 testing of preg-
nancies presenting with CH with or without hydrops or associ-
ated anomalies indicated the detection of a mutation in 16% of
cases.23 This finding was replicated in another study that found
mutations in both PTPN11 and RAF1 in pregnancies with
CH,24 leading to the recommendation that Noonan syndrome
gene testing should be considered in all cases of CH with nor-
mal karyotypes. Approximately 85% of cases of Noonan syn-
drome can be diagnosed by of a panel of genes responsible for
Noonan syndrome and other RASopathies.
Two other groups of genetic disorders should be consid-
ered in the context of CH and fetal hydrops: fetal akinesia
deformation sequence (FADS) and lethal multiple pterygium
syndrome (LMPS). Both disorders have some phenotypic
overlap with decreased fetal movements and arthrogryposis.
A review of 79 consecutive cases of FADS showed that 15%
presented with fetal hydrops.25 Both FADS and LMPS are
genetically heterogeneous and have been shown in a propor-
tion of cases to be caused by mutations in the neuromuscu-
lar junction genes: recessive mutations in CHRNA1, CHRND
and CHRNG have been described in patients with LMPS and
recessive mutations in CNTN1, DOK7, RAPSN and SYNE1 in
patients with FADS,26 and more recently recessive mutations
in RYR1 have been found in 8.3% of patients with FADS/
LMPS phenotypes.27 It has been postulated that the decreased
fetal movement in these disorders is associated with decreased
lymphatic flow, leading to the CH and fetal hydrops. With
exome sequencing studies being performed on cases of FADS
and LMPS, it is likely that numerous additional genes causing
these phenotypes will be identified.
Abnormalities in lymphatic development (chylothorax or
chyloascites) can also present in the second and third trimes-
ters. The abnormalities may be reflective of a chromosomal or
genetic syndrome or primary lymphatic dysplasia. Congenital
chylothorax is the most common type of pleural effusion in
fetal life and is associated with fetal hydrops in 60% to 70% of
CHAPTER 36  Fetal Hydrops 433
cases.28,29 In one retrospective case series with 10 cases of con-
genital chylothorax, a diagnosis of trisomy 21 was made in one
case and a diagnosis of Noonan syndrome in three cases.29 A
larger prospective German nationwide epidemiologic study of
chylothorax reported on 27 cases of chylothorax with one case
with trisomy 21 and five cases with Noonan syndrome.30 A
review of the prenatal features in a series of 47 patients with a
molecular diagnosis of Noonan syndrome reported 5 patients
nios. Mutations in PTPN11 and SOS1 were found in four and
one patients, respectively.31 Mutations in other genes of the
RAS-MAPK pathway have been reported to cause chylothorax
and fetal hydrops, including SHOC2 and CBL.32,33 Although
Noonan syndrome is the most common syndrome associated
with chylothorax and fetal hydrops, other syndromes that are
part of the RASopathies, cardio-facio-cutaneous and Costello
syndrome, can be the cause of the prenatal findings of lym-
phatic dysplasia.34
Primary generalised lymphatic dysplasia (GLD), although
rare, is a recognised cause of fetal hydrops. GLD results from
an inherent developmental abnormality of the lymphatic system
involving the viscera. The onset of lymphatic drainage failure
can be prenatal or postnatal.35 Hennekam syndrome is an exam-
ple of an autosomal recessive syndromic form of GLD that is
characterised by lymphoedema of the limbs, genitalia and face;
secondary facial dysmorphic features; intestinal lymphangiec-
tasia; and varying degrees of intellectual disability.36 In severe
cases, it may present prenatally with fetal hydrops.36,37 Recessive
mutations in CCBE1 and FAT4 are found in fewer than 50% of
Hennekam patients, indicating that the condition is genetically
heterogeneous, and some genes remain unknown.38 Another
form of recessive GLD with a high incidence of NIHF with
either demise or complete resolution of the neonatal oedema
followed by childhood onset of lymphoedema with or without
systemic involvement has recently been reported in six fami-
lies harbouring mutations in PIEZO1, a gene coding for a cal-
cium permeable mechanically activated ion channel found in
the plasma membrane of various cell types.39 A recent report
of hydrops fetalis and pulmonary lymphangiectasia caused by
autosomal dominant FOXC2 mutation which presented with
lymphoedema of the lower limbs and irregularly implanted eye-
lashes (lymphoedema-distichiasis syndrome) in the father and
numerous family members illustrates that these conditions can
have variable presentations and that a thorough analysis of the
familial pedigree is important.40 Primary congenital lymph-
oedema also known as Milroy disease, a dominant condition
caused by mutations in FLT4 inherited as autosomal dominant
or recessive, typically presents with congenital chronic swelling
of the lower limbs. However, it has also been reported to pres-
ent prenatally with oedema of the lower limbs and transient
ascites and pleural effusions, further supporting the concept of
variable presentation with the more severe cases presenting with
fetal hydrops.41 Given the growing number of genes implicated
in lymphatic anomalies,42 it is conceivable that de novo muta-
tions in these genes may account for a significant proportion
of the cases currently considered as idiopathic fetal hydrops.
Indeed, sequencing analysis of FLT4, FOXC2 and SOX18
of 12 probands who survived in utero generalised oedema or
hydrops fetalis of unknown aetiology identified mutations in
FLT4 in two patients and a mutation in FOXC2 in one patient,
although none of the patients had a positive family history of
lymphoedema.43 
434 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Infection
A variety of infectious agents have been associated with NIHF,
including parvovirus B19 (most common), cytomegalovirus
(CMV), herpes simplex virus, Toxoplasma gondii, rubella, syphi-
lis and chlamydia.44 Infectious agents produce hydrops through
effects on fetal bone marrow, myocardium or vascular endothe-
lium. They represent 7% of cases of NIHF. Fetal infections are
detailed in Chapter 42.  (9.3%) had an LSD. This finding strongly suggests that LSD and
Thoracic Masses
Thoracic masses account for 5%2 of cases of NIHF and include
any thoracic pathology that has a significant mass effect and
results in elevated central venous pressure. This includes congeni-
tal pulmonary airway malformations (CPAMs; see Chapter 30),
diaphragmatic hernia (see Chapter 31), chylothorax (discussed
under lymphatic dysplasia earlier) and intrathoracic tumours (see
Chapter 37).  ders may include obstruction of venous blood return resulting
Metabolic Conditions
Inborn errors of metabolism are a recognised cause of fetal
hydrops, although they are likely to be underdiagnosed given the
need for specialised metabolic or genetic testing that may not
be readily available. The systematic reviews of hydrops fetalis by
Bellini and colleagues (including publications between 1979 and
2007 and 2008 and 2013) found that inborn errors of metabolism
accounted for 1.1% and 1.3% of cases, respectively.2,7 However,
another systematic review of hydrops fetalis aimed at determining
the contribution of lysosomal storage disease (LSD) (the most com-
mon class of inborn error of metabolism causing NIHF) reported
that of the 54 retrospective studies and case series with cases of
NIHF with its workup described, only 15 (27.8%) reported test-
ing for LSD, and the extent of this workup varied among studies.
Amongst the 678 total NIHF cases identified in those 15 publi-
cations, 35 cases (5.2%) were diagnosed with LSD.45 In the two
studies with the most extensive workup for LSD, 8 of 86 patients
other disorders of inborn errors of metabolism may account for a
significant proportion of cases of idiopathic NIHF.
At least 15 different LSDs have been reported in series or
case reports as causing NIHF (Table 36.5). The most com-
mon conditions reported were mucopolysaccharidosis type VII
and type IVa, Gaucher disease, GM1 gangliosidosis, sialidosis,
Niemann-Pick types C and A, galactosialidosis, infantile sialic
acid storage disorder and mucolipidosis II. The mechanisms
contributing to the development of hydrops in storage disor-
from organomegaly, anaemia associated with hypersplenism or
reduction of erythropoietic bone marrow stem cells caused by
infiltrating storage cells, and hypoproteinaemia caused by liver
dysfunction.46
A number of inborn errors of metabolism other than LSD have
been reported in cases NIHF, and these are listed in Table 36.5.
Congenital disorders of glycosylation (CDG) type 1 have been
described in a number of cases of NIHF, leading to the sugges-
tion that this class of disorder should be considered in every case
of unexplained NIHF and investigations for PMM2-CDG might
be pursued given it is the most common form of CDG type 1.47 

TABLE Lysosomal Storage Diseases and Nonlysosomal Inborn Errors of Metabolism Associated With Nonimmune
36.5 Hydrops Fetalis
LYSOSOMAL STORAGE DISEASES ENZYME OR PROTEIN DEFECT GENE DEFECT

MUCOPOLYSACCHARIDOSIS
MPS I α-L-iduronidase IDUA
MPS IVA N-acetylgalactosamine-6-sulphatase GALNS
MPS VII Beta-glucuronidase GUSB

SPHINGOLIPIDOSIS OR OLIGOSACCHARIDOSES
GM1 gangliosidosis β-Galactosidase GLB1
Niemann-Pick A Sphingomyelin phosphodiesterase SMPD1
Niemann-Pick C Niemann-Pick C1 protein NPC1
Epididymal secretory protein E1 NPC2
Sialidosis Sialidase-1 NEU1
Galactosialidosis Lysosomal protective protein CTSA
Gaucher type II Glucosylceramidase GBA
Farber disease Acid ceramidase ASAH1
Multiple sulfatase deficiency Sulfatase-modifying factor 1 SUMF1

LYSOSOMAL TRANSPORT DEFECT

Infantile sialic acid storage disease Sialin SLC17A5
OTHERS
Wolman disease Lysosomal acid lipase LIPA
Mucolipidosis Type II N-acetylglucosamine-1-phosphotransferase subunits alpha/beta GNPTAB
 CHAPTER 36  Fetal Hydrops 435

TABLE Lysosomal Storage Diseases and Nonlysosomal Inborn Errors of Metabolism Associated With Nonimmune
36.5 Hydrops Fetalis—cont’d
NONLYSOSOMAL INBORN ERRORS OF
METABOLISM ENZYME OR PROTEIN DEFECT GENE

GLYCOGENOSIS
Type IV 1,4-α-Glucan-branching enzyme GBE1
CONGENITAL DISORDERS OF GLYCOSYLATION
CDG Ia Phosphomannomutase 2 PMM2
CDG Ik Chitobiosyldiphosphodolichol β-mannosyltransferase ALG1
CDG Ih Probable dolichyl pyrophosphate Glc1Man9GlcNAc2 α-1,3-glucosyltransferase ALG8
CDG Ig Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol α-1,6-mannosyltransferase ALG12

PEROXISOMAL DISORDER
Zellweger syndrome Peroxisome biogenesis factor 1 PEX1
FATTY ACID OXIDATION DEFECTS
Long-chain-hydroxyacyl CoA dehydrogenase deficiency Hydroxyacyl-CoA dehydrogenase HADHA
Primary carnitine deficiency Solute carrier family 22 member 5 SLC22A5

CHOLESTEROL BIOSYNTHESIS DEFECTS

Smith-Lemli-Opitz syndrome 7-Dehydrocholesterol reductase DHCR7
Greenberg syndrome: hydrops-ectopic calcification Lamin-B receptor LBR
moth-eaten skeletal dysplasia
Conradi Hünermann: chondrodysplasia punctata 3-β-Hydroxysteroid-δ(8),δ(7)-isomerase EBP

OTHERS
Fumarase deficiency Fumarate hydratase, mitochondrial FH
Transaldolase deficiency Transaldolase TALDO1
S-adenosylhomocysteine hydrolase deficiency Adenosylhomocysteinase AHCY

  

Other Genetic Conditions Idiopathic

A number of genetic conditions and genetic syndromes have been Approximately 18% of cases of NIHF are classified as idio-
discussed under previous aetiologic categories. Skeletal dyspla- pathic because no underlying pathology is found to explain
sias represent another group of genetic disorders that can lead to the hydrops. As discussed in the section on inborn errors of
NIHF. A number of rare skeletal dysplasias include as a consistent metabolism, the percentage of idiopathic cases in each series
feature CH or fetal hydrops in addition to severe micromelia with varies and is in part dependent on the extent of the investiga-
short ribs and a small chest. Table 36.6 reviews the most com- tions carried out. As whole-exome sequencing becomes a more
mon skeletal dysplasias associated with NIHF. Arriving at a spe- integral part of the prenatal and postnatal and postmortem
cific diagnosis of the type of skeletal dysplasia is important because workup of NIHF, it is likely that the percentage of idiopathic
some have an autosomal recessive mode of inheritance, and others cases will decrease. 
represent new mutations for a dominant gene. (More details on
skeletal dysplasias are given in Chapter 34.)  Clinical Evaluation
In 2015, the Society for Maternal-Fetal Medicine (SMFM) pub-
Other Less Common Categories lished a clinical guideline for NIHF.49 The recommendations
Malformations of the gastrointestinal tract or genitourinary tract made in this guideline are still valid. However, given progress
and extrathoracic tumours each account for a small percentage of made in DNA testing for single gene disorders using next genera-
cases of NIHF (see Table 36.2).  tion sequencing (gene panels or whole exome), these modalities
436 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
36.6 Skeletal Dysplasia Associated With Nonimmune Hydrops Fetalis
Condition and
Incidence
Achondrogenesis types
1A, 1B and 2: 1 in
40,000 for all types
of achondrogenesis
Caffey disease, severe
lethal variant: very
rare
Desbuquois dysplasia
Very rare
Greenberg dysplasia
(Hydrops-ectopic
calcification-moth-
eaten skeletal
dysplasia): extremely
rare
Short rib–polydactyly
types 1 and 3: very
rare
Short rib–polydactyly
type 2: very rare
Thanatophoric dyspla-
sia: 1 in 20,000 to 1
in 50,000
48
Gestational Age of
Presentation Ultrasound Findings Gene Inheritance
11–16 wk Considerable overlap in US findings between the differ- 1A: TRIP11 AR
ent types 1B: DTDST AR
Cystic hygroma or hydrops; short long bones; small 2: COL2A1 AD, de novo
thorax with slender; short ribs; unossified vertebral mutation
bodies; hypomineralisation of the skull in type 1A
14–20 wk Hydrops Unknown AR
Cortical hyperostosis of long bones and ribs, resulting in
limb bowing and shortening, and narrow thorax
20 wk Hydrops CANT1 AR
Micrognathia; proptosis; marked rhizomesomelic
shortening; short, narrow thorax; cleft palate; large
joint dislocations
Possible associated anomalies: CHD, omphalocele
17 wk Cystic hygroma, hydrops LBR AR
Severe micromelia, poorly ossified skull vault, abnormal
contours of the long bones with irregular hypo- and
hyperechoic areas within the bones. Flattened ver-
tebrae and irregular hyperechogenicity of vertebral
bodies; small thorax, short ribs
Postaxial polydactyly may be present
Type 1: 12–14 wk Cystic hygroma and hydrops DYNC2H1 AR
Type 3: milder SRPS1: marked micromelia; polydactyly of hands
second to third and sometimes feet; narrow thorax with short,
trimesters horizontally oriented ribs and a prominent abdomen;
occasionally absent fibula
Associated anomalies: CHD, renal dysplasia, renal cysts,
anal atresia, ambiguous genitalia
SRPS3: similar but milder with less frequent associated
anomalies
12–14 wk Hydrops NEK1 AR
Marked micromelia; polysyndactyly of hands and some- NEKI and DYNC2H1 Digenic biallelic
times feet; narrow thorax with short, horizontally mutations
oriented ribs and a prominent abdomen
Associated anomalies: midline cleft lip, cleft palate,
renal dysplasia, renal cysts, ambiguous genitalia,
cerebellar and brain anomalies
12–18 wk Increased nuchal translucency and occasionally hydrops FGFR3 AD, de novo muta-
TD I: short, curved femurs, occasionally clover leaf skull tions
TD II: straight, long femurs with cloverleaf skull
Both have narrow thorax with short ribs, trident hands
Associated anomalies: ventriculomegaly, CHD, renal
anomalies

AR, Autosomal recessive; AD, autosomal dominant; CHD, congenital heart defect; SRPS, short rib polydactyly syndrome; US, ultrasound.
  

must now be considered in the investigation of fetal hydrops that
remain unexplained after all other investigations have been carried
out.
Clinical History
The assessment of the hydropic fetus should start with a detailed
clinical history, including maternal age, current and prior obstetri-
cal history, medication (prescription and nonprescription), infec-
tious exposure, maternal and paternal blood type, single-gene
carrier screening, aneuploidy screening, ethnicity, family history
of both patient and partner to assess for consanguinity, inherited
conditions, history of stillbirths, recurrent spontaneous losses and
infants with birth defects. 
Fetal Assessment
Imaging techniques. Real-time fetal ultrasonography, fetal vascular
blood flow Doppler, fetal echocardiography and ultrafast fetal
magnetic resonance imaging are the common imaging techniques
 CHAPTER 36  Fetal Hydrops 437

TABLE 51
36.7 Distribution of Fluid Collection in HF of Different Aetiologies and Timing of Hydrops
Cause of HF (n = 220) # of Cases by Pleural Pericardial Effusions Cystic
Trimester: First/Second/Third Ascites (%) Effusions (%) (%) Skin Oedema (%) Hygroma (%)
Aneuploidy (n = 85) 40 35.3 9.4 69.4 51.8
44/31/10
CHD (n = 32) 68.8 31.3 12.5 78.1 12.5
5/18/9
SD (n = 13) 69.2 15.4 30.8 76.9 15.4
5/5/3
PV B19 (n = 9) 66.7 44.5 66.7 55.6 11.2
2/7/0
Immune HF (n = 2) <1 0 <1 <1 0
0/2/0
Idiopathic (n = 54) 64.8 48.1 9.3 81.5 7.4
17/24/13
Other (n = 25) 56 64 36 68 24
8/15/2

CHD, Congenital heart defect; HF, hydrops fetalis; PV B19, parvovirus B19; SD, skeletal dysplasia.
  

used to assess the extent of the hydrops and cardiovascular status
of the fetus as well as to identify an underlying aetiology. Hydrops
is only a clinical sign. Reaching a diagnosis means establishing the
underlying cause. Imaging is also an important tool for ongoing
fetal surveillance.
The identification of fetal hydrops should result in urgent
detailed obstetric ultrasound and fetal echocardiography:
• Detailed obstetric ultrasound examination for assessment of
fetal biometry with estimation of fetal weight, amniotic fluid
index, biophysical profile, site and number of fluid collections,
fetal structural evaluation, measurement of placental thickness
and assessment of fetal venous and arterial circulation by Dop-
pler of middle cerebral artery, ductus venosus and umbilical
artery
• Fetal echocardiography to assess for an underlying cardiac struc-
tural defect and arrhythmia in addition to cardiac function
• Other measurements: cardiothoracic ratio (CTR defined as the
area occupied by the heart in diastole divided by the thoracic
area in a standard axial image of the fetal thorax), combined
ventricular output indexed to fetal weight (CVOi = veloc-
ity time integral × heart rate × semilunar valve area for left
and right ventricle outflows) and cardiovascular profile score
(CVPS). CVPS is a clinical tool that accounts for both cardiac
function and Doppler velocimetry by calculating the extent of
derangement in five parameters: fetal hydrops, heart size, car-
diac function and arterial and venous Doppler flow through
the umbilical vessels and ductus venosus.50
In a study of 220 cases of fetal hydrops, Hartge and coworkers51
­analysed the distribution of fluid collections and the gestational
age at ultrasound diagnosis in fetuses with different causes (Table
36.7). CH with skin oedema was found to be a strong indicator of
aneuploidy. Cases with congenital heart defect showed generalised
skin oedema and ascites as common signs of heart failure. Another
finding to consider is the high frequency of pericardial effusion in
cases of fetal hydrops caused by parvovirus B19. Based on a study
by Whybra and coworkers,52 LSD should be considered in unex-
plained NIHF with ascites. 
Testing for underlying aetiology. The findings from the his-
tory and fetal imaging will help guide the aetiologic workup. A
stepwise process for investigating fetal hydrops is discussed in a
number of publications.49,53-56 The evaluation and diagnostic pro-
cess is summarised as:
Step 1: maternal blood workup (urgent)
• Complete blood count (CBC)
• Kleihauer-Betke, ABO blood type and antigen status, indirect
Coombs (antibody status)
• Serology for syphilis, parvovirus, toxoplasmosis, CMV and
rubella
• Liver function tests, uric acid and coagulation tests (if sus-
pected mirror syndrome; see next section)
• SS-A, SS-B antibodies (if fetal bradycardia present)
• Dependent on maternal ethnic origin: If CBC shows mean
RBC volume is less than 80 fL, DNA testing for α-thalassemia
indicated. Glucose-6-phosphate dehydrogenase deficiency
screen should also be considered in populations at risk. 
Step 2: invasive testing
• Amniocentesis: (because fetal hydrops is usually detected at or
after 15 weeks of gestation)
•  Chromosomal analysis: rapid aneuploidy screening by
quantitative fluorescence polymerase chain reaction (QF-
PCR) or fluorescence in situ hybridisation (FISH) followed
by microarray analysis if QF-PCR negative. Karyotype is an
option if microarray is not available.
• DNA for next generation, sequencing by panel or whole-
exome if other investigations failed to find an etiology
• DNA banking
• Infection (polymerase chain reaction [PCR] for CMV, par-
vovirus B19, toxoplasmosis)
• Glycosaminoglycans and enzymatic analysis for lysosomal stor-
age disorders57 if next generation sequencing is not an option
438 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Cordocentesis: fetal blood (on a case-by-case basis depending
on the differential diagnosis)
• CBC, white blood cell count, platelets
• Direct Coombs test, blood group and type
• PCR for CMV, parvovirus B19 and toxoplasmosis
• Liver function tests, protein, albumin, haemoglobin elec-
trophoresis
• Fetal body cavity aspiration
• Lymphocyte count (pleural)
• Protein, albumin and creatinine (ascites)
• PCR for CMV and viral and bacterial cultures
Ultrasound-guided amniocentesis and cordocentesis are impor-
tant invasive techniques for the investigation of fetal hydrops. The
technique of choice will be depend on the gestational age at the
time of presentation (amniocentesis can be done from 15 weeks
of gestation to term, but cordocentesis can only be done from 18
weeks of gestation to term) and whether or not anaemia is present
and an intrauterine transfusion (IUT) is being considered. Chori-
onic villi sampling is a further option for cases of NIHF presenting
between 11 and 14 weeks’ gestation. Before any invasive diagnos-
tic procedure, the informed consent process requires the discus-
sion of maternal and fetal risks and benefits. The benefits include
establishing a specific diagnosis, possible treatment and predicting
prognosis based on natural history of the condition. The sponta-
neous pregnancy loss rate in the context of fetal hydrops is unde-
termined. An invasive diagnostic procedure adds to this loss rate.
There are no specific postprocedure fetal loss rates in cohorts with
fetal hydrops, and evidence-based risk rates quoted are from fetal
cohorts undergoing invasive testing for variable indications.
Before invasive testing, reproductive genetic counselling
should occur to ensure informed consent to microarray testing,
gene sequencing panels or whole-exome sequencing. Patients
and their partners should understand the benefits of identifying
a pathogenic variant explaining the hydrops as well as the risks of
identifying a variant of unknown clinical significance or an inci-
dental finding that may have implications for the fetus, one of the
parents and other family members.  indications and survival rates.65 For each indication (total num-
Maternal Assessment for Mirror Syndrome and
Other Obstetric Complications
Evaluation and follow-up of the pregnancy after the finding of
fetal hydrops requires attention for mirror syndrome. It is defined
as the development of maternal oedema in association with fetal
hydrops.58 A systematic review of cases published between 1956
and 2009 illustrated that mirror syndrome is associated with both
IHF and NIHF and is caused by diverse aetiologies, including
TTTS, viral infection, structural anomalies, arrhythmias, fetal
anaemia, inherited metabolic disorders and fetal or placental
tumours.58 However, this review did not allow one to estimate the
incidence of mirror syndrome in cases of fetal hydrops. One small
retrospective study of 35 singleton ongoing pregnancies with fetal
hydrops found that 10 out of the 35 (29%) pregnant women
developed mirror syndrome.59 The cases of fetal hydrops with mir-
ror syndrome had an earlier onset and were more severe compared
with the cases of fetal hydrops without mirror syndrome. It was
noted that the treatment of the fetal hydrops in 2 cases by pleu-
roamniotic shunting showed improvements in the fetal hydrops
and maternal oedema. Another retrospective study of 75 cases of
NIHF in singleton pregnancies assessed the incidence of mirror
syndrome in their population but defined it as development of
maternal preeclampsia associated with fetal hydrops. Using this
definition, mirror syndrome was seen in 5.3% of cases.60 Given
these risks, ongoing pregnancies with fetal hydrops should be
followed for the development of excessive maternal weight gain,
peripheral oedema, increased blood pressure, proteinuria and
abnormal liver function test results (maternal HELLP [haemoly-
sis, elevated liver enzymes, low platelet count] syndrome).
Ongoing pregnancies with fetal hydrops are also at risk of
hydramnios, which may contribute to fetal malpresentation,
preterm labour, premature rupture of membranes with placental
abruption and chorioamnionitis. 
Fetal Therapy
After a possible aetiology and diagnosis for the NIHF have been
identified, then selected fetal treatments may be considered or ini-
tiated to improve fetal physiology. The fetal therapies associated
with increased maternal–fetal risk may be delayed until results
of the diagnostic workup are available. All treatments require
informed consent counselling of the parents. Established prena-
tal treatment options for different aetiologies associated with fetal
hydrops are summarised in Table 36.8 based on published fetal
hydrops cohorts but with no randomised controlled trials (RCTs)
for evidence.
Maternal–fetal surgery for NIHF including the principles, indi-
cations and treatment evidence are summarised by Adzick61 and
Wenstrom and Carr.62 Therapeutic options for selected aetiologies of
NIHF are summarised (more detail can be found in other chapters):
• Fetal intravascular or maternal directed antiarrhythmic drugs for
treatment of fetal arrhythmia (tachyarrhythmia; atrioventricular
heart block-anti SS-A/SS-B). Recent reviews and scientific con-
sensus for fetal arrhythmias and fetal cardiac disease with clear
definition, incidence, diagnosis, management and outcome for
specific diagnostic assessment but no specific comments for car-
diac associated hydrops and treatment were given.63,64
• IUT with a diagnosed fetal anaemia (immune and nonim-
mune). A 2014 systematic review of IUT reviewed the current
ber of cohorts reported–total number of cases), the survival rate
with IUT were RBC alloimmunisation (40–491) with a sur-
vival rate of 80.5% to 93.5%, parvovirus B19 (16–73) with
a survival rate of 66.7% to 72.7% and TTTS (4–13) with a
survival rate of 75-76.9%.
• IUT for α-thalassemia has been used with surviving children
requiring lifelong transfusion therapy, haematopoietic stem cell
transplantation or both. Opinions at present are that because
of the high maternal and fetal morbidity secondary to the dis-
ease and the cost and ethical dilemma, this fetal therapy still
requires considerable thought and discussion. This treatment
option is not supported by the SMFM 2015 guidelines.49
Informed consent counselling includes information that IUT
is considered a safe method to correct severe fetal anaemia. Acute
procedure-related complications include abnormal fetal heart
tracing secondary to cord accidents (rupture, spasm, haematoma
tamponade, excessive bleeding), fetal volume overload or preterm
labour. Fetal demise can occur because of compromised fetal
physiology related to the anaemia or because of the procedure.
Procedure-related fetal loss is estimated at 0.9% to 4.9% per pro-
cedure with the highest loss rates caused by fetal hydrops, early
gestational age, failing to use fetal paralysis, transfusion in free
loop or artery, operator experience and severity of the anaemia.
Premature rupture of membranes and chorioamnionitis are rare
IUT complications.
 CHAPTER 36  Fetal Hydrops 439

TABLE Evidence-Based Therapies for Different Oetiologies of Hydrops Fetalis and Quality of the Evidence Based on
36.8 Cohort Studies (No Randomised Controlled Trials)
Therapy Cause of Hydrops Level of Evidence
MATERNAL AND/OR FETAL MEDICAL ADMINISTRATION

Maternal IM corticosteroids similar to fetal lung CPAM: large-volume (increased risk for hydrops) solid or Moderate
maturity protocol (two doses) macrocyst CPAM Low
Cardiac heart block
Maternal oral digoxin SVT Moderate
Fetal intravascular or intraperitoneal SVT Moderate to strong
Intrauterine transfusion with ultrasound guidance RBC alloimmunisation (mainly Rhesus D) Strong
α-Thalassemia (haemoglobin Bart’s disease) Low
Parvovirus B19 Moderate
Post TTTS laser vascular occlusion TAPS Moderate
Percutaneous ultrasound–guided laser for vascular TTTS recipient fetal hydrops Strong
occlusion under direct vision
TRAP hydropic TRAP twin Moderate
Placental chorioangioma Moderate
Solid CPAM with attempt at occlusion of a feeding vessel Low
(after corticosteroid use)
RFA TRAP Strong
Bronchopulmonary sequestration Strong
Sacrococcygeal or mediastinal teratoma (feed vessel size dependent) Low
Percutaneous ultrasound–guided pig-tailed shunt Macrocystic CPAM (after percutaneous drainage and Moderate
corticosteroid use)
Pleural effusions (pleurodesis or thoracoamniotic shunt) Moderate
Lower urinary tract obstruction: Moderate
• Posterior urethral valves Moderate
• Urethral stenosis Low
• Urethral atresia
Percutaneous ultrasound–guided fetal cardiac Aortic stenosis with possible development of hypoplasia left Low
balloon valvuloplasty heart syndrome, prematurely closed foramen ovale
Open maternal fetal therapy CPAM solid or cystic Moderate
Sacrococcygeal or mediastinal teratoma Moderate
EXIT delivery Cervical teratoma with hydrops and polyhydramnios Moderate
secondary to esophageal compression
CHAOS caused by laryngeal obstruction Moderate
CHAOS, Congenital high airway obstruction; CPAM, congenital pulmonary adenomatoid malformation; EXIT, ex utero intrapartum treatment; IM, intramuscular; RBC, red blood cell; RFA, radiofrequency
ablation; SVT, supraventricular tachycardia; TRAP, twin-reversed arterial perfusion; TTTS, twin-to-twin transfusion syndrome.
  

Long-term complications after IUT include the need for an
increased number of neonatal RBC transfusions likely caused by
prolonged fetal initiated erythropoiesis suppression. In addition,
transplacental IUT access and small fetomaternal haemorrhage
afer IUT has created a 19% to 26% prevalence of new maternal
RBC antibodies, which may complicate future crossmatching and
blood transfusions in subsequent pregnancies.
• Percutaneous laser or radiofrequency vessel ablation
for fetal vascular shunting malformations. The proactive
therapy for stage IV TTTS, based on RCT and systematic
reviews, is endoscopic laser coagulation with interruption of
the vascular placental anastomoses or selective termination of
the hydropic twin with bipolar cord cautery occlusion. Peri-
natal survival outcomes for stage IV range from 4% to 17%
with high morbidity. Fetal therapy for twin-reversed arterial
perfusion (TRAP) are amnioreduction, inotropic agents and
surgical approaches directed at the ‘acardiac twin’ (hysterot-
omy with selective delivery, umbilical cord occlusion, radio-
frequency ablation, intrafetal laser ablation).66-71
• Single or repeat thoracocentesis or percutaneous thora-
coamniotic shunt insertion for fetal pleural effusions (PEs).
A literature review (Table 36.9) identified 477 ongoing preg-
nancies (terminated pregnancies in ascertained cohorts were
excluded) with percutaneous thoracoamniotic shunts for iden-
tified unilateral or bilateral PE with 354 of /477 (74%) cases
having associated fetal hydrops. Various fetal therapy cath-
eters and techniques were used, including Rocket, Cook and
double-basket catheters. The overall survival rate for hydropic
fetuses with PE undergoing thoracoamniotic shunting was
58% (range, 33%–100%) compared with a survival rate for
440 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
36.9 Antenatal Percutaneous Shunting for Pleural Effusions
Survival Rate for Hydropic Survival Rate for Nonhydropic
Reference Shunted PE Cases (n) Hydropic Cases (n) Cases (%) Cases (%)
Rodeck et al72 8 5 3/5 (60%) 3/3 (100%)
2 NND
Nicolaides and Azar73 44 28 14/28 (50%) 15/16 (94%)
2 IUFD; 12 NND
Bernaschek et al74 8 8 3/8 (38%) NA
4 IUD; 1 NND
Picone et al75 47 47 31/47 (66%) NA
9 IUD; 7 NND
Smith et al76 21 16 7/16 (44%) 3/5 (60%)
7 IUD; 2 NND
Rustico et al77 51 41 25/41 (61%) 9/10 (90%)
5 IUD; 11 NND
Yinon et al78 88 59 Resolved: 20/28 (71.4%);8 21/29 (72%)
NND
Unresolved: 11/31 (35.5%)
10 IUD;10 NND
Caserio et al79 6 4 2/4 (50%) 2/2 (100%)
2 NND
Walsh et al80 15 5 3/5 (60%) 5/10 (50%)
1 IUD; 1 NND
Takahashi et al81 24 17 12/17 (71%) 7/7 (100%)
Pellegrinelli et al82 25 18 8/18 (44%) 6/7 (86%)
Peterson et al83 6 3 1/3 (33%) 3/3 (100%)
1 IUD; 1 NND
Miyoshi84 15 11 5/11 (46%) 4/4 (100%)
White et al85 5a 5 Resolved: 5/5 (100%) NA
Derderian et al86 17 16 9/16 (56%) 1/1 (100%)
Nowakowska et al87 2 0 N/A 2/2 (100%)
Peranteau et al88 35 21 Resolved: 10/15 (67%) 11/14 (79%)
Nonresolved: 0/6 (0%)
Jeong et al89 65 50 Resolved: 25/29 (86.2%) 14/15 (93%)
Nonresolved: 10/21 (47.6%)
Total 482 354/477 (74%) 204/354 (58%) 106/128 (83%)
aSeldinger-based percutaneous approach.
IUD, Intrauterine demise; NA, not available; NND, neonatal death.
  

nonhydropic shunted fetuses for PE of 83% (range, 50%–
100%).
• Open maternal fetal therapy. The fetal conditions that
are indicated for ‘open laparotomy’ maternal fetal surgery
include CPAM, mediastinal and sacrococcygeal teratoma
(SCT) and congenital high airway obstruction syndrome
associated with developing or overt hydrops and gestational
ages of less than 30 weeks. Adzick reports on 24 cases of
massive multicystic or predominately solid CPAM with
fetal lobectomy at gestational ages of 21 to 31 weeks.90
There were 13 healthy survivors (54%) with 1 to 16 years’
follow-up. In the 13 fetuses that survived, the resection
allowed hydrops resolution over 1 to 2 weeks, return of the
mediastinum to the midline in 3 weeks and impressive in
utero lung growth. Neurodevelopment has been normal in
all survivors evaluated.
• Open maternal–laparotomy fetal therapy for isolated SCT uses
a triage process based on high-output cardiac and a gestational
age of less than 32 gestational weeks for in utero SCT resec-
tion. Practice points include that most fetuses with an antenatal
congenital anomaly diagnosis can be treated at an appropriate cen-
tre with maternal transport, planned delivery and prompt therapy
 CHAPTER 36  Fetal Hydrops 441

TABLE Overall Fetal and Infant Mortality Rates for Pregnancies With Nonimmune Hydrops Fetalis and Mortality Rate
36.10 in Live-Born Infants With Nonimmune Hydrops Fetalis in Studies Published from 1979 to 2009 Reviewed by
Randenberg93
Studies Reporting Fetal and Infant Studies Reporting Infant Mortality
Cause (n) Pregnancies (n) Mortality Rate (%) (n) Live Births (n) Rate (%)
Chromosomal 19 280 98 18 72 58
Genetic 15 72 92 13 33 61
Haematologic 11 101 88 11 49 57
Lymphatic 14 192 66 17 139 24
Cardiovascular
• CHD 22 181 92 24 168 61
• Arrhythmia 19 101 39 22 118 26
Infections 20 94 68 23 82 37
Placental 16 78 71 17 118 51
Idiopathic 23 295 77 28 323 51

CHD, Congenital heart defect.

Adapted from Randenberg AL. Nonimmune hydrops fetalis part II: does etiology influence mortality? Neonatal Netw 29(6):367–380, 2010.
  

after birth. Fetal surgery is currently reserved for a small subgroup
of patients with extremely poor prognosis.91 This strict approach of
balancing maternal risk with fetal and neonatal benefit is demon-
strated by Roybal and associates with high risk SCT cases between
27 and 32 weeks undergoing early surgical delivery.92  ment (subcutaneous oedema, PE, pericardial effusion, ascites) and
Prognosis and Recurrence Risk Counselling
The natural history of the NIHF is entirely dependent on the aeti-
ology, and as such, the prognosis for any given patient will be
dependent on the underlying aetiology and whether treatment is
available for the underlying cause. Earlier gestational age at diag-
nosis is associated with an increased fetal and infant mortality
rates, but this is because of a higher proportion of fetuses with
a chromosomal abnormality amongst the cases of fetal hydrops
diagnosed before 26 weeks.93 Outcome data for NIHF of differ-
ent aetiologies is essential for counselling of couples having to
make decisions regarding the pregnancy or neonatal care. A ret-
rospective review of 36 studies published from 1979 and 2009
with outcome of pregnancies by aetiologies of hydrops included a
total of 2,453 cases found overall high mortality rates of 80% for
combined fetal and infant NIHF and 49% for live-born infants
with NIHF.94 For the subset of studies published from 1998 to
2009, the overall prognosis remained poor with a 72% mortality
rate for combined fetal and infant NIHF cases and a 47% mortal-
ity rate for live-born infants. Excluding the aetiologies with small
number of cases, the fetal and infant mortality as well as mortality
rate for live-born infants for the different aetiologies as reported
by Randenberg for studies published between 1979 and 2009 (to
increase sample size and given little difference between the entire
study and subset published between 1998 and 2009) is presented
in Table 36.10. Although the mortality rates among many of the
aetiologic categories are very high and not significantly different,
some aetiologies have a better prognosis such as NIHF as a result
of a lymphatic congenital anomaly or fetal arrhythmia.94
Ultrasonography and echocardiography are used to predict
outcomes and guide management of fetuses with an identified
cause for their hydrops. Kim and associates95 have published an
ultrasound NIHF severity scoring tool for prediction of perinatal
mortality. The tool used the presence of the abnormal fluid collec-
tion in each body compartment (1 point per each body compart-
0 points with absence of abnormal fluid collection). The number
of abnormal collections was totalled and used as the ultrasono-
graphic severity score for nonimmune hydrops (USNIH). The
perinatal death rate in this cohort was 47% (20 of 43) with a
significant USNIH score difference between nonsurvivors and
survivors (≥3 was 67% and =2 was 13%; P < .005). The signifi-
cant difference remained even with an adjustment for confound-
ing variables and with an idiopathic or unknown NIHF aetiology.
This tool may be useful for counselling and triage in the early
part of the NIHF investigations. Two previous studies that had
looked at survival of newborns based on number of compartments
involved had published similar findings.96,97 However, a larger
prenatal cohort of 167 pregnancies followed in a fetal therapy
centre found an overall mortality rate of 56% (varying from 25%
to 100% depending on the aetiology), and the absolute number
of involved compartments did not correlate with the mortality
rate: 46% of patients survived when two compartments were
involved compared with 42% when three or more compartments
were involved, suggesting that increasing fluid does not necessar-
ily indicate a more severe disease state.55 In that study, among the
patients with the more common aetiologies, survival was highest
(50%–55%) among those with CPAM, primary hydrothorax and
anaemia, and it was the lowest (10%) among those with SCT.
Resolution of the hydrops and delivery at a later gestation age for
fetuses whose hydrops persisted were both associated with a bet-
ter survival regardless of the underlying disease process. Improved
survival was also seen in patients who were able to undergo fetal
treatment for diseases in which a treatment exists (as discussed in
previous section). Finally, the study demonstrated that the CTR
(the area occupied by the heart in diastole divided by the tho-
racic area in a standard axial image of the fetal thorax) identified
two distinct groups of patients: those with cardiomegaly (SCT,
442 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
anaemia, cardiac disease and TTTS) and those without (primary
hydrothorax, congenital diaphragmatic hernia, congenital high
airway obstruction syndrome and CPAM). Among those with a
larger CTR, survival was better when the cardiovascular profile
score (CVPS) was higher; CVPS was not predictive of survival in
fetuses with diseases that do not increase the CTR.
Studies of outcome of survivors of NIH are limited, each study
has a small cohort of children and none has long-term outcomes.
One cohort of 28 survivors of NIHF of diverse aetiologies with
follow-up information from parents at 1 to 84 months (mean, 29
months) found that 39% (11 cases) had comorbidity that varied
from some mild conditions (e.g. feeding difficulty and episodes
of tachyarrhythmias) to significant neurodevelopmental delay,
the latter being present in 11% of the cohort.44 In this cohort
of NIHF, nine cases were caused by a parvovirus infection. Most
aetiologic categories had too few live births to analyse outcome by
aetiologic category. However, it is worth noting that there were
eight live-born infants with NIHF caused by parvovirus infec-
tion. One live-born infant was lost to follow-up, and the other
seven cases were found to have normal development. There were
also eight live-born infants with hydrothorax. Two infants died in
the newborn period, three infants were lost to follow-up and the
other three infants had normal development. Another report of 33
infants of NIHF who survived to 1 year of age shows that, exclud-
ing the 5 cases of trisomy 21, only 15 of 28 (53%) infants were
developmentally normal.98 These two reports show significant dif-
ferences in outcome and illustrate our limitations in counselling
parents regarding long-term neurodevelopmental outcome based
on limited data and a large number of underlying aetiologies that
will affect the composition of any small cohort and likely affect
prognosis.
Counselling of couples should address risk of recurrence
in future pregnancies. This will entirely be dependent on the
underlying aetiology for the fetal hydrops. NIHF caused by a
tumour has an extremely low risk of recurrence. When an iso-
lated structural fetal abnormality is the cause of the hydrops such
as a cardiac defect, the multifactorial mode of inheritance would
give a small risk of recurrence (3%–5%). This is also true for
cases caused by trisomies (1%–2%). However, when NIHF is
caused by an inherited autosomal recessive metabolic disorder
or genetic syndrome, the recurrence risk will be 25%. In some
cases, the recurrence risk may be 50% because of the inheri-
tance of a dominant mutation found in a parent with a milder
phenotype. This illustrates the importance of counselling and
extensive investigations prenatally, neonatally or at the time of
an autopsy to reach a diagnosis. Fetal hydrops is only a sign that
does not allow to provide parents accurate recurrence risk coun-
selling. Determining the underlying cause of the fetal hydrops
is critical. For cases of NIHF that remain idiopathic, the pos-
sibility of a recurrence based on an undiagnosed recessive condi-
tion must be shared with couples as reports of exome sequencing
studies in cases of NIHF have demonstrated that a proportion
of ‘idiopathic’ NIHF are genetic disorders. Prenatal diagnosis
in subsequent pregnancies depends on whether or not a specific
diagnosis was made to explain the fetal hydrops. Without a spe-
cific diagnosis, enhanced prospective prenatal surveillance and
imaging is recommended with ultrasound examinations at 12,
16, 20, 24 and 28 weeks of gestation. 
Conclusion
Hydrops fetalis is the common end point for a wide variety of
conditions. It is an ultrasound finding that requires an urgent
directed stepwise investigation to arrive at a specific diagnosis
of the underlying aetiology. This is best pursued in a tertiary
centre by a multidisciplinary team with expertise in maternal
fetal medicine, imaging and clinical genetics. Evaluation is
time sensitive because some aetiologies are amenable to treat-
ment, resulting in improved prognosis. With the introduction
of immunoprophylaxis for women at risk Rhesus-D alloim-
munisation, the proportion of fetal hydrops with an immune
rather than nonimmune cause has decreased significantly. In
addition, the expansion of biochemical testing and NGS is
uncovering a growing number of single-gene disorders present-
ing with NIHF and previously considered ‘idiopathic NIHF’.
Arriving at a specific diagnosis is critical for appropriate man-
agement of the current pregnancy, better understanding of
prognosis and counselling regarding risk of recurrence and
options for prenatal diagnosis in future pregnancies.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 25. Hellmund A, Berg C, Geipel A, et al. Prenatal diagnosis of fetal akine-
sia deformation sequence (FADS): a study of 79 consecutive cases. Arch
Gynecol Obstet. 2016. [Epub ahead of print].
1. Randenberg AL. Nonimmune hydrops fetalis part I: etiology and patho-
26. Chen CP. Prenatal diagnosis and genetic analysis of fetal akinesia defor-
physiology. Neonatal Netw. 2010;29(5):281–295.
mation sequence and multiple pterygium syndrome associated with
2. Bellini C, Donarini G, Paladini D, et  al. Etiology of non-immune
neuromuscular junction disorders: a review. Taiwan J Obstet Gynecol.
hydrops fetalis: an update. Am J Med Genet A. 2015;167A(5):1082–1088.
2012;51(1):12–17.
3. Kontopoulos E, Chmait RH, Quintero RA. Twin-to-twin transfusion
27. McKie AB, Alsaedi A, Vogt J, et  al. Germline mutations in RYR1 are
syndrome: definition, staging, and ultrasound assessment. Twin Res Hum
associated with foetal akinesia deformation sequence/lethal multiple pte-
Genet. 2016;19(3):175–183.
rygium syndrome. Acta Neuropathol Commun. 2014;2:148.
4. García-Díaz L, Carreto P, Costa-Pereira S, et  al. Prenatal management
28. Caserio S, Gallego C, Martin P, et al. Congenital chylothorax: from foetal
and perinatal outcome in giant placental chorioangioma complicated
life to adolescence. Acta Paediatr. 2010;99:1571–1577.
with hydrops fetalis, fetal anemia and maternal mirror syndrome. BMC
29. Downie L, Sasi A, Malhotra A. Congenital chylothorax: associations and
Pregnancy Childbirth. 2012;12:72.
neonatal outcomes. J Paediatr Child Health. 2014;50(3):234–238.
5. Knilans TK. Cardiac abnormalities associated with hydrops fetalis. Semin
30. Bialkowski A, Poets CF, Franz AR. Erhebungseinheit für seltene pädia-
Perinatol. 1995;19(6):483–492.
trische Erkrankungen in Deutschland Study Group. Congenital chylo-
6. Pruksanusak N, Suntharasaj T, Suwanrath C, et  al. Fetal cardiac rhab-
thorax: a prospective nationwide epidemiological study in Germany. Arch
domyoma with hydrops fetalis: report of 2 cases and literature review. J
Dis Child Fetal Neonatal Ed. 2015;100(2):169–172.
Ultrasound Med. 2012;31(11):1821–1824.
31. Baldassarre G, Mussa A, Dotta A, et  al. Prenatal features of Noonan
7. Bellini C, Hennekam RC, Fulcheri E, et  al. Etiology of nonimmune
syndrome: prevalence and prognostic value. Prenat Diagn. 2011;31(10):
hydrops fetalis: a systematic review. Am J Med Genet A. 2009;149A(5):
949–954.
844–851.
32. Gargano G, Guidotti I, Balestri E, et al. Hydrops fetalis in a preterm new-
8. Beke A, Joó JG, Csaba A, et al. Incidence of chromosomal abnormalities
born heterozygous for the c.4A>G SHOC2 mutation. Am J Med Genet A.
in the presence of fetal subcutaneous oedema such as nuchal oedema, cys-
2014;164A(4):1015–1020.
tic hygroma and non-immune hydrops. Fetal Diagn Ther. 2009;25(1):83–
33. Bülow L, Lissewski C, Bressel R, et al. Hydrops, fetal pleural effusions
92.
and chylothorax in three patients with CBL mutations. Am J Med Genet
9. Schwanitz G, Zerres K, Gembruch U, et al. Rate of chromosomal aber-
A. 2015;167A(2):394–399.
rations in prenatally detected hydrops fetalis and hygroma colli. Hum
34. Myers A, Bernstein JA, Brennan ML, et  al. Perinatal features of the
Genet. 1989;84(1):81–82.
RASopathies: Noonan syndrome, cardiofaciocutaneous syndrome and
10. Nadel A, Bromley B, Benacerraf BR. Nuchal thickening or cystic hygro-
Costello syndrome. Am J Med Genet A. 2014;164A(11):2814–2821.
mas in first- and early second-trimester fetuses: prognosis and outcome.
35. Connell FC, Kalidas K, Ostergaard P, et al. CCBE1 mutations can cause
Obstet Gynecol. 1993;82(1):43–48.
a mild, atypical form of generalized lymphatic dysplasia but are not a
11. Anandakumar C, Biswas A, Wong YC, et al. Management of non-immune
common cause of non-immune hydrops fetalis. Clin Genet. 2012;81(2):
hydrops: 8 years’ experience. Ultrasound Obstet Gynecol. 1996;8(3):
191–197.
196–200.
36. Bellini C, Mazzella M, Arioni C, et al. Hennekam syndrome presenting
12. McCoy MC, Katz VL, Gould N, et al. Non-immune hydrops after 20
as nonimmune hydrops fetalis, congenital chylothorax, and congenital
weeks’ gestation: review of 10 years’ experience with suggestions for man-
pulmonary lymphangiectasia. Am J Med Genet A. 2003;120A(1):92–96.
agement. Obstet Gynecol. 1995;85(4):578–582.
37. Shah S, Conlin LK, Gomez L, et al. CCBE1 mutation in two siblings,
13. Sohan K, Carroll SG, De La Fuente S, et  al. Analysis of outcome in
one manifesting lymphoedema-cholestasis syndrome, and the other, fetal
hydrops fetalis in relation to gestational age at diagnosis, cause and treat-
hydrops. PLoS One. 2013;8(9):e75770.
ment. Acta Obstet Gynecol Scand. 2001;80(8):726–730.
38. Alders M, Al-Gazali L, Cordeiro I, et  al. Hennekam syndrome can be
14. Fukushima K, Morokuma S, Fujita Y, et al. Short-term and long-term
caused by FAT4 mutations and be allelic to Van Maldergem syndrome.
outcomes of 214 cases of non-immune hydrops fetalis. Early Hum Dev.
Hum Genet. 2014;133(9):1161–1167.
2011;87(8):571–575.
39. Fotiou E, Martin-Almedina S, Simpson MA, et al. Novel mutations in
15. Smrcek JM, Baschat AA, Germer U, et  al. Fetal hydrops and hepato-
PIEZO1 cause an autosomal recessive generalized lymphatic dysplasia
splenomegaly in the second half of pregnancy: a sign of myeloprolif-
with non-immune hydrops fetalis. Nat Commun. 2015;6:8085.
erative disorder in fetuses with trisomy 21. Ultrasound Obstet Gynecol.
40. de Bruyn G, Casaer A, Devolder K, et  al. Hydrops fetalis and pulmo-
2001;17(5):403–409.
nary lymphangiectasia due to FOXC2 mutation: an autosomal dominant
16. Arcasoy MO, Gallagher PG. Hematologic disorders and nonimmune
hereditary lymphoedema syndrome with variable expression. Eur J Pedi-
hydrops fetalis. Semin Perinatol. 1995;19(6):502–515.
atr. 2012;171(3):447–450.
17. Gallagher PG, Petruzzi MJ, Weed SA, et al. Mutation of a highly con-
41. Boudon E, Levy Y, Abossolo T, et al. Antenatal presentation of hereditary
served residue of betaI spectrin associated with fatal and near-fatal neona-
lymphoedema type I. Eur J Med Genet. 2015;58(6-7):329–331.
tal hemolytic anemia. J Clin Invest. 1997;99(2):267–277.
42. Brouillard P, Boon L, Vikkula M. Genetics of lymphatic anomalies. J
18. Magor GW, Tallack MR, Gillinder KR, et al. KLF1-null neonates dis-
Clin Invest. 2014;124(3):898–904.
play hydrops fetalis and a deranged erythroid transcriptome. Blood.
43. Ghalamkarpour A, Debauche C, Haan E, et al. Sporadic in utero gen-
2015;125(15):2405–2417.
eralized oedema caused by mutations in the lymphangiogenic genes
19. Balta G, Topcuoglu S, Gursoy T, et  al. Association of nonimmune
VEGFR3 and FOXC2. J Pediatr. 2009;155(1):90–93.
hydrops fetalis with familial hemophagocytic lymphohistiocytosis in
44. Santo S, Mansour S, Thilaganathan B, et al. Prenatal diagnosis of non-
identical twin neonates with perforin His222Arg (c665A>G) mutation. J
immune hydrops fetalis: what do we tell the parents? Prenat Diagn.
Pediatr Hematol Oncol. 2013;35(8):e332–e334.
2011;31(2):186–195.
20. Iwatani S, Uemura K, Mizobuchi M, et  al. Familial hemophagocytic
45. Gimovsky AC, Luzi P, Berghella V. Lysosomal storage disease as an etiol-
lymphohistiocytosis presenting as hydrops fetalis. AJP Rep. 2015;5(1):
ogy of nonimmune hydrops. Am J Obstet Gynecol. 2015;212(3):281–290.
e22–e24.
46. Staretz-Chacham O, Lang TC, LaMarca ME, et  al. Lysosomal storage
21. Bechara E, Dijoud F, de Saint Basile G, et al. Hemophagocytic lympho-
disorders in the newborn. Pediatrics. 2009;123(4):1191–1207.
histiocytosis with Munc13-4 mutation: a cause of recurrent fatal hydrops
47. Léticée N, Bessières-Grattagliano B, Dupré T, et  al. Should PMM2-
fetalis. Pediatrics. 2011;128(1):e251–e254.
deficiency (CDG Ia) be searched in every case of unexplained hydrops
22. Da Costa L, Chanoz-Poulard G, Simansour M, et al. First de novo muta-
fetalis? Mol Genet Metab. 2010;101(2-3):253–257.
tion in RPS19 gene as the cause of hydrops fetalis in Diamond-Blackfan
48. Hall CM, Offiah AC, Forzano F, et  al. Fetal and perinatal skeletal dys-
anemia. Am J Hematol. 2013;88(2):160.
plasia: an atlas of multimodality imaging. London: Radcliffe Publishing;
23. Lee KA, Williams B, Roza K, et  al. PTPN11 analysis for the prenatal
2012.
diagnosis of Noonan syndrome in fetuses with abnormal ultrasound find-
49. Society for Maternal-Fetal Medicine (SMFM), Norton ME, Chauhan SP,
ings. Clin Genet. 2009;75(2):190–194.
Dashe JS. Society for Maternal-Fetal Medicine (SMFM) clinical guide-
24. Croonen EA, Nillesen WM, Stuurman KE, et al. Prenatal diagnostic test-
line #7: nonimmune hydrops fetalis. Am J Obstet Gynecol. 2015;212(2):
ing of the Noonan syndrome genes in fetuses with abnormal ultrasound
127–139.
findings. Eur J Hum Genet. 2013;21(9):936–942.

442.e1
442.e2 References
50. Peranteau WH, Wilson RD, Liechty KW, et  al. Effect of maternal
betamethasone administration on prenatal congenital cystic adeno-
matoid malformation growth and fetal survival. Fetal Diagn Ther.
2007;22(5):365–371.
51. Hartge DR, Weichert J, Gembicki M, et al. Confirmation of etiology in
fetal hydrops by sonographic evaluation of fluid allocation patterns. Eur J
Obstet Gynecol Reprod Biol. 2015;195:128–132.
52. Whybra C, Mengel E, Russo A, et al. Lysosomal storage disorder in non-
immunological hydrops fetalis (NIHF): more common than assumed?
Report of four cases with transient NIHF and a review of the literature.
Orphanet J Rare Dis. 2012;7:86.
53. Desilets V, Audibert F, Wilson R, et al. Investigation and management
of non-immune fetal hydrops. J Obstet Gynaecol Can. 2013;35(10):
e1–e14.
54. Yeom W, Paik ES, An JJ, et al. Clinical characteristics and perinatal out-
come of fetal hydrops. Obstet Gynecol Sci. 2015;58(2):90–97.
55. Derderian SC, Jeanty C, Fleck SR, et al. The many faces of hydrops. J
Pediatr Surg. 2015;50(1):50–54.
56. Bellini C, Hennekam RCM, Banjola E. A diagnostic flow chart for non-
immune hydrops fetalis. Am J Med Genet A. 2009;149A:852–853.
57. Gort L, Granell MR, Fernández G, et al. Fast protocol for the diagno-
sis of lysosomal diseases in nonimmune hydrops fetalis. Prenat Diagn.
2012;32(12):1139–1142.
58. Braun T, Brauer M, Fuchs I, et al. Mirror syndrome: a systematic review
of fetal associated conditions, maternal presentation and perinatal out-
come. Fetal Diagn Ther. 2010;27(4):191–203.
59. Hirata G, Aoki S, Sakamaki K, Takahashi T, et al. Clinical characteristics
of mirror syndrome: a comparison of 10 cases of mirror syndrome with
non-mirror syndrome fetal hydrops cases. J Matern Fetal Neonatal Med.
2016;29(16):2630–2634.
60. Gedikbasi A, Oztarhan K, Gunenc Z, et al. Preeclampsia due to fetal non-
immune hydrops: mirror syndrome and review of literature. Hypertens
Pregnancy. 2011;30(3):322–330.
61. Adzick NS. Prospects for fetal surgery. Early Hum Dev. 2013;89:
881–886.
62. Wenstrom KD, Carr SR. Fetal surgery: principles, indications and evi-
dence. Obstet Gynecol. 2014;123:817–835.
63. Donofrio MD, Moon-Grady AJ, Hornberger LK, et  al. Diagnosis
and treatment of fetal cardiac disease: a scientific statement from the
American Heart Association. Circulation. 2014;129(21):2183–2242.
64. Sekarski N, Jeijboom EJ, Di Bernardo S, et al. Perinatal arrhythmias. Eur
J Pediatr. 2014;173:983–996.
65. Lindenburg LTM, van Kamp IL, Oepkes D. intrauterine blood trans-
fusion: current indications and associated risks. Fetal Diagn Ther.
2014;36:263–271.
66. Ilagan JG, Wilson RD, Bebbington M, et  al. Pregnancy outcomes fol-
lowing bipolar umbilical cord cauterization for selective termination
in complicated monochorionic multiple gestations. Fetal Diagn Ther.
2008;23:153–158.
67. Lee H, Bebbington M, Crombleholme TM, et al. The North American
Fetal Therapy Network Registry data on outcomes of radiofrequency
ablation for twin-reversed arterial perfusion sequence. Fetal Diagn Ther.
2013;33:224–229.
68. Lu J, Ting YH, Law KM, et  al. Radiofrequency ablation for selective
reduction in complicated monochorionic multiple pregnancies. Fetal
Diagn Ther. 2013;34:211–216.
69. Peeters SHP, Devlieger R, Middeldorp JM, et al. Fetal surgery in compli-
cated monoamniotic pregnancies: case series and systematic review of the
literature. Prenat Diagn. 2014;34:586–591.
70. Prefumo F, Fichera A, Zanardini C, Frusca T. Fetoscopic cord transaction
for treatment of monoamniotic twin reversed arterial perfusion sequence.
Ultrasound Obstet Gynecol. 2014;43:233–235.
71. Stephenson CD, Temming LA, Pollack R, Iannitti DA. Microwave abla-
tion for twin-reversed perfusion sequence: a novel application of technol-
ogy. Fetal Diagn Ther. 2015;38(1):35–40.
72. Rodeck CH, Fisk NM, Fraser DI, Nicolini U. Long-term in utero drain-
age of fetal hydrothorax. N Engl J Med. 1988;319:1135–1138.
73. Nicolaides KH, Azar GB. Thoraco-amniotic shunting. Fetal Diagn Ther.
1990;5:153–164.
74. Bernaschek G, Deutinger J, Hansmann M, et al. Feto-amniotic shunt-
ing—report of the experience of four European centres. Prenat Diagn.
1994;14:821–833.
75. Picone O, Benachi A, Mandelbrot L, et  al. Thoracoamniotic shunting
for fetal pleural effusions with hydrops. Am J Obstet Gynecol. 2004;191:
2047–2050.
76. Smith RP, Illanes S, Denbow ML, Soothill PW. Outcome of fetal pleural
effusions treated by thoracoamniotic shunting. Ultrasound Obstet Gynecol.
2005;26:63–66.
77. Rustico MA, Lanna M, Coviello D, et al. Fetal pleural effusion. Prenat
Diagn. 2007;27:793–799.
78. Yinon Y, Grisaru-Granovsky S, Chadha V, et al. Perinatal outcome fol-
lowing fetal chest shunt insertion for pleural effusion. Ultrasound Obstet
Gynecol. 2010;36:58–64.
79. Caserio S, Gallego C, Martin P, et al. Congenital chylothorax: from foetal
life to adolescence. Acta Paediatr. 2010;99:1571–1577.
80. Walsh J, Mahony R, Higgins S, McParland P, Carroll S, McAuliff F.
Thoraco-Amniotic shunting for fetal pleural effusion—a case series. Ir
Med J. 2011;104:205–208.
81. Takahashi Y, Kawabata I, Sumie M, et  al. Thoracoamniotic shunting
for fetal pleural effusions using a double-basket shunt. Prenat Diagn.
2012;32:1282–1287.
82. Pellegrinelli JM, Kohler A, Hohler M, et al. Prenatal management and
thoracoamniotic shunting in primary fetal pleural effusions: a single cen-
tre experience. Prenat Diagn. 2012;32:467–471.
83. Petersen S, Kaur R, Thomas JT, et al. The outcome of isolated primary
fetal hydrothorax: a 10-year review from a tertiary center. Fetal Diagn
Ther. 2013;34:69–76.
84. Miyoshi T, Katsuragi S, Ikeda T, et  al. Retrospective review of thora-
coamniotic shunting using a double-basket catheter for fetal chylothorax.
Fetal Diagn Ther. 2013;34:19–25.
85. White SB, Tutton SM, Rilling WS, et al. Percutaneous in utero thora-
coamniotic shunt creation for fetal thoracic abnormalities leading to non-
immune hydrops. J Vas Interv Radiol. 2014;25:889–894.
86. Derderian SC, Trivedi S, Farrell J, et al. Outcomes of fetal intervention
for primary hydrothorax. J Pediatr Surg. 2014;49:900–903.
87. Nowakowska D, Gaj Z, Grzesiak M, et al. Successful treatment of fetal
bilateral primary chylothorax—report of the two cases. Ginekol Pol.
2014;85:708–712.
88. Peranteau WH, Adzick NS, Boelig MW, et al. Thoracoamniotic shunts
for the management of fetal lung lesions and pleural effusions: a single
institution review and predictors of survival in 75 cases. J Pediatr Surg.
2015;50:301–305.
89. Jeong BD, Won HS, Shim MY, et  al. Perinatal outcomes of fetal
pleural effusion following thoracoamniotic shunting. Prenat Diagn.
2015;35:1365–1370.
90. Adzick NS. Management of fetal lung lesions. Clin Perinatol.
2009;36:363–376.
91. Adzick NS. Open fetal surgery for life-threatening fetal anomalies. Semin
Fetal Neonatal Med. 2010;15:1–8.
92. Roybal JL, Moldenhauer JS, Khalek N, et al. Early delivery as an alterna-
tive management strategy for selected high risk fetal sacrococcygeal tera-
tomas. J Pediatr Surg. 2011;46:1325–1332.
93. Fukushima K, Morokuma S, Fujita Y, et al. Short-term and long-term
outcomes of 214 cases of non-immune hydrops fetalis. Early Hum Dev.
2011;87(8):571–575.
94. Randenberg AL. Nonimmune hydrops fetalis part II: does etiology influ-
ence mortality? Neonatal Netw. 2010;29(6):367–380.
95. Kim SA, Lee SM, Hong JS, et  al. Ultrasonographic severity scoring of
non-immune hydrops: a predictor of perinatal mortality. J Perinat Med.
2015;43(1):53–59.
96. Wafelman LS, Pollock BH, Kreutzer J, et  al. Nonimmune hydrops
fetalis: fetal and neonatal outcome during 1983-1992. Biol Neonate.
1999;75(2):73–81.
97. Wy CA, Sajous CH, Loberiza F, et al. Outcome of infants with a diagno-
sis of hydrops fetalis in the 1990s. Am J Perinatol. 1999;16(10):561–567.
98. Ota S, Sahara J, Mabuchi A, et al. Perinatal and one-year outcomes of
non-immune hydrops fetalis by etiology and age at diagnosis. J Obstet
Gynaecol Res. 2016;42(4):385–391.
37
Fetal Tumours
THOMAS R. EVERETT, ROSALIND PRATT, COLIN R. BUTLER, RICHARD J. HEWITT,
PAOLO DE COPPI AND PRANAV P. PANDYA
KEY POINTS
• Fetal tumours are rare and should be managed by an experi-
enced multidisciplinary team.
• Magnetic resonance imaging is an important imaging mo-
dality in the diagnosis and management of fetal tumours.
• Fetal neck masses may result in airway obstruction and the
ex utero intrapartum treatment (EXIT) procedure may need
to be planned for delivery.
• In utero treatment of sacrococcygeal teratomas can be
­performed in fetuses showing signs of compromise.
• Good outcomes can be achieved in many cases of fetal
­tumours.
Introduction
Fetal tumours are a rare and heterogeneous group of fetal mal-
formations, ranging from benign lesions that may cause fatal air-
way obstruction without appropriate management, through those
with mass effect and low malignant potential, to very rare fetal
malignancies. The unifying feature is that they are rare and require
complex antenatal, intrapartum and postnatal management from
highly specialist services. With improvements in antenatal detec-
tion and assessment, involvement of the multidisciplinary team
(MDT) and individualised care planning and management from
the point of diagnosis, devastating and fatal outcomes such as
unexpected airway obstruction or massive fetal or neonatal haem-
orrhage can be avoided, and potentially, the outcomes for these
infants can be improved.  tial. Anomalies of thyroid development, including tumours and
Neck Masses
Introduction
Neck masses are most commonly noted in the second trimester at
the time of the detailed anatomy scan, although they may present
as an incidental finding on later scans or be referred due to poly-
hydramnios and measuring large for gestational age. The majority
of neck masses detected at this gestational age are mixed cystic and
solid in nature and arise in the anterior triangle. Posterior cystic
neck lesions and those evident in the first trimester commonly
have a different aetiology as outlined later and in Chapter 19.
The two most common neck masses are lymphangiomas and
teratomas. A lymphangioma will be a well-circumscribed, pre-
dominantly avascular mass consisting of thin walled cystic areas.
Lymphangiomas arise laterally but often crosses the midline, and
although not invasive, they can extend into the mediastinum and
thoracic cavity and can obstruct the airway (Fig. 37.1).
A teratoma has a heterogeneous appearance with varying
degrees of cystic and solid components (Fig. 37.2). They are often
well vascularised. There is no pathognomonic ultrasound appear-
ance,1 and teratomas can be difficult to differentiate from lymph-
angiomas. However, areas of calcification on ultrasound (or fat
signal on magnetic resonance imaging (MRI)) make the diagnosis
of teratoma more likely. As with lymphangiomas, fetal teratomas
are rarely invasive but can cause oropharyngeal obstruction and
deviation. Compressive effects can also cause significant disrup-
tion to the bony skull and the skull base; ultrasound (US) and
MRI should be used to detect signs of this. Care should be taken
to differentiate a cervical teratoma from an epignathus, a rare
teratoma arising from the palate-pharyngeal region around the
basisphenoid (Rathke pouch), which fills the buccal cavity. This is
associated with poor prognosis because of the location and poten-
tial for invasion into the skull base and brain tissue. 
Differential Diagnoses
Before confirmation of a cystic neck mass, other differentials must
be considered. Posteriorly, this would primarily be gross nuchal
oedema. Careful US examination of the skull and brain structure
should be performed to exclude encephalocele or cervical myelo-
meningocele. Anteriorly, cervical teratoma is a primary differen-
goitres can present similarly. Masses with a more cystic appearance
may represent thyroid or branchial cleft cysts or thyroglossal duct
cysts. 
Cystic Lymphangioma
Embryologically, the lymphatic system develops after the forma-
tion of blood vessels. The most widely accepted model of lymphatic
development to date was developed more than a century ago. It pro-
poses that the endothelial cells bud from the veins to form primary
lymphatic sacs. These then sprout towards the periphery. The two
main lymphatic sacs develop near the junction of the subclavian
and anterior cardinal veins, and the lymphatic capillaries spread
443
444 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Voluson Voluson
E8 E8

A B
• Fig. 37.1  A, Ultrasound image of a lymphangioma at 25 weeks’ gestation. Note the thin-walled cystic
nature of the lesion. B, Ultrasound images of a teratoma at 23 weeks’ gestation measuring 61.8 × 41.8
mm. Note the mixed solid and cystic component and massive size.

Voluson Voluson
E8 E8

2
1

A B
Voluson Voluson
E8 E8

C D
• Fig. 37.2  A and B, Teratoma at 20 weeks’ gestational age, demonstrating the relationship with the nose,
mouth, neck and thorax. The mass measures 37.6 × 37mm. C, Teratoma at 32 weeks’ gestational age.
Doppler can be used to help identify the airway. Movement of fluid is suggestive, although not diagnostic,
of airway patency. D, Vascular teratomas. Doppler can be used to assess the vascularity of the lesion and
the blood flow within it.

from these towards the head, neck, arm and thorax. The failure of
these jugular lymph sacs to drain into the developing lymphatic
system is thought to cause abnormal lymphatic sprouting, lymph
accumulation and the development of lymphangiomas.1,4
The terms cystic hygroma and lymphangioma are synonymous
and have been used to describe congenital dilated cystic malfor-
mations, predominantly in the region of the neck. However, it
should be recognised that these masses may arise from or extend
into the thorax and mediastinum or axilla. Less commonly,
lymphangiomas have been reported elsewhere in fetuses, includ-
ing the extremities and abdominal wall.
In the first trimester, the term cystic hygroma is used to describe
a significantly increased nuchal translucency, often with inter-
nal septations (see Chapter 19). Such cases are more frequently
associated with underlying chromosomal aneuploidies, particu-
larly monosomy X (Turner syndrome), trisomy 18 and trisomy
21.5 In the absence of aneuploidy, there is a higher incidence of
underlying genetic disorders that may not be detected antenatally.
However, in the era of microarray and improved targeted testing
for Noonan syndrome (in which >80% can be diagnosed),6 the
antenatal detection rate is improving.7 A detailed first trimester
anomaly scan, including cardiac anatomy, should be performed
in such cases and further investigations offered. In the event of
a normal karyotype or microarray, if the findings do not resolve,
and particularly if there is a further anomaly found on ultrasound,
genetic counselling should be considered. If antenatal testing is
declined, careful assessment of the infant should be performed in
the neonatal period.
Increased nuchal translucency is also associated with cardiac
abnormalities, and in some cases, ultrasound signs of fluid in
other fetal compartments such as the thorax, abdomen and gen-
eralised skin oedema may be noted; this is known as fetal hydrops
or hydrops fetalis. In cases of hydrops, investigations as described
earlier should be performed, as well as consideration of other
causes such as red cell alloimmunisation or nonimmune causes,
including viral infection and metabolic disease (see Chapter 36).
In cases of persistent hydrops presenting in the first trimester, the
outlook is very poor with few fetuses surviving to delivery.
 CHAPTER 37  Fetal Tumours 445

TABLE
37.1 Table of Differential Diagnosis of Fetal Neck Masses
Diagnosis Ultrasound features Incidence Outcome
Cervical lymphangiomas Well-circumscribed or diffuse cystic 1 in 1775 live births2 Can cause complex airway
mass, laterally located, and often obstruction; referral to ENT and
arising from cervical area, floor of consideration of EXIT procedure
mouth or the tongue recommended; presence in the
first trimester is associated with
chromosomal abnormalities
Cervical teratomas Can be massive, usually solid with 1 in 40,000 live births3 Can cause complex airway
some cystic areas, and well obstruction; referral to ENT and
defined; may have internal calcifi- consideration of EXIT procedure
cations, positioned in the anterior are recommended
neck; 3:1 female:male ratio
Haemangioma Typically posterolateral, well-defined, Rare May be associated with cortical mal-
solid masses with slow vascular formations; brain MRI indicated,
flow can be part of PHACES syndrome
Cervical thymic cyst Commonly multiloculated but can be Very rare
uniloculated, most commonly to
the left, splaying the carotid artery
and jugular vein
Congenital goitre Symmetric thyroid enlargement; Rare Unlikely to cause airway obstruction;
associated with maternal propyl- when present, intubation is usually
thiouracil use (for Graves’ disease) successful
and maternal thyroid stimulation-
blocking antibody
Brachial cleft cysts Unilateral, uniloculated anterolateral Rare Not reported to cause airway
thin-walled cyst obstruction
Vascular malformations Multiloculated cystic structure often Rare
laterally located
Neuroblastoma Retropharyngeal, solid mass, with or Very rare
without calcification, extending
into mediastinum or skull

ENT, Ear, nose and throat; EXIT, ex utero intrapartum treatment; MRI, magnetic resonance imaging; PHACES, posterior fossa malformation, haemangiomas, arterial anomalies, cardiac defects, eye abnor-
malities and sternal cleft.
  

In cases of first trimester cystic hygromas that have been appro-
priately investigated and no genetic abnormality found, particu-
larly in those cases that resolve, the prognosis is very good.
The development of a cystic neck mass in the second trimes-
ter is likely to have a different aetiology (Table 37.1) from those
seen in the first trimester and may carry a better prognosis unless
associated with hydrops or generalised oedema, in which cases the
prognosis remains poor.  of gestation, that are able to proliferate to form disorganised struc-
Teratomas
Fetal teratomas are rare tumours with an incidence of 1 in
40,000 live births. Although most commonly found in the
region of the lower spine and pelvis (sacrococcygeal teratomas
(SCTs); discussed in detail later), 6% are related to the neck.
Teratomas are (almost always) formed of tissues derived from
the three germ cell layers: endoderm, mesoderm and ecto-
derm. Although teratomas are predominantly (>80%) benign
tumours with well-differentiated tissues, the rapid growth and
the mass effect can cause significant complications; primarily
by obstruction or deviation of the trachea and oesophagus. In
cases of immature teratoma, with more poorly differentiated
tissues, there in increased risk for invasion and metastases;
however, the prognosis remains good with a greater than 80%
5-year survival rate.8
The origin of fetal teratomas remains poorly understood. The
most widely accepted hypothesis is that aberrant pluripotent cells
are sequestered during embryogenesis, in the fourth to fifth week
tures comprising tissue types derived from the three embryonic
germ layers.1 
Antenatal Management
Tertiary-level ultrasound assessment and multidisciplinary coun-
selling are important in any neck mass. The ultrasound assess-
ment should include details of the site and size of the mass, solid
or cystic components, calcification, associated vascularity and
assessment of invasion into or deviation of adjacent structures.
Attempts should be made to determine the nature of the mass
to aid counselling regarding postnatal management, likelihood of
complications and long-term outcomes.
446 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
• Fig. 37.3 Teratoma at 31 weeks’ gestational age. Three- and four-
dimensional imaging can help the parents visualise the mass and help with
parent counselling.
A routine element of ultrasound assessment of fetal neck
masses should be assessment for evidence of tracheal deviation or
oesophageal occlusion. Indirect markers of these include polyhy-
dramnios or a small or nonvisible stomach bubble. In some cases,
the oropharynx may be fluid filled and readily visible, indicating
significant partial or total occlusion. Ultrasound assessment is also
important in the determination of fetal well-being, particularly in
identifying development of cardiac compromise or hydrops, and
to regularly assess the growth and size of the mass. The utility of
three-dimensional (3D) US imaging in neck masses is unclear and
becomes difficult to achieve in larger tumours at later gestations,
particularly in the absence of polyhydramnios. Although its clini-
cal benefit may be limited in such cases, we have found that it is
useful to the parents (Fig. 37.3).
After 24 weeks’ gestation, ultrasound assessment should be per-
formed every 2 weeks. These assessments should focus on assessing
the size of the mass, any changes in characteristics including vas-
cularity, fetal neck extension, amniotic fluid volume and signs of
cardiac compromise. To aid delivery planning, fetal presentation
and placental site should be carefully mapped.
Increasing polyhydramnios and features of cardiac compro-
mise, including hydrops or Doppler abnormalities, are indications
for more frequent assessment. In cases of significant polyhydram-
nios, particularly associated with maternal discomfort, amniod-
rainage may be indicated, although this is associated with rupture
of membranes and preterm labour. In view of the anticipated air-
way difficulties and the advantages of a planned delivery, amniod-
rainage should be kept to a minimum and performed in a centre
where ex utero intrapartum treatment (EXIT) is possible.
Magnetic resonance imaging. Fetal MRI is generally accepted
to be safe up to 3 Tesla and is being increasingly exploited. The
advantages of the technique are its excellent soft tissue definition
and the large field of view it provides, allowing global imaging of
the fetal head and neck at any gestation. In addition, it is a use-
ful adjuvant to ultrasound when imaging is limited, for example,
in cases of oligohydramnios, poor fetal position or maternal obe-
sity. The difficulty with fetal MRI is in imaging an unpredictably
mobile fetus. To overcome this, fast MRI sequences are used to
obtain single-slice acquisitions, producing a motion frozen stack
of images, free from artefact. A variety of fast MRI sequences are
used to obtain T1- and T2-weighted images (depending on the
manufacturer of the hardware), which acquire 15 to 20 3-mm
slices in less than 25 seconds.
The most common and important indication for fetal MRI in
the context of neck masses is to assess airway patency to inform
decisions on mode of delivery (Fig. 37.4). Because the fetal airway
is fluid filled, it appears bright on T2-weighted sequences. This
allows the trachea to be traced through the neck using imaging in
three orthogonal planes. This may confirm patency or conversely
assist surgeons in planning the best approach for tracheostomy.9
After patency is confirmed, the passage of the trachea can be
mapped and displacement assessed. As an aid to determining the
degree of airway displacement, the tracheoesophageal displace-
ment index (TEDI) has been developed, which is defined as the
sum of the lateral and ventral displacement of the tracheoesopha-
geal complex from its normal anatomical location.10 A TEDI
score greater than 12 mm has been reported to be predictive of a
complicated airway (100% vs 46%; P = .04)10 (Fig. 37.5).
As well as assessing airway patency, fetal MRI can help iden-
tifying the underlying cause of neck masses,11 as the improved
soft tissue definition and differences in signal from T1- and
T2-weighted imaging can help distinguish pathologies.12,13 MRI
can also help describe the relationship among the mass, airway
and other structures of the neck, head and thorax14 and help assess
for neck extension secondary to the tumour, which may necessi-
tate delivery by caesarean section. Additional information on the
depth of invasion and structures involved can aid surgical plan-
ning.11,14 MRI has been reported to obtain additional findings
or make an alternative diagnosis to ultrasound in 83% of cases of
fetal neck masses.11
Magnetic resonance imaging is also useful for assessing the fetal
lungs, which are clearly visualised in T2-weighted imaging. They
can be collapsed, hypoplastic or hyperinflated in cases of fetal neck
masses. There is potential for MRI derived total lung volume mea-
surements to help predict lethal pulmonary hypoplasia.15
Determining airway patency remains challenging with MRI
because of the relative thickness of the slices compared with
the size of the fetal airway structures. There is ongoing research
in reconstructing the two-dimensional stacks of slices in three
orthogonal planes into anatomically relevant 3D volumes. This
would improve spatial resolution and therefore the diagnostic
ability of imaging in the future. Reconstructing virtual bronchos-
copies as an aid in evaluating airway patency is an exciting future
possibility.16 
Prognosis. The antenatal prognosis for a fetus with a cystic
hygroma and normal karyotype and cardiac structure is good with
few cases dying in utero.17 The in utero mortality rate in cases of
teratoma is higher because of increased vascularity and associ-
ated cardiac demands.18 At present (because of limited worldwide
cases), there is no predictive model for adverse in utero outcomes. 
In utero therapies. There are a few reports of in utero therapy,
including sclerotherapy,19,20 for cystic hygroma and fetal surgery21
for teratoma. The numbers are very limited, and these therapies
are not routinely practised, although they may have a role in the
case of the previable hydropic fetus, in which the mortality rate is
close to 100% if delivered or left untreated.
Fetoscopy is used in some centres to assess the patency of the
fetal airway; however, there are only currently two reports in the

A
B Axial magnetic resonance image of fetal neck demonstrating a large mass
C for ventilation.
• Fig. 37.4  A, Magnetic resonance images of a lymphangioma at 30
weeks’ gestational age. A right-sided lymphangioma crosses the midline
anterior to the airway; the airway can be seen and was thought to be pat-
ent. Also note the flow artefact coming from the fetal nose in (thick arrow),
similar to Doppler this demonstrates flow of fluid in the fetal airway, and is
suggestive but not diagnostic of airway patency (thin arrow). B, Teratoma
at 31 weeks’ gestational age. This teratoma originated from the anterior
and left side of the neck. The airway could be followed and appeared pat-
ent; however, note the displacement of the soft tissue and trachea (thin
arrow) to the right of the cervical spine on the axial view (thick arrow). C,
A teratoma at 29 weeks’ gestational age. Although the nasal airway and
nasopharynx appear patent the remainder of the airway cannot be fol-
lowed, so airway patency could not be confirmed.
CHAPTER 37  Fetal Tumours 447
Ventral
TEDI = L + V
T
L
CS
Lateral
A
V
B
• Fig. 37.5  A, Measurement of the tracheoesophageal displacement index
(TEDI). TEDI was defined as the sum of the lateral (L) and ventral (V) dis-
placements of the tracheoesophageal complex (T) from the ventral aspect
of the cervical spine (CS) on fetal magnetic resonance imaging (MRI). B,
(M) and significant displacement of the tracheoesophageal complex (thin
arrow) from the ventral aspect of the cervical spine (thick arrow). (Repro-
duced from Lazar DA, Cassady CI, Olutoye OO, et al. Tracheoesophageal
displacement index and predictors of airway obstruction for fetuses with
neck masses. J Pediatr Surg 47:46–50, 2012.)
literature of operative intervention for neck masses. One report
details the in utero removal of a pedunculated nasopharyngeal
tumour using YAG (yttrium aluminum garnet) laser excision.22
A second report details a fetal endoscopic tracheal intubation
technique (FETI).23 In this case, a fetus with a giant cervical
teratoma was successfully intubated at 35 weeks’ gestation. This
allowed a standard elective lower segment caesarean section to be
performed with immediate use of the endotracheal tube at birth
 
Intrapartum Management: The EXIT Procedure
The EXIT procedure is performed to secure the fetal airway before
stopping placental circulation. By maintaining the fetus on pla-
cental circulation, oxygenation is assured, allowing additional
time to establish an airway. The first successful case was described
in 199024,25 for the insertion of a tracheostomy in a fetus with an
epignathus. However, the term EXIT was coined, and the pro-
cedure was first routinely used for management of pregnancies
complicated with congenital diaphragmatic hernia treated with
fetoscopic endotracheal balloon occlusion26 (FETO) (see Chap-
ter 31). The EXIT procedure is now a widely used technique in
448 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
centres managing the delivery of fetuses with neck masses with
a high suspicion of airway complications. Although developed
from the routinely performed lower segment caesarean section, an
EXIT procedure should not be regarded as such and comes with
its unique complexities and risks to both the mother and fetus.
The EXIT procedure is used for a variety of fetal anomalies
which obstruct the fetal airway, preventing spontaneous respira-
tion and making intubation of the airway either very difficult
or impossible. The anomalies can be extrinsic, including terato-
mas and lymphangiomas, or intrinsic, such as laryngeal atresia,
congenital upper airway obstruction, obstructive malformations
of the upper airways and intrathoracic lesions such as congeni-
tal hydrothorax. It is also sometimes used in cases of FETO in
which the balloon has not been electively removed before delivery.
Interventions performed with the EXIT procedure include intra-
tracheal intubation, tracheotomy and tracheoplasty.
It remains difficult to predict which fetuses will have a com-
plicated airway. Ultrasound and MRI may be unable to demon-
strate airway patency throughout its course, and in such cases, it
is sensible to assume EXIT is required. A large mass, a suspected
diagnosis of teratoma or signs of obstruction such as polyhydram-
nios or a small or absent stomach bubble all increase the chance
of airway obstruction; therefore, if any of these are present, an
EXIT should be planned. When the airway is visible but deviates
from its normal course, the TEDI (see earlier) provides a guide to
when EXIT may be appropriate. In masses that are not teratomas,
a TEDI of greater than 12 mm has 100% sensitivity and 86%
specificity for a complicated airway.10
However, all cases should be evaluated on a case-by-case basis
by an experienced multi-MDT. In our experience, neck masses can
change rapidly after delivery, with sudden postpartum increases in
vascularity and size, so even in cases in which antenatal imaging
suggests a patent airway, careful consideration should be given by
an MDT on the possible benefits of an EXIT procedure to safely
establish a secure airway in the early neonatal period.
Detailed antenatal planning with an MDT, which should
include the obstetricians; neonatologists; anaesthetists (both
maternal and neonatal); paediatric ear, nose and throat surgeons;
radiologists; theatre scrub teams; and critically the parents, is
essential for each case. The theatre space itself should be sufficient
to accommodate the various teams and ‘zones’ established for each
specialty. A detailed brief and ‘talk through’ of the procedure,
including plans for foreseeable complications and confirmation
that all necessary equipment and drugs are available, should be
performed immediately before the procedure. Ideally, delivery
should be as close to term gestation as possible; however, because
of increasing polyhydramnios, up to 76% of cases are delivered at
a late preterm gestation (median, 35weeks).27
The parents of the baby should be involved in the antenatal
planning process and a nominated midwife assigned to the case.
Discussion should be undertaken antenatally with the parents and
plans made for cases in which it is not possible to gain airway
access and other worst-case scenarios such as severe hypoxia or
prolonged fetal bradycardia. Psychological support for the parents
should be offered.
To provide adequate fetal perfusion, maternal anaesthesia
should aim to provide optimal uterine relaxation and suit-
able maternal blood pressure. EXIT procedures are usually
performed under deep maternal general anaesthesia because
volatile anaesthetics assist in uterine relaxation. Regional
anaesthesia has been reported but requires intravenous infusion
of glyceryl trinitrate (GTN) and remifentanil. Rapid-sequence
induction and general endotracheal anaesthesia with maternal
paralysis and epidural for postoperative analgesia is a standard
technique.28 After delivery of the fetus, the concentration of
volatile agents is reduced and/or converted to intravenous
anaesthesia, and oxytocin is given to establish uterine tone.
More recently, the SIVA (supplemental intravenous anaes-
thesia) technique has been used.29 This exploits the tocolytic
effects of propofol and allows a reduction in the use of vola-
tile anaesthetic agents. This reduced exposure to volatile agents
may reduce fetal myocardial suppression and may also be a
more suitable technique in cases in which prolonged maternal
exposure to volatile agents is not desirable.
Before uterine incision, particularly in cases of extensive ante-
rior placentation, intraoperative sterile ultrasound mapping of the
placenta should be performed to avoid inadvertent placenta inci-
sion. The head, neck and upper torso of the fetus are delivered
along with the fetal right arm (Fig. 37.6). The fetus should have
10 μg/kg fentanyl and 0.1 mg/kg of vecuronium immediately into
the deltoid muscle. Monitoring of the fetal saturations and heart
rate should be performed with sterile monitoring equipment. Atro-
pine should be available and usage determined by a senior neona-
tologist. After this, attempts should be made to secure the airway
with direct laryngoscopy. If this is not successful, examination of
the airway with a rigid bronchoscope, flexible bronchoscopy, or
both should be performed and guided intubation attempted. If
this is unsuccessful, an attempt at tracheostomy should be con-
sidered. After the airway is secured, the baby can be delivered and
transferred to the neonatal unit for further assessment. If there are
prolonged episodes of desaturation or bradycardia or compromise
to the maternal health (bleeding, hypotension), the baby should
be delivered immediately even if a definitive airway has not been
secured.
The EXIT procedure carries increased risks to the mother, pre-
dominantly the risk for haemorrhage and risks associated with
longer operative time. Approximately 10% of cases need blood
transfusions, with haemorrhage caused by placental abruption,
uterine atony and bleeding at the incision site.28
With prior planning, operative risks can be minimised, and the
EXIT procedure should be considered a safe and effective proce-
dure. In experienced centres, a secured airway can be obtained in
close to 100% of planned EXIT procedures. Without the EXIT
procedure, the neonatal mortality rate for patients with cervical
neck masses has been reported at greater than 50%; however, with
the EXIT procedure, this is now less than 10%.17,28,30,31
A review of our own cases in the past 5 years includes seven
EXIT procedures (one case of presumed congenital high airways
obstruction (CHAOS); six delineate neck masses). In two cases,
the fetus died: one baby had a very large anterior teratoma with
bronchoscopic evidence of invasion into the upper airway. Endo-
tracheal intubation was not possible via direct laryngoscopy, and
a tracheostomy was successfully performed; however, there was
early neonatal death secondary to presumed severe hypoxic brain
injury; postmortem examination was declined. The other was a
case of presumed CHAOS, which was in fact a rare lethal congeni-
tal bronchopulmonary disorder (congenital alveolar dysplasia).
In the five patients with giant cervical masses who survived
delivery, two patients had teratomas which were fully resectable
with excellent outcomes. In the three lymphatic malformations,
one patient was treated by sclerotherapy alone, and one patient
required an initial surgical debulk of cervical compressive disease
and sclerotherapy for a compressive mediastinal comment. The lat-
ter patient was treated with sclerotherapy and has required surgical

A ally required.
B Full endoscopic evaluation of the airway may not always be pos-
C ease can readily invade neighbouring structures.18,25 Lymphatic
• Fig. 37.6  Pictures demonstrating the processes involved in an ex utero
intrapartum treatment (EXIT) procedure. A, The head, neck and upper
torso of the baby are delivered along with the right arm. Fentanyl and
vecuronium (with or without atropine) are injected into the deltoid muscle.
B, The fetal airway is secured with direct laryngoscopy, visualising the
vocal cords under direct vision to ensure correct placement of the endo-
tracheal tube. C, The baby is then delivered, and the umbilical is cord cut.
debulk of the tongue for an invasive component. The latter two
children with lymphatic malformations have ongoing tracheos-
tomy because of extensive disease into the upper airway (medi-
astinal and tongue base invasion) and related tracheomalacia.
Otherwise the children do not have any neurologic or swallowing
deficits.  is generally safe with minimal side effects, it is worth noting that
Postnatal Management and Outcomes
The EXIT procedure will secure an airway either by an endo-
tracheal route or tracheostomy. Immediate postnatal manage-
ment includes a brief evaluation of the airway through direct
CHAPTER 37  Fetal Tumours 449
endoscopy if not already done during the EXIT procedure. If
the tracheostomy was performed in an expedited fashion, for-
malisation of the tracheostomy tract is required to ensure the
tract remains established and secure in the postnatal period.
Otherwise, further management is largely dependent on the
cause of the airway obstruction (i.e., whether the pathology
is intrinsic or extrinsic) and identifying the extent of disease.
Additional factors have to be considered, including other con-
genital malformations, particularly cardiac, which may need
urgent correction at the same time as the airway. The combined
factors may have implications towards strategy of timing of
surgery, and input from a dedicated aerodigestive MDT is usu-
 
Postnatal Investigations
After delivery of the infant and subsequent airway stabilisation,
the initial priority is to evaluate the extent of airway disease.
Essential investigations include direct endoscopic evaluation with
microlaryngoscopy and tracheobronchoscopy; bronchography;
and imaging with ultrasound, computed tomography (CT) and/
or MRI. Adjunct investigations may be necessary and include
oesophagoscopy, optical coherence tomography (OCT) imaging
of the airway, echocardiography and cardiac catheterisation, and
genetic analysis. Direct evaluation of the airway may be performed
as part of the EXIT procedure itself or immediately after delivery.
sible because of the pathology itself, and imaging with CT or MRI
will further delineate the location and extent of the lesion. Further
management focuses on determining if the airway obstruction is
amenable to surgical correction. In the case of neck masses, the
key question to managing the lesion is whether it is resectable. 
Postnatal Management
In general, MRI will determine the resectability of a lesion. Cervi-
cal teratomas are often amenable to full excision, and other lesions,
including cervical thymic cysts, neuroblastomas and haemangio-
endotheliomas, can also be considered for surgical removal. How-
ever, lymphovascular malformations have to be considered more
carefully because the borders are less well defined, and the dis-
malformations are either macro- or microcystic disease or hetero-
geneous, consisting of both forms. Options include immediate
decompression via aspiration (for large interconnecting cysts),
sclerotherapy, surgical debulking and excision, or a combination
of these.32-34
Sclerotherapy aims to deliver an agent into the lesion, thereby
inducing a cascade of thrombitis and fibrosis. The agents used for
this include ethanol, bleomycin, doxycycline and sodium tetra-
decyl sulphate.35 Multiple applications may be required to induce
resolution of the lesion. Because the sclerosant induces an inflam-
matory reaction, there is often a temporary increase in the size of
lesion followed by a resolution phase. This has implications for air-
way compromise if adjacent to the airway. Although sclerotherapy
posttreatment infections can occur. Sclerosant therapy is normally
confined to the lesion but can occasionally leak into surrounding
tissues, leading to cutaneous necrosis, damage to muscle fibres and
injury to peripheral nerves. In cases in which there is resistance to
sclerotherapy or the disease is predominantly microcystic, surgery
may be warranted.36
450 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
Surgery aims to remove the compressive pathology, but the extent
of surgery often needs to be balanced against the potential morbid-
ity that may occur. In most cases with significant airway compres-
sion, surgery is the best modality. It particularly offers the advantage
of being immediate; sclerotherapy may require repeated administra-
tion of sclerosants and is often complicated by temporary oedema and
increased size of the lesion. Conversely, when the pathology infiltrates
the tongue base and encases major nerves and vessels, surgery is more
challenging and not always indicated. The risks from surgery include
permanent cranial nerve injury, dysphagia and problems associated
with disturbed lymphatic drainage. In the literature, these have been
reported between 10% and 30%.37 Despite surgery and sclerother-
apy, recurrence rates can be 15% to 50%.38 Much debate has centred
around the optimal treatment, particularly sclerotherapy versus sur-
gery. It is generally accepted that given the heterogeneity of disease,
individual cases are preferentially discussed within a dedicated MDT
and treatment tailored to the individual child.32
The timing of such surgery is also dependent on concomitant
pathology. Congenital cardiac disease may need to be addressed
at the same time, if not before corrective airway surgery. If the
airway is considered established and uncompromised, repair can
be delayed to allow for growth in the child. More often, air-
way surgery is required immediately to relieve the obstruction
and is typically performed as soon as practically possible after
EXIT delivery. In general, most lesions can be reduced or fully
removed, and outcomes are favourable. Outcome reports from
the EXIT procedure for 12 infants with giant neck masses found
a mortality rate of 8% (n = 1), and 8% (n = 1) had evidence of
mild developmental delay. The remaining patients had no func-
tional deficits.39  may have some benefits in prediction of long term functional uri-
Sacrococcygeal Teratomas
Sacrococcygeal teratomas are the commonest form of congenital
tumours. However, SCTs are rare with an incidence of 1 in 35,000
to 1 in40,000 in pregnancies and an incidence of 1 in 27,000
at birth. There is an unexplained fourfold greater incidence in
females than males. There are data to suggest that antenatally diag-
nosed SCTs have a poorer prognosis than those diagnosed postna-
tally, for which the prognosis is good.40
Traditionally, SCTs have been classified into four subtypes
according to the Altman classification system41 (Table 37.2).
Although useful as a descriptive term, it is doubtful whether
the Altman classification has an impact on in utero outcome or
postnatal surgical outcomes. A national registry study from the
Netherlands did not find an association among Altman class, fetal
sex or tumour pathology and functional outcome,42 although a
recent study showed that there were no urologic or anorectal com-
plications in type 1 SCTs.43
Antenatal Imaging
The initial diagnosis of SCT is most commonly made by ultra-
sound after 20 weeks’ gestation. It is of interest that neonates with
a poor prognosis are reported to be diagnosed with SCT at an ear-
lier median gestational age than those with good prognosis (21.1
weeks (18.50–29.86 weeks) vs 23.9 weeks (16.60–34.14 weeks);
P = .030),44 although the overlap in gestations limits it clinical utility
in counselling parents. At midtrimester gestations, good assessment
of the tumour size can be made (Fig. 37.7). The appearance of the
internal component should be described as well as any deviation or
obstruction of normal structures. Assessment of the degree to which
TABLE Sacrococcygeal Teratoma Subtypes (Altman
37.2 Classification)
Subtype Description Frequency (%)
I Predominantly external tumours with 46
only a small presacral component
II External tumour mass but with a 34
significant intrapelvic extension
III External tumour mass with a pre- 9
dominantly pelvic or intraabdominal
mass
IV Presacral tumour with no external 10
component
  
the tumour consists of solid and cystic components should be per-
formed. The size of the tumour should be measured, and 3D imaging
techniques in both US and MRI can be used for volume calculations,
which allow for individual assessment of the proportions of cystic
and solid components. Vascularity should be assessed, and particular
attention should be taken to identify any large feeding vessels.
Magnetic resonance imaging should be seen as a complimen-
tary imaging modality to US. MRI has benefits over US in imag-
ing internal components, particularly in the third trimester. MRI
assessment for signs of invasion, obstruction or deviation of inter-
nal structures may aid counselling regarding postnatal surgery and
nary or bowel complications.43 
Antenatal Management
Prediction of outcome. Several models have been proposed to predict
outcomes of fetuses with SCT. It was already known that features
such as high levels of vascularity and fetal hydrops predict adverse
outcomes because of cardiac overload. Rodriguez and colleagues45
further hypothesised that the tumour volume to fetal weight ratio
(TFR) in the second trimester may be predictive of outcome, with
poor outcomes being defined as development of hydrops, fetal demise
or neonatal death. Tumour volume was calculated as a prolate ellipsoid
using the greatest dimensions of length, height and depth as measured
by ultrasound or MRI; estimated weight was calculated with the
Hadlock formula. Measured before 24 weeks’ gestation, all fetuses
with a TFR of 0.12 or less had a good outcome. Further studies,44,46
including a multi-institutional review including 50 fetuses with
SCT, have validated that a TFR greater than 0.12 before 24 weeks’
gestation is predictive of poor prognosis (area under the curve, 0.913;
sensitivity, 91.7%; specificity, 76.2%; positive predictive value (PPV),
86.8%; negative predictive value (NPV), 84.2%).
The component elements of the tumour may also be predic-
tive. Tumours that had a 50% or greater solid component were
seven times more likely to have a poor outcome compared with
those with a predominantly cystic mass (70.7% vs 9.1%44). A
predictor of adverse outcome that accounts for the cystic:solid
nature of SCTs, the solid tumour volume index (STVI), has
been proposed.47 Using MRI, the volume of the tumour was
assessed, and the maximum dimensions of the solid component
of the tumour were used to calculate solid tumour volume and
then divided by total tumour volume. Fetuses with an STVI
greater than 0.09 were significantly more likely to develop
 CHAPTER 37  Fetal Tumours 451

Voluson
Voluson E10
E10

A B

Voluson
E10

C D
• Fig. 37.7  A, Ultrasound of a mostly external sacrococcygeal tumour (SCT) at 26 weeks’ gestational age,
invading into the pelvis. Note the mixed cystic and solid component. B, A different case at 28 weeks’
gestational age, clearly showing the extent of pelvic invasion, and the separate bladder (thin arrow), spine
(thick arrow) and cord insertion. C, Doppler image demonstrating the vascularity of the lesion in A. Notice
the two large feeding vessels (thin arrows). This pregnancy had no evidence of cardiac failure, so in utero
treatment was not thought to be appropriate. D, MRI of a different case at 35 + 6 weeks’ gestation of an
Altman type 2 SCT with cystic and solid components. The tumour is mostly external but with a significant
intrapelvic mass (thin arrow). Normal bladder (thick arrow) and bowel (star) can be seen separate from
the tumour.

hydrops or high-output cardiac compromise (PPV, 81.25%;
NPV, 100%). It should be noted that this is a complex index
to calculate and requires further validation.
The tumour growth rate has also been associated with adverse
outcomes.48,49 Further investigation of tumour growth rate has
shown that an increase tumour volume of more than 61 cm3 per
week is associated with adverse outcomes, including hydrops,
high-output cardiac failure and in utero death (likelihood ratio
(LR), 4.52). Tumour growth rates of more than 165 cm3 per week
are highly associated with in utero demise (LR, 18.4).50 A recent
review of a UK-based registry showed increased growth rate to
be associated with a poor prognosis.51 However, as commented
on in this study, it remains unclear as to whether outcomes are
associated with growth rate of the whole tumour or growth rate of
the solid component, which is likely to have increased metabolic
demands and higher vascularity contributing the phenomenon of
vascular steal and its subsequent adverse effects on the fetal cardiac
function.50
After 24 weeks’ gestation, ultrasound assessment should be
performed every 2 weeks. These assessments should focus on
assessing the size of the mass and any changes in characteris-
tics, including increasing vascularity or signs of cardiac com-
promise, which may require in utero intervention or prompt
delivery.  follow-up was poor in many studies). These included a fetus
In utero therapies. Several strategies for in utero therapy,
including radiofrequency ablation (RFA), laser (both vascular
and interstitial), sclerosing therapies and coiling of the main
vessels have been attempted with a view to improving fetal out-
comes, particularly in hydropic fetuses and fetuses with high out-
put cardiac failure. Because of the heterogeneity of interventions,
the risk for selection bias in small cohorts or case reports and
incomplete descriptions, drawing conclusions as to the success
of each individual intervention is not possible. A case series and
systematic review by Van Meigham and colleagues52 described
results from 34 cases of SCT treated with minimally invasive
procedure. The survival rate was 30% (6 of 20) in patients with
evidence of cardiac failure and 67% (8 of 12) in those without.
In the subset of hydropic fetuses treated with RFA or interstitial
laser, the survival rate was 45% (5 of 11). Because previability
cardiac compromise is usually fatal, this result is encouraging,
suggesting that in utero intervention, particularly RFA and laser
treatment, may improve outcomes; however, the data are very
limited and of mixed quality. Prematurity was a common occur-
rence after intervention with a mean gestational age at delivery
of 29.7 ± 4.0 weeks in this series. More concerning, perhaps,
are the complications arising from the interventions, mostly
reported in RFA (although the reporting of complications and
452 SE C T I O N 7     Diagnosis and Management of Fetal Malformations
with perineal skin necrosis; another with gluteal necrosis, ischial
and femoral head hypoplasia and sciatic nerve trauma; and a
third fetus with a leg palsy (although the association with inter-
vention was not clear in the latter case). Any in utero treatment
should therefore be considered experimental and should only be
performed by experienced operators in fetal therapy centres after
adequate patient counselling.
Comparison has also been made between the use of ‘interstitial’
techniques, which aim to directly ablate the tumour and ‘vascular-
targeted’ techniques that aim to ablate the major feeding vessel.53
The data analysed are a reassessment of many of those included in
the previous paper, plus a further five cases from two expert centres
(totalling 33 cases). There is a tendency towards improved survival
in the vascular-targeted group (64%, 7 of 11) compared with inter-
stitial ablation (41%, 9 of 22). In hydropic fetuses, resolution of
hydrops was seen more often in the vascular targeted group than in
those that underwent interstitial intervention (75% (6 of 8) vs 33%
(2 of 6)). Again, the authors express the same limitations of data and
the need for multicentre prospective data collection.
Historically, open fetal surgery has been reported. This is not
discussed in detail here because of improvements in minimally
invasive techniques, and no data have been published for more
than a decade. In view of the complexity and uncertainty regard-
ing in utero therapy and the improvements in neonatal care, early
delivery from 28 weeks’ gestation should be considered in rap-
idly growing tumours or deteriorating fetuses that are not yet
hydropic.54  rate in excess of 90%.44,64 Most of the tumours are benign, and
Intrapartum Management
Planned delivery should take place in a unit with neonatal and
paediatric surgical experience in teratomas. In cases of SCT with a
predominantly internal component or with only a small external
component, a vaginal delivery can be anticipated. In SCTs with a
large external component (>5 cm) or in cases of fetal compromise
or prematurity, a caesarean section should be planned. It should be
anticipated that the SCT may be vascular and friable. Care should
be taken to minimise trauma to the SCT, and a classical caesarean
section may be required in larger cases. In larger vascular cases,
neonatal blood should be available in case of bleeding and urgent
need for transfusion. A consultant paediatric surgeon should be
present in the delivery suite in cases of SCT with a large external
component for the risk for bleeding. There may also be a role for
interventional radiology in such cases.  of these risks at prenatal consultation.57,65
Postnatal Management and Outcomes
At birth, if the patient is stable, postnatal imaging should be
obtained in preparation for surgery which should be ideally per-
formed in a semi-elective setting during the first week of life.40,55
An ultrasound is usually enough to evaluate the extension of the
mass and distinguish the four SCT subtypes. MRI can be used to
better define the anatomy but is usually not necessary.56
Care should be taken in managing the teratoma before the oper-
ation, and cling film should be used to avoid ulcerations and mini-
mise the risk for bleeding.57 α-Fetoprotein (AFP) and β-human
chorionic gonadotropin should be obtained both before surgery
and in the early postoperative phase. Their values would be particu-
larly important in the follow-up for early detection of recurrence.58
The SCT subtype it useful in deciding the surgical approach.
Specifically, types I and II are mostly resectable using a posterior
approach with the patient in a prone position.59 Conversely, if
the tumour has a predominant intraabdominal component, a
two-step procedures will be needed with an anterior approach fol-
lowed by a posterior one. In cases in which it is unclear if the
mass can be removed using exclusively the posterior approach, this
should be attempted first. In our experience, a good proportion
of tumour with intraabdominal or intrapelvic components can be
removed via a posterior approach, particularly if there is a large
cystic component.60,61
In terms of the surgery, after introducing a urinary catheter, the
child should be positioned in the prone position and a skin inci-
sion made with the principle of preserving as much skin as pos-
sible.62 The mass should be removed following its capsule, without
leaving residual tumour. However, careful dissection is needed to
minimise damage, particularly at the level of the sphincter and
the rectum.57,63 The latter can also be identified by introducing
a Hegar dilator into the anus. If the rectum is damaged, closure
with absorbable sutures should be conducted. In case of extensive
damage, a stoma formation may become necessary, but it is usu-
ally avoidable. The coccyx should be removed together with the
mass to avoid recurrence.55,59 Anterior to the coccyx, careful dis-
section is required to ligate the vascular supply to the tumour.61
Skin closure should be done, trying to give an acceptable cosmetic
appearance. In our experience, the best results are obtained using
the technique described by Fishman and associates.62
Fetuses with tumours diagnosed in utero without fetal hydrops
and severe prematurity have an excellent prognosis with a survival
when they are completely excised, recurrence is rare. They may
contain immature elements but are rarely malignant if removed in
the neonatal period. If undetected and resected later in life, there
is a higher risk for malignant recurrence, particularly if the AFP
is high.56,63
In terms of follow-up, slightly different approaches have been
suggested but in general should consist of physical examination
(including rectal) and serum markers every 3 months for at least 2
years and then every 6 months for the next 2 years. Ultrasound can
be conducted every 3 or 6 months depending on the local proto-
col.58,65 Recurrence of the tumour is usually local, but metastatic
lesions can be present in cases of malignancy. Surgical follow-up
should be maintained for at least the first 4 to 5 years to monitor
bowel and urinary function. In general, about 30% of children
undergoing SCT resection present with problems, ranging from
constipation to incontinence, and parents should be made aware
 
Conclusion
Fetal tumours are extremely rare, but with more widespread ultra-
sound screening in pregnancy, the incidence of prenatal diagnosis
is increasing. Management of such cases should be in fetal medi-
cine centres working with an MDT of specialists to optimise peri-
natal outcome.
Masses arising anteriorly from the fetal neck are most com-
monly lymphangiomas or teratomas. Differentiation can be dif-
ficult antenatally. MRI can be a useful imaging modality in these
cases and is of particular use for soft tissue definition. Fetal neck
masses can cause significant airway obstruction, and careful ante-
natal surveillance is required. In large masses, especially teratomas,
an EXIT procedure should be planned at delivery. The treatment
for teratomas is usually surgical resection, but lymphovascular
abnormalities may require a combination of treatments, including
sclerotherapy, aspiration and surgical debulking.

Sacrococcygeal teratomas are the commonest form of congeni-
tal tumour. Careful antenatal assessment is required with serial
ultrasound. High vascularity, rapid growth or a large solid compo-
nent to the teratoma are associated with poor antenatal outcomes.
In fetuses with hydrops at less than 28weeks’ gestation, in utero
therapies may improve outcomes but are associated with high rates
of comorbidity. Postnatally, resection of the SCT is the preferred
treatment. Follow-up into childhood is required because there is
a risk for recurrence, and up to one third of children may have
complications, particularly related to bladder and bowel function.
CHAPTER 37  Fetal Tumours 453
The outlook for fetuses with these rare tumours is improving.
Key to this is the management of these pregnancies in an expe-
rienced fetal medicine centre with easy access to antenatal input
from the associated paediatric and adult specialties.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 27. Laje P, Johnson MP, Howell LJ, et  al. Ex utero intrapartum treat-
ment in the management of giant cervical teratomas. J Pediatr Surg.
2012;47:1208–1216.
1. Kadlub N, Touma J, Leboulanger N, et al. Head and neck teratoma: from
28. Lin EE, Moldenhauer JS, Tran KM, et al. Anesthetic management of 65
diagnosis to treatment. J Cranio-Maxillofacial Surg. 2014;42:1598–1603.
cases of ex utero intrapartum therapy. Anesth Analg. 2016;123:411–417.
2. Howarth ES, Draper ES, Budd JL, et al. Population-based study of the
29. Boat A, Mahmoud M, Michelfelder EC, et al. Supplementing desflurane
outcome following the prenatal diagnosis of cystic hygroma. Prenat
with intravenous anesthesia reduces fetal cardiac dysfunction during open
Diagn. 2005;25:286–291.
fetal surgery. Paediatr Anaesth. 2010;20:748–756.
3. Forrester MB, Merz RD. Descriptive epidemiology of teratoma in infants,
30. Ryan G, Somme S, Crombleholme TM. Airway compromise in the fetus
Hawaii, 1986-2001. Paediatr Perinat Epidemiol. 2006;20:54–58.
and neonate: prenatal assessment and perinatal management. Semin Fetal
4. Oliver G. Lymphatic vasculature development. Nat Rev Immunol.
Neonatal Med. 2016;21:230–239.
2004;4:35–45.
31. Barthod G, Teissier N, Bellarbi N, et  al. Fetal airway management on
5. Molina FS, Avgidou K, Kagan KO, et al. Cystic hygromas, nuchal edema,
placental support: limitations and ethical considerations in seven cases.
and nuchal translucency at 11–14 weeks of gestation. Obstet Gynecol.
J Obstet Gynaecol. 2013;33:787–794.
2006;107:678–683.
32. Cheng J, Bastidas N. Considerations for management of head and neck
6. Croonen EA, Nillesen W, Schrander C, et al. Noonan syndrome: com-
lymphatic malformations in children. J Craniofac Surg. 2016;27:908–
paring mutation-positive with mutation-negative Dutch patients. Mol
912.
Syndromol. 2013;4:227–234.
33. Morgan P, Keller R, Patel K. Evidence-based management of vascular
7. Shaffer LG, Rosenfeld JA, Dabell MP, et al. Detection rates of clinically
malformations. Facial Plast Surg. 2016;32:162–176.
significant genomic alterations by microarray analysis for specific anoma-
34. Bagrodia N, Defnet AM, Kandel JJ. Management of lymphatic malfor-
lies detected by ultrasound. Prenat Diagn. 2012;32:986–995.
mations in children. Curr Opin Pediatr. 2015;27:356–363.
8. Aubin A, Pondaven S, Bakhos D,L, et  al. Oropharyngeal teratomas in
35. Horbach SE, Lokhorst MM, Saeed P, et al. Sclerotherapy for low-flow
newborns: management and outcome. Eur Ann Otorhinolaryngol Head
vascular malformations of the head and neck: a systematic review of scle-
Neck Dis. 2014;131:271–275.
rosing agents. J Plast Reconstr Aesthetic Surg. 2016;69:295–304.
9. Kathary N, Bulas DI, Newman KD, Schonberg RL. MRI imaging of fetal
36. Hoff SR, Rastatter JC, Richter GT. Head and neck vascular lesions.
neck masses with airway compromise: utility in delivery planning. Pediatr
Otolaryngol Clin North Am. 2015;48:29–45.
Radiol. 2001;31:727–731.
37. Elluru RG, Azizkhan RG. Cervicofacial vascular anomalies. II. Vascular
10. Lazar DA, Cassady CI, Olutoye OO, et al. Tracheoesophageal displace-
malformations. Semin Pediatr Surg. 2006;15:133–139.
ment index and predictors of airway obstruction for fetuses with neck
38. Adams MT, Saltzman B, Perkins JA. Head and neck lymphatic mal-
masses. J Pediatr Surg. 2012;47:46–50.
formation treatment: a systematic review. Otolaryngol Head Neck Surg.
11. Santos XM, Papanna R, Johnson A, et  al. The use of combined ultra-
2012;147:627–639.
sound and magnetic resonance imaging in the detection of fetal anoma-
39. Lazar DA, Olutoye OO, Moise Jr KJ, et al. Ex-utero intrapartum treat-
lies. Prenat Diagn. 2010;30:402–407.
ment procedure for giant neck masses—fetal and maternal outcomes. J
12. Lall NU, Meyers ML, Mirsky DM. MRI of fetal and maternal diseases in
Pediatr Surg. 2011;46:817–822.
pregnancy. Springer International Publishing, Switzerland; 2016:119–137.
40. Peiró JL, Sbragia L, Scorletti F, et  al. Management of fetal teratomas.
13. Saleem SN. Fetal MRI: an approach to practice: a review. J Adv Res.
Pediatr Surg Int. 2016;32:635–647.
2014;5:507–523.
41. Altman RP, Randolph JG, Lilly JR. Sacrococcygeal teratoma: American
14. Hubbard A, Crombleholme T, Adzick N. Prenatal MRI evaluation of
Academy of Pediatrics Surgical Section Survey—1973. J Pediatr Surg.
giant neck masses in preparation for the fetal EXIT procedure. Am J
1974;9:389–398.
Perinatol. 2008;15:253–257.
42. Derikx JP, De Backer A, van de Schoot L, et al. Long-term functional
15. Wolfe K, Lewis D, Witte D, et  al. Fetal cervical teratoma: what is the
sequelae of sacrococcygeal teratoma: a national study in the Netherlands.
role of fetal MRI in predicting pulmonary hypoplasia? Fetal Diagn Ther.
J Pediatr Surg. 2007;42:1122–1126.
2013;33:252–256.
43. Partridge EA, Canning D, Long C, et al. Urologic and anorectal compli-
16. Werner H, Lopes dos Santos JR, Fontes R, et al. Virtual bronchoscopy
cations of sacrococcygeal teratomas: prenatal and postnatal predictors. J
for evaluating cervical tumors of the fetus. Ultrasound Obstet Gynecol.
Pediatr Surg. 2014;49:139–143.
2013;41:90–94.
44. Akinkuotu AC, Coleman A, Shue E, et al. Predictors of poor prognosis in
17. Laje P, Peranteau WH, Hedrick HL, et al. Ex utero intrapartum treat-
prenatally diagnosed sacrococcygeal teratoma: a multiinstitutional review.
ment (EXIT) in the management of cervical lymphatic malformation. J
J Pediatr Surg. 2015;50:771–774.
Pediatr Surg. 2015;50:311–314.
45. Rodriguez MA, Cass DL, Lazar DA, et al. Tumor volume to fetal weight
18. Sheikh F, Akinkuotu A, Olutoye OO, et  al. Prenatally diagnosed
ratio as an early prognostic classification for fetal sacrococcygeal teratoma.
neck masses: long-term outcomes and quality of life. J Pediatr Surg.
J Pediatr Surg. 2011;46:1182–1185.
2015;50:1210–1213.
46. Shue E, Bolouri M, Jelin EB, et al. Tumor metrics and morphology pre-
19. Ogita K, Suita S, Taguchi T, et al. Outcome of fetal cystic hygroma and
dict poor prognosis in prenatally diagnosed sacrococcygeal teratoma: a
experience of intrauterine treatment. Fetal Diagn Ther. 16:105–110
25-year experience at a single institution. J Pediatr Surg. 2013;48:1225–
20. Chen M, Chen CP, Shih JC, et al. Antenatal treatment of chylothorax
1231.
and cystic hygroma with OK-432 in nonimmune hydrops fetalis. Fetal
47. Coleman A, Kline-Fath B, Keswani S, Lim FY. Prenatal solid tumor vol-
Diagn Ther. 2005;20:309–315.
ume index: novel prenatal predictor of adverse outcome in sacrococcygeal
21. Hirose S, Sydorak RM, Tsao K, et  al. Spectrum of intrapartum man-
teratoma. J Surg Res. 2013;184:330–336.
agement strategies for giant fetal cervical teratoma. J Pediatr Surg.
48. Westerburg B, Feldstein VA, Sandberg PL, et al. Sonographic prognostic fac-
2003;38:446–450.
tors in fetuses with sacrococcygeal teratoma. J Pediatr Surg. 2000;35:322–326.
22. Kontopoulos EV, Gualtieri M, Quintero RA. Successful in utero treat-
49. Benachi A, Durin L, Vasseur Maurer S, et  al. Prenatally diagnosed
ment of an oral teratoma via operative fetoscopy: case report and review
­sacrococcygeal teratoma: a prognostic classification. J Pediatr Surg. 2006;
of the literature. Am J Obstet Gynecol. 2012;207:e12–e15.
41:1517–1521.
23. Cruz-Martinez R, Moreno-Alvarez O, Garcia M, et al. Fetal endoscopic
50. Coleman A, Shaaban A, Keswani S, Lim FY. Sacrococcygeal teratoma
tracheal intubation: a new fetoscopic procedure to ensure extrauterine
growth rate predicts adverse outcomes. J Pediatr Surg. 2014;49:985–989.
tracheal permeability in a case with congenital cervical teratoma. Fetal
51. Ayed A, Tonks AM, Lander A, Kilby MD. A review of pregnancies com-
Diagn Ther. 2014;38:154–158.
plicated by congenital sacrococcygeal teratoma in the West Midlands
24. Kelly MF, Berenholz L, Rizzo KA, et al. Approach for oxygenation of the
region over an 18-year period: population-based, cohort study. Prenat
newborn with airway obstruction due to a cervical mass. Ann Otol Rhinol
Diagn. 2015;35:1037–1047.
Laryngol. 1990;99:179–182.
52. Van Mieghem T, Al-Ibrahim A, Deprest J, et al. Minimally invasive ther-
25. Catalano PJ, Urken ML, Alvarez M, et  al. New approach to the man-
apy for fetal sacrococcygeal teratoma: case series and systematic review of
agement of airway obstruction in “high risk” neonates. Arch Otolaryngol
the literature. Ultrasound Obstet Gynecol. 2014;43:611–619.
Head Neck Surg. 1992;118:306–309.
53. Sananes N, Javadian P, Schwach Werneck Britto I, et al. Technical aspects
26. Deprest J, Gratacos E, Nicolaides KH. Fetoscopic tracheal
and effectiveness of percutaneous fetal therapies for large sacrococcygeal
occlusion(FETO) for severe congenital diaphragmatic hernia: evolu-
teratomas: cohort study and literature review. Ultrasound Obstet Gynecol.
tion of a technique and preliminary results. Ultrasound Obstet Gynecol.
2016;47:712–719.
2004;24:121–126.

453.e1
453.e2 R E F E REN C ES
54. Roybal JL, Moldenhauer JS, Khalek N, et al. Early delivery as an alterna-
tive management strategy for selected high-risk fetal sacrococcygeal tera-
tomas. J Pediatr Surg. 2011;46:1325–1332.
55. Flake AW. Fetal sacrococcygeal teratoma. Semin Pediatr Surg. 1993;
2:113–120.
56. Schmidt B, Haberlik A, Uray E, et al. Sacrococcygeal teratoma: clinical
course and prognosis with a special view to long-term functional results.
Pediatr Surg Int. 1999;15:573–576.
57. Malone PS, Spitz L, Kiely EM, et al. The functional sequelae of sacrococ-
cygeal teratoma. J Pediatr Surg. 1990;25:679–680.
58. Yao W, Li K, Zheng S, et al. Analysis of recurrence risks for sacrococ-
cygeal teratoma in children. J Pediatr Surg. 2014;49:1839–1842.
59. Cohen L, Lauer SJ, Ablin A, et al. Complete surgical excision is effective
treatment for children with immature teratomas with or without malig-
nant elements: a Pediatric Oncology Group/Children’s Cancer Group
Intergroup Study. J Clin Oncol. 1999;17:2137–2143.
60. Hedrick HL, Flake AW, Crombleholme TM, et al. Sacrococcygeal ter-
atoma: prenatal assessment, fetal intervention, and outcome. J Pediatr
Surg. 2004;39:430–438. -8.
61. Kremer ME, Wellens LM, Derikx JP, et al. Hemorrhage is the most com-
mon cause of neonatal mortality in patients with sacrococcygeal tera-
toma. J Pediatr Surg. 2016;51:1826–1829.
62. Fishman SJ, Jennings RW, Johnson SM, Kim HB. Contouring buttock
reconstruction after sacrococcygeal teratoma resection. J Pediatr Surg.
2004;39:439–441.
63. Rescorla FJ, Sawin RS, Coran AG, et al. Long-term outcome for infants
and children with sacrococcygeal teratoma: a report from the Children’s
Cancer Group. J Pediatr Surg. 1998;33:171–176.
64. Brace V, Grant SR, Brackley KJ, et al. Prenatal diagnosis and outcome
in sacrococcygeal teratomas: a review of cases between 1992 and 1998.
Prenat Diagn. 2000;20:51–55.
65. Shalaby MS, Walker G, O’Toole S, et  al. The long-term outcome of
patients diagnosed with sacrococcygeal teratoma in childhood. A study of
a national cohort. Arch Dis Child. 2014;99:1009–1013.
38
Open Fetal Surgery
LUC JOYEUX, FRANK VAN CALENBERGH, ROLAND DEVLIEGER, LUC DE CATTE AND
JAN DEPREST
KEY POINTS
• Most fetuses with a prenatal diagnosis of a congenital abnor-
mality can be managed expectantly. For some conditions, in
utero referral is mandatory for planned delivery and manage-
ment after birth.
• Fetal surgery is only required for conditions that cannot await
therapy after birth and when there is enough evidence that
prenatal surgery partly reverses the natural course.
• Open fetal surgery (OFS) is one modality to perform opera-
tions on fetuses. OFS is invasive, with maternal morbidity and
an impact on the uterus in the index and future pregnancies,
and it increases the risk for preterm delivery.
• Open spina bifida is the only nonlethal condition operated in
utero, yet it became the most frequent indication based on
level I evidence. OFS improves the outcome of children with
spina bifida, but it is not a cure.
Introduction
Definition
Open fetal surgery (OFS) is the type of fetal surgery that is per-
formed via hysterotomy. It is also referred as open maternal-fetal
surgery or fetal surgery by open access.  for SBA improves outcome compared with standard postnatal
Rationale of Open Fetal Surgery for
Congenital Abnormalities
Pathophysiology and Natural History
Since the introduction of ultrasound (US) and later other imag-
ing modalities and the increased interest for congenital anomalies
(CAs) and their outcomes in relation to prenatal findings, it has
become possible to define the natural history of several potentially
correctable CAs.1 Also, researchers developed appropriate animal
models for the disease of interest to study both the pathophysi-
ology as well as the surgical interventions contemplated. These
studies, particularly in primates, have been used for developing
the anaesthetic, tocolytic and surgical protocols for hysterotomy
and OFS.  adenomatoid malformation (CCAM) and its related malformation,
454
Selection Criteria for Open Fetal Surgery
Open fetal surgery is currently being offered in highly specialised
multidisciplinary fetal centres for highly selected fetuses with a
condition that is, without any intervention, either lethal or in
which the subsequent organ function loss leads to an extremely
poor quality of life after birth. The International Fetal Medicine
and Surgery Society (IFMSS) defined five criteria required to jus-
tify fetal surgery (Table 38.1).2 
Indications
A Nonlethal Condition: Spina Bifida Aperta
The epidemiology, pathophysiology and natural history have
been addressed in Chapter 28. Within routine screening US
programs neural tube defects should be diagnosed prenatally. An
invasive fetal procedure for spina bifida aperta (SBA) seems to be
justified because of the significant lifelong neurologic disabilities,
the prenatal progression of findings and the experimental vali-
dation of the two-hit pathophysiology.3 Experimental research
and early clinical experience suggest that ongoing damage to the
exposed malformed spinal cord and developing brain is allevi-
ated by prenatal repair.1,4 The Management of Myelomeningo-
cele Study (MOMS) was a multicentre randomized controlled
trial that unequivocally demonstrated that prenatal surgery
repair.5 Table 38.2 displays the initial and current indications
and contraindications for in utero SBA repair. Fetal surgery was
shown to lessen or reverse hindbrain herniation, reduce the
postnatal ventriculoperitoneal shunt rate at 1 year of age and
improve neurofunctional outcome at the age of 30 months. 
Lethal Conditions
Congenital Thoracic Malformations Complicated With Fetal
Hydrops. Congenital thoracic malformations (CTMs) are a
heterogeneous group of rare disorders that may involve the airways
or lung parenchyma. As an accurate pathological prenatal diagnosis is
not possible; a detailed prenatal description of the appearance of the
lesion is sufficient and should follow the new CTM classification and
nomenclature (see Chapter 30).6 This section focuses on the two
most frequent CTMs that are also amenable to OFS: congenital cystic

TABLE Five Criteria for Maternal-Fetal Surgery From
38.1 the International Fetal Medicine and Surgery
Society
1. Accurate diagnosis and staging possible, with exclusion of associ-
ated anomalies
2. Natural history of the disease is documented, and individualised progno-
sis is established
3. Currently no effective postnatal therapy (i.e., improving the condition
or curing it)
4. In utero surgery proven feasible in animal models, reversing the
deleterious effects of the condition
5. Interventions performed in specialised multidisciplinary fetal treatment
centres within strict protocols and approval of the local ethics committee
with informed consent of the mother or parents
Adapted Harrison MR, Filly RA, Golbus MS, et  al. Fetal treatment 1982. N Engl J Med
307(26):1651–1652, 1982.
  
bronchopulmonary sequestration (BPS). It is important to realise that
the exact nature of the condition (or their combination) may only be
possible after resection. In fact, lung parenchymal malformations,
although superficially heterogeneous in appearance, have significant
overlap and seem to share a common embryologic origin.7 According
to the European Surveillance of Congenital Anomalies (EUROCAT)
registry, the prevalence of CTM between 2008 and 2012 was 4.13
per 10,000 live births. Among the CTMs, the estimated prevalence
of CCAM was 1.05 per 10,000 live births, that is, about one quarter
of all CTMs.8 The exact prevalence of BPS is unknown and probably
lower than for CCAM.9 CTMs may spontaneously regress before
birth; the ones that deteriorate in utero and may even lead to fetal
death are of relevance to this chapter (see Table 38.2).
Congenital Cystic Adenomatoid Malformation. Congenital
cystic adenomatoid malformation is a benign cystic intrapulmo-
nary nonfunctioning lung mass that is usually localised in one lobe
of the lung and mainly unilateral. CCAM contains cysts ranging
from smaller than 1 mm to larger than 10 cm in diameter. Most
CCAMs derive their blood supply from the pulmonary circula-
tion. CCAM is histologically characterised by an overgrowth of
terminal respiratory bronchioles that form cysts and lack normal
alveoli. Many pathologists consider it a hamartoma (i.e., a devel-
opmental abnormality with excess of one or several tissue compo-
nents). Although nonfunctional for normal gas exchange, CCAM
parenchyma has connections with the tracheobronchial tree as
evidenced by air trapping that can develop during postnatal resus-
citative efforts.10 There are different prenatal classifications; how-
ever, postnatally, typically four Stocker types (I–IV) are described.
Congenital cystic adenomatoid malformation growth usually
reaches a plateau by 28 weeks of gestation and may even nearly
disappear by birth.10 Others may cause fetal hydrops and in utero
death. The impact on normal lung development and postnatal
function has not been properly studied. Prenatally, the size of the
lesion, the cyst size, the growth and perfusion and the secondary
signs have all been used to describe the severity of the impact on
the fetus. In general, fetal hydrops is the single accepted criterion
for fetal therapy. A CCAM volume ratio (CVR) (CCAM volume
by sonographic measurement using the formula for an ellipse,
length × height × width × 0.52 divided by head circumference to
correct for differences in fetal size) greater than1.6 has been shown
to predict hydrops in 80% of fetuses with CCAM.10  OFS for this condition are detailed in Table 38.2.
CHAPTER 38  Open Fetal Surgery 455
Bronchopulmonary Sequestration. Bronchopulmonary
sequestration is defined as a nonfunctioning lung mass that
receives a systemic blood supply rather than from a branch of
the pulmonary artery. It belongs to the spectrum of congenital
foregut malformations arising as an aberrant outpouching from
the developing foregut. BPS is believed to be aberrantly located
pulmonary mesenchyma that develops apart from the normal
lung.7
Bronchopulmonary sequestration is classified into intralo-
bar and extralobar forms. The extralobar (25%) form consists
of pulmonary tissue located outside the lung and enveloped in
its own pleura without communication with the normal tra-
cheobronchial tree. Around 90% are supradiaphragmatic and
10% infradiaphragmatic, usually left suprarenal. The intralobar
form is found within the normal lung tissue with or without
communication.9 
Other Rarer Lethal Conditions. We will not discuss these even
rarer indications, some of which have been operated in utero10:
• Hybrid lesions (CCAM and BPS)
• Bronchogenic and enteric cysts
• Mediastinal cystic teratoma
• Congenital lobar emphysema
• Haemangioma
• Bronchial atresia
• Pulmonary leiomyofibroma
• Intrathoracic gastric duplication cyst 
Sacrococcygeal Teratoma With Hydrops Fetalis. Sacrococcy-
geal teratoma (SCT) is the most common tumour of newborns
with a prevalence of about 0.37 to 0.93 per 10,000 live births.11,12
SCT is uniformly attached to the coccyx and has been classified
into four types (I–IV) by the relative amounts of intrapelvic and
external tumour. SCTs presenting postnatally have excellent long-
term outcomes; conversely, prenatally diagnosed SCTs have a
significant perinatal mortality ranging from 25% to 37%. Death
occurs mainly in fetuses with fast-growing, solid and highly vas-
cularised teratomas that lead to high-output cardiac failure or
haemorrhage.13 The pathophysiological mechanism behind this is
explained by the ‘vascular steal’ from the placenta and the fetus
and by the mass effect10:
1. SCT acts as a large arteriovenous malformation that deviates
high volumes of blood from the fetus and the placenta. Also,
bleeding inside the tumour can cause anaemia. The conse-
quence is high-output cardiac failure, which can then lead to
placentomegaly, hydrops fetalis, intrauterine fetal demise, pre-
term birth and neonatal death. This may also cause Ballantyne
syndrome (maternal mirror syndrome), which is a dangerous
maternal complication.
2. SCT compresses the abdominal and thoracic organs, leading to
polyhydramnios (oesophageal and gastric compression) induc-
ing uterine irritability, premature rupture of the membranes
(PPROM) and preterm delivery. The tumour may also have an
effect on nearby organs, such as obstructive uropathy. Dystocia
in undiagnosed large masses is frequently associated with trau-
matic tumour rupture and haemorrhage during delivery, which
is usually fatal.
Assessment of tumour size, growth rate and fetal cardiac func-
tion by fetal imaging allows the identification of fetuses at partic-
ular risk for decompensation. Ultrafast fetal magnetic resonance
imaging (MRI) is superior to US in delineating the intrapelvic
extent of the tumour, yet this does not contribute to the indi-
cation for fetal surgery.14 The currently accepted indications for
 
456 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
38.2 Indications for Open Fetal Surgery
Indications Type of
for OFS Malformation Rationale for In Utero Therapy Inclusion Criteria Exclusion Criteria
Spina bifida MMC or myeloschisis • Untethering and covering of • Maternal age ≥18 yr • Multiple-gestation pregnancy
aperta with CM exposed malformed spinal cord • Gestational age 19 + • Uterine anomaly or corporeal uterine
to prevent or reverse functional 0-25 + 6weeks surgery
damage to the cord and • Isolated lesion • Additional fetal anomalies unrelated to SBA
nerves54,55 • Normal karyotype (chromosomal or not)
• Cessation of CSF leakage to • Level lesion from • Fetal kyphosis ≥30 degrees
prevent or reverse hydroceph- T1–S1 • Previous spontaneous singleton delivery at
aly and CM56-60 • Confirmed CM on <37 wk gestation
prenatal US and MRI • History of incompetent cervix or short
cervix <20 mm by US scan
• Placenta previa
• Obesity defined by BMI ≥35 (later shifted
to 40)
• IDP diabetes (later changed to uncontrolled)
• Other serious maternal medical condition
• Maternal-fetal Rh isoimmunisation
• Positive maternal HIV or haepatitis B or
known haepatitis C positivity
• No support person to stay with the preg-
nant woman at the centre
• Psychosocial limitations
• Inability to comply with travel and follow-up
protocols
Congenital Large solid lesion • Prevention of fetal death • Isolated lesion • Multiple-gestation pregnancy
thoracic complicated with • Reversal of heart and great • Fetal hydrops • Chromosomal abnormality
malfor- fetal hydrops vessels compression and thus • <32 wk of gestation • Other significant anatomic abnormalities
mations of nonimmune hydrops and • No dominant cyst • Placentomegaly
(CCAM cardiac failure61,62 • CVR >1.6 • Maternal mirror syndrome
and BPS) • Reversal of lung compression • Short cervix
and thus of lung hypoplasia61 • History of heavy cigarette smoking
• Reversal of oesophageal • Maternal psychosocial difficulties
compression and thus of • Other maternal medical risk factors
polyhydramnios
• Prevention of placentomegaly
and maternal mirror syndrome
Sacrococ- Large, fast-growing • Prevention of fetal death • Isolated lesion • Multiple-gestation pregnancy
cygeal solid lesion com- • Cessation of vascular steal • Type I or II (more • Chromosomal abnormality
teratoma plicated with fetal phenomenon, reversing high- external forms) • Other significant anatomic abnormalities
hydrops output cardiac failure • Fetal hydrops • Placentomegaly
• Reversal of mass effect • Progressive evolu- • Maternal mirror syndrome
tion of high output • Short cervix
cardiac failure • History of heavy cigarette smoking
• <28 wk of gestation • Maternal psychosocial difficulties
• Other maternal medical risk factors

BMI, Body mass index; BPS, bronchopulmonary sequestration; CCAM, congenital cystic adenomatoid; CM, Chiari II malformation; CSF, cerebrospinal fluid; CVR, cystic adenomatoid malformation volume
ratio; HIV, human immunodeficiency virus; IDP, insulin-dependent pregestational; MMC, myelomeningocele; MOMS, Management of Myelomeningocele Study; MRI, magnetic resonance imaging; OFS,
open fetal surgery; SBA, spina bifida aperta; US, ultrasonography.
  

Surgical Technique
Pre- and Intraoperative Management
Open fetal surgery is recommended to take place in tertiary
medical centres that have a multidisciplinary team experienced
with the anaesthetic and surgical techniques described later;
familiar with the management of the potential postoperative
complications such as amniotic fluid leakage, fetal membrane
separation and preterm labour; and with proven track record
of managing the condition of interest postnatally.15 Any candi-
date OFS centres should follow a specific training with expert
centres. We did so in a mixed training model, in-house training
by physicians familiar with OFS, as well as exported training
(i.e., short stay of selected team members at a large volume
OFS centre).16 We performed our first five local surgeries
under direct supervision.15,16

Preoperative Management. A multidisciplinary OFS team
of specialists will perform a complete prenatal evaluation and
counselling of the parents.17 Surgery should not proceed until
full informed consent is obtained. The maternal-fetal evaluation
includes detailed prenatal US to confirm the diagnosis and sever-
ity and exclude other abnormalities as well as technical aspects
such as placental location and maternal assessment. A fetal echo-
cardiogram is mandatory to measure the haemodynamic impact of
the condition while also ruling out congenital heart defects. Ultra-
fast fetal MRI today seems to be standard of care for most condi-
tions eligible for fetal surgery. If no genetic testing was done yet,
this should be undertaken first. Moreover, the expectant mothers
undergo psychological evaluations and extensive counselling on
the postnatal impact of the condition as well as that of OFS.10,18,19
Any nonmedical, socioeconomic issues should also be discussed
and when necessary dealt with. Some centres organise a consult
with the whole multidisciplinary team present.  abdominal skin incision is made. If the placenta is posterior, an
Intraoperative Management
Control of Labour. The OFS team usually consists of one
anaesthesiologist and an assistant, two general surgeons (either
general paediatric surgeons or obstetricians, depending on the
centre), another paediatric surgeon from the discipline of rel-
evance to the condition, a maternal-fetal specialist, a specialist in
echocardiography, one to two scrub nurses or midwifes, a circulat-
ing nurse and a perfusionist.10,20
Patients are admitted before the operation for obstetric moni-
toring, including measurement of the cervix, and initiation of
tocolysis. Most centres use an aggressive prophylactic tocolytic
regimen. Drugs used are indomethacin (50 mg per rectum or
orally) and atosiban in countries where it is available. Adequate
analgesia in the peri- and postoperative period is also important;
this is why an epidural anaesthesia is combined with the general
anaesthesia.5 Prophylactic antibiotics are also given (e.g., cephazo-
lin 1000 mg IV).  uterine stapling device loaded with absorbable polyglycolic acid
Maternal-Fetal Anaesthesia and Analgesia. The general
anaesthesia ensures good uterine relaxation and is also anaesthetic
for the fetus. Preoxygenation, rapid-sequence induction with cri-
coid pressure is used before intubation. Maintenance anaesthesia
is with a combination of volatile agents, nitrous oxide and intra-
venous (IV) anaesthetics (e.g., propofol). Systolic arterial blood
pressure is maintained over 100 mm Hg, and IV fluids are limited
to 0.9% sodium chloride (rate 100 mL/hr), the latter to avoid pul-
monary oedema. Maternal neuromuscular blockade is provided
with a short-acting muscle relaxant (rocuronium 0.6mg/kg). After
hysterotomy, fetal analgesia and immobilisation is performed
using intramuscular injection of fentanyl (10 ug/kg), atropine sul-
phate 0.01 mg/kg and a muscle relaxant (cisatracurium, 0.4 mg/
kg).5,16  perfuses Ringer lactate solution at 38° to 40°C and keeps the fetus
Maternal Conditioning and Installation. The mother is posi-
tioned supine on a mouldable mattress usually slightly on her left
side to avoid compression of the vena cava. Maternal periopera-
tive monitoring, cannulisation and catheterisation includes blood
pressure, electrocardiogram leads, two large IV catheters, a blad-
der catheter, leg compression boots, a transcutaneous pulse oxim-
eter and if required a central radial arterial catheter and central
venous catheter.5,20  Closure of Uterus and Skin. A watertight two-layer uterine
Fetal Resuscitation. Drugs for fetal resuscitation should be pre-
pared and transferred in a sterile fashion into individually labelled
syringes held by the scrub nurse for immediate fetal administra-
tion by the surgeon in case of fetal distress. These drugs include
single-unit doses of atropine (20 mcg/kg), epinephrine (10 mcg/
CHAPTER 38  Open Fetal Surgery 457
kg) and crystalloid (10 mL/kg). In the rare case of maternal circu-
latory collapse, if fetal resuscitation has been unsuccessful after 4
minutes, the fetus should be delivered immediately and managed
by a neonatology team according to her or his gestational age and
the country’s legislation.21,22 
The Surgical Procedure
Elective Open Fetal Surgery for Spina Bifida Aperta Repair. In
the MOMS trial, the operation was scheduled between 19 weeks +
0 days to 25 + 6 days. Subsequent experience showed that the risk
for membrane rupture was lower when the operation is deferred
until 23 weeks (see Table 38.2 and Video 38.1).5,23 The duration of
the surgery should be as short as possible because operating time
is directly related to risks of prematurity.
Skin Opening and Uterus Exposition. A low transverse
anterior hysterotomy will be required. In these, a vertical midline
fascial incision is sufficient because the uterus can remain in the
abdomen. If the placenta is anterior, a posterior hysterotomy will
be necessary. To enable comfortable exteriorisation of the uterus,
the rectus muscles may need division. This will prevent compres-
sion on the lateral uterine vessels because the uterus is tilted out
of the abdomen. Then a large abdominal ring retractor maintains
exposure. 
Determining the Incision. Sterile intraoperative US delineates
fetal position and placental location.17,20 The fetus may need to
be manipulated. The edge of the placenta is marked under US
guidance using electrocautery. The position and orientation of the
hysterotomy are planned to stay parallel to, and at least 6 cm from,
the placental edge. 
Hysterotomy. Standard full-thickness haemostatic hysterot-
omy is performed, facilitated by the placement of two 0 mono-
filament slowly resorbable polydioxanone sutures (PDS), with a
staples (Poly CS 57 mm stapler; Covidien, Mansfield, MA, USA).
This is to minimise blood loss and anchor the membranes, and
in contrast with the use of metal staples, does not impair sub-
sequent fertility.24-26 Since the MOMS trial, some modifications
have been made in the United States such as clamping of the myo-
metrium before stapling.27,28 A Brazilian group uses a homemade
trocar to enter the uterus, and a Polish group has good outcomes
with nonstapled hysterotomy (Table 38.3).29,30 
Fetal Monitoring and Amniotic Fluid Replacement. Contin­
uous intraoperative fetal echocardiography is performed to
monitor fetal myocardial performance (i.e., fetal heart rate and
ventricular function). Initially, a strict maintenance of amniotic
fluid volume was kept by using a ‘level I rapid infusion device’. This
warm and buoyant. Others have meanwhile moved to replacing the
amniotic fluid with warmed normal saline manually. 
Standard Spina Bifida Aperta Fetal Repair. Most surgeons
perform this surgery using loupes or even a microscope. The tech-
nique consists of eight steps whatever the type of SBA (i.e., myelo-
meningocele in two thirds and myeloschisis in one third of the
cases; Figs. 38.1 and 38.2).5,20,27,31-33 
closure is performed with double-armed full-thickness 0 PDS
interrupted stay sutures followed by a running 2-0 PDS suture.
Warmed Ringer lactate is infused until normal fluid levels are
obtained, and 500 mg of oxacillin or cefazolin is instilled into
the amniotic cavity just before completing the running layer. The
458 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE
38.3 Modification of the Hysterotomy Technique in Comparison With the MOMS Trial
Fetal Centre Aim Steps Results
MOMS Reference point • Two monofilament stay sutures are placed through the full-thickness PPROM rate: 44%
uterine wall under US guidance. CMS rate: 30%
• Electrocautery is used to desiccate the tissue between the sutures
through the myometrium to identify and enter the fetal membranes
under control and make a 1-cm hysterotomy.
• Under US and manual guidance, the uterine stapler is placed and
palpated to exclude the presence of fetal tissue.
• The stapler is then fired. The number of staple lines is decided on
the fetal anatomy and exposure to create a 6- to 8-cm uterine inci-
sion large enough to expose the fetal defect.
• Any detached membranes are fixed with monofilament sutures.
Philadelphia To avoid jamming of the • The staple line in the stapler is pretreated with mineral oil to facili- Lower rate of PPROM
technique27 uterine stapler in thick tate staple disengagement. (32.3%)
uterine tissue • An atraumatic Glassman intestinal clamp (V. Mueller, San Diego, Lower CMS rate (23%)
CA, USA) is placed to compress the uterine tissue in the line of the
planned stapler deployment followed by removal of the Glassman
clamp.
Vanderbilt tech- To control uterine bleeding • Allis-Adair clamps are placed at the cut edges of the coagulated Lower rate of PPROM (22%)
nique28 and avoid CMS muscle, exposing the amniotic membrane. Lower rate of CMS (0%)
• Full-thickness running locked O chromic sutures to secure the
membrane to the uterine wall both sides of the incision.
• Under US and manual guidance, one uterine stapler is used to create
a 6-cm incision.
Brazilian tech- To perform a faster and • After membrane fixation, insertion of the Almodin-Moron trocar NS
nique29 less traumatic uterine placed by a modified Seldinger technique under US guidance.
entry
Polish technique30 To substitute the stapler • A double full-thickness running suture is placed on both sides of the NS
hysterotomy.

CMS, Chorioamniotic membrane separation; MOMS, Management of Myelomeningocele Study; NS, nonspecified; PPROM, preterm premature rupture of the membranes; US, ultrasonography.
  

stay sutures are then tied, and an omental flap can be mobilised
and secured over the hysterotomy site. The maternal laparotomy
incision is closed in layers. A subcuticular maternal skin closure
covered with a transparent dressing is used, allowing US.20,27,28  postnatally. Hence, the SCT is exposed through an appropriate
Other Procedures in Open Fetal Surgery for Lethal Conditions.
The procedures discussed in this section have a higher impact on
fetal haemodynamics, so additional measures need to be taken.
A fetal IV line is placed to check fetal blood gases and haemato-
crit, and if necessary, resuscitate the fetus with medication (atro-
pine), fluid and fresh warm blood.10 If possible, this access can
be complemented with intraoperative fetal monitoring. When a
fetal limb can be exposed, a miniaturised pulse oximeter is placed
around it. This oximeter is wrapped around the fetal palm or foot
and protected with aluminium foil and Tegaderm (3M Company,
St. Paul, MN, USA) to minimise light exposure.10
Congenital Thoracic Malformations. Complete mass resec-
tion by fetal lobectomy is performed. The fetal chest is entered by a
fifth intercostal space thoracotomy. The CTM readily decompresses
out through the incision, consistent with increased intrathoracic
pressure from the mass. The appropriate pulmonary lobe(s) con-
taining the lesion is resected. The fetal thoracotomy is closed in lay-
ers, the fetus is returned to the uterus and warmed Ringer lactate
containing antibiotics is instilled into the amniotic cavity.10  of muscular function recovery is needed because MgSO4
Sacrococcygeal Teratomas. A fetal debulking procedure
for external SCT is performed. The objective of this OFS is to
occlude the tumour vascular supply and arrest the steal effect.
No attempt is made to dissect the intrapelvic SCT component
or to remove the coccyx. Completion of the removal is done
hysterotomy, and a Hegar dilator is placed in the rectum. The
skin is incised circumferentially around the base of the SCT, and
a tourniquet applied to constrict blood flow. The tumour is deb-
ulked externally usually with a 90-mm-thick tissue stapler (US
Surgical Corporation). The fetal sacral wound is finally closed in
layers.10,34 
Postoperative Management
Postoperative Prevention of Preterm Labour. In the United
States, the tocolytic regimen for SBA repair is complex and
includes IV magnesium sulphate (MgSO4), which is started from
closure of the hysterotomy and continued for about 18 to 24
hours (6-g loading dose followed by a continuous infusion at
2–4 g/hr), together with indomethacin rectal suppositories
(50 mg) every 6 hours postoperatively for 48 hours. After
the MgSO4, nifedipine (10–20 mg every 6 hours) is started
and continued until the time of delivery. Careful monitoring
potentiates nondepolarising neuromuscular blockers. Adverse
effect screening comprises monitoring of serum magnesium
 CHAPTER 38  Open Fetal Surgery 459

A B

C D

E F

G H I
• Fig. 38.1  Technique of open fetal surgery for spina bifida aperta (SBA) as performed in Leuven, Belgium.
Exposition of a myelomeningocele (MMC) in a 25 weeks’ gestation fetus through hysterotomy (A). 1,
Circumferential sharp dissection at the junction line of the full-thickness skin (blue circle) and the SBA sac
(green circle) with attention to the vascular supply and with preservation of all neural components (B). 2,
Circumferential sharp dissection of the neural placode (B). 3, Resection of all pathological elements still
attached to the placode (i.e., SBA sac, zona epitheliosa and junction line). The three first steps completed
allow complete untethering of the neural placode leading to spontaneous repositioning of the placode
into the spinal canal (C). The next five steps correspond to the anatomical closure in three layers in this
case (occasionally, two layers or in 20% a patch may be required).27  4, Closure of the dural sac (D) to
cover the placode with a 6/0 running suture or with a dural patch (Duragen, Integra Life Sciences Corpo-
ration, Plainsboro, NJ; or Duraform, Codman & Shurtleff, Inc.) if there is insufficient dura. Reneurulation
(pia arachnoid closure) was not under taken in this case; it is a controversial step. 5, Circumferential skin
undermining. 6, Mobilisation of paraspinal myofascial flaps. 7, Closure of paraspinal myofascial flaps over
the closed dura (E, G, H). 8, Closure of skin (F) using a 4/0 running monofilament suture. Alternatively,
relaxing incisions can be made or the defect can be covered with acellular human cadaveric dermal
allograft (AlloDerm Life Cell, Branchburg, NJ or Integra Regeneration Dermal Template, Integra Life Sci-
ences Corporation, Plainsboro, NJ, USA; or Flex HD, Musculoskeletal Transplant Foundation) (I). (Copy-
right UZ Leuven, Leuven, Belgium.)
460 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

E
C

D G
• Fig. 38.2  Schematic drawings of the steps of open fetal surgical repair: exposition of a myelomeningo-
cele (MMC) (A); MMC (B); myeloschisis (C); dissection and untethering of the neural placode (D); standard
anatomical closure in three layers: closure of dural sac (E), myofascial flaps (F) and skin (G). (Drawings by
Myrthe Boymans [http://www.myrtheboymans.nl] for and copyright UZ Leuven, Leuven, Belgium.)

levels and observing for clinical signs of magnesium toxicity morbidity due to the hysterotomy, the general anaesthesia and the
and daily fetal echocardiography to detect adverse fetal effects medication used. To our knowledge, no maternal deaths have been
of indomethacin, including ductal constriction, tricuspid reported. There are also fetal risks. The most frequent events are
regurgitation and oligohydramnios.10,33,35 In Europe, atosiban reduced ventricular contractility of the heart, with subsequent brady-
is used, which has fewer side effects.16,22  cardia and cardiac arrest. The main complications of OFS are listed
Postoperative Maternal-Fetal Monitoring. Cardiotocography in Table 38.4. 
externally records fetal heart rate and uterine activity. Daily US Outpatient Follow-Up. For the first 2 weeks after discharge,
is performed to screen for fetal movements, anatomic evaluation, the mother is prescribed modified bed rest (mealtime and bath-
fetal membrane position and amniotic fluid volume status.10  room privileges). Subsequently, she is allowed graduate to moder-
Postoperative Complications. Open fetal surgery is inva- ate activity if the uterus is quiescent. Twice-weekly fetal US and
sive hence has inherent maternal-fetal risks. There is the maternal obstetric assessments are performed.10 
 CHAPTER 38  Open Fetal Surgery 461

TABLE Maternal, Fetal and Paediatric (Until 2.5 Years) Outcomes Comparing Open Fetal Surgery With Postnatal
38.4 Surgery for Spina Bifida Aperta: Summary of Results of the Randomised MOMS Trial5,a

Results of the MOMS Trial Open Fetal Repair Postnatal Repair Statistical Analysis
Number of pregnancies and fetuses 78 (MOMS trial) 80 (MOMS trial) Relative risk (95% CI) P valueb
91 (complete trial cohort) 92 (complete trial cohort)
Fetal Profile
Gestational age at randomisation 23.7 ± 1.4 23.9 ± 1.3 NS ≥0.5
Lesion level on ultrasonography
• Thoracic 5.1% (4/78) 3.7% (3/80) NS ≥0.5
• L1–L2 27% (21/78) 12.5% (10/80) NS ≥0.5
• L3–L4 38.5% (30/78) 56.2% (45/80) NS ≥0.5
• L5–S1 29.5% (23/78) 27.5% (22/80) NS ≥0.5
Operative Outcomes
Mean operation time (min) 105.2 ± 21.8c NS NA NA
Intraoperative incomplete closure 0% (0/91) 0% (0/92) NA NA
Bradycardia during repair 10.3% (8/78) 0% (0/80) NA 0.003
Maternal Outcomes
Placental abruption 6.6 (6/91) 0% (0/92) NA 0.01
Pulmonary oedema 5.5% (5/91) 0% (0/92) NA 0.03
Chorioamnionitis 2.2% (2/91) 0% (0/92) NA 0.25
Oligohydramnios 20% (19/91) 3.3% (3/92) 6.40 (1.96–20.89) <0.001
Chorioamniotic membrane separation 30% (33.0) 0% (0/92) NA <0.001
Preterm premature rupture of membranes 44% (40/91) 7.6% (7/92) 5.78 (2.73–12.22) <0.001
Haemorrhage requiring transfusion at delivery 8.8% (8/91) 1.1% (1/92) 8.09 (1.03–63.37) 0.02
Hysterotomy scar thinning or dehiscence at 35.3% (31/88) NS NA NA
delivery
Fetal and Neonatal Outcomesd
Perinatal mortality rate 2.2% (2/91) 2.2% (2/92) 1.01 (0.1–9.34) 1.00
Mean gestational age at birth (wk) 34.0 ± 3.0 37.3 ± 1.1 NA <0.001
Preterm birth <30 wk 11% (10/91) 0% (0/92) NA 0.001
Mean birth weight (g) 2383 ± 688 3039 ± 469 NA <0.001
Partial dehiscence at repair site not requiring 13% (10/77) 6.3% (5/80) 2.05 (0.73–5.73) 0.16
reoperation
Postnatal additional SBA recoveragee 2.6% (2/77)c NS NA NA
Respiratory distress syndrome 20.8% (16/77) 6.3% (5/80) 3.32 (1.28–8.63) 0.008
Periventricular leukomalacia 5.2% (4/77) 2.5% (2/80) 2.08 (0.39–11.02) 0.44
Necrotising enterocolitis 1.3% (1/77) 0% (0/80) NA 0.49
Paediatric Outcomes
At 1 year Improvement of structures of CNS
Primary outcome (fetal or neonatal death or the 72.5% (66/91)g 97.8% (90/92)g 0.74 (0.65–0.85)g <0.001g
need for CSF shunt)
Placement of CSF shunt 44% (40/91)g 83.7% (77/92)g 0.52 (0.42–0.67)g <0,001g
Any hindbrain herniation 64.3% (45/70) 95.6% (66/69) 0.67 (0.56–0.81) <0.001
Complete reversal of Chiari Malformation 35.7% (25/70) 4.3% (3/69) NS <0.001
Chiari Malformation decompression surgery 1.3% (1/77) 5% (4/80) 0.26 (0.03–2.24) 0.37
Surgery for tethered cord 7.8% (6/77) 1.2% (1/80) 6.15 (0.76–50.00) 0.06
Continued
462 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Maternal, Fetal and Paediatric (Until 2.5 Years) Outcomes Comparing Open Fetal Surgery With Postnatal
38.4 Surgery for Spina Bifida Aperta: Summary of Results of the Randomised MOMS Trial5,a—cont’d

Results of the MOMS Trial Open Fetal Repair Postnatal Repair Statistical Analysis
At 2.5 years Improvement of functions of peripheral nervous system
Primary outcomeh 148.6 ± 57.5 122.6 ± 57.2 NA 0.007
Bayley Mental Development Indexf 89.7 ± 14.0 87.3 ± 18.4 NA 0.53
Difference between motor function and anatomi- 0.58 ± 1.94 -0.69 ± 1.99 NA 0.001
cal levelsh 32.3% (20/62) 11.9% (8/67)
• >2 Levels better 11.3% (7/62) 9% (6/67)
• 1 Level better 22.6% (14/62) 25.4% (17/67)
• No difference 21% (13/62) 25.4% (17/67)
• 1 Level worse 12.9% (8/62) 28.4% (19/67)
• >2 Levels worse
Independent walking (ability to walk without 41.9% (26/62) 20.9% (14/67) 2.01 (1.16–3.48) 0.01
orthotics or devices)
Bayley Psychomotor Development Indexf (mean) 64.0 ± 17.4 58.3 ± 14.8 NA 0.03
Peabody Developmental Motor Scales (locomo- 3.0 ± 1.8 2.1 ± 1.5 NA 0.001
tion)
WeeFIM score (degree of disability)i 19.9 ± 6.4 16.5 ± 5.9 NA 0.003
• Mobility 20.5 ± 4.2 19.0 ± 4.2 NA 0.02
• Self-care 23.9 ± 5.2 24.1 ± 5.9 NA 0.67
• Cognitive
At 2.5 years Urologic outcomes
Number of children 56 59 RR (95% CI) P value
Primary outcome (death or need for CIC by 30 52% (29/56) 66% (39/59) 0.78 (0.57–1.07) 0.133
months)
Death at 2.5 years 0% (0/56) 0% (0/59) NA 1.00
Patients on CIC use 38% (21/56) 51% (30/59) 0.74 (0.48– 1.12) 0.189
Bladder trabeculations on urodynamics and US 8% (4/51) 33% (17/52) 0.39 (0.19–0.79) 0.003
Open bladder neck on urodynamics 26% (13/51) 44% (23/52) 0.71 (0.46–1.09) 0.063
At 2.5 years Impact on family and parental stress
Number of women 87 88 RR (95% CI) P value
Total parental stress (PSI-SF) 61.3 ± 21.3 60.3 ± 15.4 NA 0.89
Familial-social impact (IFS) 14.0 ± 3.8 15.3 ± 3.7 NA 0.004
IFS score 24.6 ± 6.5 26.8 ± 6.6 NA 0.02

CI, Confidence interval; CIC, clean intermittent catheterisation; CNS, central nervous system; CSF, cerebrospinal fluid; GA, gestational age; IFS, 15-item Impact on Family Scale; NA, nonapplicable; NS,
nonspecified; PSI-SF, 36-item Parenting Stress Index-Short Form; RR, relative risk; SBA, spina bifida aperta; US, ultrasonography.
aWhen data are available from the complete trial cohort, only the latter ones are displayed,39,63 two substudies evaluating urologic outcomes40 and the impact on family and parental stress.38
bBold P values indicate statistically significant values.
cData from our previous systematic review.53
dBased on the number of liveborn infants.
ePostnatal reoperation in case of dehiscence of all layers.
fOnthe Bayley Scales of Infant Development II, the Mental Development Index and the Psychomotor Development Index are both scaled to have a population mean (± standard deviation) of 100 ± 15, with
a minimum score of 50 and a maximum score 150. Higher scores indicate better performance.
gOne-year outcomes for the complete MOMS trial: only ventricular size is associated with the need for shunting, and prenatal surgery does not improve shunt outcome when ventricular size is ≥15mm;
lesion level and degree of Chiari II malformation (CM) had no effect on the eventual need for shunting.63
hPrimary outcome at 2.5 years: score derived from the Bayley Mental Development Index and the difference between functional and anatomical level of the lesion.
iThe WeeFIM score (Functional Independence Measure for Children) measures the degree of disability in children. On the WeeFIM evaluation, the score on the self-care measurement ranges from 8 to 56,
and scores on the mobility and cognitive measurements range from 5 to 35, with higher scores indicating greater independence.
jFor the difference between the motor function level and the anatomical level, positive values indicate function that is better than expected on the basis of the anatomical level.
  

TABLE Paediatric Pros and Cons of Open Fetal Surgery
38.5 for Spina Bifida Aperta When Compared With
Postnatal Surgery Based on Level 1 Evidence
(MOMS Trial)
Paediatric
outcomes Pros Cons
At 1 yr Lower rate of: Higher rate of:
• Need for CSF shunt • Preterm birth
• Hindbrain herniation • Low birthweight
• Respiratory distress
syndrome
• Surgery for tethered
cord
At 2.5 yr Improvement in: No improvement in
• Motor function level of lower urologic outcomes
limbs
• Independent walking
• Psychomotor development
CSF, Cerebrospinal fluid; MOMS, Management of Myelomeningocele Study.
  
Delivery. Delivery should take place in a tertiary centre.
Because the midgestation hysterotomy for OFS is not in the lower
uterine segment, patients cannot labour and require elective cae-
sarean delivery for all future deliveries. Delivery is scheduled at 37
weeks. Some perform an amniocentesis at 36 weeks to confirm
lung maturity.20  and open bladder neck on video urodynamics (marker of bladder
Maternal and Paediatric Outcomes After
Open Fetal Surgery
Outcomes of Spina Bifida Aperta
Potential benefits of SBA OFS must be balanced against the risks
of fetal and maternal morbidity (Table 38.5). Here we report on
short-term (after a mean follow-up of <1 year), medium-term
(from ≥1 to <5 years) and long-term (from ≥5 years) outcomes
(see Tables 38.4 to 38.8).
Maternal Outcomes. Even if the choice of a well-informed
couple for OFS is evidence-based,5 the mother is at added risk
for obstetric long-term complications, both in her index and fol-
lowing pregnancies.25 Eventually, she has two uterine incisions. In
other words, OFS may cause short-term maternal morbidity with-
out any direct benefit.36 These complications related to prenatal
surgery include oligohydramnios, chorioamniotic separation, pla-
cental abruption and PPROM (see Table 38.4).
In subsequent pregnancy, medium-term complications include
uterine dehiscence (14%)25 and rupture (14%)25 versus 6% after
a classic caesarean section and abnormal placentation (attachment
resulting in spontaneous abortion, placenta accreta or abruptio).37
Other reproductive outcomes, specifically fertility and gynaeco-
logic factors, do not appear to be compromised in women under-
going OFS.25
Furthermore, researchers of the MOMS trial studied the impact
of SBA surgery on families and parental stress 12 and 30 months
after delivery.38 The overall negative familial and social impact of
caring for a child with SBA was significantly lower in the prenatal
surgery group. This was not the case for parental stress, which was
comparable in both groups. Moreover, the ambulation status of
the child with SBA and family resources were also predictive of
CHAPTER 38  Open Fetal Surgery 463
impact on family and parental stress. In fact, when adjusting for
demographics and other potential confounders, walking indepen-
dently at 30 months and greater family resources at 12 months
were associated with both lower parental stress at 30 months
(Parenting Stress Index, i.e., overall level of experienced parent-
ing stress; P = .0375 and P < .0001, respectively) and lower fam-
ily impact at 30 months (Impact on Family Scale, i.e., impact of
chronic childhood illnesses on families; P = .006 and P < .0001,
respectively) (see Table 38.4). 
Paediatric Outcomes
Perinatal Outcomes. In the MOMS trial, the perinatal
mortality rate of OFS for SB is comparable to that for post-
natal surgery. However, perinatal morbidity is increased with
higher rates of preterm birth, low birth weight and respiratory
distress syndrome. Some of the survivors (2.6%) required post-
natal reoperation in case of dehiscence of all layers (see Tables
38.4 and 38.5). 
Improved Short- and Medium-Term Outcomes Until 2.5
Years. The MOMS trial provided level I evidence that OFS for
SBA compared with postnatal surgery reduces the need for ven-
triculoperitoneal shunting and improves motor outcomes at 30
months of age (see Table 38.4).5,39 Children were more likely to
have a level of function that was two or more levels better than
expected, according to the anatomical level.
Later on, also some urologic outcomes were improved such as
bladder trabeculation (marker of bladder outlet obstruction; rela-
tive risk [RR], 0.39; 95% confidence interval [CI], 0.19– 0.79)
dysfunction; RR, 0.61; 95% CI, 0.40–0.92) after adjustment by
child’s sex and lesion level. These findings could mean that ulti-
mately, bladder control may be improved and the need for rein-
terventions in the long term reduced. However, the rate of clean
intermittent catheterisation at 30 months was not different (RR,
0.74; 95% CI, 0.48–1.12) (see Table 38.4).40
One of the major postoperative complications of SBA repair
is tethered cord syndrome, a secondary attachment of the spinal
cord to the scar that can cause spinal cord anomalies including
stretching and dysfunction. In the pre-MOMS studies, there was a
trend towards a higher incidence of tethered cord syndrome com-
pared with historical controls.41 This trend was confirmed in the
MOMS trial (see Table 38.4). 
Probably Improved Medium-Term Outcomes at 5 Years.
Knowledge of determinants of future independence of children
with SBA is of utmost importance for parental counselling on
long-term management and to provide realistic expectations.
Data from the MOMS trial are not yet available at 5 years, yet the
Philadelphia team reported on a prospective cohort of 58 children
who met the majority of the MOMS criteria (the gestational age
window was 20–26 weeks, ventricular size <17 mm, conserved
motor function in the lower limbs at the time of fetal surgery) and
were operated before the MOMS trial.42
Medical Outcomes. When operated prenatally, brainstem
function and lower extremity neuromotor function are not
comparable to those of normal children. To our knowledge, the
comparison with historical controls operated postnatally has not
been made yet.42 Conversely, all preschool neurodevelopmental
outcomes, especially intelligence quotient, are within the range
of that in normal children; again, no control data were available
in that study.43 In a subgroup analysis of SBA children operated
prenatally, total, neurocognitive and mobility independence were
higher in nonshunted than shunted patients and in children with
464 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Paediatric 5-Year Medical and Social Outcomes of Children With Spina Bifida Aperta After Open Fetal Surgery
38.6 in a Prospective Cohort of 54 Patients Followed Up From the Children’s Hospital of Philadelphia
Children with SBA
Children with SBA Undergoing Open Normal Children Undergoing Postnatal
5-Year Medical Outcomes Fetal Surgery (Population Norms) Surgery (Historical Data)
Hindbrain herniation and brainstem function n = 48/54 (89%) (64) NS NS
• Shunt rate 50% NS NS
• HH-related death 0% NS NS
• HH symptoms of brainstem dysfunction 48% NS NS
• Persistent cyanotic apnoea 0% NS 20%–100%
• Neurogenic dysphagia 23% NS 60%–100%
• Mild gastroesophageal reflux disease 17% NS 17%–84%
• Neuro-ophthalmologic disturbances 40% NS 100%
Lower extremity neuromotor function and ambula- n = 54/54 (100%) (65) NS NS 
tory potential
• Better than predicted (by a median of 2 functional 57.4% NS NS
levels)
• Worse than predicted (by a median of 1 functional 18.5% NS NS
level)
• Independent ambulatory status 69% NS NS
• Wheelchair dependent 7% NS NS
Preschool neurodevelopmental outcomes42 n = 30/54 (56%)
• Cognitive testing (WPPSI-III) 87.3–101.4 100 ± 15% NS
• Mean VIQ 100.8 ± 18.9 100 ± 15% NS
• VIQ shunted 93.6 ± 22.3 100 ± 15% NS
• Mean PIQ 93.1 ± 15.1 100 ± 15% NS
• PIQ shunted SBA patients 87.9 ± 19.9 100 ± 15% NS
• Mean FIQ scores 95.4 ± 16.0 100 ± 15% NS
• FIQ shunted SBA patients 88.6 ± 20.4 100 ± 15% NS
• Mean PS 87.3 ± 16.8 100 ± 15% NS
• PS shunted SBA patients 79.3 ± 20 100 ± 15% NS
• Achievement testing (WJP-BR16 test) 101.6–113.4 100 ± 15% NS
• Tests of VMI 88.3 ± 19.0 100 ± 15% NS
• Test of Differential Abilities Scales (DAS assess- 40.6 ± 11.2 50 ± 10% NS
ment)
Preschool functional independence status (WeeFIM) n = 26/30 (87%)43 NS (66, 67) NS46,68-71
• Total independence 58% 87.3% NS
• Neurocognitive independence 84% 85.7% 79.5%–80.6%
Self-care independence 38% 82.1% 30.4%–40%
• Independent in toileting 35% 85.7% NS
• Independent in bladder management 23% 85.7% NS
• Independent in bowel management 27% 100% NS
Mobility independence 62% 97.1% 28%–45.1%
• Independent walking 69%–81% 100% 53%
Continued
 CHAPTER 38  Open Fetal Surgery 465

TABLE Paediatric 5-Year Medical and Social Outcomes of Children With Spina Bifida Aperta After Open Fetal Surgery
38.6 in a Prospective Cohort of 54 Patients Followed Up From the Children’s Hospital of Philadelphia—cont’d
Children with SBA
Children with SBA Undergoing Open Normal Children Undergoing Postnatal
5-Year Medical Outcomes Fetal Surgery (Population Norms) Surgery (Historical Data)
5-year social outcomes
• Preschool neurobehavioural outcome n = 22/30 (73%)44 NS NS
• Total problems 14% 18% NS
• Anxious or depressed behaviour Higher in shunted SBA NS NS
• Withdrawn behaviour Higher in shunted SBA NS NS
• Pervasive developmental behaviour Higher in shunted SBA NS NS

FIQ, Full Intelligence Quotient; HH, hindbrain herniation; NS, nonspecified; OFS, open fetal surgery; PIQ, Performance Intelligence Quotient; PS, processing speed; VIQ, Verbal Intelligence Quotient; VMI,
Visual Motor Integration; WeeFIM, global Functional Independence Measure in children; WJP-BR16, Woodcock-Johnson Psychoeducational Battery-Revised16 test; WPPSI-III, Wechsler Preschool and
Primary Scale of Intelligence, 3rd Edition.
aIndependence means complete independence or partial independence under minimal supervision to complete tasks. Nonshunted and children with spina bifida aperta (SBA) with normal neurodevelop-
mental outcome are more likely to be independent in daily living activities than shunted children with SBA for progressive hydrocephalus.
  

TABLE Global Functional Independence Measure (WeeFIM) in Children With Spina Bifida Aperta at 5 Years of Age,
38.7 Either After Fetal or Postnatal Surgery: Comparison of Independence in Nonshunted Versus Shunted Children
With Spina Bifida and in Children With Spina Bifida With Average Versus Neurodevelopmental Scores
OPEN FETAL SURGERY (43) POSTNATAL SURGERY (46, 72-74)
Average vs Lower Average vs Lower
WeeFIM Results in Children with Neurodevelopmental Neurodevelopmental
SBA (%) Nonshunted vs Shunted Scores Nonshunted vs Shunted Scores
Total independence Higher in non-S (P < .01) Higher (P < .01) Higher in non-S72 —
Neurocognitive independence Higher in non-S (P = .02) Higher) (P < .001 Higher in non-S Higher46,72-74
Self-care independence Higher in non-S (P = .07) Higher (P = .09) Higher in non-S46,72 Higher46
Mobility independence Higher in non-S (P = .02) Higher (P = .01) Higher in non-S72 —

non-S, Nonshunted children with spina bifida aperta; OFS, open fetal surgery SBA, spina bifida aperta.
  

average neurodevelopmental scores. In the same situations, self-
care independence tends to be higher. Finally, functional indepen-
dence does not fall within the range of that of normal children; a
comparison with historical controls operated after birth was not
possible for these authors (see Tables 38.6 and 38.7).43  On the other hand, because OFS for SBA increases indepen-
Social Outcomes. Open fetal surgery and subsequent preterm
delivery are not associated with increased behavioural problems,
impaired social interactions or restricted behaviour patterns (see
Table 38.6).44 Quality of life of families and their children has not
yet been reported in this population.  best indicators of daily life function and quality of life.46 Further-
Potentially Improved Long-Term Outcomes at 10 Years
Medical Outcomes. Open fetal surgery for SBA improves long-
term neurodevelopmental and neurofunctional outcomes as well
as long-term ambulatory status, at least when compared with his-
torical controls ( Table 38.8).45 
Social Outcomes. Although it has not been studied yet, it is
reasonable to expect that despite improved medical outcomes, the
persisting dependence in everyday self-care tasks of SBA patients
may induce a heavy burden on these children, parents and caregiv-
ers. After all, prenatal surgery does not cure this disease. Children
and young adults with SBA are known to experience social disad-
vantages and have poor self-care capability, including decreased
opportunity for peer relationships, prolonged caregiver depen-
dency and decreased community acceptance.
dent mobility, and under the assumption, this persists, a long-term
benefit could be expected. A cross-sectional study including 122
children with SBA operated postnatally showed that good mental
ability, muscle strength and being independent in mobility are the
more, being independent in mobility appeared to contribute more
to health-related quality of life than being independent in self-care
or being wheelchair dependent. 
Outcomes of Congenital Thoracic Malformations
The Children’s Hospital of Philadelphia (CHOP) has the largest
experience with fetal lobectomy. CHOP reported on the outcomes
of 24 fetuses with massive multicystic or predominantly solid masses
undergoing fetal lobectomy at 21 to 31 weeks of gestation. Resections
involved a single lobe in 18 cases, right middle and lower lobectomies
466 SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Paediatric Outcomes of Children With Spina Bifida Aperta at the Age of 10: Comparison of a Cohort of 42
38.8 Children From the Initial Pre-MOMS Prospective Cohort With Results From Other Studies: Assessment of
Neurofunctional Outcomes, Executive Functioning and Behavioural Adaptive Skills45 a
SBA Children Undergoing
Children with SBA Normal Children (Population Postnatal Surgery
10-Year Outcomes Undergoing OFS Norms) (Historical Data)
Neurofunctional Outcomes
Need for shunt 43% NS NS
Spinal cord tethering 33% NS NS
Community ambulator 79% NS NS
Normal bladder function 26% NS 10%
Need for CIC 74% NS NS
Normal bowel function 31% NS NS
Enrolled in bowel movement program 59% NS NS
Executive Functioning (Measured by the BRIEF Scale)
GEC mean T-scoreb 55% <60% 62%
GEC proportion of impaired T-score 24% 7% NS
MCI mean T-score 57% <60% 63-65%
MCI proportion of impaired T-score 33% 6-7% 40-60%
BRI mean T-score 51% <60% 56-57
BRI proportion of impaired T-score 14% 7%–11% 10%–30%
Adaptive Behaviour (Measured by the ABAS II Scale)
CCS mean score (SD) 92% 100 ± 15% NS
CCS proportion of impaired score 15% 5% NS
SCS mean score 98% 100 ± 15% NS
SCS proportion of impaired score 7% 5% NS
PCS mean score 82% 100 ± 15% NS
PCS proportion of impaired score 25% 5% NS
GACS mean score 87% 100 ± 15% NS
GACS proportion of impaired score 27% 5% NS

ABAS II, Adaptive Behaviour Assessment System-Second Edition; BRI, Behaviour Regulation Index; BRIEF, Behaviour Rating Inventory of Executive Function; CCS, Conceptual Composite Score; GACS,
General Adaptive Composite Score; GEC, Global Executive Composite; MCI, Metacognition Index; PCS, Practical Composite Score; SCS, Social Composite Score; SD, standard deviation.
aThe majority of children with spinal bifida aperta (SBA) can successfully complete everyday tasks at home and at school. Nonshunted children with normal early neurodevelopmental outcome are less likely

to experience problems with executive functioning and behaviour adaptive skills. Symptomatic spinal cord tethering is associated with functional loss. More than expected children with SBA are continent,
but bowel and bladder control continue to be an ongoing challenge. Although direct comparison of open fetal surgery (OFS) SBA outcomes with previously published data in postnatally managed children
with SBA is not possible, it is encouraging that following OFS SBA mean scores across executive skills and behavioural regulation domains appear to be improved.45
bT-score is derived for each scale and index, with higher T-scores indicating greater impairment: a normal T-score is <60 (average range), and an impaired score is ≥65 (clinically significant score). Scores
are presented as percentage of sample with T-scores ≤65.
  

in 4 cases, and 1 left pneumonectomy for CCAM and extralobar BPS
resection in one case. All cases were confirmed on pathology. Fetal
resection led to resolution of hydrops within 1 to 2 weeks, return of
the mediastinum to the midline within 3 weeks and impressive in
utero lung growth. Eleven fetuses died in utero, either intraoperatively
because of cardiac arrest (n = 6) or uncontrolled intraoperative uterine
contractions (n = 1), or postoperatively because of maternal mirror
syndrome (n = 1), bradycardia (n = 3). Eventually, survival at 1 to 16
years was 54% all children having normal development (n = 13).10
Apart from single or multiple courses of maternal betamethasone
recommended for CCAM cases with hydrops and CVR greater than
1.4,47,48 alternative therapeutic options include minimally invasive
approaches such as thoracoamniotic shunting for single dominant
CCAM cyst, US-guided radiofrequency thermal ablation for multicys-
tic CCAM or laser ablation of the systemic arterial supply for BPS.10,49 
Outcomes of Sacrococcygeal Teratoma
The CHOP team also reported on five fetuses with type I or II
SCT undergoing OFS between 22 and 26 weeks of gestation.34,50
Two of them (40%) died, one intraoperatively because of intra-
operative cardiac failure and another one postoperatively because
 CHAPTER 38  Open Fetal Surgery 467

of cardiac failure caused by preexisting in utero ductus arteriosus For CTM, fetal lobectomy by OFS before 32 weeks is feasi-
constriction and subsequent ventricular hypertrophy. ble. Along the same lines, OFS for debulking high-risk SCT has
Outcomes of OFS for SCT are often compared with tumour occasionally been done in previable fetuses with progressive high-
ablation by minimally invasive therapy (coiling or ablation). The output cardiac failure. At present, minimally invasive alternatives
latter may cause collateral damage and hence cause access-specific for these procedures are being explored.
complications. When reviewing the available literature, the sur- Premature rupture of the membranes, preterm labour and pre-
vival rate in hydropic SCT fetuses after OFS is 55% (6 of 11) term birth remain the Achilles’s heel of any fetal surgery. Multi-
and after minimally invasive therapy is 30% (6 of 20). Mean centre clinical research programs should be developed to answer
gestational age at delivery is comparable, respectively, 29.8 ± clinical questions in light of the small number of fetuses undergo-
2.9 and 29.7 ± 4.0 weeks. Survivors have normal developmental ing OFS for rare indications.
testing.13,34 Finally, another alternative could be ‘early delivery’ For SBA repair, the numbers are much higher, and the guide-
between 27 and at 32 weeks.50  lines for these centres have been described in a position state-
ment paper.15 This surgery should be performed in established
Conclusions and experienced fetal therapy centres using a multidisciplinary
team approach and having an adequate annual volume of open
Open fetal surgery can be offered in selected patients with the fetal and ex utero intrapartum treatment procedures to maintain
aforementioned congenital abnormalities in highly specialised competency.
centres. OFS has some important limitations, such as obstetric
side effects (uterine scar problems, need for abdominal delivery, Access the complete reference list online at ExpertConsult.com.
PPROM and preterm delivery, placental abruption) as well as Self-assessment questions available at ExpertConsult.com.
maternal complications (haemorrhage, pulmonary oedema and
morbidity caused by the large abdominal incision).
For SBA, OFS should be offered to all eligible patients as an
option, the operation taking place between 19 and 26 weeks of
gestation and consisting of a primary anatomical repair. In rare
cases, skin augmentation by patch may be required.
The debate about whether similar results can be obtained by
fetoscopic surgery is beyond the scope of this chapter. At pres-
ent, however, neither the safety or efficacy of fetoscopic repair has
been demonstrated, either in experimental or in clinical condi-
tions.51,52 Whether it reduces PPROM and premature delivery
remains controversial. The obstetric advantages are an important
incentive for further preclinical experimentation with this alterna-
tive modality.53
References 19.  Saylors 3rd RL, Cohn SL, Morgan ER,
Brodeur GM. Prenatal detection of neuro-
Harrison MR, ed. Philadelphia: WB
Saunders, 2001.
blastoma by fetal ultrasonography. Am J Pedi- 36. Golombeck K, Ball RH, Lee H, et al. Mater-
1. Deprest JA, Flake AW, Gratacos E, et  al.
atr Hematol Oncol. 1994;16(4):356–360. nal morbidity after maternal-fetal surgery. Am
The making of fetal surgery. Prenat Diagn.
20. Heuer GG, Adzick NS, Sutton LN. Fetal J Obstet Gynecol. 2006;194(3):834–839.
2010;30(7):653–667.
myelomeningocele closure: technical consid- 37. Wilson RD, Johnson MP, Crombleholme
2. Harrison MR, Filly RA, Golbus MS,
erations. Fetal Diagn Ther. 2015;37(3):166– TM, et al. Chorioamniotic membrane separa-
et  al. Fetal treatment 1982. N Engl J Med.
171. tion following open fetal surgery: pregnancy
1982;307(26):1651–1652.
21.  Ferschl M, Ball R, Lee H, Rollins MD. outcome. Fetal Diagn Ther. 2003;18(5):314–
3. Adzick NS. Fetal surgery for spina bifida:
Anesthesia for in utero repair of myelo- 320.
past, present, future. Semin Pediatr Surg.
meningocele. Anesthesiology. 2013;118(5): 38. Antiel RM, Adzick NS, Thom EA, et  al.
2013;22(1):10–107.
1211–1123. Impact on family and parental stress of prena-
4. Danzer E, Johnson MP. Fetal surgery for neu-
22.  De Buck F, Deprest J, Van de Velde M. tal vs postnatal repair of myelomeningocele.
ral tube defects. Semin Fetal Neonatal Med.
Anesthesia for fetal surgery. Curr Opin Am J Obstet Gynecol. 2016;215(4):522.e1–e6.
2014;19(1):2–8.
Anaesthesiol. 2008;21(3):293–297. 39. Johnson MP, Bennett KA, Rand L, et al. The
5. Adzick NS, Thom EA, Spong CY, et  al. A
23. Soni S, Moldenhauer JS, Spinner SS, et  al. Management of Myelomeningocele Study:
randomized trial of prenatal versus postnatal
Chorioamniotic membrane separation and obstetrical outcomes and risk factors for
repair of myelomeningocele. N Engl J Med.
preterm premature rupture of membranes obstetrical complications following prenatal
2011;364(11):993–1004.
complicating in utero myelomeningocele surgery. Am J Obstet Gynecol. 2016;215(6):
6. Kotecha S, Barbato A, Bush A, et al. Antena-
repair. Am J Obstet Gynecol. 2015;214(5): 778.e1–e9.
tal and postnatal management of congenital
647.e1–e7. 40. Brock 3rd JW, Carr MC, Adzick NS, et  al.
cystic adenomatoid malformation. Paediatr
24. Adzick NS, Harrison MR, Glick PL, et  al. Bladder function after fetal surgery for myelo-
Respir Rev. 2012;13(3):162–1670; quiz 170-
Fetal surgery in the primate. III. Maternal meningocele. Pediatrics. 2015;136(4):e906–
171.
outcome after fetal surgery. J Pediatr Surg. e913.
7. Davenport M, Eber E. Long term respira-
1986;21(6):477–480. 41. Johnson MP, Sutton LN, Rintoul N, et  al.
tory outcomes of congenital thoracic mal-
25. Wilson RD, Lemerand K, Johnson MP, Fetal myelomeningocele repair: short-term
formations. Semin Fetal Neonatal Med.
et  al. Reproductive outcomes in subsequent clinical outcomes. Am J Obstet Gynecol.
2012;17(2):99–104.
pregnancies after a pregnancy complicated by 2003;189(2):482–487.
8. European Surveillance of Congenital Anom-
open maternal-fetal surgery (1996-2007). Am 42. Danzer E, Gerdes M, Bebbington MW, et al.
alies (EUROCAT). http://www.eurocat-
J Obstet Gynecol. 2010;203(3):209.e1–e6. Preschool neurodevelopmental outcome of
network.eu.
26. Farrell JA, Albanese CT, Jennings RW, et al. children following fetal myelomeningocele
9. Joyeux L, Mejean N, Rousseau T, et  al.
Maternal fertility is not affected by fetal sur- closure. Am J Obstet Gynecol. 2010;202(5):
Ectopic extralobar pulmonary sequestra-
gery. Fetal Diagn Ther. 1999;14(3):190–192. 450.e1–e9.
tions in children: interest of the laparoscopic
27. Moldenhauer JS, Soni S, Rintoul NE, 43. Danzer E, Gerdes M, Bebbington MW, et al.
approach. J Pediatr Surg. 2010;45(11):2269–
Spinner SS, et  al. Fetal myelomeningocele Fetal myelomeningocele surgery: preschool
2273.
repair: the post-MOMS experience at the functional status using the Functional Inde-
10. Adzick NS. Open fetal surgery for life-
Children’s Hospital of Philadelphia. Fetal pendence Measure for children (WeeFIM).
threatening fetal anomalies. Semin Fetal Neonatal
Diagn Ther. 2015;37(3):235–240. Child Nerv Syst. 2011;27(7):1083–1038.
Med. 2010;15(1):1–8.
28. Bennett KA, Carroll MA, Shannon CN, 44. Danzer E, Gerdes M, Bebbington MW, et al.
11. Swamy R, Embleton N, Hale J. Sacrococ-
et  al. Reducing perinatal complications and Preschool neurobehavioral outcome following
cygeal teratoma over two decades: birth
preterm delivery for patients undergoing in fetal myelomeningocele surgery. Fetal Diagn
prevalence, prenatal diagnosis and clinical
utero closure of fetal myelomeningocele: fur- Ther. 2011;30(3):174–179.
outcomes. Prenat Diagn. 2008;28(11):1048–
ther modifications to the multidisciplinary 45. Danzer E, Thomas NH, Thomas A, et  al.
1051.
surgical technique. J Neurosurg Pediatr. Long-term neurofunctional outcome, execu-
12. Pauniaho SL, Heikinheimo O, Vettenranta K,
2014;14(1):108–114. tive functioning, and behavioral adaptive
et  al. High prevalence of sacrococcy-
29. Hisaba WJ, Cavalheiro S, Almodim CG, skills following fetal myelomeningocele sur-
geal teratoma in Finland—a nationwide
et  al. Intrauterine myelomeningocele repair gery. Am J Obstet Gynecol. 2016;214(2):269.
population-based study. Acta Paediatr.
postnatal results and follow-up at 3.5 years e1–e8.
2013;102(6):e251–e256.
of age—initial experience from a single ref- 46. Schoenmakers MA, Uiterwaal CS, Gulmans
13. Van Mieghem T, Al-Ibrahim A, Deprest J,
erence service in Brazil. Childs Nerv Syst. VA, et  al. Determinants of functional inde-
et  al. Minimally invasive therapy for fetal
2012;28(3):461–467. pendence and quality of life in children with
sacrococcygeal teratoma: case series and sys-
30. Zamlynski J, Olejek A, Koszutski T, et  al. spina bifida. Clin Rehabil. 2005;19(6):677–
tematic review of the literature. Ultrasound
Comparison of prenatal and postnatal treat- 685.
Obstet Gynecol. 2014;43(6):611–619.
ments of spina bifida in Poland—a non- 47. Peranteau WH, Boelig MM, Khalek N, et al.
14. Danzer E, Hubbard AM, Hedrick HL, et al.
randomized, single-center study. J Matern Fetal Effect of single and multiple courses of mater-
Diagnosis and characterization of fetal sacro-
Neonatal Med. 2014;27(14):1409–1417. nal betamethasone on prenatal congenital
coccygeal teratoma with prenatal MRI. AJR
31. Albright AL, Pollack I, Adelson P. Principles lung lesion growth and fetal survival. J Pediatr
Am J Roentgenol. 2006;187(4):W350–W356.
and Practice of Pediatric Neurosurgery. 2nd ed. Surg. 2016;51(1):28–32.
15. Cohen AR, Couto J, Cummings JJ, et  al.
Albright AL, ed. New York: Thieme, 2007. 48. Peranteau WH, Wilson RD, Liechty KW,
Position statement on fetal myelome-
32. Kim D, Betz R, Huhn S, Newton P. Surgery et  al. Effect of maternal betamethasone
ningocele repair. Am J Obstet Gynecol.
of the Pediatric Spine. New York: Thieme; administration on prenatal congenital cystic
2014;210(2):107–111.
2008. adenomatoid malformation growth and fetal
16. Ovaere C, Eggink A, Richter J, et al. Prenatal
33. Sutton LN. In utero surgery for myelome- survival. Fetal Diagn Ther. 2007;22(5):365–
Diagnosis and patient preferences in patients
ningocele and hydrocephalus. In: Albright 371.
with neural tube defects around the advent of
AL, ed. Principles and Practice of Pediatric 49. Baud D, Windrim R, Kachura JR, et al. Mini-
fetal surgery in Belgium and Holland. Fetal
Neurosurgery. 2nd ed. New York: Thieme; mally invasive fetal therapy for hydropic lung
Diagn Ther. 2015;37(3):226–234.
2007:313–322. masses: three different approaches and review
17. Harrison M, Evans M, Adzick NS, Holzgreve W.
34. Hedrick HL, Flake AW, Crombleholme of the literature. Ultrasound Obstet Gynecol.
The Unborn Patient: The Art and Science
TM, et al. Sacrococcygeal teratoma: prenatal 2013;42(4):440–448.
of Fetal Therapy. 3rd ed. Harrison M, ed.
assessment, fetal intervention, and outcome. 50. Roybal JL, Moldenhauer JS, Khalek N,
St. Louis: Saunders; 2001.
J Pediatr Surg. 2004;39(3):430–438; discus- et  al. Early delivery as an alternative man-
18. Endo M, Van Mieghem T, Eixarch E, et  al.
sion 438. agement strategy for selected high-risk fetal
The prenatal management of neural tube
35. Harrison MR, Evans ME, Adzick NS, sacrococcygeal teratomas. J Pediatr Surg.
defects: time for a re-appraisal. Fetal and
Holzgreve W. The Unborn Patient. 3rd ed. 2011;46(7):1325–1332.
Matern Med Rev. 2012;23:158–186.

467.e1
467.e2 R E F E REN C ES
51. Kohl T. Percutaneous minimally-invasive
fetoscopic surgery for spina bifida aperta—
part I: surgical technique and periopera-
tive outcome. Ultrasound Obstet Gynecol.
2014;44(5):515–524.
52. Pedreira DA, Zanon N, Nishikuni K, et  al.
Endoscopic surgery for the antenatal treat-
ment of myelomeningocele: the CECAM
trial. Am J Obstet Gynecol. 2016;214(1):111.
e1–e11.
53. Joyeux L, Engels AC, Russo FM, et al. Feto-
scopic versus open repair for spina bifida
aperta: a systematic review of outcomes. Fetal
Diagn Ther. 2016;9(3):161–171.
54. Heffez DS, Aryanpur J, Hutchins GM, Free-
man JM. The paralysis associated with myelo-
meningocele: clinical and experimental data
implicating a preventable spinal cord injury.
Neurosurgery. 1990;26(6):987–992.
55. Meuli M, Meuli-Simmen C, Hutchins GM,
et  al. In utero surgery rescues neurological
function at birth in sheep with spina bifida.
Nat Med. 1995;1(4):342–347.
56. Korenromp MJ, van Gool JD, Bruinese HW,
Kriek R. Early fetal leg movements in myelo-
meningocele. Lancet. 1986;1(8486):917–
918.
57. Sival DA, Begeer JH, Staal-Schreinemachers
AL, et al. Perinatal motor behaviour and neu-
rological outcome in spina bifida aperta. Early
Hum Dev. 1997;50(1):27–37.
58. Sival DA, Guerra M, den Dunnen WF, et al.
Neuroependymal denudation is in progress
in full-term human foetal spina bifida aperta.
Brain Pathol. 2011;21(2):163–179.
59. Stiefel D, Copp AJ, Meuli M. Fetal spina
bifida in a mouse model: loss of neural func-
tion in utero. J Neurosurg. 2007;106(suppl
3):213–221.
60. McLone DG, Dias MS. The Chiari II malfor-
mation: cause and impact. Childs Nerv Syst.
2003;19(7-8):540–550.
61. Harrison MR, Jester JA, Ross NA. Correc-
tion of congenital diaphragmatic hernia in
utero. I. The model: intrathoracic balloon
produces fatal pulmonary hypoplasia. Surgery.
1980;88(1):174–182.
62. Adzick NS, Harrison MR, Flake AW,
et  al. Fetal surgery for cystic adenomatoid
malformation of the lung. J Pediatr Surg.
1993;28(6):806–812.
63. Tulipan N, Wellons 3rd JC, Thom EA, et al.
Prenatal surgery for myelomeningocele and
the need for cerebrospinal fluid shunt place-
ment. J Neurosurg Pediatr. 2015;16(6):613–
620.
64. Danzer E, Finkel RS, Rintoul NE, et  al.
Reversal of hindbrain herniation after mater-
nal-fetal surgery for myelomeningocele sub-
sequently impacts on brain stem function.
Neuropediatrics. 2008;39(6):359–362.
65. Danzer E, Gerdes M, Bebbington MW, et al.
Lower extremity neuromotor function and
short-term ambulatory potential following in
utero myelomeningocele surgery. Fetal Diagn
Ther. 2009;25(1):47–53.
66. Msall ME, DiGaudio K, Duffy LC, et  al.
WeeFIM. Normative sample of an instrument
for tracking functional independence in chil-
dren. Clin Pediatr. 1994;33(7):431–438.
67. Wong V, Wong S, Chan K, Wong W. Func-
tional Independence Measure (WeeFIM)
for Chinese children: Hong Kong Cohort.
Pediatrics. 2002;109(2):E36.
68. Dennis M, Barnes MA. The cognitive pheno-
type of spina bifida meningomyelocele. Dev
Disabil Res Rev. 2010;16(1):31–39.
69. Barnes MA, Wilkinson M, Khemani E, et al.
Arithmetic processing in children with spina
bifida: calculation accuracy, strategy use,
and fact retrieval fluency. J Learn Disabil.
2006;39(2):174–187.
70. Dahl M, Ahlsten G, Butler A, et al. Self-care
skills in young children with myelomenin-
goceles. Eur J Pediatr Surg. 2000;10(suppl
1):52–53.
71. Norrlin S, Strinnholm M, Carlsson M, Dahl
M. Factors of significance for mobility in chil-
dren with myelomeningocele. Acta Paediatr.
2003;92(2):204–210.
72. Verhoef M, Barf HA, Post MW, et al. Func-
tional independence among young adults
with spina bifida, in relation to hydrocepha-
lus and level of lesion. Dev Med Child Neurol.
2006;48(2):114–119.
73. Buran CF, Sawin KJ, Brei TJ, Fastenau PS.
Adolescents with myelomeningocele: activi-
ties, beliefs, expectations, and perceptions.
Dev Med Child Neurol. 2004;46(4):244–252.
74. Tsai PY, Yang TF, Chan RC, et al. Functional
investigation in children with spina bifida—
measured by the Pediatric Evaluation of Dis-
ability Inventory (PEDI). Childs Nerv Syst.
2002;18(1-2):48–53.
39
Fetal Growth and Growth Restriction
EMILY J. SU AND HENRY L. GALAN
KEY POINTS
• Fetal growth restriction (FGR) is practically defined as a sono-
graphic estimated fetal weight of less than the 10th percen-
tile for gestational age. In actuality, a growth-restricted fetus
is one that is unable to meet its inherent growth potential
secondary to an underlying pathologic process. Distinguish-
ing between a pathologically growth-restricted fetus and a
constitutionally small one is imprecise, and using an estimat-
ed fetal weight cutoff of less than the 10th percentile as the
definition is more inclusive and less likely to miss abnormally
small fetuses.
• The causes underlying FGR are heterogeneous, including
maternal, placental and fetal factors.
• In pregnancies with an increased a priori risk for FGR, ultraso-
nographic biometry remains the mainstay of screening.
• Confirmation of gestational age and dating are key factors in
the diagnosis of FGR.
• Workup of FGR includes consideration of aneuploidy, infec-
tious causes such as cytomegalovirus and toxoplasmosis and
structural anomalies. If severe, leading to delivery before 34
weeks’ gestation, evaluation for antiphospholipid antibody
syndrome should also be considered.
• Use of umbilical artery Doppler velocimetry in the setting of
FGR has been rigorously shown to decrease perinatal mortal-
ity.
• Management of a growth-restricted fetus should include
serial assessment of biometry, amniotic fluid volume and fetal
Doppler studies with the goal of maximising fetal maturity
and minimising injury secondary to exposure to an abnormal
in utero environment.
Introduction
Fetuses that are unable to meet their inherent growth potential
are at substantial risk for perinatal and long-term morbidity and
mortality. Delivery, oftentimes preterm with its attendant conse-
quences of prematurity, is the only known treatment for avert-
ing in utero injury or stillbirth for these fetuses. Furthermore, the
combination of fetal growth restriction (FGR) and preterm deliv-
ery has been associated with even worse neonatal and long-term
outcomes compared with appropriately grown infants who were
born prematurely. This chapter reviews key definitions, a etiolo-
gies, screening, diagnosis and clinical management of FGR while
highlighting emerging areas of investigation in the field of normal
fetal growth and FGR.  upon population growth charts, 10% of fetuses will be diagnosed
Terminology
Fetal growth restriction, also known as intrauterine growth restriction
(IUGR), occurs when a fetus is unable to achieve its inherent growth
potential secondary to an underlying pathologic process. Antenatally
distinguishing between a pathologically growth-restricted fetus and a
constitutionally small one, however, is imprecise. In fact, only approx-
imately 30% of fetuses with an estimated fetal weight (EFW) less
than the 10th percentile are pathologically growth restricted.1,2 On
the other hand, there remains an increased risk for adverse outcome
in a fetus that measures greater than the 10th percentile but is still not
meeting its innate growth trajectory.3
For practical purposes, the American Congress of Obstetricians
and Gynecologists (ACOG) defines FGR when the sonographic
EFW is less than the 10th percentile for gestational age on a stan-
dardised population growth curve.4 Controversy surrounding this
term remains, however, as to whether this is the optimal definition
when many of these fetuses will be constitutionally small and not
pathologically growth restricted. The phrase ‘small for gestational
age (SGA)’ has been utilised in a variety of contexts, ranging from
interchangeable use with FGR to denoting only neonates whose
birth weight is less than the 10th percentile for gestational age. This
latter definition of SGA is used by ACOG and is what the term SGA
will represent in this chapter.4 Low birth weight (LBW) is defined
by the World Health Organization as a birth weight of less than
2500 g.5 This definition does not take into account gestational age
and thus has less relevance to fetal growth and growth restriction. 
Normal Fetal Growth
Most classic growth curves, including the Lubchenco, Brenner and
Williams curves, show similar growth trajectories with advancing
gestational age.6 More recent data derived from a large, racially
diverse US cohort, however, demonstrate that birth weights of this
modern population differ from those that generated the classic
Lubchenco curve.7,8 For example, the percentage of SGA infants
would tend to be underestimated by Lubchenco curves between
32 and 40 weeks’ gestation (Tables 39.1 and 39.2).7,8 In contrast,
the percentage of large for gestational age (LGA) infants would be
overestimated by the Lubchenco population at term (see Tables
39.1 and 39.2).7,8 These findings have been supported by a meta-
analysis including nearly 4 million births from six countries.9 
Epidemiology
Overall, the incidence of FGR depends upon the definition and
population being used. Using the ACOG definition of FGR based
469
470 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE
39.1 Female Birth Weight Percentiles by Gestational Age

PATIENTS (N) 10TH PERCENTILE (G) 50TH PERCENTILE (G) 90TH PERCENTILE (G)
Lubchenco Lubchenco Lubchenco Lubchenco
GA et al. Olsen et al. et al. Olsen et al. et al. Olsen et al. et al. Olsen et al.
24 11 438 490 524 760 651 1295 772
26 25 773 700 645 935 827 1350 1004
28 54 1187 870 807 1140 1061 1530 1310
30 48 1606 1025 1052 1380 1373 1880 1693
32 58 3007 1250 1352 1675 1731 2330 2116
34 71 5936 1550 1730 2155 2187 2920 2661
36 84 4690 1960 2028 2630 2664 3335 3339
38 282 5755 2405 2526 2940 3173 3545 3847
40 588 5529 2630 2855 3160 3454 3720 4070

GA, Gestational age.

Adapted from Lubchenco LO, Hansman C, Dressler M, Boyd E. Intrauterine growth as estimated from liveborn birth-weight data at 24 to 42 weeks of gestation. Pediatrics 32:793–800, 1963 and Olsen
IE, Groveman SA, Lawson ML, et al. New intrauterine growth curves based on United States data. Pediatrics 125:e214–e224, 2010.
  

TABLE
39.2 Male Birth Weight Percentiles by Gestational Age

PATIENTS (N) 10TH PERCENTILE (G) 50TH PERCENTILE (G) 90TH PERCENTILE (G)
Lubchenco Lubchenco Lubchenco Lubchenco
GA et al. Olsen et al. et al. Olsen et al. et al. Olsen et al. et al. Olsen et al.
24 13 451 610 561 830 690 1230 813
26 43 881 760 704 965 890 1330 1065
28 64 1281 915 884 1205 1141 1570 1385
30 61 1992 1085 1114 1465 1443 1875 1761
32 66 3677 1320 1433 1760 1829 2280 2218
34 74 7291 1645 1810 2220 2285 2920 2763
36 118 7011 2105 2170 2745 2792 3385 3432
38 354 8786 2505 2652 3080 3306 3665 3986
40 576 7235 2700 2950 3290 3579 3880 4232

GA, Gestational age.

Adapted from Lubchenco LO, Hansman C, Dressler M, Boyd E. Intrauterine growth as estimated from liveborn birth-weight data at 24 to 42 weeks of gestation. Pediatrics 32:793–800, 1963 and Olsen
IE, Groveman SA, Lawson ML, et al. New intrauterine growth curves based on United States data. Pediatrics 125:e214–e224, 2010.
  

as growth restricted.4 There is no specific definition at this time,
however, that is able to take into account a fetus’s inherent growth
potential. Although there are investigations into individualised
(vs population-based) growth standard, none have been shown to
improve outcomes to date.10-13  cranial dimensions), has conventionally been ascribed to placental
Classification of Fetal Growth Restriction
Fetal growth restriction has often been segregated into two sepa-
rate classifications: Symmetric versus asymmetric FGR. Symmet-
ric FGR, in which there is a proportional reduction in all of the
biometric parameters, traditionally has been attributed to insults
that occur early in pregnancy when the main component of fetal
growth is cellular hyperplasia. In contrast, asymmetric FGR, in
which estimated fetal weight is below normal primarily because of
a decrease in abdominal circumference (with normal skeletal and
disorders, which is thought to impair the normal process of cel-
lular hypertrophy in fetal growth and deposition of glycogen in
the fetal liver.
The clinical utility of this stratification is unclear for several
reasons. For instance, early-onset placental disease may lead to
symmetric FGR. More important, though, both symmetric and
asymmetric FGR have been associated with increased risk for

TABLE
39.3 Causes of Fetal Growth Restriction
Maternal factors
Maternal disease (e.g. hypertension, cyanotic cardiac disease,
antiphospholipid antibody syndrome)
Severe, inadequate nutrition
Toxins
Certain prescribed medications
Placental factors
Abnormal placental disk diameter and thickness
Placental abruption
Placental infarction
Chorioangioma
Umbilical cord abnormalities (e.g. velamentous cord insertion)
Fetal factors
Genetic factors
Structural anomalies
Infection
Multiple gestation
poor perinatal outcome, and antenatal surveillance and Doppler
velocimetry appear to be better predictors of pregnancy outcome
regardless of the classification of FGR.14  Similarly, abnormal placental lobation, abruption, chorioangio-
Causes of Fetal Growth Restriction
There are several potential causes of FGR, and they can be divided
into three basic categories: maternal, placental and fetal factors
(Table 39.3).
Maternal Factors
Maternal disease. Several maternal medical conditions,
especially ones that lead to alterations in uteroplacental perfusion,
may contribute to the phenotype of FGR. For instance, one frequent
cause of FGR is maternal hypertensive disease in pregnancy,
including preeclampsia, chronic hypertension and preeclampsia
superimposed upon chronic hypertension.15-18 Similarly,
preexisting diabetes, renal disease and autoimmune disease have
all also been associated with an increased risk for development of
FGR. The currently presumed mechanistic aetiology underlying
the association between these medical conditions and FGR is
thought to be impaired trophoblastic invasion of the maternal
spiral arterioles in conjunction with maternal vascular and
endothelial derangements.19 This is supported clinically by
uterine artery Doppler studies demonstrating that in pregnancies
complicated by hypertension, there is a higher incidence of FGR
in pregnancies where abnormal waveforms were recorded.20,21  Infection. Most cases of FGR that are attributable to congeni-
Inadequate nutrition. In general, minor alterations in mater-
nal nutrition are unlikely to result in growth restriction. Extreme
undernourishment, however, affects fetal development. The
majority of our understanding regarding malnutrition and fetal
growth comes from data during the 1940s. The Dutch famine,
CHAPTER 39  Fetal Growth and Growth Restriction 471
which lasted for approximately 6 months, showed that significant
malnutrition only in the third trimester resulted in decreased fetal
growth. Specifically, pregnant women who took in an average of
less than 1500 kcal/day during the third trimester delivered infants
with birth weights that declined about 10%.22,23 In contrast, the
Siege of Leningrad during World War II, which lasted more than
2 years, demonstrated that when both pre- and intragestational
weight gain were poor, birth weights were reduced by approxi-
mately 400 to 600 g.24 
Toxins. Maternal cigarette smoking is a well-established risk
factor for FGR, with studies demonstrating that smoking dur-
ing pregnancy confers a 3- to 10-fold increased risk for deliver-
ing an SGA neonate.25-27 In fact, tobacco use in pregnancy is
the leading preventable cause of FGR.28 In a Cochrane review,
smoking cessation was found to reduce LBW by 17%, but the
large Danish National Birth Cohort database demonstrated that
nicotine replacement did not affect birth weight or the rate of still-
birth.29-31 Use of illicit substances such as cocaine, amphetamines
and heroin also increases the risk for development of FGR.32 Vari-
ous therapeutic agents have also been implicated in the a etiology
of FGR. These include antiepileptic medications, β-blockers, che-
motherapy and chronic steroid use.33 
Placental Factors
The placenta, as the maternal–fetal interface that mediates nutri-
ent and oxygen exchange, plays a key role in fetal growth. From
a gross perspective, decreased placental weight and disk diameter
have been associated with impaired fetal growth.34 There also
appears to be an optimal placental disk thickness, in which exces-
sively thin or thick placentas have been correlated with FGR.35,36
mas and velamentous cord insertions also increase risk for devel-
opment of FGR.34,37-39
Histopathologic findings, such as chronic villitis, massive
chronic intervillositis, maternal floor infarction with fibrin or
fibrinoid deposition and fetal thrombotic vasculopathy reflect the
potential mechanisms underlying placental-mediated FGR.40,41
These include deficient endovascular trophoblast invasion of the
implantation site, inadequate extravillous trophoblast invasion of
maternal spiral arterioles and maldevelopment of the villous and
fetoplacental vascular tree. 
Fetal Factors
Genetic factors. Fetal aneuploidy is one cause of FGR, with
19% of growth-restricted fetuses demonstrating an abnormal
karyotype at a tertiary referral centre.42 FGR is often present in
fetuses with trisomy 18, although the risk is still elevated with other
chromosomal abnormalities, including triploidy, sex chromosome
abnormalities, other trisomies, deletions and duplications.43,44
Less frequently, other abnormalities such as confined placental
mosaicism or uniparental disomy also can result in FGR.43,45 
Structural anomalies. More than 22% of infants with congeni-
tal anomalies manifest concurrent FGR.46 Furthermore, the more
defects that are present, the higher the frequency of FGR. 
tal infection arise from either viral or parasitic infections. Cyto-
megalovirus (CMV), rubella, toxoplasmosis and malaria are most
often implicated, with malaria being the most common cause of
FGR worldwide.47-50 Other data suggest that a primary outbreak
of herpes simplex virus (HSV) may also increase risk for FGR.51
472 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
Traditionally, these infections, especially when they occur early
in pregnancy, have been thought to result in FGR secondary to
insults to cellular proliferation. More recent data suggest that the
mechanisms are more complex than simple cytopathic effects
and can include arrest in placental vascularisation, impairment
of placental transport and an altered immunologic milieu.52,53 As
an example of the potential role of immunologic derangements
in FGR, HIV infection itself is not necessarily associated with
FGR.54 Instead, there is evidence suggesting that CD4 counts less
than 200 cells/mm3 in the first trimester are strongly linked to risk
for FGR rather than viral load itself.55  10th percentile.68
Multiple gestation. Beyond the aetiologic factors that can lead
to FGR in singletons, the risk for FGR is further increased in mul-
tiple gestation. It is related in part to chorionicity and number of
fetuses, with greater incidences of FGR in higher order multiples
and monochorionic fetuses.56 There is controversy as to whether
these findings are a result of pathologic compromised growth ver-
sus fetal adaptation to shared resources.  rationale for this is uncertain, although low first trimester levels
Consequences of Fetal Growth Restriction
The consequences of FGR are extensive, traversing the antenatal
period to adulthood. In general, FGR increases risks of perinatal
morbidity and mortality, with a substantial increase in both as
birth weight falls below the 6th percentile for gestational age.57-61
Although pregnancies complicated by FGR often undergo indi-
cated preterm delivery, the attendant consequences of prematurity
such as respiratory distress (RDS), intraventricular haemorrhage
(IVH) and necrotising enterocolitis (NEC) are significantly worse
in FGR neonates than gestational age-matched, appropriately
grown control participants.60,62 Furthermore, neonates who were
growth restricted with absent or reversed end-diastolic velocity of
the umbilical artery are at even higher risk for adverse outcome.63
Other potential neonatal complications of FGR include low
Apgar scores, hypothermia, hypoglycaemia, hypocalcaemia, poly-
cythaemia and impaired immune function.64
Beyond the neonatal consequences, children who were growth
restricted at birth also demonstrate higher incidences of chronic
medical issues such as bronchopulmonary dysplasia (BPD), pul-
monary hypertension, neurodevelopmental delay and cerebral
palsy.64 As adults, these individuals are at increased risk for cardio-
vascular disease, metabolic syndrome and obesity.65  normal pregnancies, there should be a progressive decrease in uter-
Screening
Fundal Height
Fundal height, a measure from the maternal pubic symphysis to
the uterine fundus, is a commonly used measure to screen for
FGR in routine obstetric prenatal care. Existing data, however,
suggest that there is insufficient evidence to determine whether
this measurement is effective in identifying FGR.66,67  considered an abnormal finding between 11 and 14 weeks’ gesta-
Serum Analytes
Serum analyte results from first trimester, second trimester and
sequential or integrated screening for aneuploidy and open neural
tube defects may indicate increased risks of abnormal fetal growth
and adverse pregnancy outcome regardless of their association
with structural and karyotypic abnormalities. For example, low
concentrations of pregnancy-associated plasma protein A (PAPP-
A) at the time of first trimester aneuploidy screening has been
associated with an increased risk for subsequent development of
FGR and adverse pregnancy outcome.68-70 PAPP-A, which is a
protease for insulin-like growth factor binding proteins (IGFBPs)
such as IGFBP-4, enhances IGF availability.71 Thus low PAPP-A
may mechanistically signify inadequate IGF availability for proper
placental function and fetal growth. Despite this association, the
positive predictive value (PPV) for adverse outcomes using PAPP-
A alone remains significantly limited, with data from the First
And Second Trimester Evaluation of Risk (FASTER) trial demon-
strating a 16% PPV for diagnosis of birth weight at less than the
The association between human chorionic gonadotropin
(hCG) levels and risk for FGR and adverse pregnancy outcome
varies depending upon gestational age. Decreased free β−hCG lev-
els from screening between 11 and 14 weeks, and elevated β−hCG
concentrations during second trimester screening have both been
associated with an increased risk for FGR.69,72 The mechanistic
may represent impaired placentation. Furthermore, data from the
FASTER trial did not demonstrate a specific association between
low first trimester free-βhCG and FGR.68 Thus, similar to PAPP-
A, the use of βhCG alone for prediction of FGR remains signifi-
cantly limited, with PPVs not exceeding 18%.72
Similarly, elevated maternal serum α-fetoprotein (MSAFP),
increased inhibin A and decreased unconjugated estriol (uE3)
concentrations have also been individually associated with an
increased risk for FGR.73,74 Whereas one abnormal marker
slightly increased this risk, two or more abnormal serum analytes
significantly increased the risk for FGR and adverse outcome.74
Despite this increase in odds ratio risk, however, the PPV, even
with multiple analytes, remained low and reached only about
18% for predicting birth weight less than the 10th percentile.74
A recent retrospective case-control study further suggests that the
more extreme the values, the higher the likelihood for developing
severe FGR with absent or reversed umbilical artery end-diastolic
velocities.75 
Uterine Artery Doppler
Uterine artery Doppler velocimetry is an indirect measure of uter-
ine artery vascular resistance (Fig. 39.1). As gestation advances in
ine vascular resistance, which is thought to reflect adequate tro-
phoblastic invasion into maternal spiral arterioles.76 This results in
appropriate uteroplacental blood flow, which in turn also contrib-
utes to proper maternal endothelial function.
The uterine artery is interrogated at the level of its bifurcation
from the internal iliac artery to determine flow waveform ratios
such as pulsatility index (PI) and look for the presence or absence
of diastolic notching. In the first trimester, early diastolic notching
can be seen in up to three quarters of all pregnancies and is not
tion.77 Elevated flow waveform ratios such as a PI greater than
2.35 (95th percentile regardless of crown–rump length) in one
study was associated with a 12% risk for development of FGR.77
A recent meta-analysis found that elevated uterine artery Dop-
pler flow velocity waveforms resulted in 15% sensitivity and 93%
specificity for development of FGR at any gestational age.78 This
study, in addition to others, has suggested initiation of low-dose
aspirin before 16 weeks’ gestation in women who screen positive
in an attempt to decrease adverse outcome.78-80 However, whereas
at this time, ACOG currently does not recommend uterine artery
 CHAPTER 39  Fetal Growth and Growth Restriction 473

12cm/s Lt Ut-PS 166.98cm/s

Gn 0 Voluson Lt Ut-ED 66.08cm/s
WMF 60 Hz E8 Lt Ut-S/D 2.53
SV Angle 0 Lt Ut-RI 0.60
Size 2.0mm C6 / M5
Depth 26.6mm FF3 / E3
Frq mid
SRI II 4 / CRI 2
PRF 14.0kHz

-12cm/s Gn -3.6
Frq mid
Qual norm
WMF low1
PRF 1.8kHz

180 180
Ut-PS
160 160

140 140

120 120

100 100

80 80
Ut-ED
60 60

40 40

20 20
Pa g e : 1 o f 2 1 ( 7 3 % ) IM: 7
cm/s cm/s

• Fig. 39.1  A 20-week ultrasound image showing a static two-dimensional image of the lateral margin
of the lower uterine segment and cervix, which also depicts a colour Doppler image of the uterine artery.
The pulsed-wave sample volume is shown on the uterine artery. The Doppler image below is a normal
flow velocity waveform with a gentle peak, slow decline during diastole, absence of an early diastolic
(protodiastolic) notch and good forward flow at the end of diastole. Ut-ED, uterine artery end diastolic
velocity; Ut-PS, uterine artery peak systolic velocity.

-140 A also appears to be a factor in a high-risk population. In a pro-

-120
spective study of women who had low PAPP-A analyte levels
during first trimester aneuploidy screening, the PPV and NPV
-100
for uterine artery Dopplers were higher at 22 weeks compared
-80
with 18 weeks. A recent open-label, randomised controlled trial
-60
(RCT) of second trimester uterine artery Doppler screening in
-40 an unselected population found that 60% of early-onset FGR
20 1 of 19 (98%)
Page: cases could be identified with a false-positive rate of 11%.83
cm/s
However, implementation of this screening modality did not
120 B improve short-term perinatal morbidity and mortality even
though the screened population underwent more medical inter-
100
ventions, including antenatal corticosteroid administration.83
80 Thus, as mentioned previously, uterine artery Doppler velocim-
60 etry is not recommended for routine screening in any trimester,
N and the ACOG and RCOG disagree about the utility of second
40
trimester screening in high-risk populations. 
20
Page: 18 of 19
cm/s
Ultrasonographic Biometry
• Fig. 39.2  A normal (A) and an abnormal (B) flow velocity waveform of
the uterine artery. Note the sharp rise and fall of the first component of Ultrasonographic biometry remains the mainstay of screening
the abnormal waveform compared with the normal waveform. In addition, for FGR. Routine third trimester screening for growth in low-
note the early diastolic notch (N) and the low end-diastolic (arrow) flow of risk populations has not been shown to improve outcome.84,85
the abnormal waveform. More recently, though, the Pregnancy Outcome Prediction
(POP) study, a prospective observational study, found that
screening in any trimester, the Royal College of Obstetricians and universal third trimester biometry in an unselected popula-
Gynaecologists (RCOG) recommends screening high-risk popu- tion of nulliparous women increased the detection rate of SGA
lations between 20 and 24 weeks.4,81 infants nearly threefold.86 This increase in detection occurred
With regard to second trimester uterine artery Doppler velo- in conjunction with a 10% false-positive rate with universal
cimetry, one meta-analysis has suggested that Doppler notching screening compared with a 2% false-positive rate with selec-
or an elevated PI was more predictive of FGR in the second tive screening of women with risk factors.86 However, despite
trimester (Fig. 39.2).82 However, this meta-analysis was based the increase in detection rate, there is no evidence that this
solely upon two studies with heterogeneous populations. Timing improves outcome. 
474 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
Diagnosis
Ultrasonographic Biometry
Based upon the ACOG Practice Bulletin on Fetal Growth Restric-
tion, the diagnosis of FGR is made when the combined biometric
measurements result in an EFW of less than the 10th percentile
for gestational age compared with an appropriate reference popu-
lation.4 A crucial issue, however, is how to optimally define the
‘reference population’, which remains a controversial topic. Birth
weight standards, which are created using cross-sectional data of
newborn birth weight at each gestational age, have several limi-
tations. First, as mentioned previously, more recent birth weight
curves demonstrate evolving differences in our contemporary
population in comparison with classic growth curves, and their
utility is only as accurate as the population it is capturing.8,9 For
instance, one epidemiologic study found that both modal birth
weights and the lowest birth weight at which perinatal mortality
nadirs vary between different regions in Europe.87 This suggests
that race, ethnicity and other regional factors influence ‘ideal’
birth weights. Second, using birth weight data to generate fetal
growth references will underestimate the number of FGR fetuses,
especially in preterm gestations before 34 weeks.88,89 Third, fetal
weight references have been shown to better predict outcomes
such as RDS, IVH, retinopathy of prematurity (ROP) and BPD,
and birth weight-derived growth curves appear more prognostic
of neonatal death.90 Finally, a systematic review of 83 observa-
tional studies whose goal was to create ultrasound (US)-based fetal
weight references found significant heterogeneity in the studies of
fetal biometry.91 This variability highlights the difficulty in inter-
preting and extrapolating existing data to diverse populations.
Given these limitations, some have advocated creating cus-
tomised growth curves that take into account specific variables
that are known to influence birth weight, such as race or ethnic-
ity, maternal height, maternal weight, parity and fetal sex.11-13
In fact, the RCOG suggest that ‘use of a customised fetal weight
reference may improve prediction of a SGA neonate and adverse
perinatal outcome’.81 Existing global data demonstrate that indi-
vidualised references may be better able to predict adverse peri-
natal outcome than noncustomised fetal weight or birth weight
standards. For example, several studies have found that use of a
customised growth model is better able to predict factors such as
stillbirth, neonatal death, low Apgar score and neonatal intensive
care unit admission compared with either population-based birth
weight or fetal weight references.92-94 This was also demonstrated
in a US population at high risk for FGR and adverse pregnancy
outcome. Specifically, customised norms were associated with
higher rates of SGA and better identification of SGA neonates
at risk for adverse outcome.95 In contrast, within a term nul-
liparous population in the United States, defining neonates as
SGA using a customised standard did not improve prediction
of adverse pregnancy outcome compared with using population-
based norms.96 This suggests that more studies are required to
define customisation within specific populations, and notably,
no RCT comparing customised to population-based growth
charts exist.10
Recently, a project was undertaken by the International Fetal
and Newborn Growth Consortium for the 21st Century (INTER-
GROWTH-21st), whose overall objective was to study growth,
health, nutrition and neurodevelopment from the first trimester of
pregnancy to 2 years of age.97 Within the ­INTERGROWTH-21st
design, there were three main studies, with one of them being the
Fetal Growth Longitudinal Study (FGLS). The goal of this arm of
the study was to develop universal fetal growth standards based
upon the supposition that optimal conditions for the mother will
lead to similar patterns of fetal growth regardless of differences
in race, ethnicity and other cultural factors. Unlike several other
prior studies designed to create fetal weight references, the study
consortium aimed to rigorously define a cohort of healthy, well-
nourished pregnant women from eight diverse populations who
were at low risk for adverse maternal and perinatal outcomes.98
These women were also all extremely well dated. They were
scanned every 5 weeks with standardised procedures for obtain-
ing biometric measurements, and sonographers were unable to
see measurements on the screen in an attempt to reduce expected
value bias.98 Baseline demographics of the study cohort were simi-
lar across all eight countries and sites, and there were very low
rates of maternal and perinatal morbidity and mortality, confirm-
ing the ‘low-risk’ nature of the participants.98 Using a statistical
analysis strategy derived from the World Health Organization
Multicentre Growth Reference Study (WHO MGRS) protocol,
the authors found that data from the eight international sites
were able to be pooled for construction of international standards
for fetal head circumference (HC), biparietal diameter, abdomi-
nal circumference (AC), femur length (FL) and occipitofrontal
diameter (Fig. 39.3).98
Despite the rigorous study design, there continue to be con-
cerns surrounding universal adoption of this global fetal growth
standard. For example, 12% of healthy women were ineligible
secondary to low maternal height (<153 cm (60 inches)), which
could very well be a factor in the definition of optimal fetal growth
for that fetus. Similarly, there are large variations among countries,
with mean birth weights after 37 weeks’ gestation varying among
countries, ranging from as low as 2900 g in India to as high as
3500 g in the United Kingdom.99 This suggests risk for overdiag-
nosis of FGR in certain populations. When INTERGROWTH-
21st standards are applied to a multiethnic population in New
Zealand, fewer SGA infants were identified compared with use of
customised growth references.100 Perhaps even more important,
infants who were categorised as SGA by INTERGROWTH-
21st standards alone did not demonstrate an increased risk for
adverse perinatal outcome. In contrast, those who were found to
be SGA by customised standards alone had a twofold increase,
but infants who were SGA by both criteria demonstrated a nearly
fivefold increase in risk for composite adverse perinatal outcome
(Fig. 39.4).
Interestingly, the POP study investigating the efficacy of uni-
versal versus selective third trimester US screening in a nulliparous
population found that using customised growth curves did not
enhance the association between EFW and neonatal morbidity
compared with EFW derived from Hadlock reference standards.86
These investigators also noted that EFW below the 10th percen-
tile (regardless of definition used) in conjunction with the lowest
decile of AC growth velocity carried the strongest interaction with
adverse perinatal outcome. They repeated analyses of AC growth
velocity using INTERGROWTH-21st fetal growth standards
and found nearly an identical relative risk for adverse perinatal
outcome as using Hadlock references.86 In total, discrepancies
in findings of these studies indicate that further investigation is
needed of both customised and universal fetal growth standards
to validate their use in diverse populations and to determine their
true utility in predicting adverse perinatal outcome. Furthermore,
it is important to acknowledge that these tools specifically attempt
to assess growth but do not address other parameters that may
reflect placental function. 
 CHAPTER 39  Fetal Growth and Growth Restriction 475

400 110
100
350

Head circumference (mm)

90

Biparietal diameter (mm)

300
80
250 70

200 60
50
150
40
100
30
50 20
0 0
0 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 14 16 18 20 22 24 26 28 30 32 34 36 38 40
A Gestational age (wk) Gestational age (wk)
B

140
400
120
Occipitofrontal diameter (mm)

Abdominal circumference (mm)

350
100 300

80 250

200
60
150
40
100
20 50
0 0
0 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 14 16 18 20 22 24 26 28 30 32 34 36 38 40
C Gestational age (wk) D Gestational age (wk)

85

75

65
Femur length (mm)

55

45

35

25

15

5
0
0 14 16 18 20 22 24 26 28 30 32 34 36 38 40

E Gestational age (wk)

• Fig. 39.3  Gestational age–specific observed and smoothed centiles for various biometric parameters.
Fitted 3rd (bottom dashed line), 50th (middle dashed line) and 97th (top dashed line) smoothed centile
curves plotted against observed (open grey circles) values for fetal head circumference (A), biparietal
diameter (B), occipitofrontal diameter (C), abdominal circumference (D) and femur length (E) demon-
strate close agreement between smoothed and empirical centiles (open red circles). (Reproduced with
permission of Elsevier and Papageorghiou AT, Ohuma EO, Altman DG, et  al. International standards
for fetal growth based on serial ultrasound measurements: the Fetal Growth Longitudinal Study of the
INTERGROWTH-21st Project. Lancet 384:874, 2014.)
476 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Non-SGA (referent) SGA-cust only SGA-IG only SGA-both

1974/47,031 (4.2%)
Composite adverse 6/172 (3.5%)
neonatal outcome 338/4,000 (8.5%)
379/2,184 (17.4%)

9/47,031 0.2/1000
0/172 0
Neonatal death
0/4,000 0
3/2,184 1.4/1000

1390/47,031 (3.0%)
6/172 (3.5%)
NICU admission >48 hr
283/4000 (7.0%)
353/2,184 (16.0%)

975/47,031 (2.1%)
3/172 (1.7%)
Respiratory support >4 hr
136/4,000 (3.4%)
93/2,184 (4.2%)

457/47,031 (1.0%)
1/172 (0.6%)
Apgar score <7 at 5 min
60/4,000 (1.5%)
36/2,184 (1.7%)

59/47,090 1.3/1000
0/172 0
Stillbirth
15/4,015 3.7/1000
23/2,207 10.4/1000

0.01 0.1 1 10 100

Relative risk (95% CI)
• Fig. 39.4  Adverse perinatal outcome by small for gestational age (SGA) classification. Diagnosis of SGA
by INTERGROWTH-21st standards (SGA-IG only) alone was not associated with an increased risk for
composite adverse neonatal outcome or stillbirth. In contrast, the diagnosis of SGA by customised charts
alone (SGA-cust only) and by customised charts in conjunction with INTERGROWTH-21st parameters
(SGA-both) demonstrated a significant increase in risk for adverse neonatal outcome. CI, Confidence
interval; NICU, neonatal intensive care unit. (Reproduced with permission of Elsevier and Anderson NH,
Sadler LC, McKinlay CJD, McCowan LME. INTERGROWTH-21st vs customised birthweight standards
for identification of perinatal mortality and morbidity. Am J Obstet Gynecol 214:509.e1–e7, 2016.)

TABLE Accuracy of Fetal Transcerebellar Diameter

Evaluation After the Diagnosis of Fetal Growth 39.4 Measurements in Predicting Gestational
Restriction Age in Fetal Growth Restriction Fetuses
Confirmation of Gestational Age Actual-to-expected FGR <28 weeks FGR ≥28 weeks
To ensure an accurate diagnosis of FGR, confirmation of ges- GA (days) (n = 40) (n = 15)
tational age is imperative. When a patient presents for her first ±0 47.5% 13.3%
US later in pregnancy and is found to have a fetus that is growth
restricted based upon her proposed gestational age, the fetal tran- ±1 82.5% 63.3%
scerebellar diameter (TCD) can be used to help stratify risk. The ±2 95.0% 73.0%
TCD has been shown to be accurate in predicting GA in single-
ton and twin gestations.101-103 Furthermore, these same investi- ±3 97.5% 93.3%
gators have also demonstrated concordance between actual and ±4 100% 100%
predicted GA using their TCD nomogram in both FGR and LGA
fetuses (Table 39.4).104 Mean (SD), days -3 (1) -3 (1)
One other possible consideration to determine the risk for FGR, Fetal growth restriction; SD, standard deviation.
FGR, although not absolutely diagnostic, is to take into account Adapted from Chavez MR, Ananth CV, Smulian JC, Vintzileos AM. Fetal transcerebellar diam-
the fetal AC, especially compared with the HC. Asymmetric eter measurement for prediction of gestational age at the extremes of fetal growth. J Ultra-
growth with an AC (especially <5th percentile for gestational age) sound Med 26:1167–1171, 2007.
may indicate pathologic FGR.105,106    

Detailed Anatomic Ultrasound
If not completed earlier in gestation, a detailed anatomical US
should be performed to rule out congenital anomalies. Although
these may be found in isolation, anatomical abnormalities may
also be secondary to aneuploidy or congenital infection, both of
which are causes of FGR.
Up to 96% of fetuses with fluid pockets less than 1 cm in
depth may be growth restricted.107 Evaluation of amniotic fluid
volume can be performed by either measuring the amniotic fluid
index (AFI) or the single maximum vertical pocket (MVP). Dye
dilution with spectrophotometric calculation of amniotic fluid
volume demonstrate that both techniques do not reliably identify
true amniotic fluid volumes.108 Several studies have found that
the AFI method increases the diagnosis of oligohydramnios and
the incidence of labour induction for low fluid without improv-
ing perinatal outcome.109-111 Thus most experts recommend
using the MVP method to estimate amniotic fluid volume at this
point.111  2-week intervals considered in rare circumstances.4,119
Evaluation for Aneuploidy
Aneuploidy should be considered especially in cases with
early-onset FGR or when anomalies are present.112 Despite
the advent of noninvasive prenatal testing, amniocentesis for
karyotype (and microarray if anomalies are present) remains
the gold standard.  by these low velocities correlate with placental structural and his-
Evaluation for Infection
Although various viral and parasitic infections have been
associated with FGR, the ones most commonly associated
are CMV and toxoplasmosis. Thus the RCOG recommends
that maternal serologies (immunoglobulin (Ig) M and IgG)
should be offered for CMV and toxoplasmosis in severe
cases of FGR.81 Rubella immunity and screening for syphi-
lis should also be considered if not already performed earlier
in pregnancy. Varicella should also be considered, starting
with inquiry regarding history of chickenpox in the past, and
malaria should also be considered in high-risk populations.
Of note, HSVs are part of the traditional TORCH (toxo-
plasmosis, other (syphilis, varicella-zoster, parvovirus B19)
rubella, CMV and herpes infections and viral serologies) panel
with associations between FGR and severe HSV.51,113 How-
ever, at this point, neither the ACOG nor the RCOG recom-
mends routine screening or testing for HSV in the setting of
FGR.4,81 If infection is suspected as the cause of FGR, amnio-
centesis should also be offered for polymerase chain reaction
testing.  evidence for use of umbilical artery Dopplers in management of
Identification of Risk Factors
Fetal growth restriction may predate clinically evident pre-
eclampsia, and evaluation for this is warranted in the setting of
a new diagnosis of FGR. Identification of any modifiable risks
factors such as cigarette smoking or substance abuse should also
be undertaken. Although no evidence to our knowledge exists to
suggest that a growth-restricted fetus will demonstrate ‘catch-up’
growth after discontinuation of the implicated substance, the
risks of FGR appear to be decreased with cessation earlier in
pregnancy.114,115  flow velocity waveforms in the MCA. Several human studies, as
CHAPTER 39  Fetal Growth and Growth Restriction 477
Management
Ultrasound
Serial biometry. Serial growth US examinations are used to assess
both interval and overall fetal growth in pregnancies complicated
by FGR. Although birth weight and fetal growth standard curves
appear continuous and smooth, fetal and postnatal studies suggest
that growth is actually uneven, and there are periods without
growth interspersed with short spurts of growth.116,117 Specifically,
investigators have found that even in normal pregnancies in which
overall fetal growth is appropriate, fetal growth may arrest for
more than 2 weeks.117 In contrast, all biometric parameters show
some degree of growth within a 4-week time period in normal
pregnancies.117 Thus there is a substantial false-positive rate for
diagnosis of FGR when growth US examinations are performed
every 2 weeks.118 Although the optimal interval for repeat growth
US examinations has not been established, experts suggest that
repeat assessment should be performed between 2 to 4 weeks, with
 
Umbilical artery Doppler. Umbilical artery velocity waveforms
are a reflection of placental vascular resistance, and secondary to
ongoing angiogenesis of the fetoplacental vascular tree, these
waveforms demonstrate a progressive increase in diastolic flow as
gestation progresses.120-122 In growth-restricted fetuses, however,
umbilical artery end-diastolic velocities are frequently lower than
expected for gestational age, and this elevated resistance represented
topathologic abnormalities, as well as with adverse pregnancy out-
comes.123-131 From a physiologic perspective, as placental vascular
resistance increases, end-diastolic velocities decrease, although for-
ward flow is still present. As pathology further worsens, absent or
reversed end-diastolic velocities can be seen by umbilical artery
Doppler (Fig. 39.5). By this time, more than half of fetuses may
demonstrate some degree of hypoxemia based upon percutaneous
umbilical vein sampling and by cord blood gas measurements.132,133
Umbilical artery Doppler velocimetry has undergone rigor-
ous clinical testing. When used in women at risk for a potentially
compromised fetus, most trials have found that use of umbilical
artery Doppler examination improves outcomes, ranging from
fewer emergency deliveries to less death and serious neonatal mor-
bidities.134-137 A key meta-analysis and a Cochrane review have
shown that using umbilical artery Doppler in high-risk pregnan-
cies reduced the risk for perinatal death by approximately one
third, without increasing interventions such as iatrogenic preterm
delivery (Table 39.5).138,139 Thus both the Society for Maternal-
Fetal Medicine and the American Congress of Obstetricians and
Gynecologists endorse umbilical artery Doppler assessment in
high-risk pregnancies with suspected FGR.4,140 Despite level I
growth-restricted fetuses, the optimal frequency of Doppler inter-
rogation or a specified intervention protocol remains unknown. 
Middle cerebral artery Dopplers. In a normally grown fetus,
the flow velocity waveform obtained by pulsed-wave Doppler
interrogation of the middle cerebral artery (MCA) will reflect
the relatively high vascular impedance of the cerebral circulation
compared with the umbilical artery waveform. Progressive placen-
tal dysfunction that leads to development of fetal hypoxemia can
result in a decrease of the normally high blood flow resistance of
the cerebral vascular bed, a phenomenon commonly referred to as
the ‘brain-sparing effect’. Figure 39.6 shows normal and abnormal
478 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

45
40 A
30
D
15 30
cm/s
20
-45

-30 10
-15 D
cm/s
cm/s
75
B
40
60
30
20 D 45
10
cm/s
30
-20
15
-15
-10
D cm/s
-5
cm/s • Fig. 39.6  A normal (A) and an abnormal (B) flow velocity waveform of the
middle cerebral artery (MCA). Note the increase in diastolic flow (arrow) in
30 the abnormal MCA waveform.
20

10 enhances prediction of adverse outcome compared with MCA

cm/s
45
30
15
cm/s
-15
• Fig. 39.5 Serial flow velocity waveforms of the umbilical artery show-
ing progressive loss of diastolic flow (D), absent end-diastolic flow velocity
(arrow) and reversed end-diastolic flow velocity (R).
TABLE Significant Effects on Obstetric and Perinatal
39.5 Outcomes With Use of Umbilical Artery Doppler
Velocimetry
Endpoint RR
Perinatal death 0.71
Induction of labour 0.89
Caesarean delivery 0.90
CI, Confidence interval.
Adapted from Alfirevic Z, Neilson JP. Doppler ultrasound for fetal assessment in high-risk
pregnancies. Cochrane Database Syst Rev 20:CD00073, 2010.
  
well as animal studies, have shown that an increase in diastolic
flow velocity is consistent with a chronic hypoxemic state in the
fetus with a redistribution of blood flow from nonvital organ sys-
tems towards the fetal heart, brain and adrenal glands.141-144
Middle cerebral artery Doppler evaluation of the FGR fetus
has been shown in several studies to identify pregnancies at high
risk for poor perinatal outcome. In pregnancies before 34 weeks’
gestation, the prediction of poor perinatal outcome is improved
with the use of MCA Doppler velocimetry compared with umbili-
cal artery Doppler alone.145,146 Combining the use of MCA and
umbilical artery Dopplers in the form of the cerebroplacental
ratio (CPR) calculated as CPR = (MCA PI)/(Umbilical artery PI),
Doppler alone.141
Middle cerebral artery Doppler velocimetry may also be ben-
eficial when there is concern for FGR after 34 weeks’ gestation.
In the latter half of the third trimester, it is not uncommon for
R umbilical artery Doppler indices to remain normal in FGR preg-
nancies, which may be a reflection of the difference in pathology
between early- and late-onset FGR. In these FGR pregnancies with
normal umbilical artery Doppler velocimetry, studies have shown
that abnormalities in the MCA Doppler may still occur in up to
40% of these fetuses.147Additional evidence suggests that these
late preterm and early term FGR fetuses with normal umbilical
artery waveforms and abnormal MCA waveforms exhibit a higher
rate of neurodevelopmental compromise, behavioural issues and
nonreassuring fetal heart rate tracings resulting in an increased
incidence of caesarean delivery (58% vs 24%).147-150
95% CI Ultimately, MCA Doppler findings both in early- and late-
0.52–0.98 onset FGR may be useful in providing additional clinical infor-
mation on fetal risk for adverse outcomes. At this point, however,
0.80–0.99 there is insufficient evidence to endorse the use of MCA Dop-
0.84–0.97 pler velocimetry as a tool to guide timing of delivery, and neither
ACOG nor the RCOG currently recommend its use routinely in
management of FGR.4,81 
Fetal venous Doppler. Evaluation of the fetal venous system
is thought to reflect fetal ventricular function, with the premise
that fetal acidosis will compromise cardiac function.151,152 In
turn, this results in an increase in preload, and in conjunction
with increased afterload secondary to placental vascular dysfunc-
tion, an absent or reversed a-wave (atrial contraction) within the
ductus venosus (DV) or pulsations in the umbilical vein can be
seen (Fig. 39.7).153 Although not as rigorously tested as umbili-
cal artery Doppler in management of FGR, observational studies
demonstrate that assessment of fetal venous Doppler can provide
additional information on the status of growth-restricted fetuses.
For instance, various investigators have demonstrated that venous
Doppler enhances prediction of acidemia, adverse short-term
outcomes, stillbirth and neonatal death, especially in fetuses with
absent or reversed umbilical artery end-diastolic velocities.154-156
Furthermore, several studies have shown that 50% to 70% of

A S
D
-30 a
-15
cm/s
B method for assessing fetal well-being. Three of its ultrasono-
-30
-15
cm/s
C occur gradually in response to redistribution of fetal blood flow in
D
-30
-15
cm/s
15
• Fig. 39.7 Serial flow velocity waveforms of the ductus venosus (DV).
Note the triphasic waveform with individual systolic (S), diastolic (D) and
atrial (a) components and how in the normal waveform they are all with
forward flow velocities (A). B to D depict progressive abnormalities in the
DV waveform showing a reduction in a-wave velocities (arrows) leading to
absent a-wave (C) and reversed a-wave (D) velocities.
growth-restricted fetuses will demonstrate DV abnormalities after
changes in the umbilical artery and MCA but before abnormal
biophysical testing.157-159 These studies laid the foundation for
the recently completed RCT incorporating DV Doppler to inves-
tigate its use to determine timing of delivery (see Trial of Random-
ized Umbilical and Fetal Flow in Europe below).  suggests worsening of placental function, and low fluid volume
Antenatal Testing
Nonstress test and cardiotocography. Fetal heart rate (FHR)
characteristics are normally controlled through parasympathetic
innervation, in which the vagus nerve innervates both the
sinoatrial and atrioventricular nodes. This tonic influence
results in decreased rates of firing, thereby controlling the FHR.
The vagus nerve also transmits impulses that result in FHR
variability. When a fetus is exposed to prolonged periods of
uteroplacental insufficiency, a noradrenergic response through the
fetal adrenals occurs. This supersedes vagal influence, leading to
both fetal tachycardia and decreased variability. If this continues,
there is ultimately myocardial depression that manifests as late
decelerations. Thus, in theory, instituting nonstress tests (NSTs)
should identify fetuses at risk for adverse perinatal outcome.
This is supported by observational studies demonstrating lower
stillbirths in pregnancies with reactive NSTs than in those with
nonreactive NSTs in addition to a lower stillbirth rate when NSTs
are increased from once to twice per week.160,161
However, a recent Cochrane Database review found that no
clear evidence exists that antenatal cardiotocography (CTG)
improves perinatal outcome in the general population.162 In preg-
nancies complicated by impaired fetal growth, there is also insuf-
ficient data to determine whether there was any one surveillance
CHAPTER 39  Fetal Growth and Growth Restriction 479
regimen that would decrease risks for adverse pregnancy out-
come.163 Despite this evidence, however, NSTs and CTGs remain
a mainstay of management of FGR, with the rationale that non-
reactive and abnormal tracings have been associated with hypox-
emia and acidemia.164 
Biophysical profile. The biophysical profile (BPP) is another
graphic components—fetal breathing, gross body movements and
fetal tone—are acutely affected by fetal hypoxemia and acidemia,
although the absence of any of these findings is not necessarily
indicative of a pathologic in utero state. Amniotic fluid volume,
the fourth sonographic component of the BPP, is considered a
chronic parameter because decreases in volume are thought to
response to ongoing uteroplacental insufficiency.165
When compared with NSTs and CTGs, BPPs do not offer
any advantage or disadvantage in terms of predicting perinatal
deaths or low Apgar scores in a high-risk population.166 Although
RCTs of the efficacy of BPP use in FGR is lacking, observational
data have shown that low BPP scores have been associated with
reasonable diagnostic accuracy in predicting adverse perina-
tal outcome.167 Furthermore, a reassuring BPP score has a high
negative predictive value for stillbirth, similar to that of twice-
weekly NSTs, and this continues to justify its use in surveillance
of FGR.161,168,169 Of note, some consider the NST the fifth com-
ponent of the BPP. One potential advantage of incorporating the
NST is that it provides the opportunity to detect fetal heart rate
decelerations that occur intermittently, which may not yet have
transpired with sufficient frequency to impact acid–base balance
and the sonographic components of the BPP. However, it has been
shown that the predictive value is not altered by addition of the
NST to the four sonographic parameters.170 
Amniotic fluid volume. Serial evaluation of amniotic fluid vol-
ume, either with an NST (i.e., modified BPP) or as part of the
BPP, is warranted after the diagnosis of FGR. Development of
oligohydramnios or anhydramnios in a growth-restricted fetus
have been shown to be related to progressive worsening of Dop-
pler velocimetry indices.171 
Potential Interventions
Optimisation of maternal health. Maternal medical comor­
bidities such as hypertension have been associated with an increased
risk for development of FGR. Although it is reasonable to optimise
maternal health status, existing data have not had adequate power
to rule in or rule out a benefit to antihypertensive treatment in
women with chronic hypertension, gestational hypertension or
preeclampsia without severe features.172-174 In fact, a meta-analysis
found that treatment of mild to moderate hypertension in pregnancy
has been associated with a higher proportion of SGA infants.173
In contrast, treatment of patients with severe hypertension before
34 weeks’ gestation has been shown to prolong pregnancy and to
protect maternal health, but the neonatal benefits appear to be
secondary to prolongation of gestational age and gain in maturity
rather than a specific effect on the incidence of FGR.175,176
Avoidance or discontinuation of toxins associated with FGR
can also be beneficial. For example, cigarette smoking is a well-
established risk factor for FGR and is the leading preventable
cause of FGR. Studies have demonstrated that with smoking ces-
sation, birth weights are improved the earlier a woman ceases to
smoke in pregnancy.114,115,177
480 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

There has been thought that increasing nutrient delivery to

the fetus through maternal intake may be of benefit to growth-
restricted fetuses. There are no data, especially in resource-rich
areas, that this ameliorates FGR.178  improve outcome, the main goal of obstetric management of FGR
Aspirin. Aspirin has been extensively studied as an agent to
prevent preeclampsia, with SGA often investigated as a second-
ary outcome. Although a Cochrane review and a meta-analysis of
individual patient data suggest that low-dose aspirin may decrease
the risk for an SGA neonate by approximately 10%, the major-
ity of studies analysed primarily included women at risk for pre-
eclampsia.179,180 Thus the effect of aspirin in women at risk for an
SGA neonate alone cannot be determined. Furthermore, after the
diagnosis of FGR is established, there is no evidence to suggest any
benefit to aspirin therapy.  fetus’ between 24 and 36 weeks’ gestation. More than 90% of the
Heparin. Heparin and low-molecular-weight heparin have
been studied in women at risk for placental dysfunction. Vari-
ous mechanisms of action have been proposed. These include
the observation that placental vascular thromboses are more
common in women with adverse perinatal outcome.181 Addi-
tionally, heparins have antiinflammatory activity and may also
affect the invasive capability of trophoblast and decrease tro-
phoblast apoptosis.182-184 A recent Cochrane review found that
treatment with heparin in women at high risk for placental dys-
function and adverse pregnancy outcome was associated with
a risk in perinatal mortality compared with no treatment.185
However, there was no effect on FGR and SGA, and the
authors concluded that more studies, including those investi-
gating infant and long-term outcomes with heparin therapy, are
needed.  and similar to initial neonatal results, there was no difference in
Oxygen. Cordocentesis of growth-restricted fetuses indicates
that these fetuses may have impaired oxygen delivery compared
with normal pregnancies.132,186 Although some studies have sug-
gested potential benefit to maternal oxygen administration in
pregnancies complicated by FGR, methodologic issues were iden-
tified, including more mature gestational ages in fetuses that were
receiving oxygen therapy.187 Thus current evidence does not sup-
port its use in treatment of FGR, and in fact, some have also cited
risks associated with maternal hyperoxygenation.  One issue with this trial, however, is that within the immedi-
Nitric oxide donors. Nitric oxide (NO) is a powerful vasodila-
tor, and its deficiency has been implicated in various pregnancy
complications, including FGR. L-arginine, an amino acid and
sole endogenous precursor of NO, has been investigated as a treat-
ment to improve placental blood flow and fetal growth. A few
small studies have been performed with conflicting results, and at
this point, there is insufficient evidence to recommend use of NO
donors as treatment or prevention of FGR.188,189  note that this trial demonstrates that immediate delivery, even in
Phosphodiesterase type 5 inhibitors. Nitric oxide ultimately
results in increased levels of cGMP, which in turn, leads to vascu-
lar smooth muscle relaxation. Phosphodiesterase type 5 (PDE5)
inhibitors such as sildenafil inhibit degradation of cyclic gua-
nosine monophosphate (cGMP) by PDE5, thereby promoting
smooth muscle relaxation and vasodilation. A small observational
study compared women that were offered sildenafil citrate for
compassionate use for severe, early-onset FGR to participants who
declined or were not offered sildenafil.190 One stillbirth occurred
within 48 hours after starting sildenafil treatment. However, silde-
nafil was associated with increased fetal abdominal circumference
growth velocity and improved intact survival.190 The STRIDER
(Sildenafil Therapy In Dismal prognosis Early-onset intrauterine
growth Restriction) trial has commenced in Europe, Australia and
New Zealand and Canada, with availability of results planned for
2020.191  fetal DV as defined by a PI greater than the 95th percentile or (iii)
Timing of Delivery
In the absence of interventions that have been proven to truly
is to attempt to time delivery when gestational age is maximised
and exposure of a fetus to hypoxemia and acidemia is minimised.
The choice of timing remains complex but is currently guided by
a few key clinical trials that will be discussed individually.
Growth Restriction Intervention Trial (GRIT)192-194. This mul-
ticentre RCT was undertaken in 13 European countries and
sought to compare earlier delivery to avoid exposure to intrauter-
ine hypoxia with delaying delivery to gain as much maturity as
possible. It targeted pregnancies with a ‘compromised preterm
cohort was diagnosed with FGR. Furthermore, more than 75%
of the participants demonstrated abnormal umbilical artery Dop-
pler velocimetry, suggesting that the degree of growth restriction
was relatively severe. Participants were randomised to immediate
delivery, which was defined as within 48 hours to permit comple-
tion of a course of antenatal corticosteroids, or to deferred deliv-
ery, meaning that delivery would occur when test results skewed
toward favouring delivery.
This trial, which was powered for death and disability at or
after 2 years of age, demonstrated that there was no difference in
survival to discharge between the two groups. Specifically, more
stillbirths were averted in the group randomised to immediate
delivery, but these appear to be traded for more neonatal deaths.192
Very few cases were lost to follow-up in both arms at 2 years of age,
death or severe disability at this 2-year time point.193 Subanalyses
of those born between 24 and 30 weeks and those born between
31 and 36 weeks suggest that deferred delivery decreased risk for
disability in infants born before 31 weeks’ gestation. Despite this
finding, there was no significant difference in school age cognition,
language, motor skills and behaviour between the two groups.194
However, there was an overall 20% rate of loss to follow-up, and
the primary study was not powered for these outcomes.
ate delivery group, the average time to delivery was only 0.9 days
(range, 0.4–1.3), suggesting that there were other indications that
pushed physicians toward delivering before completion of a full
course of steroids. Perhaps of even bigger concern, there was no
prespecified surveillance strategy or indications for delivery, which
made criteria for moving toward delivery in both cohorts hetero-
geneous and difficult to generalise. However, it is important to
a cohort of relatively severely growth-restricted fetuses, does not
improve overall survival rates nor is it protective against adverse
neurodevelopmental outcome. 
Trial of Randomized Umbilical and Fetal Flow in Europe
(TRUFFLE).195,196 Similar to the GRIT trial, the TRUFFLE study
was a multicentre RCT performed in 20 European centres. This
trial, however, solely recruited pregnancies diagnosed with FGR,
as defined by a fetal AC less than the 10th percentile according
to local standards and an abnormal umbilical artery Doppler PI
above the 95th percentile between 26 and 31 6/7 weeks’ gesta-
tion. Furthermore, antenatal surveillance regimens and criteria
for delivery within each of the three randomisation groups were
prespecified.
Participants were randomised to deliver by (i) changes in short-
term variability by computerised CTG, (ii) early changes in the

late DV changes defined as an absent or reversed a-wave. Routine
monitoring included umbilical artery Doppler and CTG at least
once per week with safety net criteria in place for all participants
regardless of randomisation group. The primary outcome was sur-
vival without neurodevelopmental impairment at 2 years of age
corrected for prematurity.
The median gestational age was essentially the same in all three
groups (30.6–30.7 weeks). There was no difference in primary
outcome among the three groups, with about 90% of infants sur-
viving without severe neurodevelopmental impairment.196 In a
subanalysis of neurodevelopmental impairment in survivors only,
however, there was a statistically significant difference between
the three groups, with a post-hoc comparison demonstrating that
outcome was improved in survivors who had been randomised
to deliver based upon late DV changes compared with those who
were randomised to deliver based upon CTG criteria alone. There
was no statistical difference when comparing outcomes between
the CTG and early DV changes group, nor was there one when
early and late DV groups were assessed.
Although surveillance regimens and indications for delivery
were prespecified, there were still some limitations to this trial. First,
although there was no difference in the primary outcome between
the three randomisation groups, the study was ultimately under-
powered to detect their primary outcome because several were lost
to follow-up despite appropriate recruitment. Thus it is possible
that an adequate sample size may have detected a difference in
the primary outcome. Second, the survival and outcomes overall
were much better than anticipated. This may have been a result
of a milder definition of FGR used, which was solely a fetal AC of
less than 10th percentile with elevated umbilical artery Doppler
PIs. Alternatively, sample size may have been even more underesti-
mated. Third, analysis was based upon an intent-to-treat principle,
although nearly 50% of subjects delivered off-protocol. For exam-
ple, 22% of participants were delivered using off-protocol criteria
at less than 32 weeks’ gestation. Additionally, nearly one third of
all participants in each group delivered after 32 weeks, at which
point delivery indication was based upon local protocol. Fourth,
the DV was not assessed in the group that delivered based upon
CTG alone. Hence, the number of fetuses with an abnormal DV
in this group may have been high, and the comparison groups may
have been more similar than originally anticipated by the authors.
Finally, there is also concern regarding generalizability, as comput-
erised CTG is not standard-of-care in the majority of the U.S.
Current ACOG and RCOG guidelines differ, although both
were published before TRUFFLE study results. The ACOG indi-
cates that further research is required, but the RCOG suggests that
it can be used to help guide timing of delivery in preterm FGR
with abnormal umbilical artery Doppler indices.4,81  delivery is targeted for between 36 and 38 weeks’ gestation.
Disproportionate Intrauterine Growth Intervention Trial
at Term (DIGITAT).197,198 This study is also a multicentre RCT
focused upon delivery management of growth-restricted fetuses,
but unlike GRIT and TRUFFLE, participants were included only
if they had a singleton pregnancy beyond 36 0/7 weeks’ gestation.
FGR was defined as an overall EFW less than the 10th percentile,
fetal AC less than the 10th percentile, flattening of the growth
curve in the third trimester as judged by a clinician or the presence
of all three factors. Fetuses with normal and abnormal Doppler
indices were included. Women were randomised to either induc-
tion of labour, which occurred within 48 hours of randomisa-
tion, or to expectant management until the onset of spontaneous
labour or development of other standard obstetric indications at
the primary obstetrician’s discretion.
CHAPTER 39  Fetal Growth and Growth Restriction 481
The primary outcome was a composite measure of adverse neo-
natal outcome. Infants who were induced delivered an average of
10 days earlier than their expectantly managed counterparts, with
a difference in birth weight of 130 g. The investigators found no
difference in primary outcome.198 Of note, there was also no dif-
ference in rate of caesarean delivery, despite inductions occurring
at earlier gestational ages. Two-year follow-up of these infants also
demonstrated no difference in neurodevelopmental or behavioural
outcome between the two groups.197
One concern of the DIGITAT trial is the possibility of hav-
ing included constitutionally small fetuses in their study cohort
because FGR fetuses with normal umbilical artery Doppler study
results were included and MCA Doppler velocimetry was not per-
formed. However, there were equivalent (and rare) numbers of
fetuses with absent end-diastolic velocities in both groups. Fur-
thermore, the median PIs in both groups were normal and similar,
suggesting that in total, induction versus expectant management
of mild FGR does not impact neonatal outcome. 
Summary of Recommendations for
Management of Fetal Growth Restriction
Based upon existing data, which are primarily derived from level
II and level III evidence with a few randomised controlled trials,
our institutional protocol for management of FGR is as follows
(Fig. 39.8). When FGR is suspected, as indicated by an overall
EFW of less than the 10th percentile for gestational age, a detailed
anatomical survey (if not already completed), evaluation of amni-
otic fluid, umbilical artery Doppler velocimetry and MCA Dop-
pler studies should be performed. Depending upon gestational
age and degree of severity, workup for aneuploidy and infectious
causes should also be considered.
If umbilical artery Doppler indices are normal, we repeat Dop-
pler evaluation in 2 weeks with initiation of antenatal surveillance
typically around 30 weeks’ gestation. Timing of delivery is targeted
for 38 to 39 weeks’ gestation if parameters remain reassuring.
If umbilical artery Doppler velocimetry demonstrates an ele-
vated systolic/diastolic (S/D) ratio or PI beyond the 95th percen-
tile for gestational age but with continued forward end-diastolic
velocity or if the MCA Doppler CPR is abnormal (defined as
<1.08), we recommend repeating Doppler surveillance weekly.
Patients should be counselled regarding prognosis depending
upon gestational age, with institution of twice-weekly antena-
tal surveillance at the gestational age at which the patient would
choose to intervene for fetal indications. Depending upon overall
growth trajectory, amniotic fluid volume and Doppler findings,
When umbilical artery Doppler studies demonstrate absent or
reversed end-diastolic velocities, the patient is admitted to the hos-
pital for daily antenatal surveillance. Antenatal corticosteroids are
given, and we evaluate for other potential comorbidities such as
preeclampsia. Umbilical artery and DV Doppler velocimetry are
repeated every 2 to 3 days. In the setting of a DV waveform with for-
ward flow throughout the cardiac cycle, patients are delivered at 32
weeks for persistent reversal of umbilical artery end-diastolic veloci-
ties and at 34 weeks for persistent absent end-diastolic velocities.
We use 29 weeks as a cutoff to stratify timing of delivery
based upon an absent or reversed DV a-wave based upon a large
international prospective study demonstrating that beyond 29
weeks of gestation, absent or reversed DV a-wave was the only
statistically significant predictor of intact survival.155 If a fetus
482 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Sonographic EFW <10th percentile

Previable Viable

• Consider workup (e.g. • UmA Doppler

infection, aneuploidy, • CPR (MCA PI/UmbA PI)
anomalies) • Growth q 3–4 weeks
• Return at viability or at
GA when patient is
willing to intervene

Either Doppler modality

Doppler studies
abnormal (but persistent UmA AREDV
normal
forward umA EDV)

• Repeat Doppler in 2 wk • Repeat Doppler q wk • Inpatient admission

• Initiate antenatal
surveillance at 30 wk
Delivery
38–40 wk
(target, 39 wk)
• Initiate antenatal • Continuous monitoring initially,
surveillance at 30 wk then BID NST
• Antenatal corticosteroids
• Neonatology consultation
• Rule out other comorbidities
Delivery (e.g. pre-E, APS)
36–38 wk
(target, 37 wk)
• UmA Doppler q2–3 d
• If UmA AREDV, DV Doppler
q2–3 d

DV w/ A/R of a-wave (< 29 wk)

DV w/o A/R of a-wave • Prolonged monitoring DV w/ A/R of a-wave (29 wk)
• BID NSTs • Consider rescue steroids • Continuous monitoring
• UmA and DV • Re-counsel pt regarding • Consider rescue steroids
Doppler q2-3 d delivery implications • Deliver

Delivery
UmA AEDV: 34 wk
UmA REDV: 32 wk

• Fig. 39.8  General management algorithm of pregnancies with a sonographic estimated fetal weight
(EFW) less than the 10th percentile for gestational age. AEDV, absent end-diastolic velocities; APS,
antiphospholipid antibody syndrome; AREDV, absent or reversed end-diastolic velocities; A/R, absent or
reversed; BID, twice daily; CPR, cerebroplacental ratio; DV, ductus venosus; EDV, end-diastolic velocities;
EFW, estimated fetal weight; GA, gestational age; MCA, middle cerebral artery; NST, nonstress test; q,
every; REDV, reversed end-diastolic velocities; UmA, umbilical artery; w/o, without.

is less than 29 weeks’ gestation with an absent or reversed DV
a-wave, the fetus is placed on continuous monitoring with con-
sideration of rescue course steroids, depending upon timing, and
the patient is recounselled regarding prognosis and timing of
delivery.  pregnancy had a 3.4% chance of having an SGA neonate at their
Preconception Counselling
Recurrence Risk
Recurrence risk is ultimately dependent upon the underlying
cause. However, it has probably been best studied in a popula-
tion-based, retrospective study of nearly 260,000 participants
who had two sequential, singleton pregnancies.199 Five percent
of the women delivered an SGA neonate in their first pregnancy,
and these women had a 23% recurrence risk in their second preg-
nancy. In contrast, the women who had an AGA fetus in their first
second delivery (Table 39.6). This recurrence risk is also in agree-
ment with other smaller studies.200 
Counselling
Modifiable risk factors for an SGA neonate should be reviewed
with the patient. These include a short interpregnancy interval of
less than 6 months, a long interpregnancy interval of more than

TABLE
39.6 Recurrence Risk for Delivering a Small for
Gestational Age Neonate
First pregnancy Recurrence risk for an SGA neonate (%)
SGA 23
AGA 3.4
AGA, Appropriately grown for gestational age; SGA, small for gestational age.
Adapted from Voskamp BJ, Kazemier BM, Ravelli AC, et  al. Recurrence of small-for-
gestational-age pregnancy: analysis of first and subsequent singleton pregnancies in the
Netherlands. Am J Obstet Gynecol 208:374.e1–e6, 2013.
  
60 months and body mass indices less than 20 or greater than
25.201-203 As previously mentioned, smoking cessation is also an
important, modifiable risk factor.  consequences of prematurity. Improved understanding of patho-
Workup
Antiphospholipid antibody testing should be done if the prior
infant was born before 34 weeks’ gestation secondary to FGR
(or preeclampsia with severe features).4 Laboratory criteria are
CHAPTER 39  Fetal Growth and Growth Restriction 483
considered positive when values are abnormal on two occasions at
least 12 weeks apart. There is no evidence to suggest screening for
inherited thrombophilias. 
Conclusion
A growth-restricted fetus, when defined as a fetus unable to meet
his or her inherent growth potential, is at significant risk for peri-
natal morbidity and mortality in addition to long-term sequelae,
including cardiovascular disease, metabolic syndrome and obe-
sity later in life. Management of the growth-restricted pregnancy
involves the use of Doppler velocimetry of the fetal vasculature
and antepartum testing, which serve as an index of acute fetal
acid–base balance and chronic intravascular volume status. There
are few randomised clinical trials that provide guidance on tim-
ing of delivery of FGR fetuses. Currently, the only ‘treatment’
available for the FGR fetus is delivery, often with the attendant
physiologic, cellular and molecular mechanisms underlying FGR
is needed along with more investigations into various preventive
and treatment modalities if outcomes are truly to be improved.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 20. Thaler I, Weiner Z, Itskovitz J. Systolic or
diastolic notch in uterine artery blood flow
39. Mucitelli DR, Charles EZ, Kraus FT. Cho-
rioangiomas of intermediate size and intra-
velocity waveforms in hypertensive pregnant uterine growth retardation. Pathol Res Pract.
1.  Ott WJ. The diagnosis of altered fetal
patients: relationship to outcome. Obstet 1990;186(4):455–458.
growth. Obstet Gynecol Clin North Am. 15(2):
Gynecol. 1992;80(2):277–282. 40. Roberts DJ, Post MD. The placenta in pre-
237–263.
21. Fleischer A, Schulman H, Farmakides G, et al. eclampsia and intrauterine growth restriction.
2. Unterscheider J, Daly S, Geary MP, et  al.
Uterine artery Doppler velocimetry in preg- J Clin Pathol. 2008;61(12):1254–1260.
Optimizing the definition of intrauterine
nant women with hypertension. Am J Obstet 41. Faye-Petersen OM. The placenta in preterm
growth restriction: the multicenter prospec-
Gynecol. 1986;154(4):806–813. birth. J Clin Pathol. 2008;61(12):1261–1275.
tive PORTO Study. Am J Obstet Gynecol.
22. Stein Z, Susser M. The Dutch famine, 1944– 42. Snijders RJ, Sherrod C, Gosden CM, Nicolaides
2013;208(4):290.e291–e296.
1945, and the reproductive process. II. Inter- KH. Fetal growth retardation: associated mal-
3. Seeds JW, Peng T. Impaired growth and
relations of caloric rations and six indices at formations and chromosomal abnormalities.
risk of fetal death: is the tenth percentile the
birth. Pediatr Res. 1975;9(2):76–83. Am J Obstet Gynecol. 1993;168(2):547–555.
appropriate standard? Am J Obstet Gynecol.
23. Stein Z, Susser M. The Dutch famine, 43. Gross SJ. Intrauterine growth restriction:
1998;178(4):658–669.
1944-1945, and the reproductive process. a genetic perspective. Clin Obstet Gynecol.
4.  American College of Obstetricians and
I. Effects on six indices at birth. Pediatr Res. 1997;40(4):730–739.
Gynecologists. ACOG Practice bulletin no.
1975;9(2):70–76. 44. Viora E, Zamboni C, Mortara G, et  al. Tri-
134: fetal growth restriction. Obstet Gynecol.
24. Antonov AN. Children born during the somy 18: fetal ultrasound findings at dif-
2013;121(5):1122–1133.
siege of Leningrad in 1942. J Pediatr. ferent gestational ages. Am J Med Genet A.
5. UNICEF, WHO. Low birthweight: country,
1947;30(3):250–259. 2007;143A(6):553–557.
regional and global estimates. New York: World
25. Aagaard-Tillery KM, Porter TF, Lane RH, 45. Wilkins-Haug L, Quade B, Morton CC.
Health Organization; 2004.
et  al. In utero tobacco exposure is associ- Confined placental mosaicism as a risk factor
6. Alexander GR, Himes JH, Kaufman RB, et al.
ated with modified effects of maternal fac- among newborns with fetal growth restriction.
A United States national reference for fetal
tors on fetal growth. Am J Obstet Gynecol. Prenat Diagn. 2006;26(5):428–432.
growth. Obstet Gynecol. 1996;87(2):163–168.
2008;198(1):66.e61–e66. 46. Khoury MJ, Erickson JD, Cordero JF,
7. Lubchenco LO, Hansman C, Dressler M,
26. Lieberman E, Gremy I, Lang JM, Cohen AP. McCarthy BJ. Congenital malformations and
Boyd E. Intrauterine growth as estimated
Low birthweight at term and the timing of intrauterine growth retardation: a population
from liveborn birth-weight data at 24 to
fetal exposure to maternal smoking. Am J Pub- study. Pediatrics. 1988;82(1):83–90.
42 weeks of gestation. Pediatrics. 1963;32:
lic Health. 84(7):1127–1131. 47. Umbers AJ, Aitken EH, Rogerson SJ. Malaria
793–800.
27. Spinillo A, Capuzzo E, Nicola SE, et al. Fac- in pregnancy: small babies, big problem.
8. Olsen IE, Groveman SA, Lawson ML,
tors potentiating the smoking-related risk of Trends Parasitol. 2011;27(4):168–175.
et  al. New intrauterine growth curves based
fetal growth retardation. Br J Obstet Gynaecol. 48. Naeye RL. Cytomegalic inclusion disease.
on United States data. Pediatrics. 2010;
1994;101(11):954–958. The fetal disorder. Am J Clin Pathol. 1967;
125(2):e214–e224.
28. Castles A, Adams EK, Melvin CL, et al. Effects 47(6):738–744.
9. Fenton TR, Kim JH. A systematic review and
of smoking during pregnancy. Five meta-anal- 49. Peckham CS. Clinical and laboratory study of
meta-analysis to revise the Fenton growth
yses. Am J Prev Med. 1999;16(3):208–215. children exposed in utero to maternal rubella.
chart for preterm infants. BMC Pediatr.
29. Lumley J, Chamberlain C, Dowswell T, et al. Arch Dis Child. 1972;47(254):571–577.
2013;13:59.
Interventions for promoting smoking cessa- 50. Desai M, ter Kuile FO, Nosten F, et al. Epide-
10. Carberry AE, Gordon A, Bond DM, et  al.
tion during pregnancy. Cochrane Database Syst miology and burden of malaria in pregnancy.
Customised versus population-based growth
Rev. 2009;(3):CD001055. Lancet Infect Dis. 2007;7(2):93–104.
charts as a screening tool for detecting small
30. Lassen TH, Madsen M, Skovgaard LT, et al. 51. Brown ZA, Vontver LA, Benedetti J, et  al.
for gestational age infants in low-risk preg-
Maternal use of nicotine replacement ther- Effects on infants of a first episode of geni-
nant women. Cochrane Database Syst Rev.
apy during pregnancy and offspring birth- tal herpes during pregnancy. N Engl J Med.
2014;(5):CD008549.
weight: a study within the Danish National 1987;317(20):1246–1251.
11. Gardosi J. Customized fetal growth standards:
Birth Cohort. Paediatr Perinat Epidemiol. 52. Conroy AL, Silver KL, Zhong K, et al. Com-
rationale and clinical application. Semin Peri-
2010;24(3):272–281. plement activation and the resulting placen-
natol. 2004;28(1):33–40.
31. Strandberg-Larsen K, Tinggaard M, Nybo tal vascular insufficiency drives fetal growth
12. Gardosi J, Francis A. A customized standard
Andersen AM, et al. Use of nicotine replace- restriction associated with placental malaria.
to assess fetal growth in a US population. Am
ment therapy during pregnancy and still- Cell Host Microbe. 2013;13(2):215–226.
J Obstet Gynecol. 2009;201(1):25.e21–e27.
birth: a cohort study. BJOG. 2008;115(11): 53. Adams Waldorf KM, McAdams RM. Influence
13. Rizzo G, Prefumo F, Ferrazzi E, et  al. The
1405–1410. of infection during pregnancy on fetal devel-
effect of fetal sex on customized fetal growth
32. Wagner CL, Katikaneni LD, Cox TH, Ryan opment. Reproduction. 2013;146(5):R151–
charts. J Matern Fetal Neonatal Med. 2016:1–8.
RM. The impact of prenatal drug exposure on R162.
14. David C, Gabrielli S, Pilu G, Bovicelli L.
the neonate. Obstet Gynecol Clin North Am. 54. Lambert JS, Watts DH, Mofenson L, et  al.
The head-to-abdomen circumference ratio:
1998;25(1):169–194. Risk factors for preterm birth, low birth
a reappraisal. Ultrasound Obstet Gynecol.
33.  Maulik D. Fetal growth restriction: the etiol- weight, and intrauterine growth retarda-
1995;5(4):256–259.
ogy. Clin Obstet Gynecol. 2006;49(2):228–235. tion in infants born to HIV-infected preg-
15. Odegard RA, Vatten LJ, Nilsen ST, et al. Pre-
34. Salafia CM, Charles AK, Maas EM. Placenta nant women receiving zidovudine. Pediatric
eclampsia and fetal growth. Obstet Gynecol.
and fetal growth restriction. Clin Obstet Gyne- AIDS Clinical Trials Group 185 Team. AIDS.
2000;96(6):950–955.
col. 2006;49(2):236–256. 2000;14(10):1389–1399.
16. Chappell LC, Enye S, Seed P, et  al. Adverse
35. Raio L, Ghezzi F, Cromi A, et  al. The thick 55. Iqbal SN, Kriebs J, Harman C, et al. Predic-
perinatal outcomes and risk factors for pre-
heterogeneous (jellylike) placenta: a strong tors of fetal growth in maternal HIV disease.
eclampsia in women with chronic hyper-
predictor of adverse pregnancy outcome. Pre- Am J Perinatol. 2010;27(7):517–523.
tension: a prospective study. Hypertension.
nat Diagn. 2004;24(3):182–188. 56. Papageorghiou AT, Bakoulas V, Sebire NJ,
2008;51(4):1002–1009.
36.  Nordenvall M, Ullberg U, Laurin J, et al. Pla- Nicolaides KH. Intrauterine growth in mul-
17. Rey E, Couturier A. The prognosis of preg-
cental morphology in relation to umbilical tiple pregnancies in relation to fetal number,
nancy in women with chronic hypertension.
artery blood velocity waveforms. Eur J Obstet chorionicity and gestational age. Ultrasound
Am J Obstet Gynecol. 1994;171(2):410–416.
Gynecol Reprod Biol. 1991;40(3):179–190. Obstet Gynecol. 2008;32(7):890–893.
18. McCowan LM, Buist RG, North RA, Gamble
37. Ananth CV, Demissie K, Smulian JC, Vintzil- 57. Clausson B, Cnattingius S, Axelsson O. Out-
G. Perinatal morbidity in chronic hyperten-
eos AM. Relationship among placenta previa, comes of post-term births: the role of fetal
sion. Br J Obstet Gynaecol. 1996;103(2):123–
fetal growth restriction, and preterm delivery: growth restriction and malformations. Obstet
129.
a population-based study. Obstet Gynecol. Gynecol. 1999;94(5 Pt 1):758–762.
19. Everett TR, Lees CC. Beyond the placental
2001;98(2):299–306. 58. Kramer MS, Olivier M, McLean FH, et  al.
bed: placental and systemic determinants of
38. Ananth CV, Wilcox AJ. Placental abruption Impact of intrauterine growth retardation and
the uterine artery Doppler waveform. Pla-
and perinatal mortality in the United States. body proportionality on fetal and neonatal out-
centa. 2012;33(11):893–901.
Am J Epidemiol. 2001;153(4):332–337. come. Pediatrics. 1990;86(5):707–713.

483.e1
483.e2 References
59. Manning FA. Fetal Medicine: Principles and
Practice. Norwalk, CT: Appleton and Lange;
1995.
60. McIntire DD, Bloom SL, Casey BM, Leveno
KJ. Birth weight in relation to morbidity and
mortality among newborn infants. N Engl J
Med. 1999;340(16):1234–1238.
61. Piper JM, Xenakis EM, McFarland M, et  al.
Do growth-retarded premature infants have
different rates of perinatal morbidity and
mortality than appropriately grown prema-
ture infants? Obstet Gynecol. 1996;87(2):
169–174.
62. Lackman F, Capewell V, Richardson B, et al.
The risks of spontaneous preterm delivery and
perinatal mortality in relation to size at birth
according to fetal versus neonatal growth stan-
dards. Am J Obstet Gynecol. 2001;184(5):946–
953.
63. Hartung J, Kalache KD, Heyna C, et al. Out-
come of 60 neonates who had ARED flow
prenatally compared with a matched control
group of appropriate-for-gestational age pre-
term neonates. Ultrasound Obstet Gynecol.
2005;25(6):566–572.
64. Pallotto EK, Kilbride HW. Perinatal out-
come and later implications of intrauter-
ine growth restriction. Clin Obstet Gynecol.
2006;49(2):257–269.
65. Barker DJ. Adult consequences of fetal
growth restriction. Clin Obstet Gynecol.
2006;49(2):270–283.
66. Lindhard A, Nielsen PV, Mouritsen LA, et al.
The implications of introducing the symphy-
seal-fundal height-measurement. A prospec-
tive randomized controlled trial. Br J Obstet
Gynaecol. 1990;97(8):675–680.
67. Robert Peter J, Ho JJ, Valliapan J, Sivasangari
S. Symphysial fundal height (SFH) measure-
ment in pregnancy for detecting abnormal
fetal growth. Cochrane Database Syst Rev.
2015;(9):CD008136.
68. Dugoff L, Hobbins JC, Malone FD, et  al.
First-trimester maternal serum PAPP-A
and free-beta subunit human chorionic
gonadotropin concentrations and nuchal
translucency are associated with obstetric
complications: a population-based screening
study (the FASTER Trial). Am J Obstet Gyne-
col. 2004;191(4):1446–1451.
69. Kirkegaard I, Henriksen TB, Uldbjerg N.
Early fetal growth, PAPP-A and free beta-
hCG in relation to risk of delivering a small-
for-­gestational age infant. Ultrasound Obstet
Gynecol. 2011;37(3):341–347.
70. Spencer K, Cowans NJ, Avgidou K, et al. First-
trimester biochemical markers of aneuploidy
and the prediction of small-for-gestational age
fetuses. Ultrasound Obstet Gynecol. 2008;31(1):
15–19.
71. Bale LK, Conover CA. Disruption of insu-
lin-like growth factor-II imprinting during
embryonic development rescues the dwarf
phenotype of mice null for pregnancy-
associated plasma protein-A. J Endocrinol.
2005;186(2):325–331.
72. Odibo AO, Sehdev HM, Stamilio DM,
Macones GA. Evaluating the thresholds
of abnormal second trimester multiple
marker screening tests associated with intra-
uterine growth restriction. Am J Perinatol.
2006;23(6):363–367.
73. Dugoff L. Society for Maternal-Fetal Medi-
cine. First- and second-trimester mater-
nal serum markers for aneuploidy and
adverse obstetric outcomes. Obstet Gynecol.
2010;115(5):1052–1061.
74. Dugoff L, Hobbins JC, Malone FD, et al. Quad
screen as a predictor of adverse pregnancy out-
come. Obstet Gynecol. 2005;106(2):260–267.
75.  Roman A, Desai N, Krantz D, et al. Maternal
serum analytes as predictors of IUGR with dif-
ferent degrees of placental vascular dysfunction.
Prenat Diagn. 2014;34(7):692–698.
76.  Papageorghiou AT, Yu CK, Nicolaides KH.
The role of uterine artery Doppler in predict-
ing adverse pregnancy outcome. Best Pract Res
Clin Obstet Gynaecol. 2004;18(3):383–396.
77. Martin AM, Bindra R, Curcio P, et  al.
Screening for pre-eclampsia and fetal growth
restriction by uterine artery Doppler at 11-14
weeks of gestation. Ultrasound Obstet Gynecol.
2001;18(6):583–586.
78. Velauthar L, Plana MN, Kalidindi M, et  al.
First-trimester uterine artery Doppler and
adverse pregnancy outcome: a meta-analysis
involving 55,974 women. Ultrasound Obstet
Gynecol. 2014;43(5):500–507.
79. Bujold E, Morency AM, Roberge S, et  al.
Acetylsalicylic acid for the prevention of pre-
eclampsia and intra-uterine growth restriction
in women with abnormal uterine artery Dop-
pler: a systematic review and meta-analysis.
J Obstet Gynaecol Can. 2009;31(9):818–826.
80. Bujold E, Roberge S, Lacasse Y, et  al. Pre-
vention of preeclampsia and intrauterine
growth restriction with aspirin started in early
pregnancy: a meta-analysis. Obstet Gynecol.
2010;116(2 Pt 1):402–414.
81. Royal College of Obstetricians and Gynaeco-
logists. The investigation and management of
the small-for-gestational-age fetus. Green-Top
Guideline No. 312 2nd Ed. 2014:11–13.
82. Cnossen JS, Morris RK, ter Riet G, et al. Use
of uterine artery Doppler ultrasonography to
predict pre-eclampsia and intrauterine growth
restriction: a systematic review and bivariable
meta-analysis. CMAJ. 2008;178(6):701–711.
83. Garcia B, Llurba E, Valle L, et al. Do knowl-
edge of uterine artery resistance in the second
trimester and targeted surveillance improve
maternal and perinatal outcome? UTOPIA
study: a randomized controlled trial. Ultra-
sound Obstet Gynecol. 2016;47(6):680–689.
84. Bricker L, Neilson JP, Dowswell T. Rou-
tine ultrasound in late pregnancy (after 24
weeks’ gestation). Cochrane Database Syst Rev.
2008;(4):CD001451.
85. Jahn A, Razum O, Berle P. Routine screening
for intrauterine growth retardation in Ger-
many: low sensitivity and questionable benefit
for diagnosed cases. Acta Obstet Gynecol Scand.
1998;77(6):643–648.
86. Sovio U, White IR, Dacey A, et  al. Screen-
ing for fetal growth restriction with universal
third trimester ultrasonography in nulliparous
women in the Pregnancy Outcome Prediction
(POP) study: a prospective cohort study. Lan-
cet. 2015;386(10008):2089–2097.
87. Graafmans WC, Richardus JH, Borsboom GJ,
et  al. Birth weight and perinatal mortality: a
comparison of ‘optimal’ birth weight in seven
Western European countries. Epidemiology.
2002;13(5):569–574.
88. Salomon LJ, Bernard JP, Ville Y. Estimation
of fetal weight: reference range at 20-36 weeks’
gestation and comparison with actual birth-
weight reference range. Ultrasound Obstet
Gynecol. 2007;29(5):550–555.
89. Fry AG, Bernstein IM, Badger GJ. Com-
parison of fetal growth estimates based on
birth weight and ultrasound references. J
Matern Fetal Neonatal Med. 2002;12(4):
247–252.
90. Zaw W, Gagnon R, da Silva O. The risks of
adverse neonatal outcome among preterm
small for gestational age infants according to
neonatal versus fetal growth standards. Pediat-
rics. 2003;111(6 Pt 1):1273–1277.
91. Ioannou C, Talbot K, Ohuma E, et al. System-
atic review of methodology used in ultrasound
studies aimed at creating charts of fetal size.
BJOG. 2012;119(12):1425–1439.
92.  Clausson B, Gardosi J, Francis A, Cnattingius
S. Perinatal outcome in SGA births defined by
customised versus population-based birthweight
standards. BJOG. 2001;108(8):830–834.
93. Figueras F, Figueras J, Meler E, et al. Custom-
ised birthweight standards accurately predict
perinatal morbidity. Arch Dis Child Fetal Neo-
natal Ed. 2007;92(4):F277–280.
94. Mikolajczyk RT, Zhang J, Betran AP, et  al.
A global reference for fetal-weight and birth-
weight percentiles. Lancet. 2011;377(9780):
1855–1861.
95. Moussa HN, Wu ZH, Han Y, et al. Custom-
ized versus population fetal growth norms and
adverse outcomes associated with small for
gestational age infants in a high-risk cohort.
Am J Perinatol. 2015;32(7):621–626.
96. Costantine MM, Lai Y, Bloom SL, et al. Pop-
ulation versus customized fetal growth norms
and adverse outcomes in an intrapartum
cohort. Am J Perinatol. 2013;30(4):335–341.
97. Villar J, Altman DG, Purwar M, et  al. The
objectives, design and implementation of
the INTERGROWTH-21st Project. BJOG.
2013;120(suppl 2):9–26, v.
98. Papageorghiou AT, Ohuma EO, Altman DG,
et al. International standards for fetal growth
based on serial ultrasound measurements:
the Fetal Growth Longitudinal Study of the
INTERGROWTH-21st Project. Lancet.
2014;384(9946):869–879.
99. Villar J, Papageorghiou AT, Pang R, et  al.
The likeness of fetal growth and newborn
size across non-isolated populations in the
INTERGROWTH-21st Project: the Fetal
Growth Longitudinal Study and Newborn
Cross-Sectional Study. Lancet Diabetes Endo-
crinol. 2014;2(10):781–792.
100. Anderson NH, Sadler LC, McKinlay CJ,
McCowan LM. INTERGROWTH-21st vs cus-
tomized birthweight standards for identification
of perinatal mortality and morbidity. Am J Obstet
Gynecol. 2016;214(4):509.e501–e507.
101. Chavez MR, Ananth CV, Smulian JC, et  al.
Fetal transcerebellar diameter nomogram in
singleton gestations with special emphasis in
the third trimester: a comparison with pre-
viously published nomograms. Am J Obstet
Gynecol. 2003;189(4):1021–1025.
102. Chavez MR, Ananth CV, Smulian JC, et  al.
Fetal transcerebellar diameter measurement
with particular emphasis in the third trimes-
ter: a reliable predictor of gestational age. Am J
Obstet Gynecol. 2004;191(3):979–984.
103. Chavez MR, Ananth CV, Kaminsky LM, et al.
Fetal transcerebellar diameter measurement
for prediction of gestational age in twins. Am J
Obstet Gynecol. 2006;195(6):1596–1600.
104. Chavez MR, Ananth CV, Smulian JC, Vintz-
ileos AM. Fetal transcerebellar diameter mea-
surement for prediction of gestational age at
the extremes of fetal growth. J Ultrasound Med.
2007;26(9):1167–1171, quiz 1173–1164.
105. al Riyami N, Walker MG, Proctor LK, et al.
Utility of head/abdomen circumference ratio
in the evaluation of severe early-onset intra-
uterine growth restriction. J Obstet Gynaecol
Can. 2011;33(7):715–719.

106. Niknafs P, Sibbald J. Accuracy of single ultra-
sound parameters in detection of fetal growth
restriction. Am J Perinatol. 2001;18(6):325–
334.
107. Manning FA, Hill LM, Platt LD. Qualita-
tive amniotic fluid volume determination by
ultrasound: antepartum detection of intrauter-
ine growth retardation. Am J Obstet Gynecol.
1981;139(3):254–258.
108. Magann EF, Chauhan SP, Barrilleaux PS,
et al. Amniotic fluid index and single deepest
pocket: weak indicators of abnormal amniotic
volumes. Obstet Gynecol. 2000;96(5 Pt 1):
737–740.
109. Kehl S, Schelkle A, Thomas A, et  al. Single
deepest vertical pocket or amniotic fluid
index as evaluation test for predicting adverse
pregnancy outcome (SAFE trial): a multi-
center, open-label, randomized controlled
trial. Ultrasound Obstet Gynecol. 2016;47(6):
674–679.
110. Magann EF, Chauhan SP, Doherty DA,
et  al. The evidence for abandoning the
amniotic fluid index in favor of the single
deepest pocket. Am J Perinatol. 2007;24(9):
549–555.
111. Moise Jr KJ. Toward consistent terminology:
assessment and reporting of amniotic fluid
volume. Semin Perinatol. 2013;37(5):370–
374.
112. Bahado-Singh RO, Lynch L, Deren O, et al.
First-trimester growth restriction and fetal
aneuploidy: the effect of type of aneuploidy
and gestational age. Am J Obstet Gynecol.
1997;176(5):976–980.
113. Diguet A, Patrier S, Eurin D, et  al. Prenatal
diagnosis of an exceptional intrauterine her-
pes simplex type 1 infection. Prenat Diagn.
2006;26(2):154–157.
114. Blatt K, Moore E, Chen A, et al. Association
of reported trimester-specific smoking cessa-
tion with fetal growth restriction. Obstet Gyne-
col. 2015;125(6):1452–1459.
115. Yan J, Groothuis PA. Timing of prenatal
smoking cessation or reduction and infant
birth weight: evidence from the United King-
dom Millennium Cohort Study. Matern Child
Health J. 2015;19(3):447–458.
116. Lampl M, Veldhuis JD, Johnson ML. Salta-
tion and stasis: a model of human growth. Sci-
ence. 1992;258(5083):801–803.
117. Bernstein IM, Blake K, Wall B, Badger
GJ. Evidence that normal fetal growth can
be noncontinuous. Obstet Gynecol Surv.
1996;51(4):213–214.
118. Mongelli M, Ek S, Tambyrajia R. Screening
for fetal growth restriction: a mathematical
model of the effect of time interval and ultra-
sound error. Obstet Gynecol. 1998;92(6):908–
912.
119. Zelop CM, Javitt MC, Glanc P, et  al. ACR
Appropriateness Criteria growth disturbances-
risk of intrauterine growth restriction. Ultra-
sound Q; 2013;29(3):147–151.
120. Sutton MS, Theard MA, Bhatia SJ, et  al.
Changes in placental blood flow in the normal
human fetus with gestational age. Pediatr Res.
1990;28(4):383–387.
121. Trudinger BJ, Stevens D, Connelly A, et  al.
Umbilical artery flow velocity waveforms and
placental resistance: the effects of embolization
of the umbilical circulation. Am J Obstet Gyne-
col. 1987;157(6):1443–1448.
122. Su EJ. Role of the fetoplacental endothelium
in fetal growth restriction with abnormal
umbilical artery Doppler velocimetry. Am J
Obstet Gynecol. 2015;213(suppl 4):S123–S130.
123. Fleischer A, Schulman H, Farmakides G, et al.
Umbilical artery velocity waveforms and intra-
uterine growth retardation. Am J Obstet Gyne-
col. 1985;151(4):502–505.
124. Giles WB, Trudinger BJ, Baird PJ. Fetal
umbilical artery flow velocity waveforms and
placental resistance: pathological correlation.
Br J Obstet Gynaecol. 1985;92(1):31–38.
125. Groenenberg IA, Wladimiroff JW, Hop
WC. Fetal cardiac and peripheral arte-
rial flow velocity waveforms in intrauterine
growth retardation. Circulation. 1989;80(6):
1711–1717.
126. Hitschold T, Weiss E, Beck T, et al. Low target
birth weight or growth retardation? Umbilical
Doppler flow velocity waveforms and histo-
metric analysis of fetoplacental vascular tree.
Am J Obstet Gynecol. 1993;168(4):1260–1264.
127. Reuwer PJ, Bruinse HW, Stoutenbeek P,
Haspels AA. Doppler assessment of the feto-
placental circulation in normal and growth-
retarded fetuses. Eur J Obstet Gynecol Reprod
Biol. 1984;18(4):199–205.
128. Salafia CM, Pezzullo JC, Minior VK, Divon
MY. Placental pathology of absent and reversed
end-diastolic flow in growth-restricted fetuses.
Obstet Gynecol. 1997;90(5):830–836.
129. Trudinger BJ, Cook CM, Giles WB, et  al.
Fetal umbilical artery velocity waveforms and
subsequent neonatal outcome. Br J Obstet
Gynaecol. 1991;98(4):378–384.
130. Trudinger BJ, Giles WB, Cook CM, et  al.
Fetal umbilical artery flow velocity waveforms
and placental resistance: clinical significance.
Br J Obstet Gynaecol. 1985;92(1):23–30.
131. Spinillo A, Gardella B, Bariselli S, et  al. Pla-
cental histopathological correlates of umbili-
cal artery Doppler velocimetry in pregnancies
complicated by fetal growth restriction. Prenat
Diagn. 2012;32(13):1263–1272.
132. Pardi G, Cetin I, Marconi AM, et  al. Diag-
nostic value of blood sampling in fetuses
with growth retardation. N Engl J Med.
1993;328(10):692–696.
133. Weiner CP. The relationship between the
umbilical artery systolic/diastolic ratio and
umbilical blood gas measurements in speci-
mens obtained by cordocentesis. Am J Obstet
Gynecol. 1990;162(5):1198–1202.
134. Newnham JP, O’Dea MR, Reid KP, Diepe-
veen DA. Doppler flow velocity waveform
analysis in high risk pregnancies: a random-
ized controlled trial. Br J Obstet Gynaecol.
1991;98(10):956–963.
135. Trudinger BJ, Cook CM, Giles WB, et  al.
Umbilical artery flow velocity waveforms in
high-risk pregnancy. Randomised controlled
trial. Lancet. 1987;1(8526):188–190.
136. Tyrrell SN, Lilford RJ, Macdonald HN,
et  al. Randomized comparison of routine
vs highly selective use of Doppler ultra-
sound and biophysical scoring to investigate
high risk pregnancies. Br J Obstet Gynaecol.
1990;97(10):909–916.
137. Almstrom H, Axelsson O, Cnattingius S,
et  al. Comparison of umbilical-artery velo-
cimetry and cardiotocography for surveillance
of small-for-gestational-age fetuses. Lancet.
1992;340(8825):936–940.
138. Alfirevic Z, Neilson JP. Doppler ultrasonog-
raphy in high-risk pregnancies: systematic
review with meta-analysis. Am J Obstet Gyne-
col. 1995;172(5):1379–1387.
139. Alfirevic Z, Stampalija T, Gyte GM. Fetal
and umbilical Doppler ultrasound in high-
risk pregnancies. Cochrane Database Syst Rev.
2013;(11):CD007529.
References 483.e3
140. 
Society for Maternal-Fetal Medicine Publi-
cations Committee. Berkley E, Chauhan SP,
Abuhamad A. Doppler assessment of the fetus
with intrauterine growth restriction. Am J
Obstet Gynecol. 2012;206(4):300–308.
141. Gramellini D, Folli MC, Raboni S, et  al.
­Cerebral-umbilical Doppler ratio as a predictor
of adverse perinatal outcome. Obstet Gynecol.
1992;79(3):416–420.
142. Mari G, Deter RL. Middle cerebral artery flow
velocity waveforms in normal and small-for-
gestational-age fetuses. Am J Obstet Gynecol.
1992;166(4):1262–1270.
143. Wladimiroff JW, Tonge HM, Stewart PA.
Doppler ultrasound assessment of cerebral
blood flow in the human fetus. Br J Obstet
Gynaecol. 1986;93(5):471–475.
144. Arbeille P, Roncin A, Berson M, et al. Explo-
ration of the fetal cerebral blood flow by
duplex Doppler—linear array system in nor-
mal and pathological pregnancies. Ultrasound
Med Biol. 1987;13(6):329–337.
145. Bahado-Singh RO, Kovanci E, Jeffres A, et al.
The Doppler cerebroplacental ratio and peri-
natal outcome in intrauterine growth restric-
tion. Am J Obstet Gynecol. 1999;180(3 Pt 1):
750–756.
146. Odibo AO, Riddick C, Pare E, et al. Cerebro-
placental Doppler ratio and adverse perinatal
outcomes in intrauterine growth restriction:
evaluating the impact of using gestational age-
specific reference values. J Ultrasound Med.
2005;24(9):1223–1228.
147. Cruz-Martinez R, Figueras F, Hernandez-
Andrade E, et al. Fetal brain Doppler to pre-
dict cesarean delivery for nonreassuring fetal
status in term small-for-gestational-age fetuses.
Obstet Gynecol. 2011;117(3):618–626.
148. Eixarch E, Meler E, Iraola A, et al. Neurodevel-
opmental outcome in 2-year-old infants who
were small-for-gestational age term fetuses
with cerebral blood flow redistribution. Ultra-
sound Obstet Gynecol. 2008;32(7):894–899.
149. Figueras F, Eixarch E, Meler E, et al. Small-for-
gestational-age fetuses with normal umbilical
artery Doppler have suboptimal perinatal and
neurodevelopmental outcome. Eur J Obstet
Gynecol Reprod Biol. 2008;136(1):34–38.
150. Oros D, Figueras F, Cruz-Martinez R, et  al.
Middle versus anterior cerebral artery Dop-
pler for the prediction of perinatal outcome
and neonatal neurobehavior in term small-for-
gestational-age fetuses with normal umbilical
artery Doppler. Ultrasound Obstet Gynecol.
2010;35(4):456–461.
151. Hecher K, Campbell S, Doyle P, et al. Assess-
ment of fetal compromise by Doppler ultra-
sound investigation of the fetal circulation.
Arterial, intracardiac, and venous blood flow
velocity studies. Circulation. 1995;91(1):129–
138.
152. Hecher K, Snijders R, Campbell S, Nico-
laides K. Fetal venous, intracardiac, and
arterial blood flow measurements in intra-
uterine growth retardation: relationship
with fetal blood gases. Am J Obstet Gynecol.
1995;173(1):10–15.
153. Baschat AA, Harman CR. Venous Doppler
in the assessment of fetal cardiovascular sta-
tus. Curr Opin Obstet Gynecol. 2006;18(2):
156–163.
154. Baschat AA, Gembruch U, Weiner CP,
Harman CR. Qualitative venous Doppler
waveform analysis improves prediction of crit-
ical perinatal outcomes in premature growth-
restricted fetuses. Ultrasound Obstet Gynecol.
2003;22(3):240–245.
483.e4 References
155. Baschat AA, Cosmi E, Bilardo CM, et  al.
Predictors of neonatal outcome in early-
onset placental dysfunction. Obstet Gynecol.
2007;109(2 Pt 1):253–261.
156. Schwarze A, Gembruch U, Krapp M, et  al.
Qualitative venous Doppler flow waveform
analysis in preterm intrauterine growth-
restricted fetuses with ARED flow in the
umbilical artery—correlation with short-
term outcome. Ultrasound Obstet Gynecol.
2005;25(6):573–579.
157. Baschat AA, Gembruch U, Harman CR. The
sequence of changes in Doppler and biophysi-
cal parameters as severe fetal growth restric-
tion worsens. Ultrasound Obstet Gynecol.
2001;18(6):571–577.
158. Ferrazzi E, Bozzo M, Rigano S, et al. Tempo-
ral sequence of abnormal Doppler changes in
the peripheral and central circulatory systems
of the severely growth-restricted fetus. Ultra-
sound Obstet Gynecol. 2002;19(2):140–146.
159. Hecher K, Bilardo CM, Stigter RH, et  al.
Monitoring of fetuses with intrauterine
growth restriction: a longitudinal study. Ultra-
sound Obstet Gynecol. 2001;18(6):564–570.
160. Freeman RK, Anderson G, Dorchester W. A
prospective multi-institutional study of ante-
partum fetal heart rate monitoring. I. Risk of
perinatal mortality and morbidity according
to antepartum fetal heart rate test results. Am J
Obstet Gynecol. 1982;143(7):771–777.
161. Boehm FH, Salyer S, Shah DM, Vaughn WK.
Improved outcome of twice weekly nonstress
testing. Obstet Gynecol. 1986;67(4):566–568.
162. Grivell RM, Alfirevic Z, Gyte GM, Dev-
ane D. Antenatal cardiotocography for fetal
assessment. Cochrane Database Syst Rev.
2015;(9):CD007863.
163. Grivell RM, Wong L, Bhatia V. Regimens of fetal
surveillance for impaired fetal growth. Cochrane
Database Syst Rev. 2012;(6):CD007113.
164. Visser GH, Sadovsky G, Nicolaides KH. Antepar-
tum heart rate patterns in small-for-gestational-
age third-trimester fetuses: correlations
with blood gas values obtained at cordo-
centesis. Am J Obstet Gynecol. 1990;162(3):
698–703.
165. Manning FA, Menticoglou S, Harman CR,
et  al. Antepartum fetal risk assessment: the
role of the fetal biophysical profile score. Bail-
lieres Clin Obstet Gynaecol. 1987;1(1):55–72.
166. Lalor JG, Fawole B, Alfirevic Z, Devane D.
Biophysical profile for fetal assessment in high
risk pregnancies. Cochrane Database Syst Rev.
2008;(1):CD000038.
167. Manning FA, Morrison I, Harman CR, et al.
Fetal assessment based on fetal biophysical
profile scoring: experience in 19,221 referred
high-risk pregnancies. II. An analysis of false-
negative fetal deaths. Am J Obstet Gynecol.
1987;157(4 Pt 1):880–884.
168. Baskett TF, Allen AC, Gray JH, et  al. Fetal
biophysical profile and perinatal death. Obstet
Gynecol. 1987;70(3 Pt 1):357–360.
169. Dayal AK, Manning FA, Berck DJ, et al. Fetal
death after normal biophysical profile score:
an eighteen-year experience. Am J Obstet
Gynecol. 1999;181(5 Pt 1):1231–1236.
170. Manning FA, Morrison I, Lange IR, et  al.
Fetal biophysical profile scoring: selective use
of the nonstress test. Am J Obstet Gynecol.
1987;156(3):709–712.
171. Unterscheider J, Daly S, Geary MP, et al. Pre-
dictable progressive Doppler deterioration in
IUGR: does it really exist? Am J Obstet Gyne-
col. 2013;209(6):539.e531–e537.
172. Sibai BM. Treatment of hypertension in preg-
nant women. N Engl J Med. 1996;335(4):257–
265.
173. von Dadelszen P, Ornstein MP, Bull SB, et al.
Fall in mean arterial pressure and fetal growth
restriction in pregnancy hypertension: a meta-
analysis. Lancet. 2000;355(9198):87–92.
174. Sibai BM, Mabie WC, Shamsa F, et  al.
A comparison of no medication versus
methyldopa or labetalol in chronic hyper-
tension during pregnancy. Am J Obstet
Gynecol. 1990;162(4):960–966; discussion
966–967.
175. Sibai BM, Mercer BM, Schiff E, Friedman
SA. Aggressive versus expectant management
of severe preeclampsia at 28 to 32 weeks’ ges-
tation: a randomized controlled trial. Am J
Obstet Gynecol. 1994;171(3):818–822.
176. Odendaal HJ, Pattinson RC, Bam R, et  al.
Aggressive or expectant management for
patients with severe preeclampsia between
28-34 weeks’ gestation: a randomized controlled
trial. Obstet Gynecol. 1990;76(6):1070–1075.
177. Suzuki K, Sato M, Zheng W, et al. Effect of
maternal smoking cessation before and dur-
ing early pregnancy on fetal and childhood
growth. J Epidemiol. 2014;24(1):60–66.
178. Say L, Gulmezoglu AM, Hofmeyr GJ. Mater-
nal nutrient supplementation for suspected
impaired fetal growth. Cochrane Database Syst
Rev. 2003;(1):CD000148.
179. Askie LM, Duley L, Henderson-Smart DJ,
et  al. Antiplatelet agents for prevention of
pre-eclampsia: a meta-analysis of individual
patient data. Lancet. 2007;369(9575):1791–
1798.
180. Duley L, Henderson-Smart DJ, Meher S,
King JF. Antiplatelet agents for preventing
pre-eclampsia and its complications. Cochrane
Database Syst Rev. 2007;(2):CD004659.
181. Redline RW. Thrombophilia and pla-
cental pathology. Clin Obstet Gynecol.
2006;49(4):885–894.
182. Kingdom JC, Drewlo S. Is heparin a placental
anticoagulant in high-risk pregnancies? Blood.
2011;118(18):4780–4788.
183. Bose P, Black S, Kadyrov M, et al. Heparin and
aspirin attenuate placental apoptosis in  vitro:
implications for early pregnancy failure. Am J
Obstet Gynecol. 2005;192(1):23–30.
184. Ganapathy R, Whitley GS, Cartwright JE,
et al. Effect of heparin and fractionated hepa-
rin on trophoblast invasion. Hum Reprod.
2007;22(9):2523–2527.
185. Dodd JM, McLeod A, Windrim RC, Kingdom
J. Antithrombotic therapy for improving mater-
nal or infant health outcomes in women consid-
ered at risk of placental dysfunction. Cochrane
Database Syst Rev. 2013;(7):CD006780.
186. Soothill PW, Nicolaides KH, Campbell S.
Prenatal asphyxia, hyperlacticaemia, hypo-
glycaemia, and erythroblastosis in growth
retarded fetuses. Br Med J (Clin Res Ed).
1987;294(6579):1051–1053.
187. Say L, Gulmezoglu AM, Hofmeyr GJ. Mater-
nal oxygen administration for suspected
impaired fetal growth. Cochrane Database Syst
Rev. 2003;(1):CD000137.
188. Winer N, Branger B, Azria E, et al. L-Arginine
treatment for severe vascular fetal intrauterine
growth restriction: a randomized double-bind
controlled trial. Clin Nutr. 2009;28(3):243–
248.
189. Xiao XM, Li LP. L-Arginine treatment for
asymmetric fetal growth restriction. Int J Gyn-
aecol Obstet. 2005;88(1):15–18.
190. von Dadelszen P, Dwinnell S, Magee LA,
et al. Sildenafil citrate therapy for severe early-
onset intrauterine growth restriction. BJOG.
2011;118(5):624–628.
191. Ganzevoort W, Alfirevic Z, von Dadelszen P,
et  al. STRIDER: Sildenafil Therapy In Dis-
mal prognosis Early-onset intrauterine growth
Restriction—a protocol for a systematic review
with individual participant data and aggregate
data meta-analysis and trial sequential analy-
sis. Syst Rev. 2014;3:23.
192. Group GS. A randomised trial of timed deliv-
ery for the compromised preterm fetus: short
term outcomes and Bayesian interpretation.
BJOG. 2003;110(1):27–32.
193. Thornton JG, Hornbuckle J, Vail A, et  al.
Infant wellbeing at 2 years of age in the Growth
Restriction Intervention Trial (GRIT): multi-
centred randomised controlled trial. Lancet.
2004;364(9433):513–520.
194. Walker DM, Marlow N, Upstone L, et al. The
Growth Restriction Intervention Trial: long-
term outcomes in a randomized trial of tim-
ing of delivery in fetal growth restriction. Am J
Obstet Gynecol. 2011;204(1):34.e31–e39.
195. Lees C, Marlow N, Arabin B, et al. Perinatal
morbidity and mortality in early-onset fetal
growth restriction: cohort outcomes of the
trial of randomized umbilical and fetal flow in
Europe (TRUFFLE). Ultrasound Obstet Gyne-
col. 2013;42(4):400–408.
196. Lees CC, Marlow N, van Wassenaer-Leemhuis
A, et al. 2 year neurodevelopmental and inter-
mediate perinatal outcomes in infants with
very preterm fetal growth restriction (TRUF-
FLE): a randomised trial. Lancet. 2015;
385(9983):2162–2172.
197. van Wyk L, Boers KE, van der Post JA, et al.
Effects on (neuro)developmental and behav-
ioral outcome at 2 years of age of induced
labor compared with expectant management
in intrauterine growth-restricted infants: long-
term outcomes of the DIGITAT trial. Am J
Obstet Gynecol. 2012;206(5):406.e401–e407.
198. Boers KE, Vijgen SM, Bijlenga D, et  al.
Induction versus expectant monitoring for
intrauterine growth restriction at term: ran-
domised equivalence trial (DIGITAT). BMJ.
2010;341:c7087.
199. Voskamp BJ, Kazemier BM, Ravelli AC, et al.
Recurrence of small-for-gestational-age preg-
nancy: analysis of first and subsequent single-
ton pregnancies in The Netherlands. Am J
Obstet Gynecol. 2013;208(5):374.e371–e376.
200. Ananth CV, Kaminsky L, Getahun D, et  al.
Recurrence of fetal growth restriction in
singleton and twin gestations. J Matern Fetal
Neonatal Med. 2009;22(8):654–661.
201. Conde-Agudelo A, Rosas-Bermudez A,
Kafury-Goeta AC. Birth spacing and risk of
adverse perinatal outcomes: a meta-analysis.
JAMA. 2006;295(15):1809–1823.
202. Han Z, Mulla S, Beyene J, et  al. Maternal
underweight and the risk of preterm birth and
low birth weight: a systematic review and meta-
analyses. Int J Epidemiol. 2011;40(1):65–101.
203. McCowan LM, Roberts CT, Dekker GA,
et  al. Risk factors for small-for-gestational-
age infants by customised birthweight
centiles: data from an international prospec-
tive cohort study. BJOG. 2010;117(13):
1599–1607.
40
Haemolytic Disease of the Fetus and
Newborn
SAUL SNOWISE AND KENNETH J. MOISE JR.
KEY POINTS
• Fetal hydrops is defined as the accumulation of fluid in two
fetal compartments (abdominal ascites, pleural effusion,
pericardial effusion, skin or scalp oedema). It may also be
associated with polyhydramnios and placental oedema.
• Immune hydrops is the result of alloimmunisation to red
blood cell antigens.
• Rhesus (Rh) immunoglobulin has decreased the relative
frequency of RhD disease.
• Kell alloimmunisation is associated with the least predictable
and most severe degree of fetal anaemia.
• Doppler measurement of fetal middle cerebral artery peak
velocity has transformed the diagnosis and treatment of fetal
anaemia and greatly reduced the use of invasive procedures.
• Intravascular fetal blood transfusions in cases of severe anae-
mia and hydrops have dramatically improved the outcome of
these pregnancies.
History
The history of haemolytic disease of the fetus and newborn
(HDFN) is an example of how modern medicine can both treat
and prevent a disease process with significant perinatal morbidity
and mortality. In the 1950s 15% of all alloimmunised gestations
ended in stillbirth.1 Work by Bevis2 and Walker3 in Rh-sensitised
pregnancies correlated amniotic fluid levels of bilirubin with the
incidence of postnatal kernicterus and the severity of anaemia
in affected infants. William Liley in New Zealand furthered this
work and created the Liley curve, a chart that was able to pre-
dict the severity of fetal haemolytic disease and aid in determining
the appropriate time for labour induction in affected gestations.
Liley’s work decreased the perinatal mortality rate in Rh-sensitised
gestations at the National Women’s Hospital in Auckland from
22% in 1958 to 8.7% in 1962.4
To improve outcomes for the most severely affected fetuses,
Liley further developed the concept of intraperitoneal fetal trans-
fusion. In patients with suspected severe haemolytic disease who
were not likely to survive, compatible red blood cells (RBCs) were
injected intraperitoneally in the early third trimester. The initial
procedures were done by first injecting radiopaque dye into the
amniotic cavity of affected pregnancies. X-ray still images were
484
then used to localise the fetal abdomen using the radiopaque dye
that had been swallowed by the fetus. Finally, proper placement of
the needle in the peritoneal cavity was confirmed by injection of
additional radiopaque dye. The first transfusion with fetal survival
was reported by Liley and his colleagues in 1962,5 and by 1965,
Liley reported on a further 16 transfused fetuses with a 38% sur-
vival rate.6
Today, serial intrauterine transfusions (IUTs) to treat severe
HDFN in association with maternal RBC alloimmunisation result
in neonatal survival in well over 90% of cases. Advancements in
ultrasound and IUT technique have certainly played a role in this
improvement, but the prevention of Rhesus alloimmunisation
by the introduction of Rhesus immunoglobulin (RhIG) has also
decreased the disease burden of RhD disease in particular. 
Epidemiology
The adoption of antenatal and postpartum RhIG has resulted
in a marked reduction in the incidence of disease secondary to
alloimmunisation by the Rh(D) antigen. Cases of RhD alloim-
munisation still occur, however, either secondary to the failure to
administer RhIG or secondary to inadequate dosing of RhIG in
the presence of an undetected large fetomaternal haemorrhage.
Even with the perfect implementation of RhIG, alloimmunisa-
tion resulting from incompatibility to the other Rhesus antigens
and the non-Rhesus antigens will continue. For example, in east
Asia, there is a lower proportion of Rh(D)-negative individu-
als, leading to a higher relative frequency of non–Rh(D)-related
HDFN. 
Pathogenesis
By 30 days of gestation, fetal RBCs express antigens, half of which
are paternally derived and may be foreign to the maternal immune
system. The transfer of RBCs across the fetal–maternal interface
that occurs with spontaneous fetomaternal haemorrhage has been
demonstrated to occur in almost all gestations with increasing fre-
quency and volume as the pregnancy progresses.7 Although feto-
maternal haemorrhage before or during birth is thought to be the
primary factor in causing maternal alloimmunisation, a complete
list of potential precipitating events is listed in Table 40.1. The
exposure of the maternal immune system to the foreign paternal
antigen on the fetal RBCs is the impetus for the initiation of the
HDFN process.
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn
TABLE
40.1 Potential Causes of Alloimmunisation
Unprovoked Provoked
Idiopathic-spontaneous Termination of pregnancy
Delivery Amniocentesis
Spontaneous abortion Chorionic villus sampling
Ectopic pregnancy Cordocentesis
Abruption External cephalic version
Antepartum haemorrhage Fetal intervention: fetoscopy, shunt,
drainage
Trauma
Manual removal of placenta
  
Maternal Response
The maternal response to RBC foreign antigens is well documented.
Cells of the innate immune system identify and destroy foreign cells
and present the antigens to the humoural immune system, where
specialised cells, specifically B lymphocytes, can recognise the anti-
gen in future encounters and respond rapidly with the production
of IgG antibodies. The initial development of immunoglobulin (Ig)
G antibodies is a slow process, and it can take anywhere from 5
to 15 weeks for a human antiglobulin titre to be detected after a
sensitising event. With few exceptions, the maternal response to the
paternal antigen is not sufficient to have a significant effect on the
fetus in the sensitising pregnancy. Exposure in a subsequent gesta-
tion when the precipitating antigen is present will trigger the rapid
production of IgG antibodies. These IgG antibodies freely cross
the placenta and bind to fetal RBCs with the offending antigen.
These sensitised cells are then sequestered by the fetal spleen and
destroyed by macrophages, resulting in fetal anaemia.
The maternal response to paternal antigens on fetal RBCs
that have crossed the fetomaternal barrier is variable. Studies in
Rh(D)-negative men have demonstrated that some individuals
can be sensitised by an intravenous (IV) injection of as little as 0.1
mL of Rh(D)-positive blood, but 30% of individuals are still not
sensitised after serial IV injections of 10 mL and 5 mL of Rh(D)-
positive blood over a 6-month period.8,9 Volume is not the only
factor responsible for initiating a maternal response to the paternal
antigen on fetal RBCs. The maximum rate of sensitisation after
exposure to a full unit of Rh(D)-positive blood is only 80%.10
Other factors thought to play a role are the frequency of the expo-
sure, a protective effect of ABO incompatibility between the fetus
and the mother11 and the status of the maternal immune system
because immunodeficiency may prevent alloimmunisation.12  hypoalbuminaemia, neither of which develop hydrops despite low
Fetal Response
The severity of the fetal anaemia is primarily dependent on the
concentration of maternal antibody crossing the placenta into the
fetal compartment, but other factors also play a significant role.
The following factors are all known to affect the development and
severity of HDFN: (i) the subclass and glycosylation of the mater-
nal antibody; (ii) the structure, site density, maturational develop-
ment and tissue distribution of the fetal blood group antigens;
485
(iii) the efficiency of transplacental IgG transport; (iv) the func-
tional maturity of the fetal spleen; (v) polymorphisms affecting
the Fc receptor function and (vi) the presence of human leukocyte
(HLA)–related inhibitory antibodies.13 As an example, maternal
antibodies cannot cause fetal anaemia if they cannot cross the
placenta or if they are directed against an antigen that is poorly
expressed or not expressed on the fetal RBCs. All IgG antibodies
are freely transported across the placenta by pinocytosis; however,
the IgG1 subclass is more efficiently transported and can more
easily result in haemolysis of fetal RBCs. In contrast, IgM anti-
bodies, such as antibodies to the Lewis, I, and P blood groups,
do not cross the placenta and therefore do not result in HDFN.
Similarly, antibodies to the Cromer blood group bind to placen-
tal proteins, preventing their access to the fetal compartment and
protecting against the development of HDFN.14 Antibodies to
the Lutheran, Vel and Cartwright blood groups are also unlikely
to cause HDFN because these antigens are poorly developed on
the RBCs during fetal life. Finally, fetal sex may play a role in the
severity of HDFN. Ulm and colleagues demonstrated that Rh(D)-
positive male fetuses have a 13-fold increased risk for developing
hydrops and an odds ratio (OR) for perinatal mortality of 3.4
compared with Rh(D)-positive female fetuses.15
As described earlier, the fetus is not an innocent bystander in
the development of fetal anaemia. Extravascular haemolysis of
antibody-bound fetal RBCs occurs via phagocytosis by reticulo-
endothelial macrophages in the spleen and liver. In severe forms
of HDFN, intravascular haemolysis by direct lysis of antibody
bound fetal RBCs also occurs. Severe anaemia resulting in fetal
hydrops occurs when the fetal haemoglobin is greater than 7 g/
dL below the mean for the estimated gestational age. This is usu-
ally consistent with a fetal haematocrit less than 15% and a fetal
haemoglobin less than 5g/dL.6 Hydrops is associated with end-
stage disease and an increased likelihood of a poor fetal outcome.
Erythropoiesis is usually elevated in a fetus with the exception of
cases of Kell alloimmunisation. The Kell antibody destroys RBC
progenitor cells in the fetus and results in an earlier onset and
more severe anaemia than that found with other alloantibodies.
Hydrops is defined as fluid collections detected by ultrasound in
two or more of the following fetal compartments: (i) skin oedema,
(ii) ascites, (iii) pericardial effusion and (iv) pleural effusion. Plac-
entomegaly and polyhydramnios are also often included in the
diagnostic criteria. Ascites is usually the first finding, followed
later by the development of pleural effusions and finally scalp and
skin oedema. The exact mechanism for the development of fetal
hydrops is not understood. Lower serum albumin levels second-
ary to the decreased production of proteins by the haematopoi-
etically focused liver has been hypothesised to lead to decreased
colloid osmotic pressure, third spacing of fetal fluid and finally
the development of hydrops.16,17 This theory, however, is not
supported by animal models in which the fetal plasma proteins
have been replaced by saline or in human fetuses with congenital
serum colloid osmotic pressure.18,19 Proposed theories to explain
the pathophysiology of developing hydrops are (i) iron overload
from haemolysed RBCs leading to increased free radical formation
and the development of endothelial cell dysfunction,20 (ii) tissue
hypoxia from anaemia leading to increased capillary permeability
and (iii) elevated central venous pressures leading to the functional
blockage of draining lymphatics.17 The last theory is supported by
the fact that intraperitoneal transfusions (IPTs) are not as effec-
tive in hydropic fetuses. The development of hydrops is also ges-
tational age dependent. Hydrops in association with HDFN at
486 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
1p34
D gene CE gene
D gene CE gene
Homozygous
RhD positive
D gene CE gene
Heterozygous
RhD positive
D gene CE gene
RhD negative
(gene deletion)
D gene CE gene
RhD negative
(gene deletion
and ψ gene)
Stop codon
• Fig. 40.1  Schematic diagram of the Rhesus (Rh) gene locus on chro-
mosome 1. The four genotypes are shown: homozygous Rh(D) positive,
heterozygous Rh(D) positive, typical Rh(D) negative and the Rh(D) pseu-
dogene (Ψ). (From Creasy and Resnik’s Maternal-fetal Medicine: Principles
and Practice, 7th edn., 2014, Elsevier, Philadelphia, PA.)
less than 22 weeks’ gestation is rare despite the presence of severe
anaemia. Yinon and colleagues21 in their series found that 71% of
fetuses with haemoglobin less than 5 g/dL did not demonstrate
any evidence of hydrops before their first transfusion. The hyper-
bilirubinaemia resulting from the destruction of fetal RBCs does
not pose the same danger to the fetus as the neonate because the
placenta transports the bilirubin to the maternal side. Of specific
clinical importance, it is imperative for the physician managing
any HDFN case to continue to monitor the infant postnatally
for haemolysis because the presence of maternal antibodies in
the fetal circulation will continue to cause haemolysis after deliv-
ery and still poses a risk for poor neonatal outcomes.22 This is
compounded in a neonate who has received multiple antenatal
transfusions because her or his haematopoietic system will be sup-
pressed with minimal reticulocytosis at the time of birth.  cally Rh(D)-negative individuals, the Rh(D) gene is present but is
Rh(D) System
Background
The standard nomenclature for denoting a pregnant women’s
blood type is by identifying the ABO type and Rhesus (D) blood
type. Other antigens in the Rhesus system also exist, including the
C/c, E/e and G antigens. These antigens are all produced by two
genes. The RHD gene and the RHCE gene are both located on the
short arm of chromosome 1 (Fig. 40.1).23 A cytosine to thymine
change in exon 2 of the RHCE gene leads to expression of the C
antigen as opposed to the c antigen, and a single cytosine to gua-
nine change in exon 5 of the RHCE gene results in the expression
of the e antigen as opposed to the E antigen. A single amino acid
change in the extramembranous portion of the RhD and RhC
antigens results in the RhG phenotype.24
Often anti-D alloimmunisation is accompanied by a low level
of alloimmunisation to anti-C (anti-C titre < anti-D). When an
RhD-negative individual is found to have anti-D and anti-C at a
similar titre or when the anti-C titre exceeds the anti-D, the RhG
phenotype should be suspected.  can Americans, and the weak D phenotype was noted in 0.96%
TABLE Prevalence of Rh(D)-Negative Blood Type in
40.2 Different Ethnic Populations
Ethnicity % Rh(D) Negative
Basque 30–35
White: North American and 15
European
African American 8
African 4-6
Indian 5
Native North American and 1–2
Inuit Eskimo
Japanese 0.3
Thai 0.3
Chinese 0.3
  
Prevalence
The prevalence of Rh(D)-negative individuals varies greatly
among different populations with a high incidence of Rh(D)-
negative individuals in the Basque population (30%–35%) to
a relative scarcity of Rh(D)-negative individuals in the Chi-
nese population (0.3%) (Table 40.2). Of all Rh(D)-positive
individuals, 40% are homozygous (DD), guaranteeing that
any offspring with an alloimmunised partner will be at risk
for HDFN; the remainder are heterozygous (D-) and have a
50% chance of having no HDFN risk with an alloimmunised
partner. 
Rh Variants
A majority of Rh(D)-negative individuals result from a gene dele-
tion in both Rh(D) genes (see Fig. 40.1), but in some phenotypi-
either not translated or expressed. Alternatively, the RhD antigen
can be weakly or only partially expressed.25
The Rh(D) pseudogene is common in the African population
with 21% of African Americans and 69% of black South Africans
expressing it.26 The Rh(D) pseudogene contains all exons of the
normal Rh(D) gene, but there is a stop codon between exons 3
and 4 stopping the translation of the gene into mRNA and pre-
venting any Rh(D) protein from being expressed on the RBC
membrane (see Fig. 40.1). As a result, the patient is serologically
Rh(D) negative.
Depending on the reagent used, some patients undergoing
RhD typing will reveal a weak D expression. When the patient’s
RBCs are tested with an anti-D reagent, they react with no or
a 2+ or less reaction; when antihuman globulin is added, there
is then a moderate to strong reaction. Previously called Du posi-
tive, the term used today for this situation is weak D positive or
partial D. The two major explanations for this finding are either
a decreased number of intact RhD antigens on the surface of
the RBCs or the presence of RhD antigens that are missing epi-
topes (Fig. 40.2). Weak Rh(D) responses resulting from these
two phenotypes are seen in 0.3% of whites and 1.7% of Afri-
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn
of participants in a Canadian study.26 Although more than 200
mutations have been associated with alterations in the expression
of the RhD antigen, the majority of weak D phenotypes are types
1, 2 or 3. When exposed to RhD-positive RBCs, these types do
not become alloimmunised to RhD. Other types of Rh variants
can, however, form anti-D when exposed to the intact RhD anti-
gen that contains the epitopes they are missing. Alloimmunisation
leading to severe HDFN and fetal hydrops has been reported in
these individuals.27
Some patients may be tested in one laboratory (using a sensi-
tive reagent that detects the weak D phenotype) and told they
are RhD positive. Testing in a second laboratory may then report
a RhD-negative result. This can occur when the patient is being
considered as a blood donor and then is tested again later in a
pregnancy. In the United States, a guideline from the American
Association of Blood Banks (AABB) does not require that a test
for weak D or variant D be performed on Rh(D)-negative preg-
nant patients. These patients will be considered RhD negative
and be candidates for RhIg. Current guidelines from The Ameri-
can College of Obstetricians and Gynecologists (ACOG), how-
ever, recommend that patients with a serologic weak D result be
considered RhD positive. In an effort to reconcile these guide-
lines, a working group with representatives from the blood bank-
ing and obstetric community was formed to review the literature
and make new recommendations. The group recommended that
when a discrepant RhD typing was noted or a weak D type was
detected, the patient should undergo genotyping. If found to be
type 1, 2 or 3, she can be considered RhD positive, and RhIg is
not indicated.  identified. As stated earlier, the frequency and severity of HDFN
Other Rh Antigens
Rh(c) and Rh(E)
The ‘other’ Rh antibodies associated with HDFN are anti-(c) and
anti-(E). Although less frequent than anti-Rh(E) antibodies, anti-
Rh(c) antibodies have a virulence similar to anti-Rh(D) with IUT
required in 1% to 17%, neonatal transfusions needed in 10% to
30% and death in up to 10%28-32 of affected cases. A large series of
118 anti-Rh(c) pregnancies also confirmed its virulence with 10%
(n = 12) of the gestations affected with severe HDFN.33  and anti-B antibodies are also commonly present in individuals
= Normal RhD antigen = RhD antigen with missing epitope
Normal RhD
Decreased
RhD variant
RBC
expression of RhD
(mosaic) RBC
RBC
Weak D phenotype
(1.0% whites, 2.6% African descent,
2.7% Hispanics)
• Fig. 40.2 Weak D: Schematic illustration of the normal Rh(D)-positive
and weak D/partial D red blood cells (RBCs). (From Gabbe Obstetrics: Nor-
mal and Problem Pregnancies, 7th edn., 2016, Elsevier, Philadelphia, PA.)
487
Rh(G)
Rh(G) antigen expression results from a single amino acid
substitution in portions of the Rh(D) and Rh(C) proteins. A
majority of patients who are Rh(D) and Rh(C) positive are
also Rh(G) positive. If both Rh(D) and Rh(C) are absent, then
Rh(G) is also absent. In this instance, patients can become allo-
immunised to Rh(C) and Rh(G) without developing antibodies
to Rh(D), and the patient is still a candidate for anti-D immune
globulin. Of importance for the clinician is that when anti-
Rh(D) and anti-Rh(C) titres are similar, a laboratory evaluation
for anti-Rh(G) antibodies should be undertaken. An elevated
titre of anti-G has been associated with severe HDFN requiring
IUTs.34 
Non-Rh Antigens
The increasing use of RhIG and variations in the frequency of
Rh(D) negativity have led to an increasing frequency of HDFN
secondary to non-Rh(D) antigens. A prospective analysis of more
than 300,000 women in the Netherlands showed that anti-E anti-
bodies, followed in decreasing frequency by anti-D, anti-Kell and
anti-c antibodies, were the antibodies most commonly associated
with HDFN.33 The decreasing prevalence of anti-D antibodies
was also shown in a recent analysis of 638 Canadian women with
anti-E (43%), anti-c (13%), anti-Jka (9%) and anti-C (5%) anti-
bodies making up a majority of affected women, with anti-D anti-
bodies accounting for only 0.4% of women with a single antibody
for non-Rh(D) antibodies depends on the type of antibody and
the maternal and fetal response.
Naturally Occurring Antibodies
Antibodies to RBC antigens can develop without exposure to for-
eign blood. Epitopes present on microorganisms similar to those
found on RBCs can cause antibody formation in a process called
molecular mimicry. Anti-M and anti-N antibodies are two natu-
rally occurring antibodies, found in 2% to 3% of the population,
that result from maternal exposure to gut microorganisms. Anti-A
who lack the corresponding antigen secondary to exposure to gut
bacteria. These are mostly IgM and IgA antibodies that are not
transported across the placenta and thus do not pose a risk for
HDFN. 
Kell
The Kell antigen group is made up of the K, k, Kp(a), Kp(b),
Ko, Js(a) and Js(b) antigens, although it is rare for any but the
K antigen to cause HDFN. The K antigen is present in about
9% of whites and is responsible for 10% of cases of severe
HDFN. Alloimmunisation to the K antigen most often occurs
secondary to sensitisation during transfusion because testing
for the K antigen is not routinely performed outside of the
Netherlands and Australia31 before RBC transfusion. HDFN
with anti-K antibodies can be severe because anti-K causes
destruction of RBC precursors and maturing erythrocytes in
the bone marrow as well as the destruction of mature circulat-
ing fetal RBCs. A series of 19 patients sensitised to K resulted
in severe HDFN in 26%.33 Further complicating the manage-
ment of these patients is the fact that the anti-K titre correlates
488 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

poorly with the likelihood of fetal anaemia35-40 and that the P

severity of the anaemia can occur rapidly over the course of
1 week. As a result, fetal hydrops can occur earlier than with The P antigen group consists of the P1 and P2 antigens that are
other antibodies.35  present in 79% and 21% of whites, respectively. Individuals who
are P2 positive and P1 negative commonly produce anti-P1, which
does not cause HDFN because it is an IgM antibody. In the rare
Duffy case that a woman has the ‘p’ phenotype, she can produce anti-P1,
The Fy(a) and Fy(b) antigens are encoded by co-dominant alleles anti-P and anti-P(k) antibodies that have been associated not only
resulting in Fy(a+,b+), Fy(a+,b-), Fy(a-,b+) and Fy(a-,b-) combi- with severe HDFN but also with recurrent early pregnancy loss.50 
nations. Only the Fy(a) gene has been associated with HDFN,
resulting in mild to moderate disease in affected gestations.41 The ABO
importance of the Fy(b) allele, which is present in 82% of blacks,
is that it serves as the receptor for malaria.41  The A and B antigens are expressed co-dominantly, leading to the
A, B, AB and O blood types, with type O being the absence of
both the A and B antigens. IgM antibodies to the A and B antigens
MNS occur naturally after exposure to bacterial antigens in the gut, but
The MNS system includes M, N, S, s, U and 32 other rare anti- IgG antibodies to the A and B antigens also occur, especially in
gens. As stated earlier, anti-M and anti-N antibodies can occur type O mothers exposed to non-O type fetuses. Fetuses are rarely
naturally from exposure to gut bacteria and are present in 2% to affected by severe haemolysis secondary to the immature nature
3% of the population without prior exposure to blood. Anti-N of the A and B antigens in fetal life. The exception to this is the
can lead to mild haemolysis,42 but anti-M rarely causes any issues presence of the B antigen in African American fetuses in which the
for the fetus because it is typically an IgM antibody and thus not antigen is more developed at birth than in other populations.51-55 
transported transplacentally. Anti-M can, however, cause severe
HDFN if it develops as an IgG antibody in high titres.43-48 Anti- Other Rare Antigens
S, anti-s and U can all cause mild to moderate HDFN, and anti-
Mur, common in Southeast Asians, can also cause mild to severe The severity of reported cases of HDFN due to rare less frequent
disease42,49  RBC antigens can be found in Table 40.3. 

TABLE
40.3 Red Blood Cell Antibodies Associated With Haemolytic Disease of the Fetus and Newborn
Antigen Group Specific Antigen(s) Disease Severity
ABO A, B Mild
Chido-Rodgers Ch1, Ch2, Ch3, Ch4, Ch5, Ch6, WH, Rg1, Rg2 None
Colton Coa Moderate
Cob, Co3 Mild
Cromer Cra, Tca, Tcb, Tcc, Dra, Esa, IFC, WESa, WESb, UMC, GUTI, None
SERF, ZENA, CROV, CRAM
Diego Dia, Dib, Wra, ELO Moderate
Wrb, Wda, Rba, WARR, Wu, Bpa Moa, Hga, Vga, Swa, BOW, None
NFLD, Jna, KREP, Tra, Fra, SW1
Dombrock Doa, Dob, Gya, Hy, Joa, DOYA None
Duffy Fya Moderate
Fyb Mild
Fy3, Fy4, Fy5, Fy6 None
Forssman FOR None
Gerbich Ge3 Moderate
Ge2, Ge4, Wb, Lsa, Ana, Dha, GEIS None
Gill Gil None
Globoside PP1Pk Severe
H H Moderate
I I, i None
Indian Ina, Inb, INFI, INJA None
Junior Jra Mild (rare: severe)
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn 489
TABLE
40.3 Red Blood Cell Antibodies Associated With Haemolytic Disease of the Fetus and Newborn—cont’d
Antigen Group Specific Antigen(s) Disease Severity
John Milton Hagen JMH, JMHK, JMHL, JMHG, JMHM None

Kell K, k, Ku, Jsb Severe

Kpb Moderate
Kpa, Jsa, Ula Mild
K11, K12, K13, K14, K15, K16, K17, K18, K19, K20, K21, K22, K23, None
K24, VLAN, TOU, RAZ, KUCI, KANT, KASH, VONG, KALT, KTIM, KYO
Kidd Jka, Jkb Mild (rare: severe)
Jk3 Mild
Knops Kna, Knb, McCa, Sl1, Yka, Sl2, Sl3, KCAM None
Kx Kx None
Langereis Lan Mild (rare:moderate)
Landsteiner-Weiner LWa, LWab, LWb None
Lewis Lea, Leb, Leab, LebH, Aleb, Bleb None
Lutheran Lua Mild
Lub, Lu3, Lu4, Lu5, Lu6, Lu7, Lu8, Lu9, Lu10, Lu11, Lu12, None
Lu13, Lu14, Lu15, Lu16, Lu17, Aua, Aub, Lu20, Lu21
Mittenberger Mia Severe
Mib None
MNSs Vw, Mur, MUT Severe
U Moderate (rare: severe)
M Mild (rare: severe)
S, s, Mta, Mv Moderate
N, Hil, Or He, Mia, Mc, Mg, Vr, Me, Sta, Ria, Cla, Nya Hut, Far, sD, Mit, Mild
Dantu, Hop, Nob, Ena, EnaKT, ‘N’, DANE, TSEN, MINY, SAT ERIK, None
Osa, ENEP, ENEH, HAG, ENAV, MARS, ENDA, ENEV, MNTD
Ok Oka None
P1Pk P, P1, pk None
Raph MER2 None
Rhesus D, c, f, Ce, Cw, cE Severe
E, Hr0 Moderate (rare: severe)
EW,hrS, Tar, Rh32,HrB Moderate
C Mild (rare: severe)
G e, Cx, VS, CE, Bea, JAL Mild (rare: moderate)
V, Hr, CG, DW, c-like, hrH, Rh29, Goa, Rh33, hrB, Rh35, Evans, Mild
Rh39, Rh41, Rh42, Crawford, Nou, Riv, Sec, CELO, Dav, None
STEM, FPTT, MAR, BARC, JAHK, DAK, LOCR, CENR, CEST
RHAG Duclos, Ola, Duclos-like None
Scianna Rd Mild, rare moderate
SC2 Mild
SC1, SC3, STAR, SCER, SCAN None
Vel Vel Mild
Yt (Cartwright) Yta, Ytb None
Xg Xga Mild
CD99 None
Antigens not classified to a blood group

Cost Csa, Csb None

Er Era, Erb, ABTI None
High prevalence antigens Ata Mild
AnWj, Emm, MAM, PEL, Sda None
Low prevalence antigens HJK Severe
Kg, Sara Moderate
Chra, Bi, Bxa, Toa, Pta, Rea, Jea, Lia, Milne, RASM, JFV, JONES, HOFM, REIT None

  
490 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
Prevention of RhD Alloimmunisation
Anti-D immunoglobulin was historically obtained from the
pooled plasma of alloimmunised women. Ironically, the success
of anti-D prophylaxis has diminished the number of alloimmun-
ised women available for plasma donation, and the main supply
is now from male donors who undergo repeated injections of
Rh(D)-positive RBCs. More recently, a recombinant monoclo-
nal anti-RhD antibody has been developed and is currently being
evaluated in human clinical trials.56 The mechanism of action of
anti-D immunoglobulin is unknown. It has been theorised that
it binds to Rh(D)-positive RBCs, leading to their rapid clearance
from the maternal circulation. In addition, it downregulates the
maternal immune response to the Rh(D)-antigen.10,11
Before the development and implementation of routine post-
partum anti-D immunoglobulin prophylaxis, approximately 16%
of Rh(D)-negative women became sensitised after the delivery of
a Rh(D)-positive infant.57 Routine postpartum administration of
anti-D immunoglobulin reduced this rate to 1% to 2%, and the
rate of alloimmunisation was further reduced to 0.1% to 0.3%
with the addition of an anti-D immunoglobulin administration
in the third trimester.58
In the United States, the ACOG recommends the screen-
ing of all women for RBC antibodies at their first prenatal visit.
All Rh(D)-negative women without antibodies should receive
anti-D immunoglobulin at 28 weeks’ gestation and again in the
postpartum period unless the infant is demonstrated to be Rh(D)-
negative or the father of the baby is confirmed to be Rh(D)-
negative with assured paternity. In the Netherlands, 200 μg
of antenatal RhIG is administered at 30 weeks’ gestation; in Eng-
land, a dose of 100 μg is administered at 28 and 34 weeks’ gesta-
tion or a single dose of 300 μg at 28 weeks’ gestation.
Although the incidence of alloimmunisation prior to 28 weeks
is extremely low, the AABB recommends repeating the maternal
antibody titre prior to administration of anti-D immunoglobulin
at 28 weeks’ gestation. Screening would allow for the identifica-
tion of any sensitised pregnancy and allow for the monitoring and
possible intervention for a potentially anaemic fetus. The half-
life of anti-D immunoglobulin is 24 days, and case reports have
described maternal alloimmunisation that is thought to be sec-
ondary to waning antibody levels. As a result, some experts recom-
mend giving another RhIG dose if the patient has not delivered by
40 weeks’ gestation.59-61
Antenatal RhIG is unnecessary in the 38% of RhD-negative
pregnant women who carry a Rh(D)-negative fetus. Ethical and
clinical concerns have therefore been raised regarding the unneces-
sary exposure of these pregnant women to a plasma-based prod-
uct.62 Until recently, many European countries had not routinely
administered RhIG because of the limited availability of this
plasma-based product. However, cell-free DNA (cfDNA) testing
is now routinely used in Denmark, the Netherlands and parts of
Sweden to determine the fetal RHD status.63,64 Antenatal RhIG is
administered only to women with a RhD-positive fetus.
Fetomaternal haemorrhage is the main sensitising event for
Rh(D)-negative women. The Rh(D) antigen is expressed as early
as 38 days after conception, and this needs to be considered
when assessing risk factors for fetomaternal haemorrhage. With
the exception of biochemical pregnancies, all gestations meeting
criteria should be considered for anti-D immunoglobulin. Cur-
rent indications for anti-D immunoglobulin prophylaxis include
spontaneous or induced abortion, threatened abortion, invasive
diagnostic or therapeutic interventions, blunt abdominal trauma,
external cephalic version, ectopic pregnancy, fetal death in the sec-
ond or third trimester, antepartum haemorrhage in the second or
third trimester and molar pregnancies (see Table 40.1).65-71
The standard vial of anti-D immunoglobulin contains a 300-
μg dose (1 μg = 5 international units). This is sufficient immuno-
globulin to prevent maternal alloimmunisation after exposure to a
volume of 15 mL of Rh(D)-positive RBCs or the equivalent of 30
mL of fetal Rh(D)-positive whole blood. The fetal blood volume
in early gestation is small, with an estimated mean RBC volume at
12 weeks of 1.5 mL; the volume reaches a level of approximately
30 mL at 20 weeks’ gestation.72 A 50-μg dose of anti-D immu-
noglobulin is sufficient prophylaxis for first trimester events, and
a standard vial of 300 μg is sufficient for all inciting events up
until 20 weeks. After 20 weeks’ gestation, the degree of fetoma-
ternal haemorrhage needs to be quantified to ensure the appropri-
ate dosing of anti-D immunoglobulin is given.73 Most US centres
perform a rosette test74 as the initial screen for the presence of
fetal blood in the maternal circulation. If the test result is posi-
tive, a Kleihauer-Betke test or flow cytometry is recommended to
estimate the percentage of fetal cells in the maternal blood.75 The
result of the Kleihauer-Betke is then multiplied by 50 to represent
the average maternal blood volume of 5 L, and this result divided
by 30 to determine the number of anti-D vials needed to cover the
fetomaternal haemorrhage.
A special circumstance occurs when the RhG phenotype is
detected. Typically, anti-D and anti-C titres are found in equiv-
alent amounts. A determination should be undertaken to see if
anti-G and anti-C are present. If this is the case, the patient can
still become alloimmunised to RhD, and RhIg should be admin-
istered for the usual indications.
Women who demonstrate anti-D antibodies should not
receive anti-D immunoglobulin because it is not effective after an
immune response has been initiated.
Immune prophylaxis to prevent alloimmunisation to other
RBC antigens is currently not available because there is little
financial impetus for pharmacologic companies to develop immu-
noglobulins to these antigens. 
Diagnosis
Maternal antibody screening is recommended at the first prenatal
visit in all pregnancies. In a RhD-negative patient with no evi-
dence of alloimmunisation, a repeat antibody screen has been rec-
ommended by the AABB. The ACOG recommends that this be
left to the discretion of the obstetrician. Although late-onset RhD
alloimmunisation is rare,76-80 the physician will miss the opportu-
nity for intervention if repeat screening is not undertaken. How-
ever, a recent study suggested this practice was not cost-effective.81
A repeat antibody screen in all pregnant women is obtained at
27 weeks’ gestation as a national policy in the Netherlands. In
RhD-positive women, 25% of the severe cases of HDFN were
detected; the majority of these were related to anti-c.82 Risk fac-
tors for the occurrence of late onset anti-c antibody were previous
blood transfusion, parity and invasive prenatal diagnostic proce-
dures earlier in pregnancy.
Indirect Coombs Test
Identification of maternal antibodies is accomplished by perform-
ing the indirect Coombs test. In this test, maternal plasma is incu-
bated with indicator RBCs with known antigenicity. The RBCs are
then washed and suspended in antihuman globulin, or Coombs
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn
serum. Cells that are coated with maternal antibody will aggluti-
nate, resulting in a positive test result. The maternal plasma should
then undergo successive dilutions to determine the level of anti-
body response. The level of the antibody response is reported as
the reciprocal of the last dilution tube that demonstrates evidence
of agglutination. For example, a titre of 32 is equivalent to the loss
of the presence of agglutination at a 1:64 dilution of the maternal
plasma. The critical titre level necessitating further evaluation is a
titre equal to or greater than 16 and is discussed in detail later. It is
clinically important to note that interlaboratory differences in titre
levels exist because numerous laboratories use enzyme preparations
to treat the RBCs to allow the detection of low-level antibodies.
Because of the somewhat subjective determination of ‘agglutina-
tion’, intralaboratory variation in titre reporting also occurs. In
addition, indicator RBC reagents used in titre determinations have
a shelf life of 1 month, so titres performed more than 4 weeks
apart may be subject to reagent variation as well. This intrala­
boratory variation should always be within one dilution, and the
treating physician should be aware that a titre increase from 16
to 32 might not be indicative of an increased maternal immune
response. Finally, titres can be undertaken in tandem; frozen serum
saved from a previous titre determination can be thawed and ana-
lysed at the same time as the new maternal sample. This allows for
standardisation of testing because the same lot of indicator cells
and the same technologist will perform the two determinations. A
rise is titre in this situation can be interpreted as real.
A promising alternative for the quantification of maternal
antibodies that has gained widespread acceptance in the blood
banking community is the gel microcolumn assay (GMA). The
potential advantages of GMA are that it is less susceptible to inter-
and intralaboratory variability; it produces clear, objective and
stable results; it is quicker than current methods to perform; and
it can be automated. The disadvantages are that it may result in
higher titre values compared with traditional tube tests by 1 to 2
dilutions,83-85 although higher variation has been reported.85,86 At
present, more correlation between the GMA titre and severity of
disease is needed before this assay can be used to safely manage
alloimmunisation in pregnancy.  4,5,7 and 10 on the Rh(D) gene are recommended.26,64,89,91-93
Management of First Affected Pregnancies
Because it takes time for the humoural immune system to develop
an antibody response, first affected alloimmunised pregnancies
differ from subsequent ones in the fact that they usually have
lower antibody titres, and severe fetal anaemia may not develop or
does not develop until late in gestation.
Paternal Zygosity
After identifying the presence of a maternal antibody, it is impera-
tive to assess if the fetus is at risk for carrying the offending anti-
gen. If the father is homozygous for the antigen, the fetus is at
risk for HDFN, but if the father is heterozygous, there is a 50%
chance that the fetus will not be at risk if proven to be antigen
negative. Correct ascertainment of paternity is also important and
is the underlying premise of accurate paternal zygosity testing.
Nonpaternity is estimated to occur in 2% to 5%87 of cases, and
thus the subject should be addressed with the patient in a confi-
dential fashion.88
The majority of RBC antigens are inherited via co-dominant
alleles and the genotype of the allele can be determined by sero-
logic testing of the RBCs. For example, in the case of the E/e
491
antigen, an individual can be E/E, E/e or e/e. By using anti-E
and anti-e reagents, the E/e genotype can be easily determined
by blood testing for the individual in question. The determina-
tion of Rh(D) zygosity is not as simple because the absence of
the D antigen is due to a gene deletion; there is no ‘d’ antigen
and thus no anti-d reagent. An Rh(D)-negative father will only
have Rh(D)-negative children with a Rh(D)-negative partner, but
an Rh(D)-positive father poses a more complex challenge when
attempting to determine zygosity. In the past, antisera to all the
Rh antigens (D, C, c, E, e) and gene frequency tables based on
race and ethnicity were used to estimate paternal zygosity for
Rh(D). More accurate determination of Rh(D) zygosity is now
accomplished by performing quantitative polymerase chain reac-
tion (PCR) and comparing the amplitude of the DNA peaks for
the D gene with that of the RhCE gene which is present as two
copies in all individuals.89 
Fetal Genotype Testing
If paternal zygosity testing demonstrates that the fetus is at risk
for carrying the offending antigen, then fetal blood genotyp-
ing should be performed. Historically, this was accomplished by
amniocentesis and determination of the fetal blood type by test-
ing of the amniocytes. The development of cfDNA testing has
changed this approach for Rh(D)-negative women.
It is estimated that 10% to 15% of the cfDNA in the maternal
circulation in the late first and early second trimesters is of fetal
origin.90 The placenta is the main source of fetal cfDNA, and its
presence is the basis for noninvasive prenatal testing for aneuploi-
dies. Fetal cfDNA can be detected as early as 38 days and increases
with advancing gestation, rapidly disappearing after delivery. The
fetal Rh(D) status can be determined by evaluation of the fetal
cfDNA sequences in the maternal plasma by using reverse tran-
scriptase PCR with a 99.1% sensitivity for detection of the fetal
Rh(D) status after the first trimester. Testing should be performed
after 10 weeks’ gestation to ensure an adequate fetal fraction of
cfDNA, and combinations of assays for the detection of exons
If these exons are not present, then the fetus is determined to
be Rh(D)-negative, and no further testing for fetal anaemia is
required. This is based on the assumption that fetal cfDNA is pres-
ent in the sample. This can be confirmed by the identification of
the Y chromosome gene sequence denoting the presence of a male
fetus, or additionally, single nucleotide polymorphisms (SNPs)
can be used to determine that the sample is from a fetal source.94
Maternal SNPs are identified from the buffy coat of maternal
white blood cells, and these are compared with the SNPs from
the fetal cfDNA sample. If there are differences between the two
samples, it confirms that the cfDNA is from a fetal source because
the variance in the SNPs is secondary to paternal origin. Another
method of confirming fetal origin of the cfDNA is to identify the
hypermethylated RASSF1A promoter that has been reported as a
universal marker for the presence of fetal cfDNA.95-97 Assays for
Kell, Rh(E), Rh(C) and Rh(c) fetal blood typing are available in
Europe but are not yet available in the United States.98-100
False-positive results for fetal cfDNA have been reported and are
thought to be secondary to Rh(D)-negative individuals who carry
the previously discussed RHD pseudogene and who have passed
the trait on to the fetus.101 The RHD pseudogene can be detected
using primers targeting exons 4 and 10 of the RHD gene. A false-
positive fetal cfDNA result can also occur in the case of a vanishing
twin with remaining cfDNA in the maternal circulation.102
492 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
Conversely, a false-negative fetal cfDNA result can lead to the
omission of surveillance of an at-risk pregnancy. A false-negative
test resulting from a low fetal fraction can be avoided by obtain-
ing the sample after 10 weeks’ gestation and ensuring appropri-
ate sensitivity of laboratory equipment.103 To minimise this risk
for a false-negative result, it is recommended that more than one
Rh(D) region be examined to avoid missing the presence of a
Rh(D) variant.
In situations in which cfDNA testing is not available for fetal
testing, PCR of uncultured amniocytes obtained from an amnio-
centesis performed after 15 weeks’ gestation can be used to deter-
mine the fetal antigen status. All efforts should be made to avoid a
transplacental approach because this has the potential to heighten
the maternal immune response and increase the severity of allo-
immunisation.104 In addition, chorionic villus biopsy should be
avoided for fetal genotype testing because of similar concerns. 
Monitoring for Fetal Anaemia
After it has been determined that a fetus is at risk for HDFN,
maternal antibody titres should be repeated at monthly intervals.
The same laboratory should be used to ensure consistent results,
and titres that are below a critical threshold but rising should be
repeated at shorter intervals.
The critical titre is defined as the titre at which a fetus is at
risk for developing severe anaemia and hydrops and denotes when
fetal ultrasound interrogation should be initiated. The relative
infrequency of alloimmunisation precludes individual institutions
from having their own patient and laboratory based critical titres;
therefore, most units use a critical titre of either 16 or 32 for RBC
alloimmunisation cases other than those secondary to anti-Kell.
Because of the erythroid precursor suppression that occurs with
Kell alloimmunisation, a critical titre of 8 should prompt the ini-
tiation of fetal observation.35,105,106 As stated earlier, if the fetal
blood type has not been determined, amniocentesis or cfDNA for
the confirmation of the fetal genotype should be considered as
titres can rise even when the fetus is antigen negative.
A higher titre does not correlate linearly with the risk for
HDFN or the severity of fetal anaemia. This is the rationale for
additional fetal testing. Doppler interrogation of the middle cere-
bral artery peak systolic velocity (MCA-PSV) has become the
mainstay of the evaluation for fetal anaemia from any cause.107-
112 The principle of MCA-PSV Doppler interrogation is that the
anaemic fetus preserves the brain by increasing cerebral blood
flow. The low-viscosity blood, combined with the increased car-
diac output, results in an increased PSV. Mari et al. demonstrated
in a review comparing MCA-PSV with haemoglobin levels
obtained via fetal blood sampling that an MCA level greater than
1.5 MoM predicted moderate or severe anaemia with a sensitivity
of 100% (95%confidence interval, 86%–100%) with or with-
out hydrops with a false-positive rate of 12%.108 A subsequent
multicentre study compared the MCA-PSV with amniotic fluid
assessment for bilirubin using the Liley and Queenan ΔOD450
curves.110 MCA-PSV was more accurate than serial amniotic
fluid assessment for ΔOD450, which was considered the standard
for diagnosis at that time. Since then, MCA-PSV has become
widely accepted as the diagnostic tool to detect fetal anaemia.
Initiation of MCA-PSV evaluation should start at 20 weeks in
a first affected gestation. The case of Kell alloimmunisation evalua-
tion should be initiated as early as 18 weeks’ gestation. The MCA-
PSV increases throughout gestation and calculators to determine
the MoM are available at www.perinatology.com. The optimal
• Fig. 40.3  Ultrasound image showing the optimal method for obtaining
a middle cerebral artery peak systolic velocity Doppler. The pulsed wave
Doppler cursor should be placed at the position marked by the arrow.
(From Creasy and Resnik’s Maternal-fetal Medicine: Principles and Prac-
tice, 7th edn., 2014, Elsevier, Philadelphia, PA.)
interval for examinations has not been determined, but expert
opinion is that assessments should be every 1 to 2 weeks and can
be guided by both the previous MCA-PSV level and the antibody
titre.108 The technique for obtaining the MCA-PSV is important.
The Doppler angle of interrogation should be as close to zero
degrees as possible, although angle correction can be used up 30
degrees113 with good correlation with the true velocity. Addition-
ally, the Doppler cursor should be as close to the origin of the ves-
sel from the carotid siphon as possible, and the fetus should be in
a quiescent state as activity can falsely elevate the result114,115 (Fig.
40.3). Values that remain below 1.5 MoM are consistent with
the absence of moderate to severe fetal anaemia, and intervention
is not necessary. Although there are no studies on the validity,
type or frequency, weekly antenatal testing (biophysical profile or
nonstress testing) maybe initiated at 32 weeks’ gestation in these
patients in conjunction with MCA-PSV measurements.116 These
patients should also have a scheduled delivery between 37 and
38 weeks as per the Society for Maternal Fetal Medicine guide-
lines.117 MCA-PSV measurements above 1.5 MoM before 35
weeks’ gestation should be investigated with fetal blood sampling
with the intention to transfuse if fetal anaemia is confirmed. After
35 weeks’ gestation, delivery is preferred to fetal blood sampling
because of the risk-to-benefit ratio in favour of delivery compared
with prematurity. 
Management of Previously Affected
Pregnancies
The innate immune system becomes more efficient at responding
to a foreign antigen over time secondary to the maternal amnestic
antibody response. As a result, alloimmunised mothers have the
potential for increasingly severe haemolytic disease with each sub-
sequent gestation. In susceptible fetuses, the anaemia will usually
occur earlier than in prior gestations with case reports demonstrat-
ing the onset of severe anaemia as early as 15 weeks.118
Determination of the risk to the pregnancy still must be
undertaken in the case of a known heterozygous partner or with
new paternity. If paternity is assured and the father tests negative
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn 493

TABLE Nomogram of Predicted Middle Cerebral Artery Peak Systolic Velocity at the 5th, 10th, 90th and 95th
40.4 Percentiles and 1.5 MoM for 11 to 18 Weeks’ Gestational Age

MCA PSV, CM/S

GA (wk) 5th 10th 50th 90th 95th 1.5 mom
11 3.2 5.2 10.7 15.2 19.5 16.1
12 4.5 6.5 12.1 16.9 21.2 18.2
13 5.8 7.8 13.6 18.5 22.8 20.3
14 7.1 9.0 15.0 20.2 24.5 22.4
15 8.4 10.3 16.4 21.8 26.1 24.5
16 9.8 11.5 17.8 23.4 27.7 26.7
17 11.1 12.8 19.2 25.1 29.4 28.8
18 12.4 14.1 20.6 26.7 31.0 30.9

Adapted from Tongsong T, Wanapirak C, Sirichotiyakul S, et al. Middle cerebral artery peak systolic velocity of healthy fetuses in the first half of pregnancy. J Ultrasound Med of 26:1013–1017, 2007.
  

for the involved RBC antigen, then no further testing is required.
Fetal testing with cell-free fetal DNA or amniocentesis should
be undertaken as previously described to document if the fetus
is at risk for developing anaemia in the case of a known hetero-
zygous partner. In at-risk pregnancies with a previous need for
IUT, postnatal exchange transfusion or a history of a perinatal
loss secondary to HDFN, early evaluation in an experienced,
qualified centre with the initiation of weekly MCA-PSV Dop-
plers from as early as 16 weeks should be considered. MCA-PSV
calculators that are available, however, do have normative val-
ues for less than 18 weeks’ gestation. Tongsong and associates
have published data on early normal MCA-PSV measurements
in prenatal patients being screened for fetal anaemia secondary
to α-thalassemia (Table 40.4).119 These values may be helpful
in assessing the MCA-PSV before 18 weeks’ gestation. Maternal
titres are less predictive of disease severity in these cases, although
they can be used to adjust the screening intervals or, as will be
discussed later, the initiation of adjuvant therapies in severely
affected cases. The remaining management is identical to that of
first affected gestations, with the exception that rarely will these
pregnancies not require treatment with IUT at some point in
gestation. An algorithm for the management of alloimmunised
pregnancies is presented in Figure 40.4.
Immunomodulation
Pregnant patients presenting with a history of an early second tri-
mester loss secondary to HDFN pose a unique challenge. Even
with advances in ultrasound technology, IV fetal blood sampling
and transfusion are technically possible only after 18 weeks’
gestation with loss rates 10-fold higher than with procedures
undertaken after 22 weeks’ gestation.21 In early-onset, severe allo-
immunisation cases, immunomodulation, consisting of plasma-
pheresis followed by weekly IV immunoglobulin (IVIG) has been
shown to be effective in delaying the gestational age at which fetal
transfusion needs to be initiated to result in improved fetal sur-
vival.120,121 Ruma and coworkers treated nine patients, seven of
whom had a previous perinatal loss, at 12 weeks’ gestation with
plasmapheresis every other day for a total of three procedures. This
treatment was followed by the administration of IVIG 1 g/kg on
two subsequent days and then weekly IVIG 1 g/kg until 20 weeks’
gestation. All nine gestations required IUTs, but these were initi-
ated on average 3 weeks later, and the fetuses had a haematocrit
65% higher at the time of the first procedure compared with the
index gestation. Importantly, all nine fetuses survived.120
When severe anaemia is suspected before 18 weeks’ gestation,
the use of IPTs with or without immunomodulation may allow for
stabilisation of the fetus until fetal blood sampling and IV trans-
fusion can be undertaken at 18 to 20 weeks’ gestation. Fox and
coworkers reported six pregnancies, four with previous perinatal
losses, that were treated with biweekly IPTs starting at 15 weeks’
gestation. A total RBC volume of 5 mL of packed RBCs was used
up to 18 weeks’ gestation, and 10 mL was given thereafter. Four
of the patients also received IVIG 0.8 g/kg/week. They reported
fetal survival in five of the six gestations.122 The main limitation to
the efficacy of IPT in the setting of fetal anaemia is the presence
of hydrops that prevents the efficient absorption of RBCs from
the peritoneal cavity secondary to a functional disruption to the
lymphatic system. 
Intervention
Intrauterine Transfusion
When the MCA-PSV demonstrates evidence of severe fetal anae-
mia a fetal blood sampling, with the intention to transfuse if fetal
anaemia is confirmed, should be undertaken. Procedures should
be performed in or proximal to the operating theatre, especially
after a viable gestational age is attained. Conscious sedation can be
used to alleviate patient anxiety. Preoperative prophylactic antibi-
otics with a first-generation cephalosporin and tocolysis are used
by some centres, although there are no studies to support their
routine use. Direct access to an automated haemocytometer to
rapidly determine the fetal levels of haemoglobin and haematocrit
is optimal.
In the case of anterior placentation, the ideal target for IV
blood sampling and transfusion is the umbilical vein at the cord
insertion site. Veins are less likely to undergo vasospasm after
needle insertion, and the umbilical cord insertion site is the least
mobile portion of the cord. In cases of posterior placentation, the
494 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

First affected Previous affected demonstrated an 80% reduction in the incidence of perinatal
pregnancy pregnancy complications.123 Some centres use vecuronium (0.1 mg/kg of
estimated fetal weight by ultrasound); others use atracurium (0.4
mg/kg). The total volume of RBCs to be transfused can be deter-
Repeat titre every month until No need mined using formulas based on the estimated fetal weight and the
24 weeks’ gestation; repeat for titre haematocrit of the donor blood. If the haematocrit of the donor
titre q 2 weeks thereafter
unit is approximately 75% to 80%, multiplying the fetal weight in
grams by a coefficient of 0.02 will indicate a volume in millilitres
of donor RBCs to be transfused to increase the fetal haematocrit
Titre remains Titre above critical value, by 10%.124 After completing the infusion of the RBC volume to
below critical e.g. ≥32 for anti-D and be transfused, a final fetal blood sample should be drawn for a hae-
value, e.g. ≤16 other antibodies; ≥8 for Kell matocrit and Kleihauer-Betke stain. A rapid determination of the
fetal haematocrit will allow for assurance that the desired target
haematocrit has been achieved before removal of the procedure
Deliver at Determine fetal antigen status
term based on paternity, paternal needle. The final Kleihauer-Betke stain result helps to determine
zygosity testing and cffDNA the interval until the next transfusion. When this shows a pre-
testing (RhD) or dominance of adult haemoglobin-containing cells, fetal haemato-
Antigen amniocentesis for fetal DNA poiesis has been suppressed, and the interval between procedures
negative (other RBC antigens)
can be extended.
The amount of RBCs to be transfused to a severely anaemic
(haematocrit ≤10%) fetus before 24 weeks’ gestation should be
24 weeks’ Antigen 16 weeks’ limited to prevent cardiovascular strain resulting from a sudden
gestation positive gestation increase in blood viscosity. The final haematocrit should not be
increased by more than fourfold over the initial value.125 A repeat
procedure in 48 hours is undertaken to achieve the usual target
Do serial fetal MCA fetal haematocrit. 
Doppler every 1–2 weeks
Transfusion Interval
The timing of subsequent transfusions is an area of ongoing debate
Peak MCA velocity Peak MCA velocity and research. Some centres use empiric intervals based on previous
<1.5 MoM ≥1.5 MoM
experience such as 10 to 14 days between the first two transfu-
sions, 2 to 4 weeks between the second and third transfusions, and
Cordocentesis to 3 to 5 weeks between subsequent procedures. However, continued
determine fetal Hct transfusions are necessary because of the shorter life span of adult
RBCs in the fetal circulation and the increased vascular volume of
the growing fetus in the face of suppressed haematopoiesis.126-128
The use of the MCA-PSV to determine the timing of subse-
Fetal Hct Fetal Hct quent transfusions is used by some centres. Concerns about the
≤30% >30%
accuracy of this modality arise because of the differences in the size
of the adult RBCs compared with fetal RBCs and the improve-
Intrauterine Repeat ment in fetal haemodynamics caused by optimal oxygen delivery
transfusion cordocentesis to peripheral tissues. Detti and associates evaluated the MCA-
in 1–2 weeks PSV for the evaluation of moderate to severe between the first and
second IUTs. They demonstrated that a threshold of 1.32 MoM
Begin antenatal Deliver by 37–38 predicted all cases of moderate anaemia with a false-positive rate
testing at 32 weeks weeks of 37%, but using a threshold of 1.69 MoM predicted all cases of
• Fig. 40.4  Algorithm for the management of alloimmunised pregnancies. severe anaemia with a false-positive rate of only 6%.129 The accu-
cffDNA, Cell-free fetal DNA; Hct, haematocrit; MCA, middle cerebral artery; racy of the MCA-PSV for the detection of moderate to severe anae-
RBC, red blood cell. (From Creasy and Resnik’s Maternal-fetal Medicine: mia after the second transfusion has not been verified.130 There is
Principles and Practice, 7th edn., 2014, Elsevier, Philadelphia, PA.) currently a multicentre trial in Australia evaluating the MCA-PSV
as a predictor of the need for subsequent transfusions (Australian
placental cord insertion is used by most US centres, but Euro- New Zealand Clinical Trials Registry, #ACTRN1208000643370;
pean centres favour the haepatic portion of the umbilical vein. www.anzctr.org.au).
Needling of a free-floating loop of cord has been associated with In the absence of using the MCA-PSV, another method of
a threefold increase in perinatal loss or the need for emergency determining the transfusion interval is based on a theoretical
delivery because of fetal bradycardia.123 expected decline in the fetal haemoglobin between procedures.
After IV access is obtained, a sample of fetal blood should be A haemoglobin decline from the final value at the conclusion
sent to determine the initial haematocrit and reticulocyte count. of the first intrauterine procedure of 0.4 mg/dL/day, 0.3 mg/
After collection of the initial blood sample, injection of a paralytic dL/day after the second IUT and 0.2 mg/dL/day after the third
agent to prevent fetal dislodgement of the needle has been shown IUT can be used to estimate the interval necessary to prevent
to improve perinatal outcome. A reported logistic regression the development of severe anaemia in the fetus.130 These values
 CHAPTER 40  Haemolytic Disease of the Fetus and Newborn
correspond roughly to a daily decline in haematocrit of 1%. In
the case of fetal hydrops, however, the decline in fetal haemato-
crit has been demonstrated to be 1.88% per day compared with
1.08% per day.131  and these infants are usually born with absent reticulocytosis.
Complications
Procedure-related complications, apart from procedure-related
losses, can occur when performing IV transfusions. Transient
fetal bradycardia is the most commonly associated procedural
complication, but the need for caesarean delivery (2% per pro-
cedure), postprocedure infection (0.3% per procedure) and
premature rupture of membranes (0.1% per procedure) also
occur. The Netherlands group reported a total procedure-related
complication rate, including procedure related fetal losses, of
3.1% (23 of 740), with both higher procedure related com-
plications (3.8% vs 2.9%) and procedure-related death rates
(2.5% vs 1.4%) in hydropic fetuses.123 Other potential com-
plications from IV transfusion include exsanguination, fetal
needle injuries, arterial spasm, fetal brain injury and worsen-
ing of alloimmunisation from procedure-induced fetomaternal
haemorrhage.132-134
The procedural complications with IPTs can be similar to
those from IV transfusion, with the exception that complications
associated with the umbilical cord (bradycardia, vasospasm and
exsanguination) are less common. Watts and coworkers reported
on their series of 77 IPTs in 35 gestations and demonstrated 5
complications that included 2 transfusions into the fetal bowel,
2 retroperitoneal transfusions and 1 fetal abdominal wall haema-
toma.135 There were no urgent deliveries performed and no fetal
deaths reported in their series.
In high-volume centres, neonatal survival rates after perform-
ing intravascular IUTs are excellent. Overall survival rates of 94%
for nonhydropic fetuses and 74% for hydropic fetuses have been
reported.135 Zwiers and colleagues reported on 1678 procedures
performed in 589 fetuses in The Netherlands between 1988
and 2014.123 This is the largest single series in the literature and
included 798 umbilical vein at the cord insertion, 552 intrahae-
patic umbilical vein, 280 transamniotic umbilical vein (insertion
at posterior placenta or free loop), 24 umbilical artery, 10 intra-
peritoneal and 1 intracardiac transfusion. The study demonstrated
a 93% overall survival rate with a survival of 78% in the presence
of hydrops. The overall perinatal loss rate decreased from 1.6%
per procedure in the first half of the study period to 0.6% from
2001 onwards.
The need for transfusion before 20 weeks of gestational age
has also been identified as a risk factor for decreased fetal sur-
vival after IUT.123,136 The higher loss rate in this group reflects the
increased technical difficulties associated with cannulating smaller
vessels and the fetus’s decreased ability to tolerate the haemody-
namic changes after transfusion.21,136,137 IPTs have been suggested
in these earlier gestational ages when severe anaemia is suspected
based on the MCA-PSV.  pies, if proven efficacious, would negate the need for IUTs.
Timing of Delivery
Most centres will continue serial IUTs until 35 weeks’ gestation.
Delivery is then scheduled 3 weeks later. Caesarean section is
reserved for standard obstetric indications. A 10-day course of
maternal phenobarbital (30 mg orally three times daily) has been
suggested in one retrospective analysis to reduce the need for neo-
natal exchange transfusion by almost 80%.138  identified and evaluated appropriately in centres with experience
495
Neonatal Issues
Successful serial IUTs lead to suppression of fetal erythropoiesis,
IVIG is often used in neonates with HDFN in an attempt to
prevent further haemolysis and thus decrease levels of bilirubin.
A randomised study that included 53 newborns who had been
treated with IUTs failed to find that IVIG after birth decreased
the maximum bilirubin level, number of days of phototherapy or
number of exchange transfusions.139
In addition to the hypoproliferative anaemia in these neonates,
donated RBCs age and are removed from the circulation, con-
tributing to a progressive anaemia over the first few weeks after
birth. This is exacerbated by the persistence of maternal antibod-
ies in the newborn circulation for weeks after delivery that will
continue the haemolysis of any newly produced RBCs. So-called
‘top-up’ transfusions may be needed in the first 1 to 3 months
after delivery.140 It is imperative that the newborn be monitored
closely during this period to prevent a severe and potentially fatal
anaemia from developing. Weekly reticulocyte and haematocrit
levels should be monitored until the reticulocyte count is noted to
increase for 2 consecutive weeks. Threshold haematocrit values of
less than 30% in symptomatic infants or less than 20% in asymp-
tomatic infants have been suggested for transfusion. Iron therapy
will not enhance neonatal erythropoiesis because iron stores are
excessive from ongoing in utero haemolysis.141
Supplementation with folate (0.5 mg/day) may be helpful. The
use of neonatal IVIG has not been shown to decrease the need or
timing of top-up transfusions. Zuppa and associates used a course
of 400 U/kg/d of erythropoietin in neonates after IUT. Although
there was an increase in reticulocytosis, many of the infants still
required a top-up transfusion.142 One of the main concerns sur-
rounding survivors of IUTs is the potential for long-term neu-
rologic morbidity resulting from severe intrauterine anaemia.
The LOTUS trial (long-term follow-up after IUTs) evaluated the
incidence and risk factors for neurodevelopmental impairment in
291 children who were treated with IUTs for HDFN. The average
follow-up period was 8.2 years (range, 2–17 years). The authors
found an incidence of severe developmental delay that was similar
to that found in the general Dutch population (3.1% vs 2.1%).
However, the incidence of cerebral palsy was higher (2.1% vs
0.7%).143 
Future Therapy
Although anti-D immunoglobulin has decreased the incidence
of HDFN, therapies to reduce the incidence of non-Rh(D) allo-
immunisation and methods to prevent anaemia in at-risk allo-
immunised gestations are being evaluated. Therapies that may
prevent the transplacental transfer of maternal antibody or that
blunt the maternal immune response have been evaluated in an
attempt to decrease the need for invasive therapy.144 Such thera-
 
Conclusion
Haemolytic disease of the fetus and newborn is an example of
modern medicine both preventing a disease and improving out-
comes for those who are affected.
Patients with a positive antibody screen result may be at risk
for fetal anaemia and immune-mediated hydrops and need to be
496 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

in HDFN. Evaluation of the father and the fetus will determine
if further monitoring is necessary in the gestation. Fetal testing
can be via cfDNA or amniocentesis, and pregnancies that are at
risk should be followed with serial maternal antibody titres until
a critical titre is reached. MCA-PSV screening for fetal anae-
mia should then be initiated, and a, MCA-PSV greater than 1.5
MoM should prompt referral to a centre with expertise in fetal
blood sampling and IUT. Although the aforementioned algo-
rithm can prevent a majority of fetal losses, the development of
therapies such as immunomodulation may improve outcomes
even further.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References damage, and rhesus haemolytic disease. Lancet.
1990;335:933–936.
40. Weiner CP, Widness JA. Decreased fetal
erythropoiesis and hemolysis in Kell
21. Yinon Y, Visser J, Kelly EN, et al. Early intra- hemolytic anemia. Am J Obstet Gynecol.
1. Fairweather DV, Tacchi D, Coxon A, et  al.
uterine transfusion in severe red blood cell 1996;174:547–551.
Intrauterine transfusion in Rh-isoimmuniza-
alloimmunization. Ultrasound Obstet Gynecol. 41. Miller LH, Mason SJ, Clyde DF,
tion. Br Med J. 1967;4:189–194.
2010;36:601–606. McGinniss MH. The resistance factor to
2. Bevis DC. Blood pigments in haemolytic dis-
22. Gurevich P, Erina S, Gershon S, Zusman I. Plasmodium vivax in blacks. The Duffy-
ease of the newborn. J Obstet Gynaecol Br Emp.
The role of the fetal immune system in the blood-group genotype, FyFy. N Engl J Med.
1956;63:68–75.
pathogenesis of RhD-hemolytic disease of 1976;295:302–304.
3. Walker AH. Liquor amnii studies in the pre-
newborns. Hum Antibodies. 1997;8:76–89. 42. Bakhtary S, Gikas A, Glader B, Andrews J.
diction of haemolytic disease of the newborn.
23. Chérif-Zahar B, Mattéi MG, Le Van Kim C, Anti-Mur as the most likely cause of mild
Br Med J. 1957;2:376–378.
et  al. Localization of the human Rh blood hemolytic disease of the newborn. Transfusion.
4. Liley AW. Diagnosis and treatment of
group gene structure to chromosome region 2016;56:1182–1184.
erythroblastosis in the fetus. Adv Pediatr.
1p34.3-1p36.1 by in situ hybridization. Hum 43. De Young-Owens A, Kennedy M, Rose RL,
1968;15:29–63.
Genet. 1991;86:398–400. et  al. Anti-M isoimmunization: manage-
5. Liley AW. Intrauterine transfusion of foetus in
24. Avent ND, Reid ME. The Rh blood group sys- ment and outcome at the Ohio State Uni-
haemolytic disease. Br Med J. 1963;2:1107–
tem: a review. Blood. 2000;95:375–387. versity from 1969 to 1995. Obstet Gynecol.
1109.
25. Wagner FF, Gassner C, Muller TH, et  al. 1997;90:962–966.
6. Green GH, Liley AW, Liggins GC. The Place
Molecular basis of weak D phenotypes. Blood. 44. Furukawa K, Nakajima T, Kogure T, et  al.
of foetal transfusion in haemolytic disease. a
1999;93:385–393. Example of a woman with multiple intrauter-
report of 22 transfusions in 16 patients. Aust
26. Singleton BK, Green CA, Avent ND, et  al. ine deaths due to anti-M who delivered a live
N Z J Obstet Gynaecol. 1965;5:53–59.
The presence of an RHD pseudogene contain- child after plasmapheresis. Exp Clin Immuno-
7. Medearis AL, Hensleigh PA, Parks DR,
ing a 37 base pair duplication and a nonsense genet. 1993;10:161–167.
Herzenberg LA. Detection of fetal erythro-
mutation in Africans with the Rh D-negative 45. Kanra T, Yuce K, Ozcebe IU. Hydrops fetalis
cytes in maternal blood post partum with the
blood group phenotype. Blood. 2000;95:12– and intrauterine deaths due to anti-M. Acta
fluorescence-activated cell sorter. Am J Obstet
18. Obstet Gynecol Scand. 1996;75:415–417.
Gynecol. 1984;148:290–295.
27. Cannon M, Pierce R, Taber EB, Schucker 46. Wikman A, Edner A, Gryfelt G, et  al. Fetal
8. Pollack W, Ascari WQ, Kochesky RJ, et  al.
J. Fatal hydrops fetalis caused by anti-D in hemolytic anemia and intrauterine death
Studies on Rh prophylaxis. 1. Relationship
a mother with partial D. Obstet Gynecol. caused by anti-M immunization. Transfusion.
between doses of anti-Rh and size of antigenic
2003;102:1143–1145. 2007;47:911–917.
stimulus. Transfusion. 1971;11:333–339.
28. Bowell PJ, Brown SE, Dike AE, Inskip MJ. 47. Duguid JK, Bromilow IM, Entwistle GD,
9. Zipursky A, Israels LG. The pathogenesis and
The significance of anti-c alloimmuniza- Wilkinson R. Haemolytic disease of the
prevention of Rh immunization. Can Med
tion in pregnancy. Br J Obstet Gynaecol. newborn due to anti-M. Vox Sang. 1995;68:
Assoc J. 1967;97:1245–1257.
1986;93:1044–1048. 195–196.
10. Kumpel BM. On the mechanism of toler-
29. Hackney DN, Knudtson EJ, Rossi KQ, et al. 48. Matsumoto H, Tamaki Y, Sato S, Shibata
ance to the Rh D antigen mediated by pas-
Management of pregnancies complicated K. A case of hemolytic disease of the new-
sive anti-D (Rh D prophylaxis). Immunol Lett.
by anti-c isoimmunization. Obstet Gynecol. born caused by anti-M: serological study of
2002;82:67–73.
2004;103:24–30. maternal blood. Acta Obstet Gynaecol Jpn.
11. Kumpel BM. On the immunologic basis of Rh
30. Kozlowski CL, Lee D, Shwe KH, Love EM. 1981;33:525–528.
immune globulin (anti-D) prophylaxis. Trans-
Quantification of anti-c in haemolytic disease 49. Wu KH, Chang JG, Lin M, et  al. Hydrops
fusion. 2006;46:1652–1656.
of the newborn. Transfus Med. 1995;5:37–42. foetalis caused by anti-Mur in first preg-
12. Boctor FN, Ali NM, Mohandas K, Uehlinger
31. Moise KJ. Fetal anemia due to non-Rhesus-D nancy—a case report. Transfus Med.
J. Absence of D- alloimmunization in AIDS
red-cell alloimmunization. Semin Fetal Neona- 2002;12:325–327.
patients receiving D-mismatched RBCs.
tal Med. 2008;13:207–214. 50. Levine P. Comments on hemolytic disease
Transfusion. 2003;43:173–176.
32. Wenk RE, Goldstein P, Felix JK. Alloim- of newborn due to anti-PP1 P k (anti-Tj a).
13. Hadley AG. Laboratory assays for predict-
munization by hr’(c), hemolytic disease of Transfusion. 1977;17:573–578.
ing the severity of haemolytic disease of the
newborns, and perinatal management. Obstet 51. Gilja BK, Shah VP. Hydrops fetalis due
fetus and newborn. Transplant Immunol.
Gynecol. 1986;67:623–626. to ABO incompatibility. Clin Pediatr.
2002;10:191–198.
33. Koelewijn JM, Vrijkotte TG, van der Schoot 1988;27:210–212.
14. Judd WJ. Scientific Section Coordinating
CE, et  al. Effect of screening for red cell 52. McDonnell M, Hannam S, Devane SP.
Committee of the A. Practice guidelines for
antibodies, other than anti-D, to detect Hydrops fetalis due to ABO incompat-
prenatal and perinatal immunohematology,
hemolytic disease of the fetus and newborn: ibility. Arch Dis Child Fetal Neonatal Ed.
revisited. Transfusion. 2001;41:1445–1452.
a population study in the Netherlands. 1998;78:F220–F221.
15. Ulm B, Svolba G, Ulm MR, et  al. Male
Transfusion. 2008;48:941–952. 53. Miller DF, Petrie SJ. Fatal erythroblas-
fetuses are particularly affected by maternal
34. Trevett Jr TN, Moise Jr KJ. Twin pregnancy tosis fetalis secondary to abo incompat-
alloimmunization to D antigen. Transfusion.
complicated by severe hemolytic disease of the ibility. Report of a case. Obstet Gynecol.
1999;39:169–173.
fetus and newborn due to anti-g and anti-C. 1963;22:773–777.
16. Nicolaides KH, Warenski JC, Rodeck CH. The
Obstet Gynecol. 2005;106:1178–1180. 54. Sherer DM, Abramowicz JS, Ryan RM, et al.
relationship of fetal plasma protein concentra-
35. Bowman JM, Pollock JM, Manning FA, et al. Severe fetal hydrops resulting from ABO
tion and hemoglobin level to the development
Maternal Kell blood group alloimmunization. incompatibility. Obstet Gynecol. 1991;78:897–
of hydrops in rhesus isoimmunization. Am J
Obstet Gynecol. 1992;79:239–244. 899.
Obstet Gynecol. 1985;152:341–344.
36. Caine ME, Mueller-Heubach E. Kell sensi- 55. Stiller RJ, Herzlinger R, Siegel S, Whetham JC.
17. Moise KJ, Carpenter RJ, Hesketh DE. Do
tization in pregnancy. Am J Obstet Gynecol. Fetal ascites associated with ABO incompat-
abnormal Starling forces cause fetal hydrops in
1986;154:85–90. ibility: case report and review of the literature.
red blood cell alloimmunization? Am J Obstet
37. Daniels G, Hadley A, Green CA. Causes of Am J Obstet Gynecol. 1996;175:1371–1372.
Gynecol. 1992;167:907–912.
fetal anemia in hemolytic disease due to anti- 56. Yver A, Homery MC, Fuseau E, et  al. Phar-
18. Cormode EJ, Lyster DM, Israels S. Analbu-
K. Transfusion. 2003;43:115–116. macokinetics and safety of roledumab, a novel
minemia in a neonate. J Pediatr. 1975;86:862–
38. Vaughan JI, Manning M, Warwick RM, et al. human recombinant monoclonal anti-RhD
867.
Inhibition of erythroid progenitor cells by antibody with an optimized Fc for improved
19. Moïse AA, Gest AL, Weickmann PH,
anti-Kell antibodies in fetal alloimmune ane- engagement of FCgammaRIII, in healthy vol-
McMicken HW. Reduction in plasma protein
mia. N Engl J Med. 1998;338:798–803. unteers. Vox Sang. 2012;103:213–222.
does not affect body water content in fetal
39. Vaughan JI, Warwick R, Letsky E, et al. Eryth- 57. Bowman JM. The prevention of Rh immu-
sheep. Pediatric Res. 1991;29:623–626.
ropoietic suppression in fetal anemia because nization. Transfus Med Rev. 1988;2:129–
20. Berger HM, Lindeman JH, van Zoeren-
of Kell alloimmunization. Am J Obstet Gyne- 150.
Grobben D, et  al. Iron overload, free radical
col. 1994;171:247–252.

496.e1
496.e2 References
58. Koelewijn JM, de Haas M, Vrijkotte TG,
et  al. One single dose of 200 microg of
antenatal RhIG halves the risk of anti-D
immunization and hemolytic disease of the
fetus and newborn in the next pregnancy.
Transfusion. 2008;48:1721–1729.
59. Bowman JM. Controversies in Rh prophy-
laxis. Who needs Rh immune globulin and
when should it be given? Am J Obstet Gyne-
col. 1985;151:289–294.
60. Bowman JM, Pollock JM. Failures of intra-
venous Rh immune globulin prophylaxis:
an analysis of the reasons for such failures.
Transfus Med Rev. 1987;1:101–112.
61. Koelewijn JM, de Haas M, Vrijkotte TG,
et  al. Risk factors for RhD immunisation
despite antenatal and postnatal anti-D pro-
phylaxis. BJOG. 2009;116:1307–1314.
62. Kent J, Farrell AM, Soothill P. Routine
administration of Anti-D: the ethical case for
offering pregnant women fetal RHD geno-
typing and a review of policy and practice.
BMC Pregnancy Childbirth. 2014;14:87.
63. Clausen FB, Christiansen M, Steffensen R,
et  al. Report of the first nationally imple-
mented clinical routine screening for fetal
RHD in D- pregnant women to ascertain the
requirement for antenatal RhD prophylaxis.
Transfusion. 2012;52:752–758.
64.  Wikman AT, Tiblad E, Karlsson A, et  al.
Noninvasive single-exon fetal RHD determi-
nation in a routine screening program in early
pregnancy. Obstet Gynecol. 2012;120:227–234.
65. ACOG practice bulletin. Prevention of Rh
D alloimmunization. Number 4, May 1999
(replaces educational bulletin Number 147,
October 1990). Clinical management guide-
lines for obstetrician-gynecologists. American
College of Obstetrics and Gynecology. Int J
Gynaecol Obstet. 1999;66:63–70.
66. Karanth L, Jaafar SH, Kanagasabai S, et  al.
Anti-D administration after spontaneous
miscarriage for preventing Rhesus alloim-
munisation. Cochrane Database Syst Rev.
2013:CD009617.
67. Meleti D, De Oliveira LG, Araujo Júnior E,
et al. Evaluation of passage of fetal erythro-
cytes into maternal circulation after invasive
obstetric procedures. J Obstet Gynaecol Res.
2013;39:1374–1382.
68. Lipitz S, Achiron R, Horoshovski D, et  al.
Fetomaternal haemorrhage discovered after
trauma and treated by fetal intravascular
transfusion. Eur J Obstet Gynecol Reprod Biol.
1997;71:21–22.
69. Boucher M, Marquette GP, Varin J,
Champagne J, Bujold E. Fetomaternal haem-
orrhage during external cephalic version.
Obstet Gynecol. 2008;112:79–84.
70. Krause HG, Goh JT. Positive Kleihauer
result following an ectopic pregnancy. Aust N
Z J Obstet Gynaecol. 1996;36:324–325.
71. Von Stein GA, Munsick RA, Stiver K, Ryder
K. Fetomaternal haemorrhage in threatened
abortion. Obstet Gynecol. 1992;79:383–386.
72. Wylie BJ, D’Alton ME. Fetomaternal haem-
orrhage. Obstet Gynecol. 2010;115:1039–
1051.
73. Fung Kee Fung K, Eason E, Crane J, et  al.
Prevention of Rh alloimmunization. J Obstet
Gynaecol Can. 2003;25:765–773.
74. Stedman CM, Baudin JC, White CA, Coo-
per ES. Use of the erythrocyte rosette test to
screen for excessive fetomaternal haemorrhage
in Rh-negative women. Am J Obstet Gynecol.
1986;154:1363–1369.
75. Kumpel BM. Analysis of factors affect-
ing quantification of fetomaternal haem-
orrhage by flow cytometry. Transfusion.
2000;40:1376–1383.
76. Adeniji AA, Fuller I, Dale T, Lindow SW.
Should we continue screening rhesus D posi-
tive women for the development of atypical
antibodies in late pregnancy? J Matern Fetal
Neonatal Med. 2007;20:59–61.
77. Andersen AS, Praetorius L, Jorgensen HL,
et al. Prognostic value of screening for irregu-
lar antibodies late in pregnancy in rhesus
positive women. Acta Obstetricia Et Gyneco-
logica Scandinavica. 2002;81:407–411.
78.  Heddle NM, Klama L, Frassetto R, et  al.
A retrospective study to determine the risk
of red cell alloimmunization and transfu-
sion during pregnancy. Transfusion. 1993;33:
217–220.
79. Judd WJ. When should tests for unexpected
antibodies be done during pregnancy?
Transfusion. 2011;51:1366–1368.
80. Rothenberg JM, Weirermiller B, Dirig K,
et al. Is a third-trimester antibody screen in
Rh+ women necessary? Am J Manag Care.
1999;5:1145–1150.
81. Abbey R, Dunsmoor-Su R. Cost-benefit
analysis of indirect antiglobulin screening in
Rh(D)-negative women at 28 weeks of gesta-
tion. Obstet Gynecol. 2014;123:938–945.
82. Slootweg YM, Koelewijn JM, van Kamp
IL, et  al. Third trimester screening for
alloimmunisation in Rhc-negative preg-
nant women: evaluation of the Dutch
national screening programme. BJOG.
2016;123:955–963.
83. Cheng D, Hao Y. Comparative evaluation of
the microcolumn gel card test and the con-
ventional tube test for measurement of titres
of immunoglobulin G antibodies to blood
group A and blood group B. J Int Med Res.
2011;39:934–943.
84. Finck R, Lui-Deguzman C, Teng SM, et al.
Comparison of a gel microcolumn assay
with the conventional tube test for red
blood cell alloantibody titration. Transfusion.
2013;53:811–815.
85. Novaretti MC, Jens E, Pagliarini T, et  al.
Comparison of conventional tube test with
diamed gel microcolumn assay for anti-
D titration. Clin Lab Haematol. 2003;25:
311–315.
86. Thakur MK, Marwaha N, Kumar P, et  al.
Comparison of gel test and conventional
tube test for antibody detection and titra-
tion in D-negative pregnant women: study
from a tertiary-care hospital in North India.
Immunohematology. 2010;26:174–177.
87. Le Roux MG, Pascal O, Andre MT, et  al.
Non-paternity and genetic counselling.
Lancet. 1992;340:607.
88. Anderson KG, Kaplan H, Lancaster JB.
Demographic correlates of paternity con-
fidence and pregnancy outcomes among
Albuquerque men. Am J Phys Anthropol.
2006;131:560–571.
89. Pirelli KJ, Pietz BC, Johnson ST, et  al.
Molecular determination of RHD zygosity:
predicting risk of hemolytic disease of the
fetus and newborn related to anti-D. Prenat
Diagn. 2010;30:1207–1212.
90. Moise KJ, Boring NH, O’Shaughnessy R,
et al. Circulating cell-free fetal DNA for the
detection of RHD status and sex using reflex
fetal identifiers. Prenat Diagn. 2013;33:
95–101.
91. Bombard AT, Akolekar R, Farkas DH, et al.
Fetal RHD genotype detection from circulat-
ing cell-free fetal DNA in maternal plasma in
non-sensitized RhD negative women. Prenat
Diagn. 2011;31:802–808.
92. Daniels G, van der Schoot CE, Olsson ML.
Report of the First International Workshop
on molecular blood group genotyping. Vox
Sang. 2005;88:136–142.
93. Tynan JA, Angkachatchai V, Ehrich M, et al.
Multiplexed analysis of circulating cell-free
fetal nucleic acids for noninvasive prenatal
diagnostic RHD testing. Am J Obstet Gyne-
col. 2011;204–251.e1–e6.
94. Finning KM, Martin PG, Soothill PW,
Avent ND. Prediction of fetal D status from
maternal plasma: introduction of a new
noninvasive fetal RHD genotyping service.
Transfusion. 2002;42:1079–1085.
95.  Chan KC, Ding C, Gerovassili A, et  al.
Hypermethylated RASSF1A in maternal
plasma: a universal fetal DNA marker that
improves the reliability of noninvasive prenatal
diagnosis. Clin Chem. 2006;52:2211–2218.
96. van den Oever JM, Balkassmi S, Segboer T,
et  al. Mrassf1a-pap, a novel methylation-
based assay for the detection of cell-free
fetal DNA in maternal plasma. PloS One.
2013;8:e84051.
97. White HE, Dent CL, Hall VJ, et al. Evalu-
ation of a novel assay for detection of the
fetal marker RASSF1A: facilitating improved
diagnostic reliability of noninvasive prenatal
diagnosis. PloS One. 2012;7:e45073.
98. Finning K, Martin P, Summers J, Daniels
G. Fetal genotyping for the K (Kell) and
Rh C, c, and E blood groups on cell-free
fetal DNA in maternal plasma. Transfusion.
2007;47:2126–2133.
99. Gutensohn K, Muller SP, Thomann K, et al.
Diagnostic accuracy of noninvasive poly-
merase chain reaction testing for the deter-
mination of fetal rhesus C, c and E status in
early pregnancy. BJOG. 2010;117:722–729.
100. Li Y, Finning K, Daniels G, et  al. Nonin-
vasive genotyping fetal Kell blood group
(KEL1) using cell-free fetal DNA in maternal
plasma by MALDI-TOF mass spectrometry.
Prenat Diagn. 2008;28:203–208.
101. Moise KJ. Fetal RhD typing with free DNA
in maternal plasma. Am J Obstet Gynecol.
2005;192:663–665.
102. Thurik FF, Ait Soussan A, Bossers B, et  al.
Analysis of false-positive results of fetal
RHD typing in a national screening program
reveals vanishing twins as potential cause for
discrepancy. Prenat Diagn. 2015;35:754–
760.
103. Bianchi DW, Avent ND, Costa JM, van der
Schoot CE. Noninvasive prenatal diagnosis
of fetal Rhesus D: ready for Prime(r) Time.
Obstet Gynecol. 2005;106:841–844.
104. Moise Jr KJ, Carpenter Jr RJ. Increased sever-
ity of fetal hemolytic disease with known
rhesus alloimmunization after first-trimester
transcervical chorionic villus biopsy. Fetal
Diagn Ther. 1990;5:76–78.
105. McKenna DS, Nagaraja HN, O’Shaughnessy
R. Management of pregnancies complicated
by anti-Kell isoimmunization. Obstet Gyne-
col. 1999;93:667–673.
106. van Wamelen DJ, Klumper FJ, de Haas
M, et  al. Obstetric history and antibody
titer in estimating severity of Kell alloim-
munization in pregnancy. Obstet Gynecol.
2007;109:1093–1098.

107. Mari G. Middle cerebral artery peak systolic
velocity: is it the standard of care for the
diagnosis of fetal anemia? J Ultrasound Med.
2005;24:697–702.
108. Mari G, Deter RL, Carpenter RL, et  al.
Noninvasive diagnosis by Doppler ultraso-
nography of fetal anemia due to maternal
red-cell alloimmunization. Collaborative
Group for Doppler Assessment of the Blood
Velocity in Anemic Fetuses. N Engl J Med.
2000;342:9–14.
109. Moise Jr KJ. The usefulness of middle cere-
bral artery Doppler assessment in the treat-
ment of the fetus at risk for anemia. Am J
Obstet Gynecol. 2008;198–161.e1–e4.
110. Oepkes D, Seaward PG, Vandenbussche
FP, et  al. Doppler ultrasonography versus
amniocentesis to predict fetal anemia. N Engl
J Med. 2006;355:156–164.
111. Pretlove SJ, Fox CE, Khan KS, Kilby MD.
Noninvasive methods of detecting fetal anae-
mia: a systematic review and meta-analysis.
BJOG. 2009;116:1558–1567.
112. van Dongen H, Klumper FJ, Sikkel E, et al.
Non-invasive tests to predict fetal anemia in
Kell-alloimmunized pregnancies. Ultrasound
Obstet Gynecol. 2005;25:341–345.
113. Ruma MS, Swartz AE, Kim E, et  al. Angle
correction can be used to measure peak
systolic velocity in the fetal middle cerebral
artery. Am J Obstet Gynecol. 2009;200. 397.
e1–e3.
114. Sallout BI, Fung KF, Wen SW, et  al. The
effect of fetal behavioral states on middle
cerebral artery peak systolic velocity. Am J
Obstet Gynecol. 2004;191:1283–1287.
115. Shono M, Shono H, Ito Y, et al. The effect of
behavioral states on fetal heart rate and mid-
dle cerebral artery flow-velocity waveforms
in normal full-term fetuses. Int J Gynaecol
Obstet. 1997;58:275–280.
116. ACOG practice bulletin. Antepartum fetal
surveillance. Number 9, October 1999
(replaces Technical Bulletin Number 188,
January 1994). Clinical management guide-
lines for obstetrician-gynecologists. Int J
Gynaecol Obstet. 2000;68:175–185.
117. Society for Maternal-Fetal Medicine, Mari
G, Norton ME, et al. Society for Maternal-
Fetal Medicine (SMFM) Clinical Guideline
#8: the fetus at risk for anemia—diagno-
sis and management. Am J Obstet Gynecol.
2015;212:697–710.
118. MacKenzie IZ, MacLean DA, Fry A, Evans
SL. Midtrimester intrauterine exchange
transfusion of the fetus. Am J Obstet Gynecol.
1982;143:555–559.
119. Tongsong T, Wanapirak C, Sirichotiyakul
S, et  al. Middle cerebral artery peak sys-
tolic velocity of healthy fetuses in the first
half of pregnancy. J Ultrasound Med of.
2007;26:1013–1017.
120. Ruma MS, Moise KJ, Kim E, et  al. Com-
bined plasmapheresis and intravenous
immune globulin for the treatment of severe
maternal red cell alloimmunization. Am J
Obstet Gynecol. 2007;196–138.e1–e6.
121. Papantoniou N, Sifakis S, Antsaklis A. Thera-
peutic management of fetal anemia: review of
standard practice and alternative treatment
options. J Perinat Med. 2013;41:71–82.
122. Fox C, Martin W, Somerset DA, et al. Early
intraperitoneal transfusion and adjuvant
maternal immunoglobulin therapy in the
treatment of severe red cell alloimmunization
prior to fetal intravascular transfusion. Fetal
Diagn Ther. 2008;23:159–163.
123. Van Kamp IL, Klumper FJ, Oepkes D, et al.
Complications of intrauterine intravascular
transfusion for fetal anemia due to maternal
red-cell alloimmunization. Am J Obstet Gyne-
col. 2005;192:171–177.
124. Giannina G, Moise KJ, Dorman K. A
simple method to estimate volume for fetal
intravascular transfusions. Fetal Diagn Ther.
1998;13:94–97.
125. Radunovic N, Lockwood CJ, Alvarez M,
et al. The severely anemic and hydropic iso-
immune fetus: changes in fetal hematocrit
associated with intrauterine death. Obstet
Gynecol. 1992;79:390–393.
126. Egberts J, van Kamp IL, Kanhai HH, et al.
The disappearance of fetal and donor red
blood cells in alloimmunised pregnan-
cies: a reappraisal. Br J Obstet Gynaecol.
1997;104:818–824.
127. Lobato G, Soncini CS. Fetal hematocrit
decrease after repeated intravascular trans-
fusions in alloimmunized pregnancies. Arch
Gynecol Obstet. 2007;276:595–599.
128. Mari G, Detti L, Oz U, et al. Accurate predic-
tion of fetal hemoglobin by Doppler ultraso-
nography. Obstet Gynecol. 2002;99:589–593.
129. Detti L, Oz U, Guney I, et  al. Doppler
ultrasound velocimetry for timing the sec-
ond intrauterine transfusion in fetuses with
anemia from red cell alloimmunization. Am J
Obstet Gynecol. 2001;185:1048–1051.
130. Scheier M, Hernandez-Andrade E, Fonseca
EB, Nicolaides KH. Prediction of severe fetal
anemia in red blood cell alloimmunization
after previous intrauterine transfusions. Am
J Obstet Gynecol. 2006;195:1550–1556.
131. Lobato G, Soncini CS. Fetal hydrops and
other variables associated with the fetal
hematocrit decrease after the first intrauter-
ine transfusion for red cell alloimmunization.
Fetal Diagn Ther. 2008;24:349–352.
132. Dildy GA, Smith LG, Moise KJ, et al. Poren-
cephalic cyst: a complication of fetal intra-
vascular transfusion. Am J Obstet Gynecol.
1991;165:76–78.
133. Ghi T, Brondelli L, Simonazzi G, et  al.
Sonographic demonstration of brain injury
References 496.e3
in fetuses with severe red blood cell allo-
immunization undergoing intrauterine
transfusions. Ultrasound Obstet Gynecol.
2004;23:428–431.
134. Nicolini U, Kochenour NK, Greco P, et al.
Consequences of fetomaternal haemor-
rhage after intrauterine transfusion. BMJ.
1988;297:1379–1381.
135. Schumacher B, Moise KJ. Fetal transfusion
for red blood cell alloimmunization in preg-
nancy. Obstet Gynecol. 1996;88:137–150.
136. Lindenburg IT, van Kamp IL, van Zwet
EW, et al. Increased perinatal loss after intra-
uterine transfusion for alloimmune anae-
mia before 20 weeks of gestation. BJOG.
2013;120:847–852.
137. Poissonnier MH, Picone O, Brossard Y,
Lepercq J. Intravenous fetal exchange trans-
fusion before 22 weeks of gestation in early
and severe red-cell fetomaternal alloimmuni-
zation. Fetal Diagn Ther. 2003;18:467–471.
138. Trevett Jr TN, Dorman K, Lamvu G, Moise
Jr KJ. Antenatal maternal administration of
phenobarbital for the prevention of exchange
transfusion in neonates with hemolytic dis-
ease of the fetus and newborn. Am J Obstet
Gynecol. 2005;192:478–482.
139. Smits-Wintjens VE, Walther FJ, Rath
ME, et  al. Intravenous immunoglobulin
in neonates with rhesus hemolytic disease:
a randomized controlled trial. Pediatrics.
2011;127:680–686.
140. Saade GR, Moise KJ, Belfort MA, et al. Fetal
and neonatal hematologic parameters in red
cell alloimmunization: predicting the need
for late neonatal transfusions. Fetal Diagn
Ther. 1993;8:161–164.
141. Rath ME, Smits-Wintjens VE, Oepkes D,
et al. Iron status in infants with alloimmune
haemolytic disease in the first three months
of life. Vox Sang. 2013;105:328–333.
142. Zuppa AA, Alighieri G, Calabrese V, et  al.
Recombinant human erythropoietin in the
prevention of late anemia in intrauterine
transfused neonates with Rh-isoimmuniza-
tion. J Pediatr Hematol Oncol. 2010;32:e95–
101.
143. Lindenburg IT, Smits-Wintjens VE, van
Klink JM, et  al. Long-term neurodevelop-
mental outcome after intrauterine trans-
fusion for hemolytic disease of the fetus/
newborn: the LOTUS study. Am J Obstet
Gynecol. 2012;206–141.e1–e8.
144. Hall AM, Cairns LS, Altmann DM, et  al.
Immune responses and tolerance to the RhD
blood group protein in HLA-transgenic
mice. Blood. 2005;105:2175–2179.
41
Fetal Platelet Disorders
DIAN WINKELHORST AND DICK OEPKES
KEY POINTS
• Fetal platelet disorder is a potentially life-threatening
condition.
• Fetal thrombocytopenia may lead to fetal bleeding.
complications, such as an intracranial haemorrhage.
• Idiopathic thrombocytopenic purpura.
• Has an incidence of 1 to 2 in 1000 pregnancies.
• Causes severe fetal thrombocytopenia in 5% to 20% of the
cases.
• Rarely leads to bleeding problems in fetuses or neonates.
• Is treated primarily with corticosteroids.
• Fetal and neonatal alloimmune thrombocytopenia.
• Occurs in 1 in 1000 pregnancies.
• Is mainly caused by human platelet antigens 1a (80%) and
5b (10%) in Caucasians.
• Causes severe bleeding complications in 10% of cases of
severe thrombocytopenia.
• Treatment should be noninvasive with intravenous
immunoglobulins.
• Population-based screening would mean a major
improvement in the prevention of bleeding complications.
Introduction
Fetal platelet disorders, causing fetal thrombocytopenia, are rela-
tively rare but potentially life-threatening conditions. During
normal fetal life, the platelet count progressively increases, and
reaches a level of approximately 150 × 109/L by the end of the
first trimester. The normal range for platelet counts in healthy
fetuses and neonates is equal to that of adults (150–450 × 109/L).
Therefore fetal and neonatal thrombocytopenia is defined as a
platelet count less than 150 × 109/L regardless of gestational age,
which corresponds with values below the fifth percentile, calcu-
lated in adults.1,2 The degree of the thrombocytopenia can be
further classified to mild (100–150 × 109/L), moderate (50–100
× 109/L) or severe (<50 × 109/L). In contrast to neonatal throm-
bocytopenia, the exact frequency of fetal thrombocytopenia is
unknown. Of all newborns, 1% to 2% have a platelet count
below 150 × 109/L3,4 and 1 to 2 of 1000 newborns have a severe
thrombocytopenia.5
The risk for fetal thrombocytopenia is the development of
bleeding complications, varying from harmless skin bleeds to
internal organ haemorrhage, intracranial haemorrhage (ICH) or
even perinatal demise. These complications mostly present after
birth, and a diagnosis of a fetal platelet disorder occurs postnatally
in the vast majority of cases. Therefore preventive measures can
only be taken in subsequent pregnancies (Table 41.1).
Nonimmune Causes for Fetal or Neonatal
Thrombocytopenia
Nonimmune conditions that are associated with fetal and neonatal
thrombocytopenia act through increased destruction of platelets as
well as a decreased production. Non–immune-mediated increased
destruction or consumption can be caused by disseminated intra-
vascular coagulation (DIC), thrombosis, Kasabach-Merritt Syn-
drome or hypersplenism. Through increased destruction, placental
insufficiency (premature birth, preeclampsia, intrauterine growth
restriction, diabetes), several genetic abnormalities, infection and
asphyxia can lead to fetal and neonatal thrombocytopenia. 
Immune Causes of Fetal and Neonatal
Thrombocytopenia
Because immune-mediated fetal platelet disorders are the most
important cause of severe fetal thrombocytopenia, responsible
for one third of all neonatal thrombocytopenia cases, this chapter
focuses on idiopathic thrombocytopenic purpura (ITP) and fetal
and neonatal alloimmune thrombocytopenia (FNAIT).3 Also,
through an unknown mechanism, a small proportion of cases with
severe fetal anaemia caused by red blood cell (RBC) alloimmunisa­
tion is associated with fetal thrombocytopenia.6 
Autoimmune or Idiopathic
Thrombocytopenic Purpura
Maternal thrombocytopenia is encountered regularly, complicat-
ing 1 in 12 pregnancies.7 The most common cause of maternal
thrombocytopenia is a benign transient condition called ges-
tational thrombocytopenia, accounting for approximately two
thirds of all cases of maternal thrombocytopenia; ITP accounts for
3%.8,9 Other causes of maternal thrombocytopenia are preeclamp-
sia, HIV, systemic lupus erythaematosus and thyroid dysfunction.
There are two different types of ITP: the acute form and the
chronic form. Acute ITP is predominantly a condition of child-
hood that seldom occurs in pregnancy. It is mostly preceded by a
viral infection and is caused by cross-reactivity between viral anti-
gens and platelet antigens. This form usually resolves within weeks
or months. ITP in pregnancy is therefore almost always chronic
ITP, with an incidence of 1 to 2 in 1000 pregnant women.9
497
498 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
TABLE Causes of Fetal and Early Neonatal
41.1 Thrombocytopenia
Increased destruction
Immune thrombocytopenia
• Maternal autoimmune (ITP, SLE)
• FNAIT
• Severe fetal haemolytic disease caused by RBC alloimmunisation
• Alloimmune drug induced (penicillin, antiepileptics, quinidine, indo-
methacin)
Peripheral consumption
• Hypersplenism
• Kasabach-Merritt syndrome
• DIC
• Thrombosis (e.g., aortic, renal vein)
Decreased production
Genetic disorders (TAR syndrome, trisomy 13,18,21, triploidy, Turner
syndrome, amegakaryocytosis, Wiskott-Aldrich syndrome, May-Hegglin
syndrome, Bernard-Soulier syndrome, Alport syndrome)
Bacterial infection (GBS, Escherichia coli, Listeria spp., syphilis)
Viral infection (CMV, parvovirus, rubella, HIV, HSV)
Parasite infection (toxoplasmosis)
Asphyxia
Placental insufficiency (preeclampsia, IUGR, diabetes, premature birth)
CMV, cytomegalovirus; DIC, disseminated intravascular coagulation; FNAIT, fetal and neonatal
alloimmune thrombocytopenia; GBS, group-B streptococcus; HIV, human immunodeficiency
virus; HSV, herpes simplex virus; ITP, idiopathic thrombocytopenia; IUGR, intrauterine growth
restriction; RBC, red blood cell; SLE, systemic lupus erythaematosus; TAR, thrombocytope-
nia-absent radii syndrome.
  
Pathophysiology
Chronic ITP is an autoimmune disorder caused by the maternal
production of antibodies against glycoproteins present on the
membranes of maternal platelets. The majority of these auto-
antibodies are of the immunoglobulin (Ig) G class and are thus
able to cross the placental barrier, bind to fetal platelets and
cause fetal thrombocytopenia. The reported incidence of severe
neonatal thrombocytopenia in ITP varies between the 5% and
20%.10,11 Although these numbers vary widely among studies,
none of them reported any significant bleeding problems in neo-
nates as a result of the ITP. No cases of severe in utero bleeding
have been documented, and the reported incidence of ICH is
low and varies between 0% and 1.2%.8,12,13 The lowest platelet
count in the affected newborns mostly occurs within 7 days after
birth.14
In pregnancy, maternal platelet counts are usually lower
than before.8,15 Maternal symptoms can vary from none to
severe haemorrhaging but are usually mild, and pregnant
women seem to have a greater tolerance to ITP compared
with nonpregnant women because of the procoagulant state in
pregnancy.16
Unfortunately, no reliable pregnancy-specific parameters exist
to predict the severity of fetal thrombocytopenia in ITP. The
maternal platelet count, nor IgG level seems to correlate with the
fetal platelet count.17,18 The strongest correlation found so far is
the lowest platelet count in older siblings.10,14 The only maternal
factor identified to predict a low platelet count in affected neo-
nates is a history of splenectomy.18-20 
Diagnosis
The diagnosis of ITP is one of exclusion, other causes of throm-
bocytopenia during pregnancy (e.g., preeclampsia, gestational
thrombocytopenia, HELLP (haemolysis, elevated liver enzymes,
low platelet count) syndrome, DIC, massive obstetric haemor-
rhage or acute fatty liver syndrome) need to be ruled out first.20
The distinction between gestational thrombocytopenia and the
first presentation of ITP may be particularly difficult. Differences
are the gestational age at detection and platelet count. ITP is usu-
ally detected in early pregnancy because the condition is present
before conception, and it usually has a lower platelet count than
gestational thrombocytopenia. In addition, an elevated mean plate-
let volume, implicating increased platelet production, can support
the diagnosis. Bone marrow examination is not performed during
pregnancy but would reveal normal or elevated megakaryocytic
numbers. Thrombopoietin (Tpo) level can be used to distinguish
platelet production disorders from platelet destruction disorders.
In ITP, Tpo levels are normal or slightly elevated in contrast to
significant elevation of Tpo levels in patients with platelet produc-
tion disorders.
Autoantibodies can be identified using a platelet immuno-
fluorescence test (PIFT), which is based on the detection of
platelet-bound antibodies. Unfortunately, it has a relatively
low sensitivity of 70% in patients with ITP.21 Positive reactions
can be identified in case of greater than 1000 molecules of IgG
bound to a platelet; 450 molecules can already cause platelet
destruction. Also, in some patients with ITP, destruction of
platelet can be T cell mediated, so platelet-bound antibodies will
not be measurable. 
Obstetric Management
Prepregnancy. Idiopathic thrombocytopenic purpura is not a
reason to discourage pregnancy; however, women with severe
thrombocytopenia despite a splenectomy and high doses of
corticosteroids are at high risk for complications and should have
extensive preconception counselling about possible risks. When
planning pregnancy, it is wise to optimise treatment before
conception, mainly to assess the need for splenectomy before
pregnancy. 
Prenatal. Management of IPT during pregnancy requires a
multidisciplinary approach, with a team including a haematolo-
gist, paediatrician (neonatologist), obstetrician and anaesthetist. A
complete overview is displayed in Fig. 41.1.
During pregnancy, there are three indications for starting treat-
ment: symptomatic women, a platelet count below 30 × 109/L
or the need for a higher platelet count before a procedure (e.g.,
caesarean section).
When platelet counts are higher than 30 × 109/L, no treat-
ment is necessary, and monitoring of maternal platelet counts
needs to be performed every 2 weeks and more closely as delivery
approaches.
First choice of treatment during pregnancy is similar to the
approach in nonpregnant women, prednisone started at a dosage
of 1 to 2 mg/kg/day and then tapered to find the minimal effective
dose. If the thrombocytopenia shows to be resistant to corticoste-
roid treatment with prednisone or in case of serious side effects,

>30 × 109/L
Pregnancy <30 × 109/L
Symptomatic
Start steroids Start steroids
<30 × 109/L >30 × 109/L
Start IVIG; maintain Continue steroids in
>20 x 109/L lowest dose
Predelivery IVIG ± Obtain >50 × 109/L
Predelivery by increasing
platelet transfusion
steroids
Vaginal delivery or Vaginal delivery or
Delivery CS for obstetric CS for obstetric
indications indications
• Fig. 41.1 Recommendations for obstetric management in idiopathic thrombocytopenic purpura.
CS, Caesarean section; IVIG, intravenous immunoglobulin.
intravenous infusion of immunoglobulins (IVIG) is next in line.
Other treatments with limited evidence for their efficiency in ITP
during pregnancy are intravenous anti-D infusion,22 splenectomy
and azathioprine. Rituximab, danazol, Tpo receptor agonists and
most other immunosuppressive drugs should not be administered
during pregnancy because of possible teratogenicity. Platelet trans-
fusions are only to be administered in very severe thrombocyto-
penia (platelet counts <20 × 109/L) and at times when there is a
high risk for bleeding. 
Labour and Delivery. Maternal platelet counts above
50 × 109/L are considered to be safe for vaginal as well as caesarean
delivery.16,20 Therefore, if predelivery platelet counts are below
50 × 109/L, IVIG treatment should be administered at a dose
of 0.8 kg/day. Although guidelines can differ among countries
and centres, a platelet count above 80 × 109/L is considered safe
for epidural analgesia.19,23-25 Treatment during pregnancy does
not seem to have any effect on fetal platelet counts or the occur-
rence of neonatal bleeding problems; it should be administered
for maternal indications only.26
Initially, the management of labour and delivery in ITP patients
was based on the concerns of ICH in the thrombocytopenic
neonate, secondary to vaginal birth trauma. Therefore fetal scalp
blood sampling or cordocentesis was performed, and caesarean
delivery was preferred when fetal platelet count was below 50 ×
109/L. Fetal scalp blood sampling appeared to yield falsely low
counts, and predelivery cordocentesis, although producing reli-
able fetal platelet counts, has a significant risk for fetal loss and
severe morbidity. In combination with the low incidence of peri-
natal bleeding, these should not be performed as routine proce-
dures in patients with ITP.27,28 The route of delivery (vaginal or
caesarean section) does not seem to affect the incidence of ICH;
therefore, caesarean section in patients with ITP should only be
performed for obstetric indications.28 Interventions such as fetal
scalp blood sampling and vacuum extraction should be avoided
during delivery.29  born child, an important difference to RBC alloimmunisation,
Postnatal. An initial blood sample should be taken from the
umbilical cord to assess the neonatal platelet count. Second, daily
neonatal platelet counts should be done for approximately 1 week,
CHAPTER 41  Fetal Platelet Disorders 499
>30 x 109/L
asymptomatic
Check platelets
<50 × 109/L >50 × 109/L
Obtain >50 × 109/L
by increasing
steroids
Vaginal delivery or Vaginal delivery or
CS for obstetric CS for obstetric
indications indications
depending on the presence and degree of neonatal thrombocyto-
penia detected.30,31 In addition, haemoglobin and bilirubin counts
should be monitored. Cranial ultrasound is indicated only in
case of severe thrombocytopenia. Postnatal treatment with IVIG
is only indicated in case of severe neonatal thrombocytopenia
(<50 × 109/L), and platelet transfusion should only be used in case
of neonatal bleeding. Breastfeeding is not contraindicated. 
Fetal and Neonatal Alloimmune
Thrombocytopenia
Fetal and neonatal alloimmune thrombocytopenia can originate
after maternal alloimmunisation against paternally derived human
platelet antigens (HPAs) on fetal platelets. FNAIT is the main
cause of thrombocytopenia in newborns. Approximately half of
the severe neonatal thrombocytopenia cases (platelet counts <50 ×
109/L) result from FNAIT.3,8
Natural History
The maternally produced alloantibodies in FNAIT are of the IgG
subclass and are thought to enter the fetal circulation by crossing
the placental barrier using the neonatal Fc receptor (FcRn) and,
after entering, cause fetal thrombocytopenia. This thrombocyto-
penia can be completely asymptomatic or lead to a wide spectrum
of fetal and neonatal bleeding problems. These bleeding problems
can vary from relatively harmless skin bleeding, such as petechiae,
purpura or haematoma, to more severe haemorrhage (Fig. 41.2).
FNAIT is associated with a high risk for neonatal mortality or
morbidity, with many surviving children with neurodevelop-
mental impairments, such as bilateral blindness, cerebral palsy
or delayed cognitive development. An ICH is the most serious
bleeding complication. The majority of these ICHs affect the first-
which does not normally affect the first child.32 A multicentre
study that reviewed the largest series of ICHs reported that 54%
of the bleedings occurred before the 28th week of gestational age,
500 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

A B

C D
• Fig. 41.2  Clinical aspects of fetal and neonatal alloimmune thrombocytopenia (FNAIT). A, Unexpected
postnatal detection of FNAIT: large cephalic haematoma after vacuum-assisted delivery; this photo was at
2 days of age. B, Unexpected postnatal detection of FNAIT: large intraparenchymal intracranial haemor-
rhage. Axial T2-weighted magnetic resonance image (MRI) obtained at 9 days of age showing porence-
phalic cysts both the left parietal and right temporal. C and D, Unexpected antenatal detection of FNAIT:
large intraparenchymal intracranial haemorrhage. T2-weighted MRI obtained at 28 weeks’ gestational age
showing a left parietal haemorrhage.

and 67% started before 34 weeks’ gestational age.32 Although a
lot of research is being performed on identifying risk factors for
developing ICH, the only useful predictor of ICH thus far is a
sibling that had ICH. Based on retrospective data on 33 untreated
successive pregnancies, the recurrence rate is estimated at 79%.33
In addition to ICH, relatively unknown massive internal organ
haemorrhage, such as pulmonary or gastrointestinal bleeding,
seem to occur as well and are associated with a considerable risk
for neonatal death.34  HPA-5b, responsible for about 10% of cases of FNAIT (Table
Pathophysiology
Platelet Alloantigens and Alloantibodies. The platelet specific
alloantigens, or HPAs, are carried by glycoprotein complexes that
are present on the platelet membrane. Different HPA epitopes are
created by single nucleotide polymorphisms (SNPs) that result in
small changes in the glycoprotein structure through an amino acid
change. Currently, there are 37 different types of HPAs described,
located on six different glycoprotein complexes (IIb/IIIa, Ib/IX,
Ia/IIa and CD109) on the platelet membrane.
For a recent overview, see http://www.ebi.ac.uk/ipd/hpa/table1.
html. The 12 most frequently involved HPAs are clustered into
six biallelic groups (Table 41.2). The greater portion of all HPA
are localised on glycoprotein (GP) IIb/GP IIIa, also called integ-
rin αIIbβ3, the fibrinogen receptor, which is the most abundant
molecule on the surface of platelets. GP IIb contains 7 and GP
IIA 16 different HPA systems. Although all HPAs that are present
on GP IIIa can be involved in FNAIT, HPA-1a is the predomi-
nant cause of FNAIT, especially in the Caucasian population.
HPA-1a accounts for approximately 80% of cases followed by
41.3). In the Asian population, however, anti–HPA-4b is the
most frequently involved alloantibody followed by anti–HPA-3a
and anti–HPA-21b.35-37 Furthermore, alloantibodies against GP
IV/CD36 seem to have an increased presence in the African and
Asian populations.38
The immune response to alloantigens is mediated by human
leukocyte antigen (HLA) class II molecules. Antigen-presenting
cells such as dendritic cells, macrophages and B lymphocytes pro-
cess the antigens into small peptides, which are presented by the
HLA class II antigens on their surfaces. CD4-positive T-helper
lymphocytes can recognise the peptide–HLA complex, lead-
ing to the activation of the T cell. Activated T cells then interact
with B lymphocytes, initiating antibody production. After active
 CHAPTER 41  Fetal Platelet Disorders 501

TABLE
41.2 Human Platelet Antigens and Frequencies

Nucleotide Amino acid FREQUENCY (%)

Antigen Original names Glycoprotein CD change change African Allele Asian Genotype White
HPA-1a Zwa, PlA1 GP IIIa CD61 T176 Leu33 1A 90 100 1a/a 72
HPA-1b Zwb, PlA2 GP IIIa C176 Pro33 1B 10 0 1a/b 26
1b/b 2
HPA-2a Kob GP Ibα CD42b C482 Thr145 2A 71 95.2 2a/a 85
HPA-2b Koa, Siba GP Ibα T482 Met145 2B 29 4.8 2a/b 14
2b/b 1
HPA-3a Baka, Leka GP IIb CD41 T2621 Ile843 3A 68 59.5 3a/a 37
HPA-3b Bakb GP IIb G2621 Ser843 3B 32 40.5 3a/b 48
3b/b 15
HPA-4a Yukb, Pena GP IIIa CD61 G506 Arg143 4A 100 99.5 4a/a >99

HPA-4b Yuka, Penb GP IIIa A506 Gln143 4B 0 0.5 4a/b <0.1

4b/b <0.1
HPA-5a Brb, Zavb GP Ia CD49b G1600 Glu505 5A 82 98.6 5a/a 88
HPA-5b Bra, Zava, Hca GP Ia A1600 Lys505 5B 18 0.4 5a/b 20
5b/b 1
HPA-15a Govb CD109 CD109 C2108 Ser703 15A 65 53.2 15a/a 35
HPA-15b Gova CD109 A2108 Tyr703 15B 35 46.8 15a/b 42

Adapted from Immuno Polymorphism Database. HPA Allele Frequencies. http://www.ebi.ac.uk/ipd/hpa/freqs_1.html.

  

TABLE
41.3 Alloantibodies Involved in Fetal and Neonatal Alloimmune Thrombocytopenia in the Caucasian Population
Authors Patients (n) Alloantibody detected Frequency (%) Alloantibody detected Frequency (%)
Mueller-Eckhart et al.126 (1989) 106 Anti–HPA-1a 90 Anti-HPA-3a 0.8
Anti–HPA-5b 8 Anti HPA-1a + 5b 0.8
Anti–HPA-1b 0.8 Anti-B 0.8
Porcelijn129 (2004) 217 Anti–HPA-1a 73.7 Anti HPA-1b 1.4
Anti–HPA-5b 14.7 Anti HPA-15a 0.5
Anti–HPA-3a 4.6 Anti HPA-15b 0.5
Anti–Priv Ag 1.5 Anti A or anti B 2.8
Davoren et al.127 (2004) 1162 Anti–HPA-1a 79 Anti GPIV (CD36) 0.4
Anti–HPA-5b 9 Anti HPA-4a 0.1
Anti–HPA-1b 4 Anti HPA-4b 0.1
Anti–HPA-3a 2 Anti HPA-6bw 0.1
Anti–HPA-5a 1 Combinations 3.1
Anti–HPA-3b 0.8
Knight et al.128 (2011) 151 Anti–HPA-1a/b 81 Anti HPA-1a + 5b 5
Anti–HPA-5a/b 7 Other 7

  
502 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
transport, through FcRn, of the formed alloantibodies against the
fetal antigens enter the fetal circulation and induce destruction of
fetal platelets.
The GP that carries the most important alloantigen in FNAIT,
HPA-1a, is GP IIIa, which is also called ‘integrin β3’. On platelets,
integrin β3 is complexed to GP IIb. In addition to this complex,
integrin β3 can form a complex with αV as well. This αVβ3 com-
plex, still carrying the HPA-1a epitope, is scarcely expressed on plate-
lets but is prominently present on the surface of endothelial cells,
vascular smooth muscle cells and syncytiotrophoblast cells.39-41 The
presence of HPA-1a on these different cell types, other than plate-
lets, and interaction of maternal alloantibodies with these cells might
reveal new mechanisms in the pathophysiology.  well.57 In contrast to HLA class II molecules, HLA class I molecules
Vascular Integrity. The exact pathogenic mechanism leading
to devastating ICH in FNAIT has never been adequately under-
stood. Generally, alloantibodies were thought to enter the fetal
circulation and cause bleeding problems through destruction
of fetal platelets resulting in thrombocytopenia. However, only
a small proportion of the severely thrombocytopenic newborns
have a severe haemorrhage, indicating another mechanism to be
involved. Because integrin β3 that carries the HPA-1a epitope is
also present on endothelial cells (in complex with αV; αVβ3), it
has been suggested that interaction of anti–HPA-1a with endothe-
lial cells plays a key role in the development of bleeding complica-
tions. In  vitro studies already illuminated the direct interaction
between anti–HPA-1a and human umbilical vein endothelial cells
(HUVECs), demonstrated by a decreased HUVEC spreading as
well as a decreased integrity of their monolayer in electric cell–
substrate impedance sensing (ECIS) assays.42 In addition, in vivo
animal studies showed that mice without circulating platelets and
or fibrinogen do not show any bleeding problems in utero. This
supports the hypothesis that instead of the thrombocytopenia and
blood coagulation, another mechanism plays a key role in causing
bleeding complications. Recently, a large study with both active
and passive murine models of anti–HPA-1a mediated FNAIT has
been performed and showed that ICHs in these mice occurred
regardless of platelet count. Also, HPA-1a antibodies inhibited
angiogenic signalling, induced endothelial cell apoptosis and
decreased vessel density in affected brains as well as retinas. This
delivered the first direct evidence for αVβ3 integrin’s role in angio-
genesis and HPA-1a–induced impaired angiogenesis in mice, sug-
gesting this to be a key factor in causing bleeding complications
in FNAIT.43 The first analysis with a small number of human sera
containing HPA-1a antibodies concluded that the interaction of
the antibodies with endothelial cells might determine and possibly
predict the occurrence of ICH.44  are potentially an underestimation. They reported 58% of allo-
Placental Function. In addition to platelets and endothelial
cells, integrin β3 is also expressed on placental tissue by syncy-
tiotrophoblast cells in αVβ3 complex as well. Although there is
no direct evidence, it has been suggested that anti–HPA-1a might
induce placental insufficiency through interaction with these syn-
cytiotrophoblast cells, demonstrated by a strong association with
intrauterine growth restriction, as well as cases of intrauterine fetal
demise (in absence of bleeding problems).45 In addition, in a group
of 13 FNAIT cases, lymphoplasmacytic chronic villitis was found
to be significantly more present in placenta tissue of untreated
cases when compared to cases treated with IVIG.46 Another corre-
lation that has been suggested is one between FNAIT and miscar-
riages.47 In addition, the expression of HPA-1a on placental tissue
might lead to increased and early exposure to the fetal HPA-1a
and might be a possible explanation for FNAIT already affecting
first pregnancies and first-born children in FNAIT.
Overall, there is increasing evidence of more advanced patho-
physiologic mechanisms initiated by anti-HPA-1a, than solely
platelet destruction. 
Human Leukocyte Antigen. Specific HLA class II molecules
seem to be associated with the occurrence of alloimmunisation in
HPA incompatible pregnancies. HLA-DRB3*0101 was found to
be positively correlated to the occurrence HPA-1a alloimmunisa-
tion in HPA-1a–negative pregnant women.48-55 The underlying
mechanism suggested is the difference in the ability to form stable
bonds between Pro33/Leu33, the amino acids present on the integ-
rin β3 peptides of HPA-1b alleles and HPA-1a alleles, respectively.56
There is some evidence that HLA class I plays a role in FNAIT as
are expressed on platelet membranes. With an expression of approx-
imately 20,000 HLA class I molecules, platelets are the major source
of HLA class I present in blood.58 Maternal alloimmunisation
against fetal HLA on platelets, possibly causing antibody-mediated
fetal thrombocytopenia in a similar mechanism to HPA alloanti-
bodies is mentioned in a number of case reports.59-69 In contrast
to what previously has been thought, HLA class I alloantibodies
can cross the placental barrier and do enter the fetal circulation.70
Whether or not these antibodies can actually be held responsible
for fetal and neonatal thrombocytopenia is not clear yet. Strong
evidence to support such a causal relationship is currently lacking. 
Incidence
In the absence of population-based screening programs, estimates
of incidence of FNAIT are mainly based on retrospective data and
some small prospective cohort studies and therefore vary widely. It
is generally accepted that FNAIT is likely to be underdiagnosed,
with only 37% of cases of severe FNAIT detected in the absence
of screening programs.71 An Irish cohort study estimated that only
7% of expected cases are detected clinically.71 Based on a systematic
review pooling results on postnatal screening of platelet counts in
59,425 newborns, the incidence of severe FNAIT is 0.04% (1 in
2500 newborns), leading to an ICH in 25% of these (1 in 10,000).5
Focussing on the predominant cause of FNAIT, HPA-1a
alloimmunisation, a few prospective studies have attempted to
estimate its incidence. The largest prospective screening study,
including 100,488 pregnant women in Norway, reported an inci-
dence of HPA-1a–negative women of 2.1%, leading to HPA-1a
alloimmunisation in 10.7%. All alloimmunised women under-
went an elective caesarean section at 2 to 4 weeks before term, so
neonatal platelet counts and the incidence of bleeding problems
immunisations to result in FNAIT, 33% in severe FNAIT and
2% of all alloimmunisations developed an ICH.72 Another, much
smaller, prospective screening study reported an overall incidence
of FNAIT caused by HPA-1a of 1 in 1163 live births and an inci-
dence of severe FNAIT of 1 in 1695.73 The most accurate estima-
tion of the population incidence of FNAIT has been made by
Kamphuis and associates,74 who performed a systematic review
including prospective studies on HPA-1a screening. They con-
cluded that HPA-1a alloimmunisation occurred in 9.7% of preg-
nancies at risk, leading to severe FNAIT in 31% of the cases and
to perinatal ICH in 10% of the severe FNAIT cases (Fig. 41.3). 
Diagnostics
With the current lack of population-based screening, FNAIT is
only identified in case of bleeding complication, either antenatally
 CHAPTER 41  Fetal Platelet Disorders 503

in case of bleeding or fetal brain abnormalities or more commonly diagnostic tests available to assess disease severity before severe
postnatally, in case of neonatal bleeding problems or a chance find- complications occur. Fetal platelet count can only be evaluated
ing of thrombocytopenia (see Fig. 41.2). Maternal immunisation by fetal blood sampling, for which the umbilical cord needs to be
can obviously be detected by maternal serum testing. Unfortu- punctured. In particular when platelets are low, this test has a high
nately, unlike in fetal anaemia, there are no antenatal noninvasive complication rate and is now largely abandoned.
In case of suspected FNAIT after birth, blood samples of the
mother, father and child should be analysed to confirm the diag-
nosis at a specialised laboratory (Fig. 41.4).
10%
ICH
Human Platelet Antigen Pheno- or Genotyping. Human plate-
let antigen genotyping has become available as a routine labora-
30% tory technique and can be used to identify HPA incompatibilities
Severe FNAIT between the mother and child. Several techniques are known of
which the polymerase chain reaction with sequence-specific prim-
10% ers (PCR-SSP) and the TaqMan technology–based allelic discrim-
HPA-1a antibodies ination assays are most used.75-78 Genotyping is more favourable
to phenotyping because no platelets and specific antisera are nec-
2% essary. Beneficial discrepancies (i.e., false-positive results) between
HPA-1a negative genotyping and phenotyping of HPA rarely occur. Furthermore,
• Fig. 41.3  Incidence of human platelet antigen (HPA)-1a–mediated fetal paternal heterozygosity can be easily determined and used to pre-
and neonatal alloimmune thrombocytopenia (FNAIT). ICH, Intracranial dict the risk for FNAIT in the next pregnancy. New microarray
haemorrhage. technologies will support routine genotyping of all known HPAs

Genotyping
HPA-1a, -1b, -3a, -3b, Mother, father and child Screen for incompatibilities
-5a, -5b, -15a, -15b

Maternal and paternal

Phenotyping
platelets

Maternal serum with

paternal platelets Screen for HLA and HPA
(untreated and chloroquine alloantibodies
treated)

PIFT Maternal serum with Screen for HPA

typed donor platelets alloantibodies

Maternal serum and

Screen for
eluate of maternal
autoantibodies
platelets

Screen for
Paternal platelets
autoantibodies

Maternal serum with Screen for HPA

typed donor platelets alloantibodies

MAIPA
Maternal serum with Screen for (private) HPA
paternal platelets alloantibodies

Tpo Plasma child Determine Tpo level

• Fig. 41.4  Overview of laboratory assays in suspected fetal and neonatal alloimmune thrombocytope-
nia. HLA, Human leukocyte antigen; HPA, human platelet antigen; MAIPA, monoclonal antibody-specific
immobilisation of platelet antigens assay; PIFT, platelet immunofluorescence test; Tpo, thrombopoietin.
504 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
TABLE
41.4 Overview of Management in Fetal and Neonatal Alloimmune Thrombocytopenia
Prepregnancy Prenatal
Counselling at specialised In case of paternal heterozygosity: fetal
fetal medicine centre genotyping
Paternal genotyping Monitoring:
• Ultrasound monitoring every 2–4 wk
Treatment:
• First choice: IVIG
• Alternatives:
• Noninvasive: steroids
• Invasive: FBS and IUPT
FBS, Fetal blood sampling; IVIG, intravenous immunoglobulins; IUPT, intrauterine platelet transfusion.
which allows detection of incompatibilities for low-frequency
antigens and increases the sensitivity for the detection of antibod-
ies against low-frequency antigens.79  crimination assays.
Human platelet antigen antibodies (platelet-specific alloan-
tibodies). For the detection of platelet-specific antibodies (anti-
HPAs), it is necessary to use techniques in which the presence
of antibodies can be tested with isolated GP complexes. Because
there are only a limited number of copies of the GP Ia/IIa com-
plexes which carry the HPA-5 system on the platelet membrane,
techniques based on antibody binding to whole platelets are not
sensitive enough, especially not for the clinically important HPA-
5b antibodies.80 The breakthrough in HPA antibody detection
was the monoclonal antibody-specific immobilisation of plate-
let antigens assay (MAIPA) described by Kiefel and colleagues in
1987.81 This complex assay is based on a sandwich enzyme-linked
immunosorbent technique using glycoprotein specific monoclo-
nal antibodies (MoAbs) to differentiate between antibodies bind-
ing to HPA located on the glycoproteins, HLA class I or aspecific
Fc-FcγRIIa antibody Unfortunately, the MAIPA is a complex
technique and requires highly skilled technicians, the availability
of MoAbs and panels of frozen typed platelets. Several solid phase
(microtitreplate) techniques have been developed based on the
MAIPA, with the important GPs already isolated and bound on
the microtitreplate well. These techniques require fewer labora-
tory skills and can be performed within 4 hours. Therefore many
laboratories use these techniques, but, unfortunately, the sensitiv-
ity (70%–75%) and the specificity (85%) are less than for MAIPA
(both sensitivity and specificity >90%), and these techniques
should only be used by experienced laboratories with knowledge
of their limitations. Overdependence on such kits or their use by
inexperienced users may lead to failure to detect clinically signifi-
cant antibodies.82,83  Prepregnancy. In absence of routine screening, the knowledge
Donor Human Platelet Antigen Genotyping. Depending on
the clinical situation and the platelet count, acute platelet transfu-
sions may be necessary. In the Netherlands, a sufficient number of
donors are genotyped to guarantee continuous availability (within
2 hours) of HPA-1a– and -5b–negative platelets. Donors with
this HPA typing are asked to donate via platelet apheresis on a
regular basis. HPA-1a– and -5b–negative platelets are the required
for approximately 90% of the FNAIT neonates (80% anti–HPA-
1a and 10% anti–HPA-5b). For large-scale HPA genotyping, we
developed a fully automated HPA-1, -2, -3, -5, -15 genotyping
Labour and delivery Postnatal
Planned (near) term delivery, not primarily Monitoring:
caesarean section • Platelet counts in the first 5 days
• Cranial ultrasound
Avoid potential traumatic events: scalp Treatment:
electrode, assisted delivery • First choice: platelet transfusion
• Alternative: IVIG
assay based on DNA isolation with a MagNa Pure LC DNA isola-
tion robot and typing with TaqMan technology-based allelic dis-
 
Neonatal Plasma Thrombopoietin Measurement. Regulation
of platelet production strongly depends on the free plasma Tpo
levels.84-88 Tpo, mainly produced in the liver at a constant level,
binds to the MPL receptors on CD34 stem cells, megakaryo-
cytes and platelets. Measurement of plasma Tpo levels is use-
ful to discriminate thrombocytopenia caused by megakaryocyte
and platelet production failure (highly elevated Tpo levels) from
thrombocytopenia caused by elevated platelet destruction as in
ITP and FNAIT (normal or only slightly elevated Tpo levels).89-93
In a retrospective study, Tpo levels in different groups of patients
with neonatal thrombocytopenia were analysed and compared
with a control group of healthy neonates.89 Neonatal thrombo-
cytopenia caused by congenital cytomegalovirus infections, severe
asphyxia or intrauterine growth restriction correlates with high
plasma Tpo levels, strongly indicating that the thrombocytope-
nia is caused by decreased platelet production because of bone
marrow suppression. Plasma Tpo levels, together with the routine
serology tests for thrombocytopenic neonates, can be used in the
laboratory workup for suspected FNAIT. 
Obstetric Management
In contrast to the management of the neonate, there is divergence
of opinion about the optimal antenatal management in pregnan-
cies complicated by FNAIT. International guidelines considering
all available evidence and opinions are currently being developed.
For a concise overview of all aspects of management in FNAIT,
see Table 41.4.
of a high risk for FNAIT prepregnancy is mostly because of an
affected sibling after a previous pregnancy complicated by FNAIT.
Like in ITP, a previous pregnancy complicated by FNAIT is not
a reason to discourage pregnancy at all. It is, however, recom-
mended to perform prepregnancy counselling at a specialised fetal
medicine centre. To optimise this counselling and give the best
advice on expected risks for subsequent pregnancies documenta-
tion on disease severity of the index or previous cases (platelet
count, bleeding complications, response to treatment), alloanti-
body involved and zygosity of father should all be considered. 

Prenatal. First, in case of known maternal alloimmunisation, it
has to be assessed whether or not the pregnancy is at risk. When
the father is homozygous for the involved platelet alloantigen,
every consecutive pregnancy is at risk. In case of paternal hetero-
zygosity, however, fetal HPA typing has to be performed to assess
whether or not the current pregnancy is incompatible and the
fetus is truly at risk for FNAIT. In case of HPA-1, in some coun-
tries, a noninvasive fetal HPA-1a genotyping assay can be per-
formed using cell-free fetal DNA in maternal plasma.94,95 It has
been suggested that fetal HPA-3, HPA-5 and HPA-15 might be
determined by performing parallel sequencing.96 Unfortunately,
noninvasive assays for other clinically relevant HPAs have not
been developed yet. In these cases, fetal genotyping can only be
performed after amniocentesis. After it has been established that
the fetus is at risk, antenatal management can be implemented
with the goal to prevent bleeding complications from occurring,
preferably at a specialised fetal medicine centre.
Monitoring and risk stratification are also important. Most
important, after it has been established that a pregnancy is
incompatible and therefore at risk for FNAIT, close ultrasound
monitoring of the fetal brain should be performed. At this stage,
preferably, clinicians should be able to evaluate and monitor
fetal disease severity as well as predict the occurrence of severe
bleeding. However, as mentioned before, in contrast to man-
agement of pregnancies complicated by RBC immunisation, in
FNAIT no diagnostic tools are available. For this purpose, in
some centres, alloantibody titres are monitored by titration and
quantification. Publications on this subject are somewhat con-
tradictory. Although high anti–HPA-1a levels do seem to cor-
relate with a more severe disease, this is not invariably the case,
and severely affected pregnancies can occur with barely detect-
able antibody levels. No reliable cut-off values to guide individ-
ual management have been established thus far.97-99 Therefore
the monitoring of antibody levels is mostly done in a research
setting only.
Several other mechanisms and markers are being studied to
enable the prediction of the fetal and neonatal disease severity
during pregnancy. For example, the glycosylation pattern of the
Fc-part of the anti-HPA alloantibodies (e.g., fucosylation and
galactosylation) is reported to correlate with neonatal platelet
counts as well as disease severity.100,101 Cord blood CRP levels are
proven to be linked to disease severity as well.102 Most recently, as
described previously, the interaction of anti–HPA-1a antibodies
with endothelial cells might play a key role in the development of
(intracranial) haemorrhages. Unfortunately, although these results
sound promising, the exact clinical implications of these markers
are still unclear, and more research has to be performed to develop
such a diagnostic tool.
In addition to these laboratory parameters, clinical charac-
teristics have been evaluated as well. Still, the most important
predictor for the occurrence of severe bleeding complications is
the obstetric history. The only reliable parameter identified is the
occurrence of an ICH in previously affected pregnancies. Without
the administration of preventive antenatal treatment, the recur-
rence rate of these ICHs is as high is 79%.33
Invasive Treatment. The first prenatal treatment strategy
in these at-risk pregnancies became available when ultrasound-
guided fetal blood sampling (FBS), already used in pregnancies
complicated by RBC immunisation, was performed accompanied
if needed by transfusion of platelets. The first intrauterine platelet
transfusion (IUPT) was performed by Daffos and colleagues in
1983.103 This strategy has the great advantage of assessing fetal
CHAPTER 41  Fetal Platelet Disorders 505
platelet count and gaining some knowledge on the disease severity.
However, puncturing the umbilical cord in the presence of a low
platelet count may lead to prolonged bleeding and even exsan-
guination (Video 41.1). In addition, given the short life span of
the transfused platelets, transfusions are needed at least weekly,
increasing the overall risk for fetal loss considerably. Therefore the
accumulated complication risk per pregnancy, including the need
for (premature) emergency caesarean section or delivery, infec-
tions and fetal loss, can reach up to 11%.104 A recent systematic
review showed that more than half of the mortality rate in preg-
nancies managed with invasive treatment was actually because of
the treatment and not because of the disease.104 
Noninvasive Treatment. Endeavouring to avoid this invasive
and high-risk treatment, Bussel and colleagues105 first reported
the successful IVIG. This treatment was adapted from the treat-
ment of ITP in pregnancy . Still, the use of IVIG in the antenatal
treatment of FNAIT is off-label (unlicensed), and the exact work-
ing mechanism of IVIG is not adequately understood. Possible
hypotheses are that IVIG is thought to compete with the anti-HPA
for FcRn receptor as well as the destruction in the fetal spleen, that
it dilutes and thereby reduces antibody titres in maternal serum
and that it prevents the binding of anti-HPA to fetal platelets.
The adverse effects of IVIG are usually mild, most often reported
is a dose-related headache. Two small studies reported the occur-
rence of IVIG-related headache, not leading to discontinuing of
the treatment, in 1 of 11 and 1 of 18 cases, respectively.106,107
Rarely, renal failure, aseptic meningitis and thrombotic compli-
cations may occur. In addition, because IVIG is a multidonor
human blood product, there is the potential of transmission of
bloodborne infections.2 To reduce possible headache complaints
and support its efficiency, corticosteroids are sometimes added
to the IVIG treatment. Three randomised trials have been con-
ducted comparing IVIG treatment with IVIG and corticosteroid
treatment.108-110 They found no benefit of the addition of corti-
costeroids to IVIG; the authors reported no significant increase
in neonatal platelet count and no difference in the occurrence of
ICH. Not surprisingly, the studies lacked the power to detect a
difference in ICH.108,109 Furthermore, several retrospective as well
as prospective cohort studies have been performed, and none of
them found a significant effect of steroids on platelet counts or the
occurrence of ICH.97,111-113
An important aspect of evaluating treatment strategies is to
realise what the true goal of this treatment is. In pregnancies com-
plicated by FNAIT, the clinically relevant goal is to prevent bleed-
ing complications rather than prevent (severe) thrombocytopenia.
Moreover, in light of the occurrence of extremely low platelet
counts without clinical bleeding and the increasing evidence for
other pathological mechanisms playing a key role in the develop-
ment of bleeding problems in FNAIT, using platelet counts as
outcome parameter to assess the effectiveness of antenatal manage-
ment has some major limitations. When comparing noninvasive
IVIG treatment with invasive management strategies, although
some studies describe higher neonatal platelet counts at birth,
there are no studies reporting differences in effectiveness regarding
the occurrence of ICH or FNAIT related perinatal death.97,114-116
Because of the noninvasive nature and its effectiveness equal to
FBS and IUPT, IVIG rapidly gained ground and is currently in
most centres the first line of therapy in FNAIT.
The dosage used is adapted from the treatment of ITP. IVIG
was first administered at 1.0 g/kg maternal body weight per week.
Different strategies with regards to dose (0.5 g/kg/wk or 2.0 g/kg/
wk) and timing of treatment have been applied since. No studies on
506 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Diagnosis FNAIT

During Obstetric
pregnancy history

Standard risk High risk

Fetus with ICH
(sibling without ICH) (sibling with ICH)

IVIG 0.5 g/kg/wk IVIG 1 g/kg/wk start

Fetal MRI
start at 24–28 weeks between 12–16 weeks

Top or IVIG Ultrasound monitoring Ultrasound monitoring

1.0 g/kg/wk fetal brain every 4 weeks fetal brain every 4 weeks

Top or elective CS Planned delivery Planned delivery

at 34 weeks at 37–38 weeks at 36 weeks

Avoid scalp electrode, Avoid scalp electrode, Avoid scalp electrode,

blood sampling and blood sampling and blood sampling and
assisted vaginal delivery assisted vaginal delivery assisted vaginal delivery

• Fig. 41.5  Recommendations for antenatal treatment in fetal and neonatal alloimmune thrombocytope-
nia (FNAIT). CS, Caesarean section; ICH, intracranial haemorrhage; IVIG, intravenous immunoglobulin;
MRI, magnetic resonance imaging.

2.0 g/kg/wk have been published thus far, in contrast to 0.5 g/kg/
wk. Although the level of evidence is weak, the effect of 0.5 g/kg/
wk seems comparable to that of 1.0 g/kg/wk in standard-risk preg-
nancies (without a sibling that had ICH).117,118 because the only
evident risk factor for developing an ICH thus far is the occurrence
of an ICH in a sibling, it seems logical to stratify pregnancies into
two groups with two different IVIG treatment strategies regarding
dose and start. The gestational age at start of treatment is mainly
based on the estimated onset of ICHs. As previously mentioned,
the largest study describing ICH cases reported the gestational age
of onset to be less than 28 weeks in more than half of the cases. This
supports starting IVIG earlier than 28 weeks’ gestation (commonly
used in Europe), such as at 24 or even 20 weeks, which is common
in the United States. More data are needed to make a firm recom-
mendation. Because of the high recurrence rate, IVIG is commonly
started significantly earlier (i.e., 12 weeks’ or 16 weeks’ gestation_ in
women with a previous child with an ICH,
In current practice, there are a few centres left still performing
invasive procedures in the management of FNAIT pregnancies.
In most cases, fetal blood sampling is not primarily performed
to transfuse platelets or to assess the effect of noninvasive treat-
ment on fetal platelet count but to determine mode of delivery
predelivery. When predelivery FBS is applied for this cause, usu-
ally a caesarean section is performed in case of fetal platelet counts
of less than 50 × 109/L.108,119
In summary, recommendations for the antenatal management
of pregnancies complicated by FNAIT are to treat with weekly
doses of IVIG only, but there is no evidence for the effect of the
addition of corticosteroids (Fig. 41.5).104 
Labour and Delivery. A planned near-term caesarean section is
often performed to reduce the birth trauma with risk for bleeding
problems. However, evidence for this rationale is lacking. First,
specific intrapartum risk for bleeding has never been proven, and
in a small cohort analysis, vaginal delivery was not associated
with the occurrence of ICH.120 Second, in the analysis of 43
cases of ICH, no intrapartum bleeding was detected, and only 3
of 43 ICHs were thought to have occurred after delivery.32 The
majority of women are multiparous, and a nontraumatic vaginal
delivery is usually expected. So, in women with a previous vagi-
nal delivery without a sibling with ICH, a planned induction of
labour at term can be considered. In contrast to women who pre-
viously delivered a child with ICH, a near-term planned deliv-
ery or caesarean section can be offered (see Fig. 41.5). In cases
of vaginal delivery, it is recommended to avoid any potential

traumatic events such as scalp electrode, scalp blood sampling
and assisted vaginal delivery.  the prevention of Fetal/Neonatal AlloImmune Thrombocytope-
Postnatal Management. After delivery, cord blood samples
should be taken to rapidly assess neonatal platelet count. Because
of the natural fall in platelet count in the first week of life, it is
advised to evaluate the neonatal platelet count daily for the first 5
days.121 Also, every newborn needs to be evaluated through cranial
ultrasound examinations. Breastfeeding is not contraindicated. 
Screening
Because FNAIT can occur in first pregnancies, the only way to
adequately identify all HPA-alloimmunised pregnancies is through
prenatal population-based screening programs. This way, all preg-
nancies at risk for FNAIT can be identified before the occurrence of
bleeding problems. The complications can then likely be prevented
by offering antenatal treatment by IVIG and possibly also by anti–
HPA-1a prophylaxis (see later), similar to the RBC immunisation
screening programs. Although FNAIT has a reported incidence
of 1 in 1000 live births, severe thrombocytopenia or even bleed-
ing complications occur less often, which would result in a high
number needed to screen. Several studies have been performed
to assess such programs and concluded population-based screen-
ing and intervention programs to be successful in reducing mor-
bidity and mortality and likely to be cost-effective as well.122 A
national screening program as described by Kjeldsen-Kragh and
associates,72 which included performing a near-term caesarean
section in all alloimmunised pregnancies, would save up to 210 to
230 quality-adjusted life years and could reduce health care costs
by €1.7 million per 100,000 pregnant women.122 Unfortunately,
however, neither Europe nor the United States has implemented
population-based screening programs. Possible reasons could be
the absence of an effective prophylaxis, as in RBC immunisation,
or a diagnostic tool to allow for identifying pregnancies at high
risk and to avoid overtreatment.  ICH. Specific treatment regimens, including dose and start of
Prophylaxis
The RBC counterpart of FNAIT, haemolytic disease of the fetus
and newborn, a highly effective prophylactic regimen was imple-
mented in 1960. This prophylactic treatment contained allo-
antibodies against the major cause of HDFN, RhD. Anti-D
prophylaxis resulted in a major step forward in preventing related
perinatal mortality and morbidity. The exact mechanism, how-
ever, to which this prophylaxis works is not completely under-
stood. Several studies, mainly in vitro or in vivo murine studies,
have been performed to develop such a prophylactic treatment
for FNAIT.123-125 Currently, an international project, called the
CHAPTER 41  Fetal Platelet Disorders 507
PROFNAIT (Development of a PROphylactic treatment for
nia) project (www.profnait.eu), is collecting plasma from HPA-
1a immunised women to extract the HPA-1a alloantibodies to
develop a prophylactic drug similar to anti-D. 
Conclusion
Idiopathic thrombocytopenic purpura and FNAIT are the most
frequent causes of fetal and neonatal platelet disorders. In ITP
patients, the low risk for perinatal ICH does not justify routine
invasive fetal testing. Antenatal therapy is only instituted for
maternal reasons. There is no evidence that maternal therapy
leads to an increase in the fetal or neonatal platelet count. Platelet
counts of newborns in consecutive pregnancies correlate well with
those of their siblings. Caesarean section is recommended only for
obstetric reasons.
Fetal and neonatal alloimmune thrombocytopenia occurs in
approximately 1 in 1000 pregnancies. In contrast to ITP, in
pregnancies complicated by FNAIT, fetal and neonatal bleed-
ing complications and in particular perinatal ICH do occur,
often with serious sequelae. Antenatal treatment is not insti-
tuted for maternal but for fetal and neonatal reasons and is
aimed to prevent these complications from occurring. In
absence of population-based screening, preventive therapy can
only be applied after an affected sibling. The antenatal man-
agement should be noninvasive because invasive strategies,
consisting of FBS with or without platelet transfusions, result
in a high risk for complications, including fetal loss. There is
growing evidence and acceptance that the use of these invasive
procedures in the management of pregnancies complicated by
FNAIT does more harm than good. The most commonly used
noninvasive treatment is weekly administration of IVIG. Treat-
ment with IVIG seems safe and highly effective in preventing
the treatment, are based on the obstetric history, in particular
whether or not the previous affected child had an ICH. Because
of both its rarity and seriousness, the treatment of this disorder
should be undertaken in or in collaboration with a centre expe-
rienced in invasive fetal procedures and with expert laboratory
and blood bank backup.
The future of managing and understanding FNAIT will be
determined by the possibility for population-based screening,
the ability to identify the pregnancies at high risk for bleeding
complications and the availability of prophylaxis.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17.  Scott JR, Rote NS, Cruikshank DP. Anti-
platelet antibodies and platelet counts in
35.  Kunishima S, Hayakawa A, Fujita K, Saito H.
Transient macrothrombocytopenia associated
pregnancies complicated by autoimmune with maternal-neonatal hpa-21bw incompat-
1. Sola-Visner M, Saxonhouse MA, Brown RE.
thrombocytopenic purpura. Am J Obstet Gyne- ibility. Thromb Res. 2013;131:e286–e288.
Neonatal thrombocytopenia: what we do and
col. 1983;145:932–939. 36.  Ohto H. [neonatal alloimmune thrombocyto-
don’t know. Early Hum Dev. 2008;84:499–
18. Kaplan C, Daffos F, Forestier F, et al. Fetal penia]. Nihon Rinsho. 1997;55:2310–2314.
506.
platelet counts in thrombocytopenic preg- 37. Ohto H, Miura S, Ariga H, et al. The natu-
2. Cherin P, Cabane J. Relevant criteria for sel­
nancy. Lancet. 1990;336:979–982. ral history of maternal immunization against
ecting an intravenous immunoglobulin prep-
19. Guidelines for the investigation and manage- foetal platelet alloantigens. Transfus Med.
aration for clinical use. BioDrugs. 2010;24:
ment of idiopathic thrombocytopenic pur- 2004;14:399–408.
211–223.
pura in adults, children and in pregnancy. Br 38.  Curtis BR, Ali S, Glazier AM, et  al. Isoim-
3. Dreyfus M, Kaplan C, Verdy E, et al. Frequency
J Haematol. 2003;120:574–596. munization against cd36 (glycoprotein iv):
of immune thrombocytopenia in newborns:
20. George JN, Woolf SH, Raskob GE, et  al. description of four cases of neonatal isoimmune
a prospective study. Immune thrombocy-
Idiopathic thrombocytopenic purpura: a thrombocytopenia and brief review of the lit-
topenia working group. Blood. 1997;89:
practice guideline developed by explicit erature. Transfusion. 2002;42:1173–1179.
4402–4406.
methods for the American Society of Hema- 39. Sajid M, Stouffer GA. The role of alpha(v)
4. Sainio S, Jarvenpaa AL, Renlund M, et  al.
tology. Blood. 1996;88:3–40. beta3 integrins in vascular healing. Thromb
Thrombocytopenia in term infants: a popu-
21. von dem Borne AE, Helmerhorst FM, van Haemost. 2002;87:187–193.
lation-based study. Obstet Gynecol. 2000;95:
Leeuwen EF, et  al. Autoimmune thrombo- 40. Campbell S, Swann HR, Seif MW, et al. Cell
441–446.
cytopenia: detection of platelet autoantibod- adhesion molecules on the oocyte and pre-
5. Kamphuis MM, Paridaans NP, Porcelijn L,
ies with the suspension immunofluorescence implantation human embryo. Hum Reprod.
et al. Incidence and consequences of neonatal
test. Br J Haematol. 1980;45:319–327. 1995;10:1571–1578.
alloimmune thrombocytopenia: a systematic
22. Michel M, Novoa MV, Bussel JB. Intra- 41. Kumpel BM, Sibley K, Jackson DJ, et  al.
review. Pediatrics. 2014;133:715–721.
venous anti-D as a treatment for immune Ultrastructural localization of glycoprotein
6. van den Akker ES, De Haan TR, Klumper
thrombocytopenic purpura (ITP) during iiia (GPIIIa, beta 3 integrin) on placental
FJ, Lopriore E, et al. Severe fetal thrombocy-
pregnancy. Br J Haematol. 2003;123:142– syncytiotrophoblast microvilli: implications
topenia and hydrops in RHD alloimmunized
146. for platelet alloimmunization during preg-
pregnancies: independent risk factors. Am J
23. Beilin Y, Zahn J, Comerford M. Safe epi- nancy. Transfusion. 2008;48:2077–2086.
Obstet Gynecol. 2007;197:S152.
dural analgesia in thirty parturients with 42. van Gils JM, Stutterheim J, van Duijn TJ,
7. Burrows RF, Kelton JG. Thrombocyto-
platelet counts between 69,000 and 98,000 et al. Hpa-1a alloantibodies reduce endothe-
penia at delivery: a prospective survey
mm(-3). Anesth Analg. 1997;85:385–388. lial cell spreading and monolayer integrity.
of 6715 deliveries. Am J Obstet Gynecol.
24. van Veen JJ, Nokes TJ, Makris M. The risk of Mol Immunol. 2009;46:406–415.
1990;162:731–734.
spinal haematoma following neuraxial anaes- 43. Yougbare I, Lang S, Yang H, et al. Maternal
8. Burrows RF, Kelton JG. Fetal thrombocy-
thesia or lumbar puncture in thrombocyto- anti-platelet beta3 integrins impair angiogen-
topenia and its relation to maternal throm-
penic individuals. Br J Haematol. 2010;148: esis and cause intracranial hemorrhage. J Clin
bocytopenia. N Engl J Med. 1993;329:
15–25. Invest. 2015;125:1545–1556.
1463–1466.
25. Choi S, Brull R. Neuraxial techniques in 44. Santoso S, Wihadmadyatami H, Bakchoul T,
9. Gill KK, Kelton JG. Management of idio-
obstetric and non-obstetric patients with et al. Antiendothelial alphavbeta3 antibodies
pathic thrombocytopenic purpura in preg-
common bleeding diatheses. Anesth Analg. are a major cause of intracranial bleeding in
nancy. Semin Hematol. 2000;37:275–289.
2009;109:648–660. fetal/neonatal alloimmune thrombocytope-
10. Samuels P, Bussel JB, Braitman LE, et  al.
26. Moise Jr KJ. Autoimmune thrombocytope- nia. Arterioscler Thromb Vasc Biol. 2016;36:
Estimation of the risk of thrombocytopenia
nic purpura in pregnancy. Clin Obstet Gyne- 1517–1524.
in the offspring of pregnant women with pre-
col. 1991;34:51–63. 45. Tiller H, Killie MK, Husebekk A, et  al.
sumed immune thrombocytopenic purpura.
27.  Christiaens GC, Helmerhorst FM. Validity of Platelet antibodies and fetal growth: mater-
N Engl J Med. 1990;323:229–235.
intrapartum diagnosis of fetal thrombocytope- nal antibodies against fetal platelet antigen
11. Burrows RF, Kelton JG. Pregnancy in patients
nia. Am J Obstet Gynecol. 1987;157:864–865. 1a are strongly associated with reduced birth-
with idiopathic thrombocytopenic purpura:
28.  Kagan R, Laros Jr RK. Immune thrombocyto- weight in boys. Acta Obstet Gynecol Scand.
assessing the risks for the infant at delivery.
penia. Clin Obstet Gynecol. 1983;26:537–546. 2012;91:79–86.
Obstet Gynecol Surv. 1993;48:781–788.
29. Gernsheimer T, McCrae KR. Immune 46. Althaus J, Weir EG, Askin F, et al. Chronic
12. Bussel J, Kaplan C, McFarland J. Recom-
thrombocytopenic purpura in pregnancy. villitis in untreated neonatal alloimmune
mendations for the evaluation and treatment
Curr Opin Hematol. 2007;14:574–580. thrombocytopenia: an etiology for severe
of neonatal autoimmune and alloimmune
30. Cines DB, Bussel JB. How I treat idiopathic early intrauterine growth restriction and the
thrombocytopenia. The working party on
thrombocytopenic purpura (ITP). Blood. effect of intravenous immunoglobulin ther-
neonatal immune thrombocytopenia of the
2005;106:2244–2251. apy. Am J Obstet Gynecol. 2005;193:1100–
neonatal hemostasis subcommittee of the sci-
31. Gernsheimer T, James AH, Stasi R. How I 1104.
entific and standardization committee of the
treat thrombocytopenia in pregnancy. Blood. 47.  Murphy MF, Hambley H, Nicolaides K,
isth. Thromb Haemost. 1991;65:631–634.
2013;121:38–47. Waters AH. Severe fetomaternal alloimmune
13. Bussel JB, McFarland JG, Berkowitz RL.
32. Tiller H, Kamphuis MM, Flodmark O, et al. thrombocytopenia presenting with fetal hydro-
Antenatal management of fetal alloimmune
Fetal intracranial haemorrhages caused by cephalus. Prenat Diagn. 1996;16:1152–1155.
and autoimmune thrombocytopenia. Trans-
fetal and neonatal alloimmune thrombocy- 48. de Waal LP, van Dalen CM, Engelfriet
fus Med Rev. 1990;4:149–162.
topenia: an observational cohort study of 43 CP. von dem Borne AE. Alloimmunization
14. Christiaens GC, Nieuwenhuis HK, Bussel
cases from an international multicentre regis- against the platelet-specific Zwa antigen,
JB. Comparison of platelet counts in first and
try. BMJ Open. 2013;3(3). pii: e002490. resulting in neonatal alloimmune throm-
second newborns of mothers with immune
33. Radder CM, Brand A, Kanhai HH. Will it bocytopenia or posttransfusion purpura, is
thrombocytopenic purpura. Obstet Gynecol.
ever be possible to balance the risk of intra- associated with the supertypic drw52 anti-
1997;90:546–552.
cranial haemorrhage in fetal or neonatal gen including dr3 and drw6. Hum Immunol.
15. Butler JP, Durrant ST, Frost T. Successful remis-
alloimmune thrombocytopenia against the 1986;17:45–53.
sion of chronic, refractory autoimmune thrombo-
risk of treatment strategies to prevent it? Vox 49. Valentin N, Vergracht A, Bignon JD, et  al.
cytopenic purpura following non-myeloablative
Sang. 2003;84:318–325. Hla-drw52a is involved in alloimmuniza-
allogeneic stem cell transplantation. Bone Marrow
34.  Winkelhorst D, Kamphuis MM, de Kloet tion against pl-a1 antigen. Hum Immunol.
Transplant. 2003;31:621–622.
LC, et  al. Severe bleeding complications 1990;27:73–79.
16. Provan D, Stasi R, Newland AC, et al. Inter-
other than intracranial hemorrhage in neo- 50.  Reznikoff-Etievant MF, Kaplan C, Muller JY,
national consensus report on the investiga-
natal alloimmune thrombocytopenia: a case et al. Allo-immune thrombocytopenias, defini-
tion and management of primary immune
series and review of the literature. Transfusion. tion of a group at risk; a prospective study. Curr
thrombocytopenia. Blood. 2010;115:168–
2016;56(5):1230–1235. Stud Hematol Blood Transfus. 1988:119–124.
186.

507.e1
507.e2 References
51. L’Abbe D, Tremblay L, Goldman M, et  al.
Alloimmunization to platelet antigen hpa-1a
(Zwa): association with HLA-drw52a is not
100%. Transfus Med. 1992;2:251.
52. Taaning E, Antonsen H, Petersen S, et  al.
Hla antigens and maternal antibodies in allo-
immune neonatal thrombocytopenia. Tissue
Antigens. 1983;21:351–359.
53. Mueller-Eckhardt C, Mueller-Eckhardt G,
Willen-Ohff H, et  al. Immunogenicity of
and immune response to the human platelet
antigen Zwa is strongly associated with HLA-
b8 and dr3. Tissue Antigens. 1985;26:71–76.
54. Delbos F, Bertrand G, Croisille L, et al. Fetal
and neonatal alloimmune thrombocytope-
nia: predictive factors of intracranial hemor-
rhage. Transfusion. 2016;56:59–66.
55. Loewenthal R, Rosenberg N, Kalt R, et  al.
Compound heterozygosity of HLA-drb3*
01:01 and HLA-drb4*01:01 as a potential pre-
dictor of fetal neonatal alloimmune thrombo-
cytopenia. Transfusion. 2013;53:344–352.
56. Wu S, Maslanka K, Gorski J. An integrin
polymorphism that defines reactivity with
alloantibodies generates an anchor for MHC
class ii peptide binding: a model for unidi-
rectional alloimmune responses. J Immunol.
1997;158:3221–3226.
57. Taaning E. HLA antibodies and fetomater-
nal alloimmune thrombocytopenia: myth
or meaningful? Transfus Med Rev. 2000;14:
275–280.
58. Pereira J, Cretney C, Aster RH. Variation of
class I HLA antigen expression among plate-
let density cohorts: a possible index of plate-
let age? Blood. 1988;71:516–519.
59. Hutchinson AL, Dennington PM,
Holdsworth R, Downe L. Recurrent HLA-
b56 mediated neonatal alloimmune throm-
bocytopenia with fatal outcomes. Transfus
Apher Sci. 2015;52:311–313.
60. Starcevic M, Tomicic M, Malenica M,
Zah-Matakovic V. Neonatal alloimmune
thrombocytopenia caused by anti-HLA-a24
alloantibodies. Acta Paediatr. 2010;99:630–632.
61. Gramatges MM, Fani P, Nadeau K, et  al.
Neonatal alloimmune thrombocytopenia
and neutropenia associated with maternal
human leukocyte antigen antibodies. Pediatr
Blood Cancer. 2009;53:97–99.
62. Thude H, Schorner U, Helfricht C, et  al.
Neonatal alloimmune thrombocytopenia
caused by human leucocyte antigen-b27
antibody. Transfus Med. 2006;16:143–149.
63. Moncharmont P, Dubois V, Obegi C,
et  al. HLA antibodies and neonatal alloim-
mune thrombocytopenia. Acta Haematol.
2004;111:215–220.
64. Saito S, Ota M, Komatsu Y, et  al. Serologic
analysis of three cases of neonatal alloimmune
thrombocytopenia associated with HLA anti-
bodies. Transfusion. 2003;43:908–917.
65. De Tar MW, Klohe E, Grosset A, Rau T.
Neonatal alloimmune thrombocytopenia
with HLA alloimmunization: case report with
immunohematologic and placental findings.
Pediatr Dev Pathol. 2002;5:200–205.
66. Sasaki M, Yagihashi A, Kobayashi D, et  al.
Neonatal alloimmune thrombocytopenia
due to anti-human leukocyte antigen anti-
body: a case report. Pediatr Hematol Oncol.
2001;18:519–524.
67. del Rosario ML, Fox ER, Kickler TS, Kao
KJ. Neonatal alloimmune thrombocytope-
nia associated with maternal anti-HLA anti-
body: a case report. J Pediatr Hematol Oncol.
1998;20:252–256.
68. Chow MP, Sun KJ, Yung CH, et al. Neonatal
alloimmune thrombocytopenia due to HLA-a2
antibody. Acta Haematol. 1992;87:153–155.
69. Onishi S, Okubo S, Matsuzaki T, et al. [report
of two cases of neonatal alloimmune throm-
bocytopenia caused by anti-HLA antibody,
and their screening using umbilical cord
blood]. Rinsho Ketsueki. 1992;33:42–47.
70. Winkelhorst D, Porcelijn L, van de Weerd
JME, et al. HLA Class i Antibodies in FNAIT.
(poster). Stockholm, Sweden: 14th European
Symposium on Platelet and Granulocyte
Immunobiology; 2016.
71. Davoren A, McParland P, Barnes CA, Mur-
phy WG. Neonatal alloimmune thrombocy-
topenia in the Irish population: a discrepancy
between observed and expected cases. J Clin
Pathol. 2002;55:289–292.
72. Kjeldsen-Kragh J, Killie MK, Tomter G,
et  al. A screening and intervention pro-
gram aimed to reduce mortality and serious
morbidity associated with severe neona-
tal alloimmune thrombocytopenia. Blood.
2007;110:833–839.
73. Turner ML, Bessos H, Fagge T, et  al. Pro-
spective epidemiologic study of the outcome
and cost-effectiveness of antenatal screening
to detect neonatal alloimmune thrombo-
cytopenia due to anti-hpa-1a. Transfusion.
2005;45:1945–1956.
74. Kamphuis MM, Paridaans N, Porcelijn L,
et al. Screening in pregnancy for fetal or neo-
natal alloimmune thrombocytopenia: system-
atic review. BJOG. 2010;117:1335–1343.
75. Skogen B, Bellissimo DB, Hessner MJ, et al.
Rapid determination of platelet alloantigen
genotypes by polymerase chain reaction
using allele-specific primers. Transfusion.
1994;34:955–960.
76. Tanaka T, Umesaki N, Maeda K, et al. Preg-
nancy and neonatal outcomes in unexplained
recurrent/habitual aborters treated by allo-
genic leukocyte immunization. Osaka City
Med J. 1997;43:81–87.
77. Kluter H, Fehlau K, Panzer S, et  al. Rapid
typing for human platelet antigen sys-
tems-1, -2, -3 and -5 by PCR amplification
with sequence-specific primers. Vox Sang.
1996;71:121–125.
78. Livak KJ. Allelic discrimination using fluoro-
genic probes and the 5’ nuclease assay. Genet
Anal. 1999;14:143–149.
79. Kaplan C, Porcelijn L, Vanlieferinghen P,
et  al. Anti-hpa-9bw (Maxa) fetomaternal
alloimmunization, a clinically severe neona-
tal thrombocytopenia: difficulties in diagno-
sis and therapy and report on eight families.
Transfusion. 2005;45:1799–1803.
80. Santoso S, Kiefel V, Mueller-Eckhardt C.
Immunochemical characterization of the
new platelet alloantigen system bra/brb. Br J
Haematol. 1989;72:191–198.
81. Kiefel V, Santoso S, Weisheit M, Mueller-
Eckhardt C. Monoclonal antibody—specific
immobilization of platelet antigens (MAIPA):
a new tool for the identification of platelet-
reactive antibodies. Blood. 1987;70:1722–
1726.
82. Lucas GF, Rogers SE. Evaluation of an enzyme-
linked immunosorbent assay kit (GTI PakPlus)
for the detection of antibodies against human
platelet antigens. Transfus Med. 1999;9:63–67.
83. Allen DL, Murphy MF. Evaluation of an
enzyme-linked immunosorbent assay kit
(GTI PakPlus) for the detection of antibod-
ies against human platelet antigens. Transfus
Med. 1999;9:384–386.
84. Kaushansky K. Thrombopoietin. N Engl J
Med. 1998;339:746–754.
85. Debili N, Wendling F, Cosman D, et al. The
MPL receptor is expressed in the megakaryo-
cytic lineage from late progenitors to plate-
lets. Blood. 1995;85:391–401.
86.  Broudy VC, Lin NL, Sabath DF, et al. Human
platelets display high-affinity receptors for
thrombopoietin. Blood. 1997;89:1896–1904.
87. Folman CC, de Jong SM, de Haas M. von
dem Borne AE. Analysis of the kinetics of
TPO uptake during platelet transfusion.
Transfusion. 2001;41:517–521.
88.  Fielder PJ, Hass P, Nagel M, et  al. Human
platelets as a model for the binding and deg-
radation of thrombopoietin. Blood. 1997;89:
2782–2788.
89. Porcelijn L, Folman CC, de Haas M, et  al.
Fetal and neonatal thrombopoietin levels in
alloimmune thrombocytopenia. Pediatr Res.
2002;52:105–108.
90. Folman CC, von dem Borne AE, Rensink
IH, et  al. Sensitive measurement of throm-
bopoietin by a monoclonal antibody based
sandwich enzyme-linked immunosorbent
assay. Thromb Haemost. 1997;78:1262–
1267.
91. Emmons RV, Reid DM, Cohen RL, et  al.
Human thrombopoietin levels are high when
thrombocytopenia is due to megakaryocyte
deficiency and low when due to increased
platelet destruction. Blood. 1996;87:4068–
4071.
92. Kosugi S, Kurata Y, Tomiyama Y, et al. Cir-
culating thrombopoietin level in chronic
immune thrombocytopenic purpura. Br J
Haematol. 1996;93:704–706.
93. Tahara T, Usuki K, Sato H, et al. A sensitive
sandwich ELISA for measuring thrombopoi-
etin in human serum: serum thrombopoietin
levels in healthy volunteers and in patients
with haemopoietic disorders. Br J Haematol.
1996;93:783–788.
94. Scheffer PG, Ait Soussan A, Verhagen OJ,
et al. Noninvasive fetal genotyping of human
platelet antigen-1a. BJOG. 2011;118:1392–
1395.
95. van der Schoot CE, Thurik FF, Veldhuisen
B, de Haas M. Noninvasive prenatal blood
group and hpa-1a genotyping: the cur-
rent European experience. Transfusion.
2013;53:2834–2836.
96. Wienzek-Lischka S, Krautwurst A, Frohner
V, et  al. Noninvasive fetal genotyping of
human platelet antigen-1a using targeted
massively parallel sequencing. Transfusion.
2015;55:1538–1544.
97. Bertrand G, Martageix C, Jallu V, et al. Pre-
dictive value of sequential maternal anti-hpa-
1a antibody concentrations for the severity
of fetal alloimmune thrombocytopenia. J
Thromb Haemost. 2006;4:628–637.
98. Sainio S, Javela K, Tuimala J, Koskinen S.
Usefulness of maternal anti-hpa-1a antibody
quantitation in predicting severity of foeto-
maternal alloimmune thrombocytopenia.
Transfus Med. 2013;23:114–120.
99. Ghevaert C, Campbell K, Stafford P, et  al.
HPA-1a antibody potency and bioactiv-
ity do not predict severity of fetomaternal
alloimmune thrombocytopenia. Transfusion.
2007;47:1296–1305.
100. Sonneveld ME, Natunen S, Sainio S, et  al.
Glycosylation pattern of anti-platelet igg is
stable during pregnancy and predicts clinical
outcome in alloimmune thrombocytopenia.
Br J Haematol. 2016;174(2):310–320.

101. Kapur R, Kustiawan I, Vestrheim A, et  al.
A prominent lack of igg1-fc fucosylation of
platelet alloantibodies in pregnancy. Blood.
2014;123:471–480.
102. Kapur R, Heitink-Polle KM, Porcelijn L, et al.
C-reactive protein enhances IgG-mediated
phagocyte responses and thrombocytopenia.
Blood. 2015;125:1793–1802.
103. Daffos F, Forestier F, Muller JY, et al. Prena-
tal treatment of alloimmune thrombocytope-
nia. Lancet. 1984;2:632.
104. Winkelhorst D, Murphy MF, Greinacher
A, et al. Antenatal management in fetal and
neonatal alloimmune thrombocytopenia:
a systematic review. Blood. 2017;129(11):
1538–1547.
105. Bussel JB, Berkowitz RL, McFarland JG,
et  al. Antenatal treatment of neonatal allo-
immune thrombocytopenia. N Engl J Med.
1988;319:1374–1378.
106. Birchall JE, Murphy MF, Kaplan C, Kroll
H. European collaborative study of the ante-
natal management of feto-maternal alloim-
mune thrombocytopenia. Br J Haematol.
2003;122:275–288.
107. Sainio S, Teramo K, Kekomaki R. Prenatal
treatment of severe fetomaternal alloimmune
thrombocytopenia. Transfus Med. 1999;9:321–
330.
108. Bussel JB, Berkowitz RL, Lynch L, et al. Ante-
natal management of alloimmune thrombo-
cytopenia with intravenous gamma-globulin:
a randomized trial of the addition of low-
dose steroid to intravenous gamma-globulin.
Am J Obstet Gynecol. 1996;174:1414–1423.
109. Berkowitz RL, Kolb EA, McFarland JG,
et al. Parallel randomized trials of risk-based
therapy for fetal alloimmune thrombocyto-
penia. Obstet Gynecol. 2006;107:91–96.
110. Berkowitz RL, Lesser ML, McFarland JG,
et  al. Antepartum treatment without early
cordocentesis for standard-risk alloimmune
thrombocytopenia: a randomized controlled
trial. Obstet Gynecol. 2007;110:249–255.
111. Lynch L, Bussel JB, McFarland JG, et  al.
Antenatal treatment of alloimmune throm-
bocytopenia. Obstet Gynecol. 1992;80:67–71.
112. Bussel JB, Berkowitz RL, Hung C, et al. Intra-
cranial hemorrhage in alloimmune thrombo-
cytopenia: stratified management to prevent
recurrence in the subsequent affected fetus.
Am J Obstet Gynecol. 2010;203:135.e131–
e114.
113. Wenstrom KD, Weiner CP, Williamson
RA. Antenatal treatment of fetal alloim-
mune thrombocytopenia. Obstet Gynecol.
1992;80:433–435.
114. van den Akker ES, Oepkes D, Lopriore E, et al.
Noninvasive antenatal management of fetal
and neonatal alloimmune thrombocytopenia:
safe and effective. BJOG. 2007;114:469–473.
115. Tiblad E, Olsson I, Petersson K, et al. Experi-
ences with fetomaternal alloimmune throm-
bocytopenia at a Swedish hospital over a
10-year period. Acta Obstet Gynecol Scand.
2003;82:803–806.
116. Kornfeld I, Wilson RD, Ballem P, et  al.
Antenatal invasive and noninvasive man-
agement of alloimmune thrombocytopenia.
Fetal Diagn Ther. 1996;11:210–217.
117. Paridaans NP, Kamphuis MM, Taune
Wikman A, et  al. Low-dose versus stan-
dard-dose intravenous immunoglobulin to
prevent fetal intracranial hemorrhage in
fetal and neonatal alloimmune thrombo-
cytopenia: a randomized trial. Fetal Diagn
Ther. 2015;38(2):147–153.
118. 
Van Der Lugt NM, Kamphuis MM,
Paridaans NP, et  al. Neonatal outcome in
alloimmune thrombocytopenia after mater-
nal treatment with intravenous immuno-
globulin. Blood. 2015;13:66–71.
119. Kaplan C, Murphy MF, Kroll H, Waters
AH. Feto-maternal alloimmune thrombo-
cytopenia: antenatal therapy with IvIgG
and steroids—more questions than answers.
European working group on FMAIT. Br J
Haematol. 1998;100:62–65.
References 507.e3
120. van den Akker ESA, Oepkes D, Brand A,
Kanhai HHH. Vaginal delivery for fetuses
at risk of alloimmune thrombocytopenia?
BJOG. 2006;113:781–783.
121. Roberts I, Murray NA. Neonatal thrombo-
cytopenia: causes and management. Arch
Dis Child Fetal Neonatal Ed. 2003;88:F359–
F364.
122. Killie MK, Kjeldsen-Kragh J, Husebekk A,
et  al. Cost-effectiveness of antenatal screen-
ing for neonatal alloimmune thrombocyto-
penia. BJOG. 2007;114:588–595.
123. Ni H, Chen P, Spring CM, et  al. A novel
murine model of fetal and neonatal alloim-
mune thrombocytopenia: response to intrave-
nous igg therapy. Blood. 2006;107:2976–2983.
124. Chen P, Li C, Lang S, et al. Animal model of
fetal and neonatal immune thrombocytopenia:
role of neonatal fc receptor in the pathogenesis
and therapy. Blood. 2010;116:3660–3668.
125. Li C, Piran S, Chen P, et  al. The maternal
immune response to fetal platelet GPIbα
causes frequent miscarriage in mice that can be
prevented by intravenous IgG and anti-FcRn
therapies. J Clin Invest. 2011;121:4537–4547.
126. Mueller-Eckhardt C, Kiefel V, Grubert
A, et  al. 348 cases of suspected neonatal
alloimmune thrombocytopenia. Lancet.
1989;1:363–366.
127. Davoren A, Curtis BR, Aster RH, McFarland
JG. Human platelet antigen-specific alloanti-
bodies implicated in 1162 cases of neonatal
alloimmune thrombocytopenia. Transfusion.
2004;44:1220–1225.
128. Knight M, Pierce M, Allen D, et  al. The
incidence and outcomes of fetomaternal allo-
immune thrombocytopenia: a UK national
study using three data sources. Br J Haema-
tol. 2011;152:460–468.
129. 
Porcelijn I, Huiskes E, von dem Borne
AEGK. Antibodies involved in NAIT.
Unpublished data, 2004.
42
Fetal Infections
GUILLAUME BENOIST, MARIANNE LERUEZ-VILLE AND YVES VILLE
KEY POINTS
• Cytomegalovirus (CMV) can cause both primary and nonpri-
mary infections. Around 50% of the pregnant women are
seronegative; 1% will develop primary infection during
pregnancy. Prenatal diagnosis relies on CMV polymerase
chain reaction (PCR) in amniotic fluid (AF) sampled at least 6
weeks after maternal seroconversion and after 21 weeks. Ce-
rebral fetal lesions determine the prognosis. A total of 10% of
infected infants are symptomatic at birth. Prenatal treatment
(hyperimmune globulins, antiviral drugs) is under evaluation.
No vaccine is currently available.
• Parvovirus B19 (PVB19) infection during the pregnancy can
cause intrauterine fetal death (IUFD), and hydrops fetalis re-
lated to fetal anaemia. Vertical transmission occurs in around
one third of the maternal infections and can be proven by
retrieving virus’ genome in the AF. Intrauterine fetal transfu-
sion is the only available treatment. It greatly improves the
prognosis when fetal anaemia is severe.
• The number of cases of congenital rubella infection has
drastically decreased since institution of vaccination pro-
grams. The risks of fetal infection and congenital abnormali-
ties decrease with gestation. No defect could be attributed
to maternal infections occurring after 20 weeks’ gestation.
Prenatal diagnosis of fetal infection can be performed by viral
genome amplification (PCR) in the AF. No therapy is currently
available when fetal infection is diagnosed. Worldwide, the
aim is to perform an adequate primary prevention through
vaccination of childbearing age women.
• In developed countries, more than 70% of pregnant women
are immune to varicella at the onset of pregnancy. Chickenpox
pneumoniae is the most severe complication of varicella. Most
of the reported cases of varicella congenital syndrome follow
maternal infections during the first trimester or at the onset of
the second trimester. Its frequency is estimated to be around
1% after maternal varicella. Prenatal diagnosis can be made
by amplification of the varicella zoster virus (VZV) genome
by PCR in AF. Perinatal infection defined by the occurrence of
varicella in neonates within 10 days from birth is caused by
maternal infection near term. VZV vaccine is available.
• Seroprevalence for Toxoplasma gondii in pregnant women
ranges from 20% to 75% among countries. Maternal primary
infection during pregnancy is diagnosed by seroconversion.
508
The earlier the transplacental passage of the parasite, the
more severe the symptoms and the prognosis. Prenatal diag-
nosis of fetal infection is mainly based on PCR amplification of
T. gondii genome in AF. When fetal infection is confirmed, the
combination of pyrimethamine, sulfadiazine and folinic acid
is the treatment protocol recommended.
• Syphilis is a sexually transmitted disease due to Treponema pal-
lidum. T. pallidum can be diagnosed by direct detection (darkfield
examination), nontreponemal tests (Venereal Disease Research
Laboratory and rapid plasma reagin, and treponemal antibody
tests. A newborn can be infected by transplacental passage of
T. pallidum from an infected untreated or insufficiently treated
mother or at the time of birth through an infected birth canal.
Transplacental passage of T. pallidum occurs throughout gesta-
tion, but fetal clinical manifestations only occur from 16 weeks
onwards. Fetal infection is associated with spontaneous abor-
tion, IUFD, preterm delivery and syphilis congenital syndrome.
Neonates affected by T. pallidum may present with manifesta-
tions divided into early signs (appearing during the first 2 years
of life) and late signs (those appearing later over the first decades
of life). Benzathine penicillin G is the reference maternal therapy
Cytomegalovirus
Virology
Cytomegalovirus is the largest member of the Herpetoviridae family.
Its genome is made of a double-stranded DNA. Human CMV is
highly species specific. Humans are its only reservoir. Several strains
have been described based on genomic definition, possibly causing
reinfections. Like other members of the Herpesvirus group, CMV
remains latent in several locations after acute infection. Reactivation
(recurrences) can therefore occur during latent infection.
The virus is acquired at mucosal sites (community exposure) or
by bloodborne transmission (blood transfusion or organ transplant).
In community exposure, cell-free virus is transmitted by contact
with saliva, tears, urine, nasal or genital tract secretion. Cell-free
virus is abundant in breast milk of infected women. Cell-mediated
spread of the virus begins after a replication phase. The main cells
infected by CMV are endothelial cells and polymorphic nuclear leu-
kocytes (PMLs). Dissemination of the virus is then haematogenous.
This viremic phase can be diagnosed by laboratory testing. The sec-
ondary sites of replication are the spleen and the liver. Dissemina-
tion and replication is not completely controlled by host immunity. 

Epidemiology
Cytomegalovirus infection is endemic and shows no seasonal vari-
ation. It is spread worldwide.
Transmission of the virus occurs by direct or indirect person-
to-person contact via urine; oropharyngeal, cervical and vaginal
secretions; semen; milk; tears; blood products; or organ trans-
plants. This is caused by prolonged shedding of the virus after
primary infection (PI). CMV infection requires intimate contact.
The patterns of seropositivity in the population vary greatly
with geographic, ethnic and socioeconomic conditions. The prev-
alence of specific antibodies to CMV increases with age and in
lower socioeconomic strata of developed countries as well as in
developing countries. Seroprevalence among women of childbear-
ing age also varies accordingly with these epidemiologic factors.
The seropositivity ranges from 50% to 85% in the United States
and in Western Europe. The incidence of CMV PI in pregnancy
also varies with socioeconomic conditions, from 1% to 6% per
year.1
Congenital infection is the result of transplacental transmis-
sion. In the United States, around 1% of all newborns are infected
when screened at birth. Nevertheless, this rate varies greatly among
geographic areas and with seroprevalence.1 The rate of transpla-
cental transmission varies between 5% and 50% for PI and 2%
and 3% for nonprimary infection (NPI).1-4
Congenital infections are mainly caused by PI, but several
reports show a possible fetal transmission after reinfection with
another strain of the virus or after reactivation of a latent infec-
tion.1,5-7 Irrespective of the type of maternal infection, the rate
of maternal-fetal transmission is considered to be lower (around
2%).1  or intermediate AI, fetal transmission was 23.6%. In multivariate
Maternal Infection
Clinical symptoms and nonspecific biological markers are more
often present in PI than in recurrences. Most PI in immunocom-
petent hosts are nevertheless subclinical. Nigro and coworkers
reported fever in 42.1% of PI and 17.1% of recurrences, asthe-
nia (31.4% and 11.4%,), myalgia (21.5% and 6.7%), rhino-
pharyngo-tracheo-bronchitis (42.1% and 29.5%) and flulike
syndrome defined as the simultaneous occurrence of fever and at
least one of these signs (24.5% and 9.5%), lymphocytosis of 40%
or greater (39.2% and 5.7%) and increased aminotransferases
blood levels (one or both >40 iu/L) (35.3% and 3.9%). The plate-
let count was significantly lower in PI but within normal range.8
The diagnosis of PI can be easily confirmed by document-
ing seroconversion (de novo appearance of virus-specific immu-
noglobulin (Ig) G in a pregnant woman who was seronegative
before the onset of pregnancy). Nevertheless, without a screening
program adopted by public health authorities where seronegative
women would be prospectively monitored during their pregnancy,
this remains a rare situation. More often, serologic testing is per-
formed when contamination is suspected with maternal clinical
symptoms or when ultrasound fetal abnormalities are visualised.
In this context, the presence of IgG leads to assess type of CMV
infection (past, primary or recurrent infection), and to date the
time of maternal PI as precisely as possible. IgM and IgG avidity
assays have been developed for these purposes.
IgM antibody response begins within days after maternal
contamination, reaching a peak in the first month after mater-
nal contamination. High to medium levels of IgM antibodies can
therefore be detected during the first 1 to 3 months after the onset
CHAPTER 42  Fetal Infections 509
of infection, after which the titres start declining. Nevertheless,
IgM detection can remain positive more than 1 year after PI.9
IgG avidity is indicative of the low functional affinity of the
recently produced IgG class antibody. Soon after PI, antibodies
show a low avidity for the antigen, though increasing progres-
sively. This characteristic is used to discriminate between recent
and over 3 months old PI. From the publication of Macé and
coworkers, an avidity index (AI) greater than 70% reflects PI for
longer than 3 months, and AI below 30% is highly suggestive of a
recent PI (<3 months). AI between 30% and 70% is more difficult
to interpret. This method is only applicable when IgG levels are
not too low.10 Nevertheless, one limitation of IgG AI testing is the
lack of standardisation. The best-published results reported 100%
negative predictive value (NPV) for a moderate to high IgG AI
obtained before the 18th week of gestation and an NPV dropping
to 91% when performed between 21 and 23 weeks’ gestation.11,12
Overall, Mace and coworkers reported that dating maternal PI
using IgG AI associated with IgM antibody detection failed to
date the onset of infection in only 1% of cases.10
One of the most challenging situations is the presence of both
IgM and IgG in the first trimester of pregnancy. Avidity and
maternal viremia help sorting out 90% of these cases using an
incremental risk algorithm for vertical transmission of between
less than 1% and 40%. Leruez-Ville and coworkers reported a sig-
nificant association between the risk for vertical transmission and
the AI combined with CMV polymerase chain reaction (PCR)
in maternal serum or IgG titres.13 In this series, a total of 4931
consecutive women were screened; 201 presented with positive or
equivocal IgM and with high, intermediate or low IgG avidity in
58.7%, 18.9% and 22.3%, respectively. In 72 women with low
analysis, positive CMV PCR in maternal serum, decreasing AI
and low IgG titres were all associated with fetal transmission.
After PI, both the virus and viral products can be recovered
from various fluids. However, viral shedding from these sites can
occur after recurrences as well. It has also been shown that detec-
tion of CMV-DNA in blood is diagnostic of PI in immunocom-
petent individuals, but in immunocompromised patients, it is
indicative of either PI or NPI.14
Quantification of viremia (infectious CMV particles in blood,
evaluated by culture or rapid shell vial method), antigenaemia
(pp65-positive peripheral blood leukocytes), quantification of
leukoDNAemia (CMV DNA in whole blood), leukocytes or
plasma and more recently RNAemia (CMV mRNA) are available.
Revello and coworkers reported the diagnostic ability of these
methods in 52 immunocompetent individuals comprising 40
pregnant women.14 Antigenaemia was detected in 57.1%, 25%
and 0% of the patients examined at 1, 2 and 3 months after onset
of infection, respectively. Viremia was detected in 26.3% of cases
during the first month only. LeuKoDNAemia, in 100%, 89.5%
and 27.3% at each of the three first months, 26.6% were still
positive at between 4 and 6 months, but none were positive after
6 months. At the same time, none of the patients with recurrent
infection had positive test results. These results provide with a
helpful method for dating maternal infection. 
Congenital Infection
Approximately 10% of the congenitally infected newborns have
signs and symptoms at birth. Half of them present the typical
cytomegalic inclusion disease (CID) with a high mortality rate.
The other half present with atypical or no symptoms. Of these,
510 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
90% are asymptomatic, although infected as shown by the pres-
ence of the virus in their urine during the first weeks of life.
Symptomatic infected newborns are defined as presenting at
least one of these abnormalities: prematurity, hypotrophy, pete-
chiae, jaundice, hepatosplenomegaly, purpura, neurological
findings (microcephaly, hypotonia, seizures), elevated alanine
aminotransferases levels, thrombocytopenia, conjugated hyper-
bilirubinemia, haemolysis and increased cerebrospinal fluid pro-
teins.15 Long-term follow-up of these children enabled researchers
to establish the occurrence of at least one sequela in 90% of the
subjects in this subgroup. These complications were psychomotor
delay (70%), sensorineural hearing loss (SNHL; 50%) and cho-
rioretinitis (54%). The mortality rate consecutive to congenital
CMV infection was estimated to be around 6%.
The best predictor for adverse neurodevelopmental outcome
in these infants is the presence of intracranial abnormalities on
computed tomography within the first month of life.16 These
abnormalities were also associated with SNHL at birth or with
deterioration of audiometric status during the first months of life.
Among asymptomatic neonates, who are known to have a better
long-term prognosis than symptomatic ones, 10% to 15% will still
develop sequelae, more often during the first 2 years of life. These
sequelae include SNHL in 7%, chorioretinitis in 2%, intellectual
deficit in 4% and microcephaly in 2%.17-21 SNHL is the most fre-
quent deficit related to congenital CMV infection in asymptom-
atic neonates. Fowler and coworkers have reported that 50% of the
audiometric deficits were bilateral; 50% worsened during the first
years of life; and in 18% of them, the audiologic deficit was diag-
nosed on average only at 27 months. CMV congenital infection
could be the cause of one third of cases of SNHL in childhood.22
Public health authorities have not adopted screening programs
in the majority of developed countries. Therefore abnormal ultra-
sound findings related to CMV congenital infection are more
likely to be diagnosed during systematic ultrasound examination
rather than during the follow-up of maternal seroconversion. This
may also explain why severe abnormalities are described more
often than subtle findings and more often after infection in the
first trimester of pregnancy.
One should know the natural history of the infection to under-
stand which ultrasound features are evocative and should lead to
offer invasive prenatal diagnosis. It is also important to remember
that the correlation between sonographic abnormal findings and
evidence of maternal infection is made several weeks apart.9 In PI,
around 25% to 50% of infected fetuses can be diagnosed by ultra-
sound examination. This is more likely to reflect the proportion of
papers published on this particular aspect than the performance
of ultrasound as a screening test.23 In the literature, description of
fetal CMV infection ultrasound features are twofold: gross abnor-
malities leading to the diagnosis of fetal CMV infection and subtle
findings discovered after thorough serial ultrasound examination
of fetuses at high risk after vertical transmission of the virus has
been shown. This at least partly explains that the performance
of ultrasound as a screening test could not be demonstrated in
a low-risk population. Ultrasound findings are summarised in
Table 42.1. Case reports or series of abnormalities mainly reported
fetal ventriculomegaly or hydrocephalus, either obstructive (aque-
ductal stenosis) or a vacuo with or without microcephaly, posterior
fossa cysts, cerebellar hypoplasia, severe intrauterine growth restric-
tion or even hydrops fetalis. Cases diagnosed because of maternal
seroconversion followed by serial fetal ultrasound examination
often lead to diagnose more subtle findings, mainly extracerebral
findings including hyperechogenic bowel and oligohydramnios.
Hyperechogenic bowel grade 2 is often a transient finding.
However, in a series comprising of 175 fetuses with hyperecho-
genic bowel, only 1 case was related to CMV infection.24 It is the
result of viral enterocolitis, and it can present as meconium ileus
or peritonitis in conjunction with ascites.25
Oligohydramnios is more often reported than polyhydram-
nios, and considering the affinity of the CMV for the kidney, it is
the result of a fetal nephritis.
The fetal heart can also be affected, showing cardiomegaly
with a thick myocardium, which may contain punctuate calcifi-
cations. As a functional consequence, Drose and coworkers have
also described tachyarrhythmia.26 This is a rare finding that could
participate in the development of fetal hydrops.
Generalised oedema and ascites may also suggest anaemia-
related hydrops caused by the combined effect of liver failure and
bone marrow infection. This striking presentation may eventually
be transient with both ultrasound and biological normalisation at
follow-up.27
Mild or unilateral ventriculomegaly, increased pericerebral
spaces, echogenic vessels in the thalami and basal ganglia or punc-
tuate echogenicities in the periventricular area are subtle findings
especially if they are isolated and are therefore likely to slip through
nontargeted ultrasound screening. The development of fetal MRI
has become an asset in the assessment of infected fetuses.28-30 MRI
using both T1- and T2-weighted sequences could help define the
onset of fetal infection. Lissencephaly may reflect injury before 16
or 18 weeks, but polymicrogyria is likely to follow injury at 18 to
24 weeks’ gestation, and cases with normal gyral patterns would
have probably been injured during the third trimester, showing
diffuse heterogeneity within the white matter.28
Fetal infection is diagnosed when the virus or the viral DNA is
found in the fetal compartment. CMV can be detected in the AF
by conventional viral isolation, rapid culture or molecular assays.
Virus isolation has a high specificity but has a lower sensitivity
than PCR. In recent years, PCR has been established as a reliable
technique in reference laboratories. Various PCR assays have been
described with multiple modalities, including single step, nested,
nested modified using higher volume or multiples aliquots, and
the most recent real-time PCR. The efficacy of these methods has
been evaluated in several studies and is dependent on the virologic
method used: sensitivity and specificity range between 75% and
100% and between 67.3% and 100%, respectively.9,31-34
The false-negative results reported were explained in most cases
by inappropriate timing of amniocentesis. After seroconversion or
reactivation, the process leading to CMV excretion in the fetal
urine takes an average of 6 to 8 weeks, and this interval should be
recognised to avoid false-negative prenatal diagnosis.9 Amniocen-
tesis should also be performed after fetal urination is well estab-
lished and therefore not before 21 weeks. When the conditions of
sampling are ideal,35 the sensitivity of prenatal diagnosis by PCR
has been reported to be close to 100%. Another explanation for
false-negative results is a late transmission of the virus through the
placenta. This occurs in 8% to 15% of cases, and infected new-
borns have a good prognosis.36
False-positive PCR results have also been reported when the
neonate was not infected; these false-positive diagnoses may be
explained by contamination of the AF with maternal blood dur-
ing amniocentesis if the mother had a positive CMV DNAemia
result at the time of sampling. Indeed, Revello and coworkers
showed that CMV DNA may be recovered in the blood of nearly
50% of immunocompetent patients up to 3 months after CMV
PI.37 Another explanation could be laboratory contamination
 CHAPTER 42  Fetal Infections 511

TABLE Fetal Abnormalities Diagnosed In Utero by Ultrasound Examination as Reported in Seven Series in the
42.1 Literature from 2000
Enders Liesnard Lipitz Azam Gouarin
et al.34 et al.31 et al.50 et al.300 Picone et al.54 Guerra et al.32 et al.53
(2001) (2000) (2002) (2001) (2004) (2000) (2002) Total
Number of congenital 57 55 51 26 42 16 30 277
CMV infectionsa
Overall ultrasound 39 14 11 5 26 6 15 116 (42%)
findings
IUGR 12 6 6 0 10 1 10 45 (16%)
Hydrops 4 0 2 0 2 0 0 8 (3%)
Ascites 15 0 0 2 2 0 1 20 (7%)
Pericardial effusion 3 0 0 0 1 0 0 4 (1%)
Pleural effusion 0 0 1 0 0 0 0 1 (<1%)
Skin oedema 2 0 0 0 0 0 0 2 (<1%)
Hyperechogenic 2 8 3 1 14 2 6 36 (13%)
bowel
Hepatomegaly or 3 1 0 1 3 0 0 8 (3%)
splenomegaly
Liver calcifications 0 1 1 0 0 0 0 2 (<1%)
Placentomegaly 2 0 0 1 2 0 0 5 (2%)
Oligohydramnios or 6 1 4 0 4 0 0 15 (5%)
anhydramnios
Polyhydramnios 1 1 1 0 1 0 0 4 (1%)
Other findingsb 8 0 0 0 0 0 0 8 (3%)
Microcephaly 11 2 0 1 6 0 5 25 (9%)
Hydrocephaly 9 2 0 0 0 0 2 13 (5%)
Ventriculomegaly 7 1 4 1 14 4 4 35 (13%)
Brain structure abnor- 10 1 3 0 13 0 9 36 (13%)
malitiesc
aCongenital cytomegalovirus (CMV) infection proved in urine at birth or after examination of fetuses after termination of pregnancy.
bOther findings: asymmetry of cardiac ventricles, cardiomyopathy, small lungs, hyperechogenic abdominal tumour, abnormal head shape, no fetal movements and short limbs.
cBrain structure abnormalities: brain calcifications, periventricular echogenicity, porencephaly, lissencephaly, subependymal cysts, choroid plexus cysts, cystic structure in cerebellum, agenesis of cerebellar

vermis and cerebellar hypoplasia.

IUGR, Intrauterine growth restriction.
  

occurring during PCR testing. Indeed, in some of these studies, a
nested CMV PCR was used, which is known to be a very sensitive
technique but at high risk for contamination. Generalisation of
semiautomated real time PCR might help to overcome the risk for
contamination and achieve quasi-absolute specificity for prenatal
diagnosis of CMV infection.
Prognostic factors during prenatal period. Currently, the
association of positive DNA detection in AF and the presence
of cerebral abnormalities on ultrasound are sufficient to accept a
woman’s request for termination of a pregnancy. Nevertheless, this
assumption must be tempered. Indeed, the individual prognostic
value of ultrasound findings is very difficult to establish because
termination of pregnancy prevents follow-up of these infants. Fur-
thermore, we know that frequent ultrasound abnormalities such
as hyperechogenic bowel, fetal growth restriction, isolated cerebral
calcifications or mild ventriculomegaly do not appear to be consis-
tently associated with a poor outcome (Table 42.2).
Prognostic evaluation benefits from the adjunct of fetal MRI
to ultrasound. The combination of MRI and ultrasound allows for
better positive predictive value (PPV) and NPV of fetal imaging
up to 90%.38,39 MRI is best at studying the cortical development
and the temporal regions whose lesions are highly suggestive of
CMV infection on postnatal imaging.40 In the study of Doneda
and coworkers, temporal lesions were observed very commonly
(37%) during magnetic resonance imaging (MRI), even if the lat-
ter was performed early (mean gestational age at MRI, 25 weeks),
and these lesions were never visible on ultrasound.41 Reservations
may be issued for some abnormalities visible at MRI alone and
512 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE Outcome at Follow-Up in Relation to Antenatal Ultrasound Findings in Cytomegalovirus (CMV) Congenitally
42.2 Infected Newborns Reported in Six Series from 2000
CMV-infected newborns with Prenatal ultrasound
Reference US abnormalities in utero (n) abnormalities Outcome at follow-up
Liesnard et al.31 (2000) 5 HB Normal at 6 mo
Moderate FGR, oligohydramnios Normal at 3 yr
Microcephaly, CNS abnormalities, HB Mental retardation, development retardation
FGR moderate Normal at 3 yr
FGR, HMG-SMG, HB Normal at 13 mo
Lipitz et al.50 (2002) 2 FGR, VMG Birth: cerebral palsy, SNHL
HB Birth: normal
Azam et al.300 (2001) 2 Hydrops Neonatal death
PV calcifications 50 mo: SNHL bilateral
Gouarin et al.53 (2002) 2 FGR Birth: normal
FGR Birth: normal
Picone et al.54 (2004) 4 HB, VMG Birth: normal
FGR Birth: normal
HB Birth: normal
Cerebral calcifications, VMG Birth: normal
Guerra et al.32 (2000) 2 VMG Birth: VMG resolved in utero, hepatitis
VMG, HB Birth: severe VMG, cerebral calcifications. HMG-
SMG

CNS, Central nervous system; FGR, fetal growth restriction; HB, hyperechogenic bowel; HMG-SMG, hepatomegaly–splenomegaly; PV, periventricular; SNHL, sensorineural hearing loss; VMG, ventriculo-
megaly.
  

for which the prognosis remains unclear. So, the significance of
the abnormal white matter signals remains currently unclear.40,42
This sign, very common in cases of CMV infection, could suggest
the diagnosis without predicting a poor prognosis, especially if it
is isolated and associated with normal ultrasound findings.41,43 In
2016, Cannie and coworkers studied the appropriate time to per-
form the fetal brain MRI.44 In this study, brain lesions were classi-
fied into five grades. The authors conducted a correlation between
the grade and the onset of hearing loss or neurologic deficit. The
MRI study fetal brain was performed at two different gestational
ages, and the authors did not show any superiority of one gesta-
tion over another (27 and 33 weeks).
The influence of gestational age at maternal infection on the
prognosis of fetal infection has been debated over many years. Sev-
eral previous studies suggested that the prognosis could be worse
when maternal infection occurs during the first trimester.2,31,45,46
The prenatal literature weighs heavily towards a worse prognosis of
infections in the first trimester. However, the interval between later
infections in pregnancy and the likelihood of developing severe
lesions before delivery and therefore visible on prenatal imaging
is very low. Nevertheless, fetal infection occurring in the second
and third trimesters can also carry a poor neurologic outcome.47
Primary infections were thought to cause more damage
than recurrences in women with detectable IgG before preg-
nancy.34,48-50 Unlike preconceptional immunity against rubella or
toxoplasmosis, preconceptional immunity against CMV provides
only partial protection against intrauterine transmission of the
virus to the fetus. Although vertical transmission rates vary sig-
nificantly between primary and NPIs (30%–50% vs 2%–3%),1-3
it seems that the prognosis of infected fetuses could be similar in
primary and in maternal NPIs.
Maternal factors predictive of fetal outcome are still poorly elu-
cidated. It seems that neither the presence of clinical symptoms
during PI nor virologic parameters in the mother are associated
with a higher transmission rate of the virus to the fetus.37
The influence of CMV strains on the biological properties of
the virus and particularly on the outcome of congenital infection
is a topic of current interest, but the use of viral sequence informa-
tion has failed to predict outcome.51
Fetal gender of infected fetuses has been retrospectively studied.
The proportion of females with brain abnormalities was statisti-
cally different from that of males (62 of 258 infected fetuses: 24%
vs 30/251: 12%, P = .004). The risk for abnormal brain develop-
ment in infected fetuses was twice as high in females as in males
(odds ratio [OR], 2; 95% confidence interval [CI], 1.26–3.21).52
The development of real-time PCR has allowed the evaluation
of the clinical significance of CMV viral load in AF.35,53,54 The
median viral loads are higher in the AF of symptomatic fetuses
than in that of asymptomatic fetuses.53 Only one study accounts
for the increase of CMV-DNA in AF with time. However, cut-offs

for CMV-DNA in AF are not easy to validate clinically.55 Alterna-
tively, proteomic analysis of AF allowed to discriminate between
fetuses to become symptomatic at birth and others.56
Recent data are available about the prognostic value of fetal
blood parameters. Thrombocytopenia is associated with active
infections more likely to lead to symptoms at birth.57 DNAe-
mia is higher in fetuses with abnormalities than in asymptomatic
fetuses.58 The mean values of neonatal blood viral load were statis-
tically higher in newborns that developed sequelae than in those
who did not and that approximately 70% of sequelae were found
in newborns with a quantitative PCR (qPCR) higher than 10,000
copies per 105 PMNLs.59 In 82 fetuses infected with CMV mainly
in the first trimester, 41 (50%) had a normal ultrasound at pre-
natal diagnosis (median, 23 weeks).55 In this study, the NPV of
ultrasound alone was 93% for predicting asymptomatic infection
at birth. By including fetal blood parameters (platelet count and
fetal blood viral load), this rate increased to 100%. Regarding the
PPV, it was 60% for ultrasound alone and was increased to 79%
when combined with fetal blood parameters. Other markers in
fetal blood, including β2-microglobulin, have shown to be cor-
related with postnatal outcome.60,61 These recent data suggest
an important role for biological analysis to refine the prognosis
of infected fetuses, mainly when the ultrasound abnormalities
are moderate (mild ventriculomegaly or isolated extra cerebral
findings).  ment. Despite the lack of randomisation, these results allow us
Management
Several antiviral drugs are active against CMV, and the three
licensed anti-CMV drugs (ganciclovir, cidofovir and foscarnet)
are being used successfully in immunocompromised patients.
However, their potential teratogenic effects and their well-known
toxicity do not support their use in pregnancy.
Anti-CMV compounds are currently at different stages of
development; several of these compounds are very promising in
term of efficacy and lack of toxicity. To date, preliminary results
on treatment of CMV congenital infection during pregnancy are
available from two studies with promising results.
The results of a first prospective nonrandomised trial using
intravenous CMV hyperimmune globulin (HIG) for CMV
maternal PI were published in 2005.62 Nigro and coworkers
selected 181 pregnant women with primary CMV infection. Two
study groups were formed. In the first group, called the ‘therapeu-
tic group’, comprising 79 patients, amniocentesis was performed.
PCR results were positive in amniotic fluid (AF) in 55. Immuno-
globulin therapy was given in 31 of them, a medical termination
of pregnancy was performed in 10 patients and no treatment was
given in 14. The proportion of symptomatic children at birth was
significantly lower in the group in patients who received treatment
(1 of 31 vs 7 of 14; OR, 0.02; P <.001). In the ‘prevention’ group,
102 women were included. An HIG treatment was initiated in 37
of them. No treatment was taken in the 65 remaining patients. In
the latter group, the pregnancy was terminated in 18 cases. The
vertical transmission rate was significantly lower in the subgroup
of patients who received treatment (6 of 37 vs 19/47; OR, 0.32; P
= .04). The results of this study suggested that the immunoglobu-
lin treatment was effective in reducing the proportion of symp-
tomatic children at birth in case of confirmed fetal infection and
could reduce vertical transmission in case of maternal infection
from 40% to 16%.
Following these encouraging results, a randomised trial against
placebo was conducted by an Italian team. The results of the
CHAPTER 42  Fetal Infections 513
CHIP trial were published in 2014.63 In this study involving 123
women with PI, 61 received treatment with immunoglobulin, and
63 were treated with placebo. The congenital infection rates were
30% in the treated group and 44% in the placebo group (P = .13).
Besides this negative result, the authors also observed an adverse
event rate of 13% versus 2%, including premature births, cho-
lestasis of pregnancy to intrauterine growth retardation and one
case of eclampsia. To date, immunoglobulins have failed to change
the prognosis of maternal CMV infection, both in terms of verti-
cal transmission and severity of fetal infection.
Jacquemard and coworkers have shown the pharmacologic
efficacy of valaciclovir in a pilot study in 21 cases of CMV con-
genital infections with ultrasound abnormalities.64 These results
have encouraged the same French team to lead a first randomised
double-blind study against placebo and methodology modified
to be interventional without randomisation with exclusive use of
valaciclovir. The results of this study titled CYMEVAL2 were pub-
lished in 2016.65 In this study, among the 43 fetuses infected with
CMV and with moderate ultrasound abnormalities or abnormal
fetal blood parameters, the proportion of asymptomatic children
was 34 of 43 (above the threshold of 31, children required to con-
sider treatment as effective). Comparing these results with data
from literature, valacyclovir increased the proportion of asymp-
tomatic neonates from 43% without treatment to 82% with treat-
to consider valaciclovir as a control to subsequently test different
antivirals. 
Prevention
No vaccine is currently available in the routine practice. Neverthe-
less, several studies have shown encouraging results, mainly with
gB recombining vaccines.66
Furthermore, Picone and coworkers have shown that if clear
information on CMV infection during pregnancy is given,
patients frequently agree to screening and this counselling can
result in a decreasing rate of seroconversion after information.67 
Parvovirus B19
Virology
The taxonomy of the Parvoviridae’s family includes the Densoviri-
nae (insect viruses) and the Parvovirinae (vertebral viruses), which
is composed of three genders (dependovirus, parvovirus and
erythrovirus). PVB19 is a nonenveloped single-stranded DNA
virus classified as an erythrovirus. It is the only parvovirus that can
cause human disease.
The primary target for PVB19 appears to be erythroid precur-
sor cells. Host cell receptor is the globoside or P-antigen (a blood
group antigen), a glycosphingolipid; therefore, patients with-
out this antigen on their red blood cells are naturally protected
against PVB19 infection.68 Globoside is situated on the surface
of erythrocyte progenitor cells (erythroblasts) but also on that of
other cells (endothelial, myocardial and placental cells, as well as
mature erythrocytes and megakaryocytes).69 Inside the host cells,
PVB19 replicates and induces apoptosis and toxic cell injury. 
Epidemiology
PVB19 infection occurs worldwide, and the characteristics of the
disease are constant. Neither antigenic nor specific viral genotypes
514 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

are related to the forms of the disease. Transmission of the virus infection before IgG can be detected. Nevertheless, it is important
continues throughout the year with winter and spring outbreaks. to remember that after a recent contact, there will be a window
Seroprevalence of PVB19 increases with age. In children of 7 days, during which both IgG and IgM will remain negative.
younger than 5 years of age, the prevalence of IgG antibodies is Furthermore, at the time of clinically overt hydrops fetalis, IgM
less than 5%, increasing to reach a median of 45% in young adults levels may sometimes already have become undetectable. In these
and more than 85% in the geriatric population.70,71 Some studies cases, PCR analysis of the same blood sample can be informative.
have shown a greater risk for PVB19 infection in women.72 Sero- In a series of 41 fetuses with PVB19 infection and anaemia, at the
prevalence is higher in white populations.73 time of fetal blood sampling, it has been reported that all mothers
The global incidence of PVB19 infection has been reported were PVB19-DNA positive, PVB19-IgG positive and B19-IgM
to be 1 to 2 per 10,000 individuals. In women of childbearing were detected in 95% of cases.91
age, risk factors for seroconversion are elementary school workers, Serologic testing should assess pregnant women who have been
contact with 5- to 11-year-old children at home or 5- to 18-year- exposed to PVB19 infections and those developing symptoms
old children at work, and women younger than 30 years of age.73 compatible with this infection. Seronegative women should be
Overall, it can be estimated that 1% to 2% of seronegative women retested 2 weeks later. They can be reassured in the absence of
at the onset of pregnancy would become infected during preg- seroconversion. In cases with PI, serial ultrasound examination
nancy in endemic periods and more than 10% in epidemic peri- including middle cerebral artery peak systolic velocity (MCA-
ods.72,74-76 Risk factors of PVB19 in pregnant women are exposure PSV) measurements should be performed every fortnight up until
to PVB19 in their own children, elementary school teachers and 12 weeks after exposure).92 
daycare workers.77-79
Infection with PVB19 usually occurs through contact with Fetal Infection
respiratory droplets, but PVB19 can also be transmitted by blood
and blood-derived products and can be transmitted vertically Vertical transmission occurs in approximately one third of the
from mother to fetus.80 Presence of IgG antibodies gives a lifelong cases of maternal infection (17%–33%).77,90,93
protection against reinfection with PVB19.  Fetal infection with PVB19 is associated with intrauterine fetal
death (IUFD), nonimmune hydrops fetalis (NIHF) and less often
brain anomalies. Fetal infection can also be asymptomatic.94 Fetal
Maternal Infection manifestations caused by PVB19 are summarised in Table 42.3.
Approximately 20% of infected immunocompetent individuals The consequences of fetal infection are not univocal through-
are asymptomatic.81 Erythema infectiosum (fifth disease) is the out the pregnancy. In the first trimester, it is controversial whether
most common clinical manifestation during childhood.82,83 It is PVB19 maternal infection increases the rate of miscarriage.95
characterised by a rash consisting of maculae that undergo cen- NIHF develops after maternal infection mainly in the first half
tral fading over in 1 to 4 days, mainly on the trunk and limbs.
Symptoms such as erythema infectiosum, mild fever, arthralgia
and headache start approximately 10 to 14 days after contami- TABLE Ultrasound Abnormalities on Fetuses Infected
nation and in about 50% of infected women. Arthralgia and 42.3 by Parvovirus B19
arthritis are common in the adult form of the infections.82,84
It affects females more often than males (60% vs 30%) and Cardiac system Increased cardiac biventricular
children (10%). Others possible symptoms include thrombocy- outer diameter73
topenia, meningoencephalitis, hepatitis, myocarditis and vascu- Myocarditis
litis.85-88 Immunocompromised hosts can also present transient
aplastic crisis, chronic red blood cell aplasia and virus-associated Nonimmune hydrops fetalis Pleural effusion
haematophagocytic syndrome.89 Symptoms reported by preg- Pericardial effusion
nant women are nonspecific, and serologic confirmation is Ascites
required. The most characteristic symptom is symmetrical
arthralgia, sometimes arthritis often involving small joints of the Abdominal wall oedema
hands, wrists and feet. The proportion of asymptomatic women Bilateral hydroceles
is around 30%.90
Amniotic fluid disorder
Specific IgM, IgG and IgA immunoglobulins are produced
after maternal infection. Specific IgM are the first antibodies to Brain abnormalitiesa Hydrocephalus74
rise around 10 days post infection. They sharply peak at 10 to 14
Microcephaly
days. The IgM response persists for 1 month to several months.
Specific IgG rises considerably more slowly about 3 weeks Intracranial calcifications
postinfection to reach a plateau at around 4 weeks after infection. Gastrointestinal system Fetal liver calcifications75
These antibodies probably last for life.
Maternal serum should be tested when there is evidence of Meconium peritonitis76,77
maternal exposure or clinical symptoms of PVB19 infection. A Other findings Sporadic cases of contractures78
booking sample with positive IgG but negative IgM indicates pre-
vious maternal infection. When the sample is negative for both Increased nuchal translucency79
IgM and IgG, absence of maternal infection can be confirmed. The Intrauterine growth restriction80
presence of IgM enables to conclude that a recent maternal infec-
tion has occurred regardless of IgG antibody levels. The absence of aAfter brain haemorrhage.
IgG associated with the presence of IgM is indicative of a recent   

of the pregnancy.92,96,97 The proportion of all NIHF caused by
PVB19 infection is around 10% to 15%. In a series of 50 cases
of NIHF, 4 were related to PVB19 infection.98 Its rate of occur-
rence is 3.9% after maternal infection throughout pregnancy, with
a maximum of 7.1% when infection occurs between 13 and 20
weeks’ gestation. The incidence of PVB19 infection associated
NIHF peaks between 17 and 24 weeks’ gestation.80 The inter-
val between maternal PVB19 infection and the development of
NIHF ranges from 2 to 6 weeks.99
Cases of IUFD have been described mainly at around 20 to
24 weeks’ gestation but as early as 10 weeks and as late as 41
weeks.90,97,100 Furthermore, IUFD could occur without NIHF.100
NIHF is mainly related to severe anaemia, which can also lead
to high-output cardiogenic heart failure. NIHF is more frequent
during the hepatic stage (8–20 weeks of gestation) of the haema-
topoietic activity when the half-life of erythrocytes is shorter than
later during the bone marrow and splenic haematopoietic stages.69
Among 63 cases of laboratory-confirmed maternal PVB19
infection, Puccetti and coworkers reported a vertical transmission
rate of 31.7% (20 of 63).101 Of the 20 infected, 8 had NIHF, 1
had signs suggestive of meconium peritonitis and 1 had an isolated
hydrothorax. Three of eight fetuses presenting with hydrops were
treated with intrauterine blood transfusion. Two of them died, and
the last showed resolution of anaemia. Among the five untreated
hydropic fetuses, one resolved spontaneously, two died and two
had also cardiomegaly and elective termination of pregnancy was
performed. All the anaemic fetuses had MCA-PSV values more
than 1.8 MoM. No stillbirth occurred. Overall, in this series, the
outcome of uncomplicated cases with PVB19 infection was good,
but in the presence of NIHF, the prognosis was very poor.
In 539 cases reported by Soothill and coworkers, 30% preceded
IUFD, 34% resolved spontaneously, 29% resolved after intrauter-
ine transfusion and 6% died despite intrauterine transfusion.102
In 20 cases of fetal PVB19 infection complicated by anaemia or
NIHF, Mace and coworkers reported that the survival rates were
70% (14 of 20) and 76% (13 of 17) for fetuses with one or more
transfusions.103 When fetal effusion regressed after the transfusion,
all 11 fetuses survived, and neonatal outcome was favourable for all.
Nonimmune hydrops fetalis is diagnosed by ultrasound exami-
nation with the association of marked ascites, cardiomegaly and
pericardial effusion and, in advanced stages, generalised oedema
and a thick hydropic placenta. Hydrops related to anaemia usually
presents with tense ascites, as well as thin-wall cardiomegaly. Pleu-
ral effusion is a late finding in anaemia-related hydrops and can be
sometimes observed as isolated finding.104
Polyhydramnios is rare with PVB19 infections. Pasquini and
coworkers evaluated the rate of women with polyhydramnios
who were screened positive to infectious disease by serum screen-
ing testing for TORCH (toxoplasmosis, other [syphilis, varicella-
zoster, parvovirus B19] rubella, CMV and herpes infections and
viral serologies) and PVB19.105 In this series of 290 cases, only two
women were positive for parvovirus B19 and one for toxoplasmo-
sis infection. In none of them the fetus was affected. Authors con-
cluded that infectious disease screening does not seem beneficial in
pregnancies with isolated polyhydramnios.
Pasquini and coworkers reviewed 141 cases of mild ventricu-
lomegaly.106 Screening for infections, including TORCH, PVB19
and syphilis, was carried out in all cases. Maternal IgM for PVB19
resulted positive in 4.6% of cases, and one neonate was infected
without any fetal or neonatal adverse consequence. Recent CMV
infection was documented in 4.4% of cases. Only in one case was
the infection transmitted to the fetus.
CHAPTER 42  Fetal Infections 515
Isolated hyperechogenic bowel has been reported as the sole
ultrasonographic sign of fetal B19 infection.107 It is likely to reflect
resolving ascites.
The involvement of the fetal heart can be limited to dilation of
the cardiac cavities related to anaemia and hydrops or present as a
hypertrophic cardiomyopathy or myocarditis that can also develop
autonomously after spontaneous resolution of the NIHF.108,109
Fetal PVB19 infection has also been associated with paediatric
stroke, neonatal encephalitis or meningitis with perivascular cal-
cifications in the fetal cerebral cortex, basal ganglia, thalamus and
germinal layers, meconium peritonitis, fetal liver calcifications, eye
abnormalities such as cornea opacification and aphakic eyes or rare
bones lesions.110-115
Structural defects (cleft lip and palate, micrognathia, arthro-
gryposis, hypospadias) reported are likely to be coincidental find-
ings.93,116 During a large community-wide outbreak of PVB19
infection, there was no increase in congenital malformations rate
compared with the periods before and after the epidemic. This
may lead to the conclusion that the association of birth defects
could be fortuitous or the consequence of anaemia-related hypoxia
or thrombocytopenia-related haemorrhage.117
Neurological impairment can be observed related to severe and
prolonged fetal anaemia accompanied by thrombocytopenia that
can lead to intraventricular brain haemorrhage. Cerebellar haem-
orrhage, polymicrogyria, infarctions, calcifications and obstructive
hydrocephalus have also been reported in association with PVB19
infection during the first half of pregnancy.110,111,118-123
Rarely, maternal symptoms of ‘mirror hydrops’ occur second-
ary to lysis of the hydropic villi of the placenta. Maternal mirror
syndrome is a maternal preeclampsia-like syndrome with oedema,
hypertension, proteinuria and anaemia and seems to reflect the
intensity and persistence of the fetal anaemia.103,124
After confirmation of maternal infection with a suggestive sero-
logic profile, fetal ultrasound examination should be performed to
exclude the presence of fetal anaemia and hydrops. However, in most
cases, NIHF is a coincidental finding during routine ultrasound
examination.125 Thus fetal anaemia should be suspected when the
MCA-PSV Doppler is increased.126,127 These changes in blood flow
are the result of increased cardiac output and decreased viscosity of
fetal blood.128 The prediction of fetal anaemia by MCA-PSV mea-
surements also allows evaluating the severity of anaemia. Elevated
values indicate the need for fetal blood sampling and intrauterine
transfusion. Severe NIHF together with normal MCA-PSV in par-
vovirus B19 infection indicates either spontaneous resolution of
fetal anaemia or progressive and autonomous myocarditis.
Increased nuchal translucency and reversed a-wave ductus
venosus Doppler in the first trimester have also been reported to be
associated with B19 infection as early signs of cardiac failure.129-131
The virus or its genome can be retrieved in AF by PCR with a
high sensitivity. This can also be applied in pregnant women lack-
ing an adequate antibody-mediated immune response or who are
immunocompromised or immunosuppressed in whom serologic
testing for PVB19 is unreliable.132 Detection of PVB19-specific IgM
in fetal blood has a significantly lower sensitivity than PCR.133,134
Otherwise, B19 PCR in the AF is part of the etiologic diagnosis
when NIHF is observed, as well as CMV PCR and karyotype.135 
Management
Management of PVB19 infection with fetal transfusion can cor-
rect anaemia and is likely to significantly reduce perinatal mortal-
ity rate. It should be restricted to cases with increased MCA-PSV.
516 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
IUT has been reported to be effective as early as 13 weeks of gesta-
tion.131 Timely IUT of anaemic fetuses with severe hydrops reduces
the risk for fetal death.125,134,136-138 In most cases, one transfusion
is sufficient for fetal recovery, and high reticulocyte levels indicate
that anaemia is being corrected spontaneously. Hydropic changes
can take up to several weeks to resolve, and MCA-PSV should be
used to evaluate the correction of fetal anaemia.127,139,140
In a consecutive series of 27 cases in a 15-year period, Chauvet
and coworkers reported that among the 19 fetuses treated by
transfusions, 11 were liveborn compared with 2 of the 6 not
treated (57.8 vs 33.3%, not significant).125 The survival rate was
higher during the second half of the study period (23.1 vs 71.4%;
P = .02) and for less severe anaemia (P = .03). In this series, all 13
liveborn children appeared healthy at the age of 1 year.
Overall, children who survived a successful IUT in this situa-
tion have a good neurodevelopmental prognosis.74,125,141-145 Nev-
ertheless, the small number of cases limits the conclusions of these
series.  syndrome and optic neuritis, are rare complications also reported
Prevention
Exclusion of pregnant women from the workplace during endemic
periods is not recommended. If pregnant women are exposed
to individuals who are suspected or known to be infected with
PVB19, they should report this exposure to their obstetrician.146
Serologic testing is required.
Ballou and coworkers described a recombinant parvovirus B19
vaccine composed of VP1 and VP2 capsid proteins, which proved
to be immunogenic and safe to use in human volunteers.147 Vac-
cination of nonimmune pregnant women could be a highly effec-
tive method to prevent fetal infection with parvovirus B19, but
the cost-effectiveness of this strategy in the general population
remains to be determined.147  infection is difficult, testing avidity of IgG antibodies is helpful. PI
Rubella
Virology
Rubella, or ‘German measles’, is a contagious disease caused by
rubella virus, an RNA virus classified as a Togavirus, genus Rubivi-
rus. It is a worldwide human disease without any animal reservoir.
Its teratogenic effect was observed by Gregg in 1941.148
This virus spreads by means of airborne transmission or drop-
lets shed from respiratory secretions from 7 days before to 5 to
7 days or more after the onset of the cutaneous eruption. The
mean incubation time is 2 weeks. Viremia occurs 5 to 7 days after
exposure. Transplacental haematogenous transmission can occur
during this phase.  NSHL is the most frequent feature of CRS, occurring in at
Epidemiology
Rubella is a moderately contagious worldwide disease. Infec-
tions occur mostly in late winter and early spring. Before the
development of vaccination programs, epidemics used to occur
every 6 to 9 years. During the last major rubella epidemic in
the United States from 1964 to 1965, an estimated 12.5 mil-
lion people got rubella, 11,000 pregnant women lost their
babies, 2100 newborns died and 20,000 babies were born with
congenital rubella syndrome (CRS).149-152 Since the introduc-
tion of systematic vaccination in childhood, epidemiologic
characteristics have changed. The number of cases of congeni-
tal infection has drastically decreased. The National Congenital
Rubella Syndrome Registry carries out the CRS surveillance. It
has reported 121 cases between 1990 and 2001 in the United
States. A large part of these cases was diagnosed in unvacci-
nated immigrant women. 
Maternal Infection
Of people infected with rubella, 25% to 50% do not have any
symptoms. When symptomatic, rubella is usually a mild disease.
A characteristic rash appears and lasts for 1 to 5 days, extending
from the face passing down through the body to the feet after an
incubation period of 14 to 21 days. This rash is often pruriginous
and proceeded by fever, headache, conjunctivitis, malaise, coryza,
lymphadenopathy and dyspnoea. Arthralgia and sometimes frank
arthritis may develop after the rash fades away. These symptoms
have been reported in up to 70% of infected women.
Thrombocytopenia, encephalitis, myocarditis, Guillain-Barré
in maternal rubella.
Laboratory diagnosis is essential because inapparent or sub-
clinical diseases are common. Furthermore, other viral eruptions
can mimic rubella.
Acute rubella infection is characterised by the appearance of
IgG and IgM. IgM antibodies are most consistently detectable 5
to 10 days after the onset of the rash, rise rapidly to peak at around
20 days and decline thereafter until disappearance after 50 to 70
days. In a few patients, IgM remains detectable for up to 1 year.
IgG antibodies can be measured by several methods, but enzyme-
linked immunoabsorbent assays (ELISA) is most commonly used.
IgG becomes detectable within 5 to 15 days after rash onset.
The titres rapidly increase to peak at 30 days and then gradually
decline over a period of years to constant titres. When dating the
is associated with low IgG avidity. The technique used should be
accounted for because the kinetics of the antibodies can vary with
the technique used. 
Fetal Infection
Congenital rubella can cause miscarriage or stillbirth but can
also be asymptomatic. Between these two extremities, a congeni-
tal rubella spectrum (CRS) of anomalies can be observed: heart
defect, eye defects and hearing abnormalities (Table 42.4).
The cardiac features of fetal rubella infection include patent
ductus arteriosus, pulmonary artery stenosis, pulmonary valve ste-
nosis, coarctation of aortic isthmus, interventricular septal defect
and interauricular septal defects.
least 80% of infected infants. It can be uni- or bilateral, and ranges
from mild to severe.
Eye defects caused by rubella infection are described as a ‘salt
and pepper’ retinopathy caused by abnormal growth of the pig-
mentary layer of the retina,150 cataract,151 microphtalmia150,152
and more rarely primary glaucoma.152 These defects can be diag-
nosed early after birth. Others ocular manifestations can have a
late onset including abnormalities of the anterior chamber of the
eye.
Other abnormalities can be related to rubella infection: intra-
uterine growth restriction,153 encephalitis, neurologic abnor-
malities (including microcephaly153 and mental retardation),
thrombocytopenia,154 hepatosplenomegaly,155 obstructive jaun-
dice155 and radiographic changes of the long bones.150

TABLE Fetal Abnormalities Related to Congenital
42.4 Rubella Infection
Cardiac system Atrial septal defects
Ventricular septal defects
Pulmonic arterial hypoplasia116
Patent ductus arteriosus116
Coarctation of aortic isthmus116
Aortic regurgitation117
Ocular system Cataract118
Microphthalmia107
Central nervous system Microcephaly110
Intracranial calcifications109
Subependymal pseudocysts
Other abnormalities Hepatomegaly
Splenomegaly
Renal disorders119
Hyperechogenic bowel
Meconium peritonitis120
Hypospadias119
Growth restriction121
  
Very late onset complications have also been attributed to
the virus, including diabetes mellitus,156 growth hormone defi-
ciency157 and thyroid dysfunction.156,158
The risk for fetal infection and congenital abnormalities
decreases with gestation. However, fetal infection can occur at
any time during pregnancy. This has been extensively reported by
Miller and coworkers.159 This rate decreases from 81% before 12
weeks’ gestation, 67% between 13 and 14 weeks’ gestation and
25% between 23 and 26 weeks’ gestation, to increase up to 35%
between 27 and 30 weeks’ gestation, 60% between 31 and 36
weeks’ gestation and 100% after 36 weeks’ gestation. Periconcep-
tional infection until 11 days after the last menstruation period
was not associated with any risk for fetal infection.160
The assessment of risk for congenital defects is dependent upon
the methodology of investigation because most infants infected
after the first trimester are grossly asymptomatic at birth. Sero-
logic testing for these children is required for precise evaluation of
congenital infection. Furthermore, long-term follow up is needed
to evaluate the rate of sequelae attributable to rubella infection.
Peckham and coworkers reported that the overall incidence of
defects in 218 children evaluated when they were at least 2 years
of age161 was 23%, including 52% after infection before 8 weeks’
gestation, 36% at 9 to 12 weeks’ gestation and 10% at 13 to 20
weeks’ gestation; no defect could be attributed to maternal infec-
tions occurring after 20 weeks’ gestation.
Sever and coworkers reported 128 cases of proven rubella at
different gestational ages: 29 mothers had rubella at or before 14
weeks’ gestation, and 38% of their infants had sequelae; 55 moth-
ers had rubella between 15 and 28 weeks’ gestation, and 20% of
CHAPTER 42  Fetal Infections 517
their infants had sequelae. No CRS was observed when maternal
infection occurred after 20 weeks of gestation.162,163
Miller and coworkers have shown that none of 102 infants
had CRS when rubella was contracted after 18 weeks of gestation,
although 85% of the children whose mothers were infected at or
before 12 weeks’ gestation and 25% between 13 and 18 weeks’
gestation were symptomatic.159
During the first 12 weeks of pregnancy, when the fetus is
incapable of producing immunoglobulin, maximum damage can
occur in 80% of fetuses.164 In the second trimester, the risk for
fetal infection decreases significantly to 25%. This is because of
the well-developed immune response of fetuses and the structural
changes of the placenta, leading to increased resistance to the
rubella virus. In the last trimester of pregnancy, the rate of fetal
infection rises back to 100%, but fetal damage is rare because of
the fully developed immune system of the fetus.
Because a proven maternal case of rubella during pregnancy is
not always associated with a vertical transmission and a fetal infec-
tion is not always indicative of fetal defects, prenatal diagnosis is
important to distinguish the cases in which the fetus is involved.
Fetal infection can be proven by direct isolation of the virus or
genome by reverse transcriptase PCR (RT-PCR) in AF sampled
by amniocentesis at least 6 to 8 weeks after maternal infection
to avoid false-negative results. This should be done in association
with a targeted ultrasound examination.165,166 A prognosis can be
drawn considering the timing of maternal infection and virologic
diagnosis of fetal infection associated with ultrasound findings. 
Management
Maternal administration of large doses of immune globulin
in women exposed to rubella during pregnancy has been pro-
posed.167-169 This treatment does not prevent fetal infection. 
Prevention
Despite many national, rubella-containing vaccine immunisation
programmes, the burden of CRS still exists. It is estimated that
about 238000 children are born with this syndrome each year,
with the majority reported in the developing countries.170 In con-
trast, during the years 2001 to 2004, only five infants with CRS
were reported in the United States.171
Three rubella vaccines were introduced in the United States in
1969. In 1979, the RA27/3 (human diploid fibroblast) strain was
introduced that replaced the other three vaccines. It is based on a
live, attenuated, nontransmissible virus. It is safe and effective in
more than 95% of vaccinated patients who are developing long-
term immunity after a single dose. Haemagglutination inhibition
antibodies typically develop 10 to 28 days after vaccination. Nev-
ertheless, up to 5% of vaccinated women fail to seroconvert. Side
effects include arthralgia or arthritis in about 25% of the cases.
The Vaccine In Pregnancy (VIP) registry collected cases of women
exposed within 3 months before conception and up to term until
1988. No CRS cases have been reported after exposure to this
vaccine during pregnancy. Three children, however, demonstrated
serologic evidence of congenital infection with rubella, but none
of the three had malformations. Nevertheless, the vaccine is still
contraindicated in pregnancy. Effective contraception is recom-
mended around the time of vaccination. In October 2001, the
Federal Center for Disease Control and Prevention changed rec-
ommendations on delaying pregnancy after receiving the rubella
vaccine, reducing it from 3 months to 1 month.
518 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
After vaccination, seroconversion is induced in more than 95%
of the vaccinated population.172,173 However, only two thirds of
the vaccinated population continue to have lifelong immunity
against rubella infection.174 This is why a considerable proportion
of women who were vaccinated during childhood are susceptible
to rubella infection by the time they reach childbearing age.
Postpartum rubella vaccination has been recommended to
reduce the risk for congenital rubella infection in subsequent preg-
nancies.175 This theoretically increases the uptake of vaccination.
The fecundity rate is reduced during the postpartum period, giving
the mother time to build immunity against rubella.176 However, a
link has been suggested between postpartum rubella immunisation
to an increased rate of arthritis, but this remains controversial.177,178
The Cochrane database questioned the effectiveness of the post-
partum rubella vaccination program in preventing the CRS.179
Their conclusions are not yet available.  up to10 days after the appearance of the rash. Dyspnoea occurs
Chickenpox-Zoster Virus
Virology
Chickenpox (i.e., varicella) is the acute form of the disease caused
by varicella zoster virus (VZV), and zoster is the representation of
the reactivation of the same virus. VZV (Herpesvirus varicellae) is
a DNA virus, member of the Herpesviridae family.
The virus is airborne spread from cutaneous vesicles and by
respiratory droplets from patients with varicella or zoster. The oro-
pharynx is the site of entry and of initial viral replication. After the
initial replication phase, the virus reaches the local lymph nodes
and spreads by transient viremia towards the viscera. Then a sec-
ond replication phase occurs with a second and more intense vire-
mia with a cutaneous rash. Crusting and scabbing of the vesicles
represent the typical features of the chickenpox rash that occurs
after cell-mediated immunity activation. Varicella is most conta-
gious at the time of onset of the rash and for 1 to 2 days after-
wards. After clinical recovery, the patient is not contagious, and
the latency phase begins.
Zoster occurs in persons who have previously had chickenpox.
This vesicular infection is generally confined in the dermatome of
the ganglia where the VZV was in latency. The virus travels down
the axon and then reaches the skin supplied by that nerve. Occa-
sionally, a disseminated zoster can occur, particularly in immu-
nocompromised patients, probably caused by a transient viremia.  (cataract, microphthalmia, chorioretinitis, Claude Bernard Horner
Epidemiology
Chickenpox ranks as one of the most contagious infectious dis-
ease during childhood. The incidence of varicella has decreased
in developed countries after the development of vaccination cam-
paigns. Between 70% and 80% of young American adults report
a history of varicella.180 Based on serologic studies, it appears that
fewer than 25% of adults with no history of chickenpox are sus-
ceptible to an acute disease.181
The rate of chickenpox during pregnancy has been estimated
between 1 to 10 per 10,000 pregnancies.182-184 In pregnancy, the
virus may be transmitted transplacentally, resulting in the congenital
varicella syndrome (CVS), or at birth, resulting in a neonatal varicella.  5%, 10% and 25% of infants at birth, after maternal varicella dur-
Maternal Infection
The usual incubation time is of 10 to 21 days. Chickenpox is
then heralded by the simultaneous occurrence of fever and rash.
In adults, the eruption is often preceded by prodromal fever by
2 to 3 days. The rash begins typically on the face or scalp and
spreads rapidly to the trunk but sparing the extremities. Lesions
are characterised by typical evolution from red macules to vesicles,
pustules and brown crusts. All stages of the lesions can be visual-
ised simultaneously in the same anatomical region. Several crops
can develop in a period of 5 days. Sometimes mucosal lesions may
develop in the mouth or on the vulva. Residual scaring is rare.
The severity of the rash ranges from a few lesions to thousands,
especially in adults.
Chickenpox pneumoniae is the most severe complication of
varicella and develops in about 10% of adults.185 Risk factors are
smoking, third trimester pregnancy and numerous skin lesions.186
Radiologic evidence of pneumonia has been found in 16% of a
series of 110 cases.187 The onset of pneumonia is 2 to 4 days and
in 70% of cases and can be accompanied by cyanosis, pain chest,
haemoptysis and bronchic rales. The mortality rate has been
reported to be of up to 10%, but this includes immunosuppressed
individuals, and the statistics are now dated.
Specificities of chickenpox in pregnant women are surrounded
by the severity of the clinical aspect and the number of the lesions,
as is the case for all adults. Nevertheless, the severity is due to the
higher incidence of pneumonia during pregnancy.
In cases with widespread typical vesicular exanthema and a his-
tory of recent exposure, the diagnosis is made clinically. Labora-
tory diagnosis can be made by demonstrating VZV antigen using
immunofluorescence188 and VZV-DNA detection by PCR in skin
lesions.189,190 VZV infections show at least a fourfold increase in
specific antibody titres by using a sensitive test as FAMA or ELISA.
The presence of IgM suggests a recent infection. The persistence
of VZV antibodies beyond the age of 8 months is suggestive of
intrauterine varicella. 
Congenital Infection
Varicella is not associated with an increased risk for fetal loss or low
birth weight but seems to be associated with an increased risk to
deliver preterm.191,192 However, cases of IUFD have been reported
after varicella, mainly during the first half of pregnancy.193,194
Laforet and coworkers described first the CVS in 1947.195 It
is defined by skin lesions (scars and skin loss); eye abnormalities
syndrome); limb deformities (hypoplasia, equinovarus, muscle
atrophia, absent digits); neurologic abnormalities (cortical atrophy,
mental retardation, microcephaly, seizures, limb paresis); abnor-
malities of the intestinal tract, diaphragm and urinary tract (due to
autonomic nervous system injury); and zoster in infancy.184,196-213
Enders and coworkers reported the results of a prospective
study of 1373 women with varicella and 366 with zoster dur-
ing their pregnancy. Among the first group, they reported defects
attributable to CVS in 0.4% for infections between 0 and 12
weeks’ gestation and 2% at between 13 and 20 weeks’ gestation.
The overall rate of congenital defects was 0.7%.199 The rate of fetal
defects must be differentiated from the rate of vertical transmis-
sion. In the same study, authors reported a detection of IgM in
ing the first, the second and the third trimesters, respectively. In
another study, the rate of vertical transmission was 8.4% among
107 cases of maternal varicella.214 In a large prospective study per-
formed in the United States of 347 cases of maternal varicella, the
rate of congenital infection was 1.3%.215

TABLE Ultrasound Abnormalities Related to Varicella
42.5 Infection of the Fetus
Cerebral anomalies149-153 Ventriculomegaly
Hydrocephalus
Microcephaly
Polymicrogyria
Porencephaly
Ophthalmologic anomalies Congenital cataract
Microphthalmia
Musculoskeletal anoma- Limb contractures
lies142,148
Hypoplasia
Other findings Intestinal echogenic foci
Hepatic echogenic foci
Hydrops
Polyhydramnios
Growth restriction
  
As highlighted by Enders and coworkers, the timing of mater-
nal infection plays a major role in the severity of fetal damage.
Most of reported cases of CVS follow maternal infections that
occurred during the first trimester or at the onset of the second
trimester. Nevertheless, several publications have reported severe
defects in fetuses after maternal chickenpox after the 20th week of
gestation,215-218 although the most common risk to infants when
VZV is transmitted to the fetus after the 20th week is zoster dur-
ing early childhood.
Mattson and coworkers reported the neurobehavioral outcome
of 84 children born to women infected with varicella during preg-
nancy.219 Children were 3 to 15 years of age at the time of testing.
Clinical features of the CVS were present in only 1 of the 84 chil-
dren. When compared with 40 children born to women who were
not infected with varicella during pregnancy, there was no difference
in the test performance of the two groups. In addition, within the
varicella sample, no meaningful differences were found relative to
infection-related hyperthermia or the timing of infection. Overall,
the authors concluded that the children born to women infected with
VZV during pregnancy and who do not have structural features char-
acteristic of the fetal varicella syndrome are not neurodevelopmentally
different from unexposed, uninfected control children.  10 days before delivery. When maternal chickenpox is suspected,
Prenatal Diagnosis of Fetal Infection
Targeted ultrasound examination can visualise some of the features
of varicella-related embryopathy involving several organs with
variable severity. A latency period of 5 to 19 weeks between mater-
nal infection and sonographic detection of the first fetal anomalies
has been reported in serial assessment of high-risk fetuses.220
Common sonographic findings include intestinal and hepatic
echogenic foci; hydrops; and musculoskeletal, cerebral and ocu-
lar anomalies. Growth restriction and polyhydramnios have also
been reported. Skin lesions following a dermatomal distribution
CHAPTER 42  Fetal Infections 519
are typical of varicella embryopathy but are not always pres-
ent and are usually not identifiable by ultrasound examination.
Musculoskeletal anomalies may be related to scar formation and
lead to limb contractures and hypoplasia, which are accessible to
ultrasound examination.199,214 Cerebral anomalies documented
with ultrasound include ventriculomegaly, hydrocephalus,192,221
microcephaly with polymicrogyria and porencephaly.222-224 Con-
genital cataract and microphthalmia are the most common ocular
lesions. Hyperechoic foci within the fetal liver have been identi-
fied as calcifications at autopsy.214,225
Ultrasound abnormalities related to varicella infection of the
fetus are summarised in the Table 42.5.
Prenatal diagnosis can be made by amplification of the VZV
genome by PCR in AF.
Mouly and coworkers reported the results of amniocentesis
and PCR performed in 107 women who had developed clinical
varicella before 24 weeks of gestation. Nine of 107 (8.4%) were
positive by PCR, but only 2 (1.8%) of them were positive in cell
culture. PCR is therefore the method of choice for diagnosis.199
The viral culture is less sensitive.
Some authors reported the necessity to avoid false-positive
results by verifying the negativity of the maternal viremia before
performing amniocentesis. Iatrogenic transmission has been
reported in this condition.226
The indication of systematic amniocentesis after maternal vari-
cella without any fetal abnormality at ultrasound examination is
controversial. If fetal defects are detected by ultrasound examina-
tion, amniocentesis should be performed, even if abnormalities
are nonspecific (AF abnormalities, growth restriction).
Other methods such as fetal blood sampling or chorionic vil-
lous sampling have been reported to be useful to identify infected
fetuses but with less efficiency (lower sensitivity, lower specificity
mainly).221,227-229 Currently, these methods are no longer used in
this condition. 
Perinatal Infection
This is defined by the occurrence of varicella in neonates within
10 days from birth. It is caused by maternal infection near term in
which chickenpox develops in 24% to 50% of the neonates.230-235
The interval between the onset of maternal rash and that of neo-
natal infection is of 9 to 15 days. When delivery occurs within 10
days of the onset of maternal clinical infection, maternal IgG can-
not develop and cross the placenta to protect the fetus or neonate.
Neonatal chickenpox is lethal in up to 30% of the cases.234 
Management Options
Varicella zoster virus infection during pregnancy and more than
serologic testing should be performed. A positive test result rules
out acute infection. When the mother is seronegative at the time
of sampling, two management options are possible:
1. Serial ultrasound examinations alone can rule out severe fetal
infections but miss asymptomatic vertical transmission of the
virus to the fetus.
2. Amniocentesis can be performed to diagnose all fetal infec-
tions. However, fetal transmission will only occur in less than
10% of the cases.
If PCR is positive in the AF, targeted ultrasound examination
should then be performed every fortnight. MRI can also be infor-
mative in assessing the fetal brain.
520 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
When maternal infection occurs after 20 weeks of gestation,
prenatal diagnosis is not recommended because fetal varicella syn-
drome has very rarely been reported after 20 weeks.236  Parasitology
Pregnant women with varicella zoster virus infection near
term and less than 10 days before delivery. When maternal erup-
tion occurs within 10 days before delivery, every attempt should
be made to delay delivery until maternal IgG has time to be pro-
duced and cross the placenta.  eral different forms: the oocyst, the tachyzoite and the cyst.
Antiviral treatment for maternal infection. Pregnant women
who develop varicella should be treated with oral (val)acyclo-
vir and followed up carefully. Those with pneumonia should be
admitted and treated with intravenous antiviral agent. Acyclo-
vir is more effective when administrated within 1 day after the
onset of varicella, and it shortens the course of illness by about
1 day.237 In postexposure prophylaxis, antiviral treatment could
decrease the severity of the maternal disease. This management
strategy has been proven to be effective in healthy children and
adolescents, too.238  ing to cell death, invasion of neighbouring cells, dissemination
Passive immunisation for infants after possible perinatal
infection. Pooled immunoglobulins can attenuate the symptoms
without preventing chickenpox when administrated to contact
persons within 72 hours of exposure.239 For infants born between
2 and 4 days after maternal eruption, the use of zoster immu-
noglobulins could decrease the risk and severity of the neonatal
disease in one uncontrolled study.235 It is also recommended to
administrate VZV immunoglobulins to infants of mothers who
develop varicella within 5 days of delivery within 2 days after
delivery (CDC 1996). Doses of 125 UI (1.25 mL or one vial)240
have been given intramuscularly as early as possible after delivery.
For postexposure prophylaxis, passive immunisation has been rec-
ommended, too.241,242 A significant reduction of maternal com-
plications and of the risk for fetal infection has been reported with
this therapeutic option.
Infected mothers and infants should be isolated from mater-
nity and neonatal units to avoid spreading the infection. 
Prevention
A live-attenuated vaccine obtains active immunisation against
VZV. The US Centers for Disease Control and Prevention (CDC)
has approved this vaccine in 1996. The vaccine protects against
varicella in about 85% of cases. A mild and transient rash may
occur about 1 month after immunisation. A possible spread of the
vaccine-type strain to nonimmunised individuals at the time of
this rash is possible. Nevertheless, in all cases reported iatrogenic
cases of varicella were mildly symptomatic. Vaccine indications in
the United States are for susceptible women of childbearing age at
least 3 months before conception.
Vaccines that contain live-attenuated VZV are contraindi-
cated during pregnancy. To monitor the pregnancy outcomes
of women inadvertently vaccinated with VZV-containing
vaccines immediately before or during pregnancy, the Reg-
istry for VZV-Containing Vaccines was introduced in 1995.
From inception of the registry in 1995 through March 2012,
no cases of CVS and no increased prevalence of other birth
defects have been detected among women vaccinated within 3
months before or during pregnancy.243 It is recommended that
women should be counselled to avoid becoming pregnant for
1 month after each dose of a VZV-containing vaccine. Thus
even if effective to decrease the severity of the disease in post-
exposure, the anti-VZV vaccine is not recommended during
the pregnancy.244  headache and cervical lymphadenopathy. The incubation period is
Toxoplasmosis
Toxoplasma gondii is an intracellular protozoan that belongs to the
phylum Apicomplexa, subclass Coccidian. Other members of this
phylum are Plasmodium and Cryptosporidium spp. It can take sev-
Oocysts. Members of the cat family are definitive hosts of
T. gondii. Replication of the parasite happens in the intestine of
the cat, resulting in the production and shedding of oocysts in
the faeces for 7 to 21 days during acute infection. After sporula-
tion, which takes place within 1 to 21 days, oocysts containing
sporozoites are infective when ingested by mammals, including
humans, and give rise to the tachyzoite stage. Tachyzoites enter
all nucleated cells by active penetration and form a cytoplasmic
vacuole. After repeated replications, host cells are disrupted, lead-
of the tachyzoites in the bloodstream and infection of many tis-
sues (CNS, eyes and muscles as well as placenta). A strong local
inflammatory response and tissue destruction are responsible for
the clinical manifestations of the disease. The immune response
causes the transformation from tachyzoites into bradyzoites and
the cysts’ formation. Bradyzoites persist in host for life inside
cysts. They are morphologically identical to tachyzoites but mul-
tiply slowly, express stage-specific molecules and are functionally
different. Tissue cysts contain hundreds of thousands of bradyzo-
ites and form within the host cells in the brain as well as skeletal
and cardiac muscles. Bradyzoites can be released from cysts, trans-
form back into tachyzoites and cause recurrences of infection in
immunocompromised patients. Cysts are infective stages for both
intermediate and definitive hosts. 
Epidemiology
Humans can be infected with T. gondii by ingestion or handling of
undercooked or raw meat (mainly pork and lamb) containing cysts
as well as water or food containing oocysts excreted in the faeces of
infected cats. Most individuals are infected inadvertently, so the spe-
cific route of transmission cannot usually be established. Variations in
the seroprevalence of T. gondii seem to correlate with nutrition and
hygiene habits of a population. This finding supports that the oral
route is the major source of infection. Epidemics of toxoplasmosis in
humans and in sheep are attributed to the exposure to infected cats
and demonstrate the important role of oocyst excretion by cats in the
propagation of the infection. Transmission during direct human-to-
human transmission other than from mother to fetus has not been
recorded, and transmission by breastfeeding is still controversial.245,246
Seroprevalence for T. gondii increases with age and does not
vary significantly between males and females. It ranges from 20%
to 75% among countries.247,248 In the United States, the overall
seroprevalence is 22.5% to 30%.248,249
The incidence of congenital infection is 2 to 3 infants per 1000
live births in France, markedly higher than in the United States,
where the incidence is 1 in 1000 to 1 in 10,000. 
Maternal Infection
Because more than 90% of PIs in immunocompetent individuals
are asymptomatic, the diagnosis of maternal infection is difficult
and based on laboratory testing. In the remaining 10%, clinical
symptoms include mononucleosis-like illness with low-grade fever,

estimated to be within the range of 4 to 21 days.248 In immu-
nocompetent women, other rare manifestations can be observed,
including encephalitis, myocarditis, hepatitis and pneumonia.
Primary infection during pregnancy is diagnosed by sero-
conversion. IgM and IgG are detected by immunofluorescence
antibody tests, enzyme-linked immune filtration assay, immuno-
absorbent agglutination assay or other methods.250 IgG becomes
detectable 1 to 2 weeks after infection and remains elevated indefi-
nitely. IgM becomes detectable within the first days and rapidly
increases, reaching a peak and remain elevated for 2 to 3 months
before decreasing. Elevated IgM have to be interpreted with cau-
tion because it has been shown that 27% of women remain posi-
tive for IgM for more than 2 years.251 Only seroconversions place
a fetus at risk for developing congenital toxoplasmosis.  depending on the trimester of maternal seroconversion.
Fetal Infection
The incidence and severity of the infection depend on gestational
age at the time of maternal infection. The earlier the transplacen-
tal passage of the parasite, the more severe the symptoms and the
prognosis.252,253
The risk for fetal infection is multifactorial, depending on the
timing of maternal infection, immune reaction of the mother
during parasitemia, parasitic load and virulence of the strain
involved.247
The probability of fetal infection is only 1% when PI occurs
during the preconception period but increases as pregnancy pro-
gresses.254 Infection acquired during the first trimester by women
untreated with anti–T. gondii drugs results in congenital infec-
tion in 10% to 25% of cases. For infections occurring during
the second and third trimesters, the incidence of fetal infection
ranges between 30% and 54% and 60% and 65%, respectively.255
Foulon and coworkers reported that when maternal infection
occurs before the fifth gestational week, the transmission rate is
less than 5%, but this rises to more than 80% at the end of the
pregnancy.256
The consequences are more severe when fetal infection occurs
early in pregnancy. It is associated with miscarriages and severe
disease. The highest incidence of severe abnormalities at birth is
seen in children whose mothers have acquired a PI at between 10
and 24 weeks of gestation257 (Fig 42.1). Hohlfeld and coworkers
have reported that 77.9% of the infected fetuses that had ultra-
sound abnormalities after maternal infection at the first trimester,
20.4% had ultrasound abnormalities after infection in the second
trimester and no infected fetuses had ultrasound abnormalities
when infection occurred at the third trimester.253
Approximately 15% of congenitally infected newborns will be
symptomatic at birth. The classical triad including hydrocepha-
lus, chorioretinitis and intracranial calcifications is found in fewer
than 10% of cases. Other clinical manifestations are nonspecific
symptoms such as maculopapular rash, generalised lymphadenop-
athy, hepatomegaly, splenomegaly, anaemia, hyperbilirubinemia
and thrombocytopenia.258
Around 85% of infected infants are asymptomatic at birth.
Nevertheless, a large proportion of these children develop sequelae
with visual impairment, mental and cognitive abnormalities of
variable severity, seizures or learning difficulties only after several
months or years. Guerina and coworkers reported that 40% of the
asymptomatic infants show abnormalities on cranial imaging and
ophthalmologic investigations.259
Vutova and coworkers investigated eye manifestations in con-
genital toxoplasmosis in 38 infants and children.260 The most
CHAPTER 42  Fetal Infections 521
1st 2nd 3rd
trimester trimester trimester
• Fig. 42.1 Schematic representation of the severity of the fetal affec-
tion (thin line) and of the vertical transmission (thick line) of toxoplasmosis
frequent finding was chorioretinitis with characteristic retinal
infiltrates (92%), and this was associated with other ocular lesions
in 71% of the cases. The second most common finding was
microphthalmia with strabismus. Other ocular lesions included
iridocyclitis, cataract, glaucoma and visual loss.
Wallon and coworkers reported the clinical evolution of ocular
lesions and final visual function in a prospective cohort of 327
congenitally infected children in France.261 The children were
identified by maternal prenatal screening and monitored for up
to 14 years. After 6 years, 79 (24%) children showed at least one
retinochoroidal lesion. In 23 of them, additional lesions were
diagnosed within10 years, mainly in a previously unaffected loca-
tion. Normal vision was found in about two thirds of the children
with lesions of one eye and in half of the children with lesions in
both eyes, and none had bilateral visual impairment. Most moth-
ers (84%) had been treated in utero, and a combination of pyri-
methamine and sulfadiazine had also been given to all children
in 38% before and in 72% after birth. Late-onset retinal lesions
and relapse can occur many years after birth, but the overall visual
prognosis of congenital toxoplasmosis seems acceptable when the
infection is identified early and treated appropriately. Early diag-
nosis and treatment are believed to reduce the risk for sequelae.262
If toxoplasmosis is suspected at the time of birth, the diagnostic
workup includes ophthalmologic, auditory and neurologic exami-
nations; lumbar puncture; and brain imaging.258 Other laboratory
tests include a full blood count, liver function tests and specific T.
gondii diagnostic tests. The diagnosis of congenital toxoplasmosis
can be made by the detection of IgM or IgA antibodies to T. gon-
dii in the neonate with a high sensitivity. In addition, amplifica-
tion of T. gondii’s DNA by PCR is almost 100% sensitive and
can be detected in various body fluids of a congenitally infected
neonate.250
Prenatal diagnosis of fetal infection is mainly based on PCR
amplification of T. gondii’s genome in the AF, but PCR in fetal
blood, inoculation of AF and/or fetal blood into mice and/or tissue
culture was previously used.263 Antsalkis and coworkers reported
the sensitivity, specificity, NPV and PPV of the different diagnos-
tic methods. These were of 61.1%, 98.6%, 91.6%, 91.13% and
of 83.3%, 100%, 100%, 97.1% sensitivity (Se), specificity (Sp),
PPV and NPV of mouse inoculation and PCR assay, respectively.
Cordocentesis had no diagnostic value. The combination of the
techniques led to 100% specificity and PPV.264 Furthermore,
amniocentesis is easier and safer to perform than cordocentesis.
Fetal infection is suspected by serial detailed fetal ultrasound
examination.253,265 The prognosis of infected fetuses is mainly
522 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
TABLE Fetal Abnormalities Related to Fetal Infection
42.6 with Toxoplasmosis
Cerebral signs Microcephaly185
Ventricular dilation, hydrocephaly172
Intracranial calcifications172
Brain atrophy185
Hydranencephaly185
Placenta Increased placental thickness172
Calcifications172
Other findings Liver calcifications172
Ascites186,187
Pericardial effusions172
Pleural effusions172
Hepatomegaly172
Hyperechogenic bowel
Intrauterine growth restriction
  
based on serial targeted ultrasound examination. The most com-
mon findings have been described by Hohfeld and coworkers in
a study comprising 89 cases of fetal infection.266 Among these
infected fetuses, 32 had ultrasound anomalies, including 25 cases
with ventriculomegaly more often bilateral and symmetrical,
starting in the occipital region before extending to the entire lat-
eral ventricle, 6 cases of intracranial calcifications, 11 cases with
increased placental thickness, 2 with placental calcifications, 4
cases with liver calcifications as well as hepatomegaly, ascites in
5 cases, pericardial effusions in 2 and pleural effusion in 1 case.
The presence of one sign was reported in 13 cases; 2 signs or more
were reported in the other 14 cases. Neither intrauterine growth
retardation nor microcephaly was observed in this study.
Ultrasound fetal abnormalities related to toxoplasmosis infec-
tion are reported in the Table 42.6. 
Treatment
After confirmation of the diagnosis of maternal seroconversion, treat-
ment is often initiated in a reference centre after specialised coun-
selling. Treatment is usually indicated during pregnancy for both
symptomatic and asymptomatic maternal disease with or without
congenital infection, although this was not proven to be useful.267
Indeed, if maternal infection is diagnosed, it is not known if antenatal
treatment is effective as shown in the European cohort trial compris-
ing of 1208 infected pregnant women, which failed to detect any dif-
ference in the risk for congenital infection with treatment or not.267
The combination of pyrimethamine (25–100 mg/day for 3–4
weeks), sulfadiazine (1–1.5 g four times a day for 3–4 weeks) and
folinic acid (leucovorin, 10–25 mg with each dose of pyrimeth-
amine to avoid bone marrow suppression) is the treatment pro-
tocol recommended by the World Health Organization and the
CDC.268
In other countries in Europe, Asia or South Africa, spiramycin
(3 g/d for weeks) and sometimes clindamycin is recommended to
prevent transplacental infection. In the United States, spiramycin
is currently not approved by the FDA but is available as an inves-
tigational drug, requiring special approval.
Reference association comprising pyrimethamine and sulfadia-
zine to prevent fetal infection is contraindicated during the first
trimester of pregnancy because of its potential teratogenicity, and
sulfadiazine should be used alone in the first trimester. However,
both drugs should be used when the mother is immunocompro-
mised or if the disease is disseminated.
Nevertheless, it is necessary to remember the heterogeneity
of the studies published for the evaluation of these treatments.
Methodologies used were very different.250 Wallon and cowork-
ers reviewed studies comparing treated and untreated concurrent
groups of pregnant women with proved or likely acute toxoplas-
mosis.269 Outcomes were reported in the offspring. The results
showed treatment to be effective in five studies but ineffective in
four.270-276
In 2007, the SYROCOT study group published the results of
a systematic review of cohort studies based on universal screening
for congenital toxoplasmosis and conducted a meta-analysis using
individual patients’ data to assess the effect of timing and type of
prenatal treatment on mother-to-child transmission of infection
and clinical manifestations before age 1 year.277 In 1438 treated
mothers identified by prenatal screening, the authors found weak
evidence that treatment started within 3 weeks of seroconver-
sion reduced mother-to-child transmission compared with treat-
ment started after 8 or more weeks (adjusted OR, 0.48; 95%CI
0.28–.80; P = .05). In 550 infected liveborn infants identified by
prenatal or neonatal screening, they did not find any evidence
that prenatal treatment reduced the risk for clinical manifestations
(adjusted OR for treated vs not treated, 1.11, 0.61–2.02). Increas-
ing gestational age at seroconversion was associated with increased
risk for mother-to-child transmission (OR 1.15, 1.12–1.17) and
decreased risk for intracranial lesions (0.91, 0.87–0.95) but not
with eye lesions (0.97, 0.93–1.00). Only a large randomised con-
trolled clinical trial would provide clinicians and patients with
valid evidence of the potential benefit of prenatal treatment.
This study (Prevention of Congenital Toxoplasmosis with Pyri-
methamine + Sulfadiazine Versus Spiramycin During Pregnancy
[TOXOGEST]; ClinicalTrials.gov Identifier: NCT01189448)
has been conducted in France, but results are not yet available. 
Prevention
Primary prevention is based on education programs that have
been developed to avoid maternal PI during pregnancy. Such
educational programs aim at avoiding exposure to the parasite by
better culinary and hygienic practical guidelines. The Cochrane
Collaboration evaluated such educational programs based on two
randomised trials278-280 and concluded that the effectiveness has
not been adequately evaluated.
Development of a vaccine to prevent human disease by
active immunisation as well as that of animals is necessary. Sev-
eral antigens are being studied for the development of effective
and safe vaccines, mainly SAG1, a surface antigen expressed on
tachyzoites.281,282
Secondary prevention is based on routine serologic screening
of pregnant women throughout their pregnancies. Modalities of
the serologic follow-up vary among countries from no screen-
ing, three times in pregnancy up to each month as established
in France. Screening should begin before conception and be fol-
lowed up until seroconversion in seronegative women. Treatment

is recommended if one of the tests suggests definite or likely pri-
mary maternal infection. The efficacy of spiramycin is further
questioned by the interval between the onset of treatment and
maternal infection even with monthly screening.267,273 However,
this strategy is expensive, amniocentesis can induce pregnancy
losses and antenatal treatment did not show any robust evidence
for efficiency to decrease the rate of fetal damages. Conversely,
Wallon and coworkers have observed a decreased rate of symp-
toms at 3 years of age after a monthly screening policy during the
pregnancy.283
Tertiary prevention is based on neonatal testing (IgM retrieved
in dried blood spots). Poland, Denmark and some areas of the
United States have adopted it. However, this strategy lacks sensi-
tivity, and almost half of infected newborns are not detected.284
There is no evidence that treating the infected infants has any
effect, and the approach is ineffective on already existing damage
at birth.285  ease is a major public health problem, mainly in Eastern Europe
Fetal Syphilis Infection
Bacteriology
Treponema pallidum is a part of the Spirochaetales. There are three
other Treponema pathogens (T. pertenue,286 T. carateum [pinta]
and T. endemicum [endemic syphilis, or bejel]). Only T. pertenue
and T. pallidum can cause congenital infection. Treponemes are
small gram-negative bacteria that can only be visualisable by dark-
field or phase-contrast microscopy. T. pallidum contains a single
circular chromosome.
When introduced into the skin or mucosal tissues, the
pathogen attaches to a receptor(s) on the host cell surface,
multiplies locally and spreads through the perivascular lym-
phatic system and the systemic circulation. It then dissemi-
nates widely before any lesion becomes clinically apparent.
During the 3-week incubation period (range, 10–90 days),
there is an intense local inflammatory response that creates the
chancre together with cellular proliferation in regional lymph
nodes when the immune response is unable to fully eradicate
the infection. Over 2 to 10 weeks, local replication leads to
dissemination in the skin, mucosal membranes and CNS. The
secondary immune response is similar to the primary one with
the development of venereal warts in response to the pres-
ence of spirochetes. Irrespective of therapy, secondary syphi-
lis resolves with a phase of relative immunologic control. At
this stage, viable pathogens remain in low numbers. Around
60% of cases remain asymptomatic and latent. Progression of
the disease to the tertiary phase, sometimes after several years,
occurs in 40% of cases. Tertiary syphilis can involve any organ
system, and the typical lesions are gummata (focal areas of
nonsuppurative inflammatory necrosis surrounded by fibrotic
scarring).
Congenital syphilis occurs when T. pallidum crosses the pla-
centa or at the time of birth by direct contact with maternal
lesions. Transplacental transmission during maternal spiro-
chetemia can occur from as early as 9 to 10 weeks of gestation
onwards. Vertical transmission occurs more frequently during
primary or secondary syphilis than with latent disease. The risk
decreases after 4 years even when untreated. Intrauterine trans-
mission causes wide dissemination in the fetus (mainly the brain,
liver, lung and bones). Early infection can lead to spontaneous
abortion.  on neonatal serum and cerebrospinal fluid relative to RIT.288
CHAPTER 42  Fetal Infections 523
Epidemiology
Humans are the sole natural hosts of syphilis. The main mode of
contamination is by sexual contact. The risk for infection is of
approximately 30% per sexual contact with an infected partner.
Acquisition via nonsexual contact has also been reported, includ-
ing contact with infected lesions in health care workers and in
laboratory workers who handle infected animals.
Epidemiologic features of this infection have changed with the
introduction of penicillin that enabled to drastically reduce the
number of cases. However, a resurgent wave occurred in the 1980s
in the United States mainly linked with drug abuse. With wider
screening practices, a decrease was also obtained from the 1990s,
mainly linked with the prevention program for HIV. Syphilis
is more common among (mainly homosexual) men and varies
among populations and geographical regions. Worldwide, the dis-
and in the developing countries in Africa and America.
Recognised risk factors include poverty, crack cocaine use,
prostitution and HIV infection. Gestational syphilis has well-
established risk factors, including unmarried mothers, teenage
mothers, absence of prenatal care, illicit drug abuse by the mother
or sexual partner, multiple sexual partners, low socioeconomic
environment and racial or ethnic minority group. 
Maternal Infection
The clinical picture is not altered by pregnancy.287 The primary
stage illustrated by the chancre is often unrecognised because of its
location. The primary lesion appears about 3 weeks after exposure.
It is a painless, ulcerated chancre with a raised border and thick base
that persists for 2 to 8 weeks. Painless adenopathy may also be pres-
ent. Only 5% to 10% of all diagnoses are made upon clinical mani-
festations. Thus the diagnosis is mainly based on serologic testing.
The secondary disseminated stage occurs 4 to 10 weeks after the
first primary lesion. It consists of condyloma latum and a dissemi-
nated maculopapular rash involving the palms and soles. Lymph-
adenopathy, weight loss, fever, anorexia, headache and arthralgia
can precede or accompany skin manifestations. Complications
include hepatitis, glomerulonephritis, nephritic syndrome, oste-
itis, meningitis and iritis. This stage resolves spontaneously in 2
to 6 weeks when the patient enters the asymptomatic, latent stage
of syphilis. The diagnosis can be made only by serologic testing.
This latent stage can be separated in two phases: early (1 year or
less than onset of infection) and late (more than 1 year). Without
treatment, one third of patients will develop tertiary syphilis char-
acterised by lesions involving the cardiovascular, central nervous
or musculoskeletal systems as well as other organs.
T. pallidum cannot be easily cultured in vitro, and a wide vari-
ety of diagnostic tests have been developed. They can be divided
into three broad categories:
1. Direct detection: The first method consists of inoculation of
T. pallidum into a rabbit. The animal is examined serially dur-
ing 3 months for clinical symptoms of syphilis and treponemal
tests. If the rabbit develops illness, examination under darkfield
microscopy is performed to confirm syphilis. This procedure
is only performed in research laboratories. The sensitivity of
the rabbit infectivity test (RIT) approaches 100%. As an alter-
native, PCR is a useful test in clinical settings. Sanchez and
coworkers reported a 71% sensitivity and a 100% specificity
524 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
2. Nontreponemal tests: Nontreponemal serologies detect antibodies
against the cardiolipin antigen released by damaged host cells.
Two tests are available: the Venereal Disease Research Laboratory
(VDRL) and the rapid plasma reagin (RPR). They are inexpen-
sive, easy to perform and widely available, making them excel-
lent screening tests. VDRL test is the only nontreponemal test
available for the diagnosis of neurosyphilis. False-positive results
have been described with nontreponemal tests, but titres are usu-
ally low (viral or bacterial infection, other spirochetal infections,
immunisation, heroin use, malignancy, chronic illnesses, auto-
immune and connective tissue disorders, aging and sometimes
the pregnancy itself). False-negative results have been reported
with large amounts of antibodies, which could inhibit the reac-
tion (serial dilutions can overcome this phenomenon). Second-
ary to the limitations of nontreponemal tests, a positive result
should always be confirmed with a treponemal test.
3. Treponemal tests: These tests detect antibodies directed specifi-
cally against T. pallidum. The two most commonly used are the
fluorescent treponemal antibody absorption (FTA-Abs) test
and the microhaemagglutination assay for anti–T. ­pallidum
(MHA-TP) antibodies. These tests are more expensive and
more difficult to perform. This is why they are not used for
screening. Furthermore, they cannot be quantitated and are
not useful for follow-up after treatment. A positive treponemal
test result combined with a positive nontreponemal test result
is very sensitive and specific for syphilis. Test results are positive
within 4 weeks of the chancre’s appearance. However, they may
be negative at the time the chancre first appears when only the
darkfield examination can make the diagnosis.  beginning from fetal hepatic dysfunction and thickening of the
Fetal Infection
Fetal syphilis is associated with spontaneous abortion, IUFD
and neonatal demise, as well as preterm delivery. Placental pas-
sage of T. pallidum occurs throughout gestation, but fetal clinical
manifestations require immunocompetence, which only occurs
from 16 weeks onwards. It was initially thought that the inability
of T. pallidum to cross the placenta was due to the thickness of
Langhans’ cytotrophoblast layer, but first trimester passage of T.
pallidum was proven by RIT and PCR on AF or histologic iden-
tification of spirochetes in fetal tissues after spontaneous abortion
as early as 9 to 10 weeks’ gestation.289-291
Vertical transmission rates have been estimated to be 29%
(3% stillbirths, 26% livebirths) with untreated primary syphilis
at delivery, 59% (20%, 39%) with untreated secondary syphilis,
50% (17%, 33%) with early latent and 13% (5%, 8%) with late
latent syphilis.289 Transmission is more likely when the bacterial
load is high and when there is a coinfection with HIV.  • BOX 42.1  Fetal Ultrasound Abnormalities in
Congenital Syphilis
Neonates affected by T. pallidum may present with manifestations
divided into early signs (appearing during the first 2 years of life)
and late signs (those appearing later over the first decades of life).292
Early signs are hepatosplenomegaly, generalised lymphadenop-
athy, haematologic abnormalities (anaemia, leukocytosis and leu-
kopenia, thrombocytopenia), dermatologic anomalies (jaundice,
rhinitis, maculopapular eruption, mucous patches), bone lesions
on x-ray examination (diaphyseal periostitis, metaphyseal osteo-
chondritis), renal manifestations, CNS anomalies, ocular mani-
festations and growth restriction.
Late congenital syphilis is characterised by signs occurring in
around 40% of untreated survivors. Many of these features are
not reversible at this stage despite late-onset antibiotic treatment.
These manifestations are mainly dental abnormalities, cartilage
destructions and bone deformities, eye involvement and eighth
nerve deafness. These three main features constitute the Hutchin-
son triad.
Fetal infection can be suspected when abnormalities are
observed at ultrasound examination. These abnormalities are sum-
marised in Box 42.1. These abnormalities are not specific and are
only evocative of an infectious disease. Laboratory methods are
always needed to confirm the diagnosis.
Serologic testing of cord blood specimens may be performed
but is sometimes difficult to interpret. The titres of nontrepone-
mal tests should be at least fourfold those in maternal blood to
confirm a significant fetal IgG production. Indeed, the difficulty
of the interpretation of the serologic tests stems largely from the
inability to distinguish the humoral response of the mother, whose
IgG antibodies pass through the placenta, from the specific anti-
body response of the infant. Because of these difficulties, PCR
method has been developed, and its results have been shown to
be similar for sensitivity and specificity compared with RIT on
AF samples.288
Nonspecific biochemical and haematologic parameters have
also been proven to be modified by fetal syphilis, including
increased GGT, anaemia, thrombocytopenia, leukopenia and
leukocytosis.293,294
A continuum of the fetal syphilis infection has been suggested,
placenta followed by haematologous dysfunction until isolated
ascites or hydrops fetalis.293 
Treatment
The CDC recommends that patients with recent syphilis (pri-
mary, secondary or latent syphilis of less than 1 year’s duration)
receive benzathine penicillin G, 2.4 million units intramuscularly
in a single dose. Patients with older active syphilis or with HIV
infection should receive 7.2 million units benzathine penicillin
G as 2.4 million units intramuscularly weekly for 3 consecutive
weeks. Patients with CNS infection require intravenous penicil-
lin for 10 to 14 days. Penicillin-allergic patients should undergo
desensitisation before treatment.
Congenital Syphilis
Hydrops fetalis208
Placental thickening209
Polyhydramnios206
Ascites206,210
Subcutaneous oedema206
Hepatomegaly208
Splenomegaly
Hyperechogenic or dilatated small bowel211
Intrauterine fetal death210

The CDC guidelines recommend serial follow-up after treat-
ment by quantitative nontreponemal serology. A fourfold decrease
in IgG titres indicates a successful treatment. It is expected by 6 to
12 months for primary and secondary syphilis, 12 to 24 months
for early latent syphilis and greater than 24 months for late latent
stage. Pregnancy has been associated with a slower decrease in
titres.295
McFarlin and coworkers noted that there was no difference
in the rates of congenital infections among women with a dis-
ease of less than 1 year if the treatment was one or three doses of
benzathine penicillin (26.7% vs 30%).296 Inadequate therapy or
dose regimen also contributes to the development of a congenital
disease. Drug abuse and lack of or late prenatal care or failure to
complete treatment and obtain follow-up are important risk fac-
tors for congenital disease.
The Jarisch-Herxheimer reaction is common during treatment
in adults. It consists of the association of chills, fever, malaise,
hypotension, tachycardia, tachypnea, accentuation of cutaneous
lesions and leukocytosis. It is more frequent in the second stage
of the infection and could be related to the release of lipoproteins
membrane stimulating a proinflammatory response. In pregnant
women, it happens in around 40% of cases and is associated with
preterm delivery (in relation with prostaglandin release), fetal dis-
tress or both.297 No preventive therapy is available.  Access the complete reference list online at ExpertConsult.com.
CHAPTER 42  Fetal Infections 525
Prevention
Congenital syphilis is a preventable disease. High-risk women
should undergo at least one serology screening test in the first tri-
mester to be repeated at the beginning of the third trimester and
at the time of delivery.287,298,299 
Conclusion
Appropriate management of infectious diseases in the pregnant
women remains a major challenge. Strategies to diagnose these
infections remain controversial across countries (toxoplasmosis,
CMV). Indications for prenatal diagnosis differ among infectious
agents. A better knowledge of the prognostic factors is still neces-
sary (toxoplasmosis, CMV). Treatments still have limited impact
on the course of the diseases (VZV, toxoplasmosis, CMV). Pre-
vention of maternal infections based on vaccination programs is
promising for CMV but is not currently recommended. Emer-
gence of new epidemic agents (e.g., Zika virus) and their ability to
cross the placenta and to induce fetal infection underline the need
to better understand this process. Thus further research is required
in this field of fetal medicine.
Self-assessment questions available at ExpertConsult.com.
References 17. Kumar ML, Nankervis GA, Gold E.
Inapparent congenital cytomegalovirus
35. Revello MG, Zavattoni M, Furione M, et al.
Quantification of human cytomegalovirus
infection. A follow-up study. N Engl J Med. DNA in amniotic fluid of mothers of con-
1. Stagno S, Pass RF, Dworsky ME, et  al.
1973;288(26):1370–1372. genitally infected fetuses. J Clin Microbiol.
Maternal cytomegalovirus infection and
18. Saigal S, Lunyk O, Larke RP, Chernesky 1999;37(10):3350–3352.
perinatal transmission. Clin Obstet Gynecol.
MA. The outcome in children with con- 36. Bilavsky E, Pardo J, Attias J, et  al. Clinical
1982;25(3):563–576.
genital cytomegalovirus infection. A lon- implications for children born with con-
2. Stagno S, Pass RF, Cloud G, et al. Primary
gitudinal follow-up study. Am J Dis Child. genital cytomegalovirus infection following
cytomegalovirus infection in pregnancy.
1982;136(10):896–901. a negative amniocentesis. Clin Infect Dis.
incidence, transmission to fetus, and clinical
19. Melish ME, Hanshaw JB. Congenital cyto- 2016;63(1):33–38.
outcome. JAMA. 1986;256(14):1904–1908.
megalovirus infection. Developmental prog- 37. Revello MG, Z Zavattoni M, Sarasini A,
3. Yow MD, Williamson DW, Leeds LJ,
ress of infants detected by routine screening. et  al. Human cytomegalovirus in blood of
et  al. Epidemiologic characteristics of cyto-
Am J Dis Child. 1973;126(2):190–194. immunocompetent persons during primary
megalovirus infection in mothers and their
20. Fowler KB, McCollister FP, Dahle AJ, et al. infection: prognosis implications for preg-
infants. Am J Obstet Gynecol. 1988;158(5):
Progressive and fluctuating sensorineural hear- nancy. J Infect Dis. 1998;177(5):1170–1175.
1189–1195.
ing loss in children with asymptomatic con- 38. Benoist G, Salomon LJ, Mohlo M, et  al.
4. Picone O, Vauloup-Fellous C, Cordier AG,
genital cytomegalovirus infection. J Pediatr. Cytomegalovirus-related fetal brain lesions:
et al. A series of 238 cytomegalovirus primary
1997;130(4):624–630. comparison between targeted ultrasound
infections during pregnancy: description
21. Noyola DE, Demmler GJ, Williamson WD, examination and magnetic resonance imag-
and outcome. Prenat Diagn. 2013;33(8):
et al. Cytomegalovirus urinary excretion and ing. Ultrasound Obstet Gynecol. 2008;32(7):
751–758.
long term outcome in children with congeni- 900–905.
5. Stagno S, Reynolds DW, Huang ES, et  al.
tal cytomegalovirus infection. Congenital 39. Picone O, Simon I, Benachi A, et al. Com-
Congenital cytomegalovirus infection. N Engl
CMV Longitudinal Study Group. Pediatr parison between ultrasound and magnetic
J Med. 1977;296(22):1254–1258.
Infect Dis J. 2000;19(6):505–510. resonance imaging in assessment of fetal
6. Schopfer K, Lauber E, Krech U. Congeni-
22. Fowler KB, McCollister FP, Dahle AJ, et al. cytomegalovirus infection. Prenat Diagn.
tal cytomegalovirus infection in newborn
Progressive and fluctuating sensorineural hear- 2008;28(8):753–758.
infants of mothers infected before pregnancy.
ing loss in children with asymptomatic con- 40. van der Knaap MS, Vermeulen G, Barkhof F,
Arch Dis Child. 1978;53(7):536–539.
genital cytomegalovirus infection. J Pediatr. et al. Pattern of white matter abnormalities at
7. Boppana SB, Rivera LB, Fowler KB, et  al.
1997;130(4):624–630. MR imaging: use of polymerase chain reac-
Intrauterine transmission of cytomegalovirus
23. Ville Y. The megalovirus. Ultrasound Obstet tion testing of Guthrie cards to link pattern
to infants of women with preconceptional
Gynecol. 1998;12(3):151–153. with congenital cytomegalovirus infection.
immunity. N Engl J Med. 2001;344(18):
24. Al-Kouatl H, Chasen ST, Streltzoff J, Radiology. 2004;230(2):529–536.
1366–1371.
Chervenak FA. The clinical significance of 41. Doneda C, Parazzini C, Righini A, et  al.
8. Nigro G, Anceschi MM, Cosmi EV. Con-
fetal echogenic bowel. Am J Obstet Gynecol. Early cerebral lesions in cytomegalovirus
genital Cytomegalic Disease Collaborating
2001;185(5):1035–1038. infection: prenatal MR imaging. Radiology.
Group. Clinical manifestations and abnor-
25. Déchelotte PJ, Mulliez NM, Bouvier RJ, 2010;255(2):613–621.
mal laboratory findings in pregnant women
et  al. Pseudo-meconium ileus due to cyto- 42. Haginoya K, Ohura T, Kon K, et  al.
with primary cytomegalovirus infection.
megalovirus infection: a report of three cases. Abnormal white matter lesions with senso-
BJOG. 2003;110(6):572–577.
Pediatr Pathol. 1992;12(1):73–82. rineural hearing loss caused by congenital
9. Revello MG, Gerna G. Diagnosis and man-
26. Drose JA, Dennis MA, Thickman D. cytomegalovirus infection: retrospective diag-
agement of human cytomegalovirus infection
Infection in utero: us findings in 19 cases. nosis by PCR using Guthrie cards. Brain Dev.
in the mother, fetus, and newborn infant.
Radiology. 1991;178(2):369–374. 2002;24(7):710–714.
Clin Microbiol Rev. 2002;15(4):680–715.
27. Watt-Morse ML, Laifer SA, Hill LM. The 43. Lipitz S, Hoffmann C, Feldman B, et  al.
10. Macé M, Sissoeff L, Rudent A, Grangeot-
natural history of cytomegalovirus infec- Value of prenatal ultrasound and magnetic
Keros L. A serological testing algorithm
tion as assessed by serial ultrasound and fetal resonance imaging in assessment of con-
for the diagnosis of primary cmv infec-
blood sampling: a case report. Prenat Diagn. genital primary cytomegalovirus infection.
tion in pregnant women. Prenat Diagn.
1995;15(6):567–570. Ultrasound Obstet Gynecol. 2010;36(6):
2004;24(11):861–863.
28. Barkovich AJ, Lindan CE. Congenital cyto- 709–717.
11. Maine GT, Lazzarotto T, Landini MP. New
megalovirus infection of the brain: imaging 44. Cannie MM, Leyder M4 Devlieger R, et al.
developments in the diagnosis of maternal
analysis and embryologic considerations. Congenital cytomegalovirus infection: con-
and congenital CMV infection. Expert Rev
AJNR Am J Neuroradiol. 1994;15(4):703–715. tribution and best timing of prenatal MR
Mol Diagn. 2001;1(1):19–29.
29. Soussotte C, Maugey-Laulom B, Carles D, imaging. Eur Radiol. 2016;26(10):3760–
12. Lazzarotto T, Spezzacatena P, Varani S,
Diard F. Contribution of transvaginal ultra- 3769.
et  al. Anticytomegalovirus (Anti-CMV)
sonography and fetal cerebral MRI in a case 45. Ahlfors K, Forsgren M, Ivarsson SA, et  al.
immunoglobulin G avidity in identification
of congenital cytomegalovirus infection. Congenital cytomegalovirus infection: on
of pregnant women at risk of transmitting
Fetal Diagn Ther. 2000;15(4):219–223. the relation between type and time of mater-
congenital CMV infection. Clin Diagn Lab
30. Malinger G, Lev D, Zahalka N, et al. Fetal nal infection and infant’s symptoms. Scand J
Immunol. 1999;6(1):127–129.
cytomegalovirus infection of the brain: the Infect Dis. 1983;15(2):129–138.
13. Leruez-Ville M, Sellier Y, Salomon LJ, et  al.
spectrum of sonographic findings. AJNR Am 46.  Pass RF, Fowler KB, Boppana SB, et  al.
Prediction of fetal infection in cases with cyto-
J Neuroradiol. 2003;24(1):28–32. Congenital cytomegalovirus infection follow-
megalovirus immunoglobulin M in the first
31. Liesnard C, Donner C, Brancart F, et al. Pre- ing first trimester maternal infection: symp-
trimester of pregnancy: a retrospective cohort.
natal diagnosis of congenital cytomegalovirus toms at birth and outcome. J Clin Virol. 2005.
Clin Infect Dis. 2013;56(10):1428–135.
infection: prospective study of 237 pregnan- 47. Steinlin MI, Nadal D, Eich GF, et  al. Late
14. Revello MG, Zavattoni M, Sarasini A, et al.
cies at risk. Obstet Gynecol. 2000;95(6 Pt 1): intrauterine cytomegalovirus infection: clini-
Human cytomegalovirus in blood of immu-
881–888. cal and neuroimaging findings. Pediatr Neu-
nocompetent persons during primary infec-
32. Guerra B, Lazzarotto T, Quarta S, et al. Pre- rol. 1996;15(3):249–253.
tion: prognostic implications for pregnancy.
natal diagnosis of symptomatic congenital 48.  Ahlfors K, Ivarsson SA, Harris S, et  al.
J Infect Dis. 1998;177(5):1170–1175.
cytomegalovirus infection. Am J Obstet Gyne- Congenital cytomegalovirus infection and
15. Boppana SB, Pass RF, Britt WJ, et  al.
col. 2000;183(2):476–482. disease in Sweden and the relative importance
Symptomatic congenital cytomegalovirus
33. Lazzarotto T, Varani S, Guerra B, et al. Pre- of primary and secondary maternal infections.
infection: neonatal morbidity and mortality.
natal indicators of congenital cytomegalovi- preliminary findings from a prospective study.
Pediatr Infect Dis J. 1992;11(2):93–99.
rus infection. J Pediatr. 2000;137(1):90–95. Scand J Infect Dis. 1984;16(2):129–137.
16. Boppana SB, Fowler KB, Vaid Y, et  al.
34. Enders G, Bäder U, Lindemann L, et  al. 49. Fowler KB, Stagno S, Pass RF, et al. The out-
Neuroradiographic findings in the newborn
Prenatal diagnosis of congenital cytomegalovi- come of congenital cytomegalovirus infec-
period and long-term outcome in children
rus infection in 189 pregnancies with known tion in relation to maternal antibody status.
with symptomatic congenital cytomegalovirus
outcome. Prenat Diagn. 2001;21(5):362–377. N Engl J Med. 1992;326(10):663–667.
infection. Pediatrics. 1997;99(3):409–414.

525.e1
525.e2 References
50. Lipitz S, Achiron R, Zalel Y, et al. Outcome
of pregnancies with vertical transmission of
primary cytomegalovirus infection. Obstet
Gynecol. 2002;100(3):428–433.
51. Rasmussen L, Geissler A, Winters M. Inter- and
intragenic variations complicate the molecu-
lar epidemiology of human cytomegalovirus.
J Infect Dis. 2003;187(5):809–819.
52. Picone O, Costa JM, Dejean A, Ville Y. Is
fetal gender a risk factor for severe congeni-
tal cytomegalovirus infection? Prenat Diagn.
2005;25(1):34–38.
53. Gouarin S, Gault E, Vabret A, et  al.
Real-time PCR quantification of human
cytomegalovirus DNA in amniotic fluid sam-
ples from mothers with primary infection. J
Clin Microbiol. 2002;40(5):1767–1772.
54. Picone O, Costa JM, Ville Y, et al. Genetic
polymorphism of cytomegalovirus strains
responsible of congenital infections. Pathol
Biol (Paris). 2004;52(9):534–539.
55. Leruez-Ville M, Stirnemann J, Sellier Y, et al.
Feasibility of predicting the outcome of fetal
infection with cytomegalovirus at the time of
prenatal diagnosis. Am J Obstet Gynecol. 2016.
56. Desveaux C, Klein J, Leruez-Ville M,
et  al. Identification of symptomatic fetuses
infected with cytomegalovirus using amni-
otic fluid peptide biomarkers. PloS Pathog.
2016;12(1):E1005395.
57. Benoist G, Salomon LJ, Jacquemard F, et al.
The prognostic value of ultrasound abnor-
malities and biological parameters in blood
of fetuses infected with cytomegalovirus.
BJOG. 2008;115(7):823–829.
58. Revello MG, Zavattoni M, Baldanti F, et al.
Diagnostic and prognostic value of human
cytomegalovirus load and IgM antibody in
blood of congenitally infected newborns.
J Clin Virol. 1999;14(1):57–66.
59. Lanari M, Lazzarotto T, Venturi V, et  al.
Neonatal cytomegalovirus blood load and
risk of sequelae in symptomatic and asymp-
tomatic congenitally infected newborns.
Pediatrics. 2006;117(1):E76–E83.
60. Fabbri E, Revello MG, Furione M, et  al.
Prognostic markers of symptomatic congeni-
tal human cytomegalovirus infection in fetal
blood. BJOG. 2011;118(4):448–456.
61. Dreux S, Rousseau T, Gerber S, et al. Fetal
serum beta2-microglobulin as a marker
for fetal infectious diseases. Prenat Diagn.
2006;26(5):471–474.
62. Nigro G, Adler SP, La Torre R, Best AM.
Congenital Cytomegalovirus Collaborating
Group. Passive immunization during preg-
nancy for congenital cytomegalovirus infection.
N Engl J Med. 2005;353(13):1350–1362.
63. Revello MG, Lazzarotto T, Guerra B, et al.
A randomized trial of hyperimmune globu-
lin to prevent congenital cytomegalovirus. N
Engl J Med. 2014;370(14):1316–1326.
64. Jacquemard F, Yamamoto M, Costa JM,
et al. Maternal administration of valaciclovir
in symptomatic intrauterine cytomegalovirus
infection. BJOG. 2007;114(9):1113–1121.
65. Leruez-Ville M, Ghout I, Bussières L, et al.
In utero treatment of congenital cytomega-
lovirus infection with valacyclovir in a mul-
ticenter, open-label, phase II study. Am J
Obstet Gynecol. 2016.
66. Pass RF, Zhang C, Evans A, et al. Vaccine pre-
vention of maternal cytomegalovirus infection.
N Engl J Med. 2009;360(12):1191–1199.
67. Picone O, Vauloup-Fellous C, Cordier AG,
et  al. A 2-year study on cytomegalovirus
infection during pregnancy in a French hos-
pital. BJOG. 2009;116(6):818–823.
68. Brown KE, Hibbs JR, Gallinella G, et  al.
Resistance to parvovirus B19 infection due to
lack of virus receptor (erythrocyte P antigen).
N Engl J Med. 1994;330(17):1192–1196.
69. Chisaka H, Morita E, Yaegashi N, Sugamura
K. Parvovirus B19 and the pathogen-
esis of anaemia. Rev Med Virol. 2003;13(6):
347–359.
70. Koch WC, Adler SP. Human parvovirus
b19 infections in women of childbearing
age and within families. Pediatr Infect Dis J.
1989;8(2):83–87.
71. Heegaard ED, Brown KE. Human parvo-
virus B19. Clin Microbiol Rev. 2002;15(3):
485–505.
72. Nascimento JP, Buckley MM, Brown
KE, Cohen BJ. The prevalence of anti-
body to human parvovirus B19 in Rio de
Janeiro, Brazil. Rev Inst Med Trop Sao Paulo.
1990;32(1):41–45.
73. Adler SP, Manganello AM, Koch WC,
et  al. Risk of human parvovirus B19 infec-
tions among school and hospital employ-
ees during endemic periods. J Infect Dis.
1993;168(2):361–368.
74. Dembinski J, Eis-Hübinger AM, Maar J,
et al. Long term follow up of serostatus after
maternofetal parvovirus B19 infection. Arch
Dis Child. 2003;88(3):219–221.
75. Trotta M, Azzi A, Meli M, et  al. Intrauter-
ine parvovirus B19 infection: early prena-
tal diagnosis is possible. Int J Infect Dis.
2004;8(2):130–131.
76. Valeur-Jensen AK, Pedersen CB,
Westergaard T, et  al. Risk factors for par-
vovirus B19 infection in pregnancy. JAMA.
1999;281(12):1099–1105.
77. Harger JH, Adler SP, Koch WC, Harger
GF. Prospective evaluation of 618 pregnant
women exposed to parvovirus B19: risks and
symptoms. Obstet Gynecol. 1998;91(3):413–
420.
78. Cartter ML, Farley TA, Rosengren S, et  al.
Occupational risk factors for infection with
parvovirus B19 among pregnant women. J
Infect Dis. 1991;163(2):282–285.
79. Gillespie SM, Cartter ML, Asch S, et  al.
Occupational risk of human parvovirus B19
infection for school and day-care personnel
during an outbreak of erythema infectiosum.
JAMA. 1990;263(15):2061–2065.
80. Enders M, Weidner A, Zoellner I, et al. Fetal
morbidity and mortality after acute human
parvovirus b19 infection in pregnancy: pro-
spective evaluation of 1018 cases. Prenat
Diagn. 2004;24(7):513–518.
81. Levy R, Weissman A, Blomberg G, Hagay
ZJ. Infection by parvovirus B 19 during
pregnancy: a review. Obstet Gynecol Surv.
1997;52(4):254–259.
82. Ager EA, Chin TD, Poland JD. Epidemic
erythema infectiosum. N Engl J Med.
1966;275(24):1326–1331.
83. Valentin MN, Cohen PJ. Pediatric parvovi-
rus B19: spectrum of clinical manifestations.
Cutis. 2013;92(4):179–184.
84. Anderson MJ, Lewis E, Kidd IM, et al. An
outbreak of erythema infectiosum associ-
ated with human parvovirus infection. J Hyg
(Lond). 1984;93(1):85–93.
85. Saarinen UM, Chorba TL, Tattersall P,
et  al. Human parvovirus B19-induced
epidemic acute red cell aplasia in patients
with hereditary hemolytic anemia. Blood.
1986;67(5):1411–1147.
86. Brass C, Elliott LM, Stevens DA. Acad-
emy rash. A probable epidemic of ery-
thema infectiosum (‘fifth disease’). JAMA.
1982;248(5):568–572.
87. Tsuji A, Uchida N, Asamura S, et al. Aseptic
meningitis with erythema infectiosum. Eur J
Pediatr. 1990;149(6):449–450.
88. Seishima M, Kanoh H, Izumi T. The spec-
trum of cutaneous eruptions in 22 patients
with isolated serological evidence of infec-
tion by parvovirus B19. Arch Dermatol.
1999;135(12):1556–1557.
89. Muir K, Todd WT, Watson WH, Fitzsi-
mons E. Viral-associated haemophagocytosis
with parvovirus-B19-related pancytopenia.
Lancet. 1992;339(8802):1139–1140.
90. Gratacós E, Torres PJ, Vidal J, et al. The inci-
dence of human parvovirus B19 infection
during pregnancy and its impact on perinatal
outcome. J Infect Dis. 1995;171(5):1360–
1363.
91. Weiffenbach J, Bald R, Gloning KP, et  al.
Serological and virological analysis of mater-
nal and fetal blood samples in prenatal
human parvovirus B19 infection. J Infect Dis.
2012;205(5):782–788.
92. Simms RA, Liebling RE, Patel RR, et  al.
Management and outcome of pregnancies
with parvovirus B19 infection over seven
years in a tertiary fetal medicine unit. Fetal
Diagn Ther. 2009;25(4):373–378.
93. Prospective study of human parvovirus (B19)
infection in pregnancy. Public Health Labo-
ratory Service Working Party on Fifth Dis-
ease. BMJ. 1990;300(6733):1166–1170.
94.  Koch WC, Adler SP, Harger J. Intrauterine
parvovirus B19 infection may cause an asymp-
tomatic or recurrent postnatal infection. Pedi-
atr Infect Dis J. 1993;12(9):747–750.
95. Giakoumelou S, Wheelhouse N, Cuschieri K,
et  al. The role of infection in miscarriage.
Hum Reprod Update. 2016;22(1):116–133.
96. Chisaka H, Morita E, Tada K, et  al. Estab-
lishment of multifunctional monoclonal
antibody to the nonstructural protein,
Ns1, of human parvovirus B19. J Infect.
2003;47(3):236–242.
97. Norbeck O, Papadogiannakis N, Petersson K,
et al. Revised clinical presentation of parvo-
virus B19-associated intrauterine fetal death.
Clin Infect Dis. 2002;35(9):1032–1038.
98. Porter HJ, Khong TY, Evans MF, et  al.
Parvovirus as a cause of hydrops fetalis:
detection by in situ DNA hybridisation.
J Clin Pathol. 1988;41(4):381–383.
99. Yaegashi N, Niinuma T, Chisaka H, et  al.
The incidence of, and factors leading to, par-
vovirus B19-related hydrops fetalis following
maternal infection; report of 10 cases and
meta-analysis. J Infect. 1998;37(1):28–35.
100. Tolfvenstam T, Papadogiannakis N,
Norbeck O, et al. Frequency of human par-
vovirus B19 infection in intrauterine fetal
death. Lancet. 2001;357(9267):1494–1497.
101. Puccetti C, Contoli M, Bonvicini F, et  al.
Parvovirus B19 in pregnancy: possible con-
sequences of vertical transmission. Prenat
Diagn. 2012;32(9):897–902.

102. Soothill P. Intrauterine blood transfu-
sion for non-immune hydrops fetalis
due to parvovirus B19 infection. Lancet.
1990;336(8707):121–122.
103. Macé G, Sauvan M, Castaigne V, et  al.
Clinical presentation and outcome of 20
fetuses with parvovirus B19 infection compli-
cated by severe anemia and/or fetal hydrops.
Prenat Diagn. 2014;34(11):1023–1030.
104. Sarafidis K, Drossou-Agakidou V, Evdoridou
I, et al. Hydrothorax as a sole manifestation
of congenital parvovirus B19 infection. Am J
Perinatol. 2008;25(9):551–554.
105. Pasquini L, Seravalli V, Sisti G, et  al.
Prevalence of a positive torch and parvovi-
rus B19 screening in pregnancies compli-
cated by polyhydramnios. Prenat Diagn.
2016;36(3):290–293.
106. Pasquini L, Masini G, Gaini C, et  al. The
utility of infection screening in isolated mild
ventriculomegaly: an observational retro-
spective study on 141 fetuses. Prenat Diagn.
2014;34(13):1295–1300.
107. Jouannic JM, Gavard L, Créquat J, et  al.
Isolated fetal hyperechogenic bowel asso-
ciated with intra-uterine parvovirus B19
infection. Fetal Diagn Ther. 2005;20(6):
498–500.
108. von Kaisenberg CS, Bender G, Scheewe J,
et al. A case of fetal parvovirus B19 myocar-
ditis, terminal cardiac heart failure, and peri-
natal heart transplantation. Fetal Diagn Ther.
2001;16(6):427–432.
109. Fishman SG, Pelaez LM, Baergen RN, Car-
roll SJ. Parvovirus-mediated fetal cardiomy-
opathy with atrioventricular nodal disease.
Pediatr Cardiol. 2011;32(1):84–86.
110. Craze JL, Salisbury AJ, Pike MG. Prena-
tal stroke associated with maternal par-
vovirus infection. Dev Med Child Neurol.
1996;38(1):84–85.
111. Isumi H, Nunoue T, Nishida A, Takashima
S. Fetal brain infection with human parvo-
virus B19. Pediatr Neurol. 1999;21(3):661–
663.
112. Zerbini M, Musiani M, Gentilomi G, et al.
Symptomatic parvovirus B19 infection of
one fetus in a twin pregnancy. Clin Infect Dis.
1993;17(2):262–263.
113. Simchen MJ, Toi A, Bona M, et  al. Fetal
hepatic calcifications: prenatal diagnosis and
outcome. Am J Obstet Gynecol. 2002;187(6):
1617–1622.
114. Plachouras N, Stefanidis K, Andronikou S,
Lolis D. Severe nonimmune hydrops fetalis
and congenital corneal opacification second-
ary to human parvovirus B19 infection. A case
report. J Reprod Med. 1999;44(4):377–380.
115. Cantey JB, Pritchard MA, Sánchez PJ.
Bone lesions in an infant with congeni-
tal parvovirus B19 infection. Pediatrics.
2013;131(5):E1659–E1663.
116. Tiessen RG, van Elsacker-Niele AM,
Vermeij-Keers C, et al. A fetus with a parvo-
virus B19 infection and congenital anoma-
lies. Prenat Diagn. 1994;14(3):173–176.
117. Rodis JF, Rodner C, Hansen AA, et al. Long-
term outcome of children following mater-
nal human parvovirus B19 infection. Obstet
Gynecol. 1998;91(1):125–128.
118. Courtier J, Schauer GM, Parer JT, et  al.
Polymicrogyria in a fetus with human par-
vovirus B19 infection: a case with radiologic-
pathologic correlation. Ultrasound Obstet
Gynecol. 2012;40(5):604–606.
119. De Haan TR, Van Wezel-Meijler G,
Beersma MF, et al. Fetal stroke and congeni-
tal parvovirus B19 infection complicated by
activated protein C resistance. Acta Paediatr.
2006;95(7):863–867.
120. Glenn OA, Bianco K, Barkovich AJ,
et  al. Fetal cerebellar hemorrhage in par-
vovirus-associated non-immune hydrops
fetalis. J Matern Fetal Neonatal Med.
2007;20(10):769–772.
121. Pistorius LR, Smal J, de Haan TR, et  al.
Disturbance of cerebral neuronal migration
following congenital parvovirus B19 infec-
tion. Fetal Diagn Ther. 2008;24(4):491–494.
122. Katz VL, McCoy MC, Kuller JA, Hansen
WF. An association between fetal parvovirus
B19 infection and fetal anomalies: a report of
two cases. Am J Perinatol. 1996;13(1):43–45.
123. Zajicek M, Gindes L, Hoffmann C, et al. Pre-
natal diagnosis of obstructive hydrocephalus
associated with parvovirus B19 infection.
Obstet Gynecol. 2010;116(suppl 2):521–522.
124. Proust S, Philippe HJ, Paumier A, et al. Mir-
ror pre-eclampsia: Ballantyne’s syndrome.
Two cases. J Gynecol Obstet Biol Reprod
(Paris). 2006;35(3):270–274.
125. Chauvet A, Dewilde A, Thomas D, et  al.
Ultrasound diagnosis, management and
prognosis in a consecutive series of 27 cases
of fetal hydrops following maternal par-
vovirus B19 infection. Fetal Diagn Ther.
2011;30(1):41–47.
126. Mari G, Detti L, Oz U, et  al. Accurate
prediction of fetal hemoglobin by Dop-
pler ultrasonography. Obstet Gynecol.
2002;99(4):589–593.
127. Cosmi E, Mari G, Delle Chiaie L, et  al.
Noninvasive diagnosis by Doppler ultra-
sonography of fetal anemia resulting from
parvovirus infection. Am J Obstet Gynecol.
2002;187(5):1290–1293.
128. Mari G, Deter RL, Carpenter RL, et  al.
Noninvasive diagnosis by Doppler ultraso-
nography of fetal anemia due to maternal
red-cell alloimmunization. Collaborative
Group for Doppler Assessment of the Blood
Velocity in Anemic Fetuses. N Engl J Med.
2000;342(1):9–14.
129. Carraca T, Matias A, Brandão O, Montene-
gro N. Early signs of cardiac failure: a clue
for parvovirus infection screening in the first
trimester? Fetal Diagn Ther. 2011;30(2):
150–152.
130. Bizjak G, Blondin D, Hammer R, et  al.
Acute infection with parvovirus B19 in
early pregnancy. Ultrasound Obstet Gynecol.
2009;34(2):234–235.
131. Kempe A, Rösing B, Berg C, et  al. First-
trimester treatment of fetal anemia second-
ary to parvovirus B19 infection. Ultrasound
Obstet Gynecol. 2007;29(2):226–228.
132. Jordan JA, Huff D, Deloia JA. Placental
cellular immune response in women
infected with human parvovirus B19 dur-
ing pregnancy. Clin Diagn Lab Immunol.
2001;8(2):288–292.
133. Beersma MF, Claas EC, Sopaheluakan T,
Kroes AC. Parvovirus B19 viral loads in rela-
tion to Vp1 and Vp2 antibody responses
in diagnostic blood samples. J Clin Virol.
2005;34(1):71–75.
134. Enders M, Schalasta G, Baisch C, et al. Human
parvovirus B19 infection during pregnancy—
value of modern molecular and serological
diagnostics. J Clin Virol. 2006;35(4):400–406.
References 525.e3
135. Adams LL, Gungor S, Turan S, et al. When
are amniotic fluid viral PCR studies indi-
cated in prenatal diagnosis? Prenat Diagn.
2012;32(1):88–93.
136. Fairley CK, Smoleniec JS, Caul OE,
Miller E. Observational study of effect of
intrauterine transfusions on outcome of
fetal hydrops after parvovirus B19 infection.
Lancet. 1995;346(8986):1335–1337.
137. Rodis JF, Borgida AF, Wilson M, et  al.
Management of parvovirus infection in preg-
nancy and outcomes of hydrops: a survey of
members of the Society of Perinatal Obste-
tricians. Am J Obstet Gynecol. 1998;179(4):
985–988.
138. Schild RL, Plath H, Thomas P, et  al. Fetal
parvovirus B19 infection and meconium
peritonitis. Fetal Diagn Ther. 1998;13(1):15–
18.
139. Hernandez-Andrade E, Scheier M, Dezerega
V, et  al. Fetal middle cerebral artery peak
systolic velocity in the investigation of non-
immune hydrops. Ultrasound Obstet Gynecol.
2004;23(5):442–445.
140. Odibo AO, Campbell WA, Feldman D,
et  al. Resolution of human parvovirus
B19-induced nonimmune hydrops after
intrauterine transfusion. J Ultrasound Med.
1998;17(9):547–550.
141. Miller E, Fairley CK, Cohen BJ, Seng C.
Immediate and long term outcome of human
parvovirus B19 infection in pregnancy. Br J
Obstet Gynaecol. 1998;105(2):174–178.
142. Nagel HT, de Haan TR, Vandenbussche
FP, et  al. Long-term outcome after fetal
transfusion for hydrops associated with
parvovirus B19 infection. Obstet Gynecol.
2007;109(1):42–47.
143. De Jong EP, Lindenburg IT, van Klink JM,
et  al. Intrauterine transfusion for parvovi-
rus B19 infection: long-term neurodevel-
opmental outcome. Am J Obstet Gynecol.
2012;206(3):204.E1–E5.
144. Lindenburg IT, van Klink JM, Smits-Wintjens
VE, et  al. Long-term neurodevelopmental
and cardiovascular outcome after intrauter-
ine transfusions for fetal anaemia: a review.
Prenat Diagn. 2013;33(9):815–822.
145. Lindenburg IT, van Kamp IL, van Zwet EW,
et  al. Increased perinatal loss after intra-
uterine transfusion for alloimmune anae-
mia before 20 weeks of gestation. BJOG.
2013;120(7):847–852.
146. 
American College of Obstetricians and
Gynecologists. Practice bulletin no. 151:
cytomegalovirus, parvovirus b19, varicella
zoster, and toxoplasmosis in pregnancy.
Obstet Gynecol. 2015;125(6):1510–1525.
147. Ballou WR, Reed JL, Noble W, et al. Safety
and immunogenicity of a recombinant
parvovirus B19 vaccine formulated with
Mf59c.1. J Infect Dis. 2003;187(4):675–678.
148. Gregg NM. Congenital cataract following
German measles in the mother. 1941. Aust N
Z J Ophthalmol. 1991;19(4):267–276.
149. Baldanti F, Silini E, Sarasini A, et al. A three-
nucleotide deletion in the Ul97 open reading
frame is responsible for the ganciclovir resis-
tance of a human cytomegalovirus clinical
isolate. J Virol. 1995;69(2):796–800.
150. Connolly JH, Hutchinson WM, Allen IV,
et  al. Carotid artery thrombosis, encepha-
litis, myelitis and optic neuritis associ-
ated with rubella virus infections. Brain.
1975;98(4):583–594.
525.e4 References
151. Sharan S, Sharma S, Billson FA. Congenital
rubella cataract: a timely reminder in the new
millennium? Clin Experiment Ophthalmol.
2006;34(1):83–84.
152. Rowen M, Singer MJ, Moran ET. Intracra-
nial calcification in the congenital rubella
syndrome. Am J Roentgenol Radium Ther
Nucl Med. 1972;115(1):86–91.
153. Preblud SR, Gross F, Halsey NA, et  al.
Assessment of susceptibility to measles and
rubella. JAMA. 1982;247(8):1134–1337.
154. Choutet P, Binet CH, Goudeau A, et  al.
Bone-marrow aplasia and primary rubella
infection. Lancet. 1979;2(8149):966–967.
155. Klein HZ, Markarian M. Dermal erythro-
poiesis in congenital rubella. description of
an infected newborn who had purpura asso-
ciated with marked extramedullary erythro-
poiesis in the skin and elsewhere. Clin Pediatr
(Phila). 1969;8(10):604–607.
156. Floret D, Rosenberg D, Hage GN, Monnet
P. Hyperthyroidism, diabetes mellitus and
the congenital rubella syndrome. Acta Paedi-
atr Scand. 1980;69(2):259–261.
157. Preece MA, Kearney PJ, Marshall WC.
Growth-hormone deficiency in congenital
rubella. Lancet. 1977;2(8043):842–844.
158. Clarke WL, Shaver KA, Bright GM, et  al.
Autoimmunity in congenital rubella syn-
drome. J Pediatr. 1984;104(3):370–373.
159. Miller E, Cradock-Watson JE, Pollock TM.
Consequences of confirmed maternal rubella
at successive stages of pregnancy. Lancet.
1982;2(8302):781–784.
160. Enders G, Nickerl-Pacher U, Miller E,
Cradock-Watson JE. Outcome of confirmed
periconceptional maternal rubella. Lancet.
1988;1(8600):1445–1447.
161. Peckham C. Congenital rubella in the United
Kingdom before 1970: the prevaccine era.
Rev Infect Dis. 1985;7(suppl 1):S11–S16.
162. Sever JL, Hardy JB, Nelson KB, Gilkeson
MR. Rubella in the collaborative perinatal
research study. II. Clinical and laboratory
findings in children through 3 years of age.
Am J Dis Child. 1969;118(1):123–132.
163. Sever JL, Nelson KB, Gilkeson MR. Rubella
epidemic, 1964: effect on 6,000 pregnancies.
Am J Dis Child. 1965;110(4):395–407.
164. Webster WS. Teratogen update: congenital
rubella. Teratology. 1998;58(1):13–23.
165. Kobayashi K, Tajima M, Toishi S, et al. Fetal
growth restriction associated with measles
virus infection during pregnancy. J Perinat
Med. 2005;33(1):67–68.
166. Chiba ME, Saito M, Suzuki N, et  al.
Measles infection in pregnancy. J Infect.
2003;47(1):40–44.
167. McDonald JC, Peckham CS. Gammaglobu-
lin In prevention of rubella and congeni-
tal defect: a study of 30,000 pregnancies.
Br Med J. 1967;3(566):633–637.
168. McCallin PF, Fuccillo DA, Ley AC, et  al.
Gammaglobulin as prophylaxis against
rubella-induced congenital anomalies. Obstet
Gynecol. 1972;39(2):185–189.
169. Brody JA, Sever JL, Schiff GM. Prevention
of rubella by gamma globulin during an epi-
demic in Barrow, Alaska, in 1964. N Engl J
Med. 1965;272:127–129.
170. Vijayalakshmi P, Kakkar G, Samprathi
A, Banushree R. Ocular manifestations of
congenital rubella syndrome in a devel-
oping country. Indian J Ophthalmol.
2002;50(4):307–311.
171. Reef SE, Redd SB, Abernathy E, et  al. The
epidemiological profile of rubella and con-
genital rubella syndrome in the United
States, 1998-2004: the evidence for absence
of endemic transmission. Clin Infect Dis.
2006;43(suppl 3):S126–S132.
172. Weibel RE, Buynak EB, McLean AA, et al.
Persistence of antibody in human subjects
for 7 to 10 years following administration of
combined live attenuated measles, mumps,
and rubella virus vaccines. Proc Soc Exp Biol
Med. 1980;165(2):260–263.
173. Weibel RE, Carlson Jr AJ, Villarejos VM,
et al. Clinical and laboratory studies of com-
bined live measles, mumps, and rubella vac-
cines using the Ra 27/3 rubella virus. Proc Soc
Exp Biol Med. 1980;165(2):323–326.
174. Johnson CE, Kumar ML, Whitwell JK,
et  al. Antibody persistence after primary
measles-mumps-rubella vaccine and response
to a second dose given at four to six vs.
eleven to thirteen years. Pediatr Infect Dis J.
1996;15(8):687–692.
175. Watson JC, Hadler SC, Dykewicz CA, et al.
Measles, mumps, and rubella—vaccine use
and strategies for elimination of measles,
rubella, and congenital rubella syndrome
and control of mumps: recommendations
of the Advisory Committee on Immuniza-
tion Practices (ACIP). MMWR Recomm Rep.
1998;47(Rr-8):1–57.
176. Valeggia C, Ellison PT. Interactions between
metabolic and reproductive functions in the
resumption of postpartum fecundity. Am J
Hum Biol. 2009;21(4):559–566.
177. Tingle AJ, Chantler JK, Pot KH, et  al.
Postpartum rubella immunization: association
with development of prolonged arthritis, neu-
rological sequelae, and chronic rubella vire-
mia. J Infect Dis. 1985;152(3):606–612.
178. Ray P, Black S, Shinefield H, et al. Risk of
chronic arthropathy among women after
rubella vaccination. Vaccine Safety Datalink
team. JAMA. 1997;278(7):551–556.
179. Alzeidan RA, et  al. Postpartum rubella vac-
cination for sero-negative women. Cochrane
Database Syst Rev. 2013;(9).
180. Preblud SR, D’Angelo LJ. Chickenpox in
the United States, 1972-1977. J Infect Dis.
1979;140(2):257–260.
181. LaRussa P, Steinberg SP, Seeman MD,
Gershon AA. Determination of immunity to
varicella-zoster virus by means of an intra-
dermal skin test. J Infect Dis. 1985;152(5):
869–875.
182. Balducci J, Rodis JF, Rosengren S, et  al.
Pregnancy outcome following first-tri-
mester varicella infection. Obstet Gynecol.
1992;79(1):5–6.
183. Mirlesse V, Lebon P. Chickenpox dur-
ing pregnancy. Arch Pediatr. 2003;10(12):
1113–1138.
184. Sauerbrei A, Wutzler P. The congenital vari-
cella syndrome. J Perinatol. 2000;20(8 Pt
1):548–554.
185. Smith CK, Arvin AM. Varicella in the fetus
and newborn. Semin Fetal Neonatal Med.
2009;14(4):209–217.
186. Harger JH, Ernest JM, Thurnau GR, et  al.
National Institute of Child Health and
Human Development, Network of Maternal-
Fetal Medicine Units. Risk factors and out-
come of varicella-zoster virus pneumonia in
pregnant women. J Infect Dis. 2002;185(4):
422–427.
187. Weber DM, Pllecchia JA. Varicella pneumo-
nia: study of prevalence in adult men. JAMA.
1965;192:572–573.
188. Rawlinson WD, Dwyer DE, Gibbons VL,
Cunningham AL. Rapid diagnosis of vari-
cella-zoster virus infection with a monoclonal
antibody based direct immunofluorescence
technique. J Virol Methods. 1989;23(1):
13–18.
189. Koropchak CM, Graham G, Palmer J, et al.
Investigation of varicella-zoster virus infec-
tion by polymerase chain reaction in the
immunocompetent host with acute varicella.
J Infect Dis. 1991;163(5):1016–1022.
190. Koropchak CM, et  al. Investigation of var-
icella-zoster virus infection by polymerase
chain reaction in the immunocompe-
tent host with acute varicella. J Infect Dis.
1992;165(1):188.
191. Pastuszak AL, Levy M, Schick B, et al. Out-
come after maternal varicella infection in the
first 20 weeks of pregnancy. N Engl J Med.
1994;330(13):901–905.
192. Scharf A, Scherr O, Enders G, Helftenbein
E. Virus detection in the fetal tissue of a
premature delivery with a congenital vari-
cella syndrome. A case report. J Perinat Med.
1990;18(4):317–322.
193. Connan L, Ayoubi J, Icart J, et al. Intra-uter-
ine fetal death following maternal varicella
infection. Eur J Obstet Gynecol Reprod Biol.
1996;68(1-2):205–207.
194. Tanimura K, Kojima N, Yamazaki T, et  al.
Second trimester fetal death caused by
varicella-zoster virus infection. J Med Virol.
2013;85(5):935–938.
195. Laforet EG, Lynch Jr CL. Multiple con-
genital defects following maternal varicella;
report of a case. N Engl J Med. 1947;236(15):
534–537.
196. Siegel M. Congenital malformations fol-
lowing chickenpox, measles, mumps, and
hepatitis. Results of a cohort study. JAMA.
1973;226(13):1521–1524.
197. Brunell PA, Kotchmar Jr GS. Zoster in
infancy: failure to maintain virus latency
following intrauterine infection. J Pediatr.
1981;98(1):71–73.
198. Paryani SG, Arvin AM. Intrauterine infec-
tion with varicella-zoster virus after mater-
nal varicella. N Engl J Med. 1986;314(24):
1542–1546.
199. Enders G, Miller E, Cradock-Watson J, et al.
Consequences of varicella and herpes zos-
ter in pregnancy: prospective study of 1739
cases. Lancet. 1994;343(8912):1548–1551.
200. Alkalay AL, Pomerance JJ, Rimoin
DL. Fetal varicella syndrome. J Pediatr.
1987;111(3):320–323.
201. Birthistle K, Carrington D. Fetal varicella
syndrome—a reappraisal of the literature. A
review prepared for the UK Advisory Group
on Chickenpox on behalf of the British
Society for the Study of Infection. J Infect.
1998;36(suppl 1):25–29.
202. Tan MP, Koren G. Chickenpox in preg-
nancy: revisited. Reprod Toxicol. 2006;21(4):
410–420.
203. Al-Katawee YA, Al-Hasoun YA, Taha MN,
Al-Moslem K. Congenital varicella-zoster
virus infection. A rare case of severe brain
and ocular malformations without limb or
cutaneous involvement in a newborn after
maternal subclinical infection. Saudi Med J.
2005;26(5):869–871.

204. Andreou A, Basiakos H, Hatzikoumi I,
Lazarides A. Fetal varicella syndrome with
manifestations limited to the eye. Am J Peri-
natol. 1995;12(5):347–348.
205. Brice JE. Congenital Varicella resulting from
infection during second trimester of preg-
nancy. Arch Dis Child. 1976;51(6):474–476.
206. Garrido Martín J, Avilés Puigvert M, Espejo
González A, Sánchez Luque JR. Congenital
Horner’s syndrome associated to varicella.
Arch Soc Esp Oftalmol. 2002;77(4):223–225.
207. Dimova PS, Karparov AA. Congenital vari-
cella syndrome: case with isolated brain dam-
age. J Child Neurol. 2001;16(8):595–597.
208. Deasy NP, Jarosz JM, Cox TC, Hughes E.
Congenital varicella syndrome: cranial MRI
in a long-term survivor. Neuroradiology.
1999;41(3):205–207.
209. Lloyd KM. Skin lesions as the sole manifes-
tation of the fetal varicella syndrome. Arch
Dermatol. 1990;126(4):546–547.
210. Hammad E, Helin I, Pacsa A. Early pregnancy
varicella and associated congenital anomalies.
Acta Paediatr Scand. 1989;78(6):963–964.
211. Bruder E, Ersch J, Hebisch G, et  al. Fetal
varicella syndrome: disruption of neural
development and persistent inflamma-
tion of non-neural tissues. Virchows Arch.
2000;437(4):440–444.
212. Sauve RS, Leung AK. Congenital varicella
syndrome with colonic atresias. Clin Pediatr
(Phila). 2003;42(5):451–453.
213. Fujita H, Yoshii A, Maeda J, et  al. Geni-
tourinary anomaly in congenital varicella
syndrome: case report and review. Pediatr
Nephrol. 2004;19(5):554–557.
214. Mouly F, Mirlesse V, Méritet JF, et  al.
Prenatal diagnosis of fetal varicella-zoster
virus infection with polymerase chain reac-
tion of amniotic fluid in 107 cases. Am J
Obstet Gynecol. 1997;177(4):894–898.
215. Harger JH, Ernest JM, Thurnau GR, et  al.
National Institute of Child Health and
Human Development Network of Maternal-
Fetal Medicine Units. Frequency of congeni-
tal varicella syndrome in a prospective cohort
of 347 pregnant women. Obstet Gynecol.
2002;100(2):260–265.
216. Michie CA, Acolet D, Charlton R, et  al.
Varicella-zoster contracted in the second
trimester of pregnancy. Pediatr Infect Dis J.
1992;11(12):1050–1053.
217. Palmer CG, Pauli RM. Intrauterine varicella
infection. J Pediatr. 1988;112(3):506–507.
218. Bai PV, John TJ. Congenital skin ulcers fol-
lowing varicella in late pregnancy. J Pediatr.
1979;94(1):65–67.
219. Mattson SN, Jones KL, Gramling LJ, et  al.
Neurodevelopmental follow-up of children
of women infected with varicella during
pregnancy: a prospective study. Pediatr Infect
Dis J. 2003;22(9):819–823.
220. Pretorius DH, Hayward I, Jones KL, Stamm
E. Sonographic evaluation of pregnancies
with maternal varicella infection. J Ultra-
sound Med. 1992;11(9):459–463.
221. Cuthbertson G, Weiner CP, Giller RH,
Grose C. Prenatal diagnosis of second-
trimester congenital varicella syndrome by
virus-specific immunoglobulin M. J Pediatr.
1987;111(4):592–595.
222. Petignat P, Vial Y, Laurini R, Hohlfeld
P. Fetal varicella-herpes zoster syndrome
in early pregnancy: ultrasonographic and
morphological correlation. Prenat Diagn.
2001;21(2):121–124.
223. Hofmey GJ, Moolla S, Lawrie T. Prenatal
Sonographic diagnosis of congenital vari-
cella infection—a case report. Prenat Diagn.
1996;16(12):1148–1151.
224. Ong CL, Daniel ML. Antenatal diagnosis
of a porencephalic cyst in congenital vari-
cella-zoster virus infection. Pediatr Radiol.
1998;28(2):94.
225. Da Silva O, Hammerberg O, Chance GW.
Fetal varicella syndrome. Pediatr Infect Dis J.
1990;9(11):854–855.
226. Mirlesse V, Solé Y, Jacquemard F, et  al.
Persistent maternal viremia after varicella
infection during pregnancy as a possible
cause of false positive prenatal diagnosis
of fetal infection on amniotic fluid. BJOG.
2004;111(8):885–887.
227. Liesnard C, Donner C, Brancart F,
Rodesch F. Varicella in pregnancy. Lancet.
1994;344(8927):950–951.
228. Kustermann A, Zoppini C, Tassis B, et  al.
Prenatal diagnosis of congenital varicella
infection. Prenat Diagn. 1996;16(1):71–74.
229. Isada NB, Paar DP, Johnson MP, et  al. In
utero diagnosis of congenital varicella zoster
virus infection by chorionic villus sampling
and polymerase chain reaction. Am J Obstet
Gynecol. 1991;165(6 Pt 1):1727–1730.
230. Newman CG. Perinatal varicella. Lancet.
1965;2(7423):1159–1161.
231. Pearson HE. Parturition varicella-zoster.
Obstet Gynecol. 1964;23:21–27.
232. Brunell PA. Varicella-zoster infections in
pregnancy. JAMA. 1967;199(5):315–317.
233. Siegel M, Fuerst HT. Low birth weight and
maternal virus diseases. a prospective study
of rubella, measles, mumps, chickenpox, and
hepatitis. JAMA. 1966;197(9):680–684.
234. Meyers JD. Congenital varicella in term
infants: risk reconsidered. J Infect Dis.
1974;129(2):215–217.
235. Hanngren K, Grandien M, Granstrom G.
Effect of zoster immunoglobulin for varicella
prophylaxis in the newborn. Scand J Infect
Dis. 1985;17(4):343–347.
236. Koren G. Risk of varicella infection dur-
ing late pregnancy. Can Fam Physician.
2003;49:1445–1446.
237. Dunkle LM, Arvin AM, Whitley RJ, et  al.
A controlled trial of acyclovir for chick-
enpox in normal children. N Engl J Med.
1991;325(22):1539–1544.
238. Klassen TP, Hartling L, Wiebe N,
Belseck EM. Acyclovir for treating vari-
cella in otherwise healthy children and
adolescents. Cochrane Database Syst Rev.
2005;(4):CD002980.
239. Brunell PA, Ross A, Miller LH, Kuo B.
Prevention of varicella by zoster immune
globulin. N Engl J Med. 1969;280(22):
1191–1194.
240. Prevention of varicella: recommendations of
the Advisory Committee on Immunization
Practices (ACIP). MMWR Morb Mortal Wkly
Rep. 1996;45:1.
241. Marin M, Güris D, Chaves SS, et al. Centers
for Disease Control and Prevention (CDC).
Prevention of varicella: recommendations
of the Advisory Committee on Immuniza-
tion Practices (ACIP). MMWR Recomm Rep.
2007;56(Rr-4):1–40.
242. Cohen A, Moschopoulos P, Stiehm RE,
Koren G. Congenital varicella syndrome:
the evidence for secondary prevention with
varicella-zoster immune globulin. CMAJ.
2011;183(2):204–208.
References 525.e5
243. Marin M, Willis ED, Marko A, et al. Centers
for Disease Control and Prevention (CDC).
Closure of varicella-zoster virus-containing
vaccines pregnancy registry—United States,
2013. MMWR Morb Mortal Wkly Rep.
2014;63(33):732–733.
244. Macartney K, Heywood A, McIntyre P.
Vaccines for post-exposure prophylaxis
against varicella (chickenpox) in children
and adults. Cochrane Database Syst Rev.
2014;(6):CD001833.
245. Bonametti AM, Passos JN, Koga da Silva
EM, Macedo ZS. Probable transmission of
acute toxoplasmosis through breast feeding.
J Trop Pediatr. 1997;43(2):116.
246. Langer H. Repeated congenital infection
with Toxoplasma gondii. Obstet Gynecol.
1963;21:318–329.
247. TenterA M, Heckeroth AR, Weiss LM.
Toxoplasma gondii: from animals to humans.
Int J Parasitol. 2000;30(12-13):1217–1258.
248. Rorman E, Zamir CS, Rilkis I, Ben-David H.
Congenital toxoplasmosis—prenatal aspects
of Toxoplasma gondii infection. Reprod Toxi-
col. 2006;21(4):458–472.
249. Jones JL, Kruszon-Moran D, Wilson M,
et  al. Toxoplasma gondii infection in the
United States: seroprevalence and risk fac-
tors. Am J Epidemiol. 2001;154(4):357–365.
250. Montoya JG, Liesenfeld O. Toxoplasmosis.
Lancet. 2004;363(9425):1965–1976.
251. Gras L, Gilbert RE, Wallon M, et al. Dura-
tion of the IgM response in women acquir-
ing Toxoplasma gondii during pregnancy:
implications for clinical practice and cross-
sectional incidence studies. Epidemiol Infect.
2004;132(3):541–548.
252. Boyer KM. Diagnosis and treatment of con-
genital toxoplasmosis. Adv Pediatr Infect Dis.
1996;11:449–467.
253. Hohlfeld P, Daffos F, Thulliez P, et al. Fetal
toxoplasmosis: outcome of pregnancy and
infant follow-up after in utero treatment.
J Pediatr. 1989;115(5 Pt 1):765–769.
254. Dunn D, Wallon M, Peyron F, et al. Mother-
to-child transmission of toxoplasmosis: risk
estimates for clinical counselling. Lancet.
1999;353(9167):1829–1833.
255. Lynfield R, Guerina NG. Toxoplasmosis.
Pediatr Rev. 1997;18(3):75–83.
256. Foulon W, Pinon JM, Stray-Pedersen B,
et  al. Prenatal diagnosis of congenital toxo-
plasmosis: a multicenter evaluation of dif-
ferent diagnostic parameters. Am J Obstet
Gynecol. 1999;181(4):843–847.
257. Remington J. Toxoplasmosis. In: Js R, ed.
Infectious Diseases of the fetus and new-
born infant. Philadelphia: WB Saunders;
1990:89–195.
258. Swisher CN, Boyer K, McLeod R. Congenital
toxoplasmosis. The Toxoplasmosis Study
Group. Semin Pediatr Neurol. 1994;1(1):4–25.
259. Guerina NG, Hsu HW, Meissner HC,
et al. Neonatal serologic screening and early
treatment for congenital toxoplasma gon-
dii infection. the New England Regional
Toxoplasma Working Group. N Engl J Med.
1994;330(26):1858–1863.
260. Vutova K, Peicheva Z, Popova A, et al. Con-
genital toxoplasmosis: eye manifestations
in infants and children. Ann Trop Paediatr.
2002;22(3):213–218.
261. Wallon M, Kodjikian L, Binquet C, et  al.
Long-term ocular prognosis in 327 children
with congenital toxoplasmosis. Pediatrics.
2004;113(6):1567–1572.
525.e6 References
262. Wilson CB, Remington JS. What can be
done to prevent congenital toxoplasmosis?
Am J Obstet Gynecol. 1980;138(4):357–363.
263. Daffos F, Forestier F, Capella-Pavlovsky M,
et al. Prenatal management of 746 pregnan-
cies at risk for congenital toxoplasmosis. N
Engl J Med. 1988;318(5):271–275.
264. Antsaklis A, Daskalakis G, Papantoniou N,
et  al. Prenatal diagnosis of congenital toxo-
plasmosis. Prenat Diagn. 2002;22(12):1107–
11111.
265. Hohlfeld P, Daffos F, Costa JM, et al. Prena-
tal diagnosis of congenital toxoplasmosis with
a polymerase-chain-reaction test on amniotic
fluid. N Engl J Med. 1994;331(11):695–699.
266. Hohlfeld P, MacAleese J, Capella-Pavlovski
M, et  al. Fetal toxoplasmosis: ultrasono-
graphic signs. Ultrasound Obstet Gynecol.
1991;1(4):241–244.
267. Gilbert R, Gras L. Effect of timing and type
of treatment on the risk of mother to child
transmission of Toxoplasma gondii. BJOG.
2003;110(2):112–120.
268. 
World Health Organization. Drugs Used.
In: Parasitic diseases. Geneva: World Health
Organization; 1995.
269. Wallon M, Liou C, Garner P, Peyron F.
Congenital toxoplasmosis: systematic review
of evidence of efficacy of treatment in preg-
nancy. BMJ. 1999;318(7197):1511–1514.
270. Gras L, Gilbert RE, Ades AE, Dunn DT.
Effect of prenatal treatment on the risk of
intracranial and ocular lesions in children
with congenital toxoplasmosis. Int J Epide-
miol. 2001;30(6):1309–1313.
271. Gratzl R, Sodeck G, Platzer P, et  al.
Treatment of toxoplasmosis in pregnancy:
concentrations of spiramycin and neo-
spiramycin in maternal serum and amni-
otic fluid. Eur J Clin Microbiol Infect Dis.
2002;21(1):12–16.
272. C1 Neto, Rubin R, Schulte J, Giugliani R.
Newborn screening for congenital infectious
diseases. Emerg Infect Dis. 2004;10(6):1068–
1073.
273. Gilbert RE, Gras L, Wallon M, et al. Effect
of prenatal treatment on mother to child
transmission of Toxoplasma gondii: retro-
spective cohort study of 554 mother-child
pairs in Lyon, France. Int J Epidemiol.
2001;30(6):1303–1308.
274. Gilbert R, Dunn D, Wallon M, et  al. Eco-
logical comparison of the risks of mother-to-
child transmission and clinical manifestations
of congenital toxoplasmosis according to pre-
natal treatment protocol. Epidemiol Infect.
2001;127(1):113–120.
275. Bessieres MH, Berrebi A, Rolland M, et  al.
Neonatal screening for congenital toxoplas-
mosis in a cohort of 165 women infected
during pregnancy and influence of in utero
treatment on the results of neonatal tests. Eur J
Obstet Gynecol Reprod Biol. 2001;94(1):37–45.
276. Foulon W, NaessenAs. Ho-Yen D. Preven-
tion of congenital toxoplasmosis. J Perinat
Med. 2000;28(5):337–345.
277. SYROCOT (Systematic Review on Congeni-
tal Toxoplasmosis) study group, Thiébaut R,
Leproust S, Chêne G, Gilbert R. Effectiveness
of prenatal treatment for congenital toxoplas-
mosis: a meta-analysis of individual patients’
data. Lancet. 2007;369(9556):115–122.
278. Carter AO, Gelmon SB, Wells GA, Toepell
AP. The effectiveness of a prenatal educa-
tion programme for the prevention of con-
genital toxoplasmosis. Epidemiol Infect.
1989;103(3):539–545.
279. Gollub EL, Leroy V, Gilbert R, et al. Euro-
pean Toxoprevention Study Group (EURO-
TOXO). Effectiveness of health education
on toxoplasma-related knowledge, behav-
iour, and risk of seroconversion in preg-
nancy. Eur J Obstet Gynecol Reprod Biol.
2008;136(2):137–145.
280. Di Mario S, Basevi V, Gagliotti C, et  al.
Prenatal education for congenital toxo-
plasmosis. Cochrane Database Syst Rev.
2013;(2):CD006171.
281. Bhopale GM. Development of a vaccine for
toxoplasmosis: current status. Microbes Infect.
2003;5(5):457–462.
282. Buxton D, Innes EA. A commercial vac-
cine for ovine toxoplasmosis. Parasitology.
1995;110(suppl):S11–S16.
283. Wallon M, Peyron F, Cornu C, et al. Con-
genital toxoplasma infection: monthly pre-
natal screening decreases transmission rate
and improves clinical outcome at age 3 years.
Clin Infect Dis. 2013;56(9):1223–1231.
284. Gilbert RE, Thalib L, Tan HK, et  al.
European Multicentre Study on Congeni-
tal Toxoplasmosis. Screening for congenital
toxoplasmosis: accuracy of immunoglobulin
m and immunoglobulin a tests after birth. J
Med Screen. 2007;14(1):8–13.
285. Gilbert RE, Peckham CS. Congenital
toxoplasmosis in the United Kingdom:
to screen or not to screen? J Med Screen.
2002;9(3):135–141.
286. Friedman CA, Temple DM, Robbins KK,
et  al. Outbreak and control of varicella in
a neonatal intensive care unit. Pediatr Infect
Dis J. 1994;13(2):152–154.
287. Wendel GD. Gestational and congenital
syphilis. Clin Perinatol. 1988;15(2):287–
303.
288. Sánchez PJ1 Jr Wendel GD, Grimprel E,
et al. Evaluation of molecular methodologies
and rabbit infectivity testing for the diagno-
sis of congenital syphilis and neonatal central
nervous system invasion by Treponema pal-
lidum. J Infect Dis. 1993;167(1):148–157.
289. Syphilis Remington J, ed. Infectious Diseases
of the Fetus and Newborn Infant. Philadel-
phia: Elsevier Saunders; 2006:545–580.
290. Harter C, Benirschke K. Fetal syphilis in
the first trimester. Am J Obstet Gynecol.
1976;124(7):705–711.
291. Nathan L, Bohman VR, Sanchez PJ, et  al.
In utero infection with Treponema pal-
lidum in early pregnancy. Prenat Diagn.
1997;17(2):119–123.
292. Woods CR. Syphilis in children: congeni-
tal and acquired. Semin Pediatr Infect Dis.
2005;16(4):245–257.
293. Hollier LM, Harstad TW, Sanchez PJ,
et  al. Fetal syphilis: clinical and laboratory
characteristics. Obstet Gynecol. 2001;97(6):
947–953.
294. Whitaker JA, Sartain P, Shaheedy M. Hema-
tological aspects of congenital syphilis.
J Pediatr. 1965;66:629–636.
295. Sanchez PJ, Wendel GD. Syphilis in preg-
nancy. Clin Perinatol. 1997;24(1):71–90.
296. McFarlin BL, Bottoms SF, Dock BS, Isada
NB. Epidemic syphilis: maternal factors asso-
ciated with congenital infection. Am J Obstet
Gynecol. 1994;170(2):535–540.
297. Klein VR, Cox SM, Mitchell MD,
Wendel Jr GD. The Jarisch-Herxheimer
reaction complicating syphilotherapy in
pregnancy. Obstet Gynecol. 1990;75(3 Pt 1):
375–380.
298. Sanchez PJ, Wendel GD, Norgard MV.
Congenital syphilis associated with negative
results of maternal serologic tests at delivery.
Am J Dis Child. 1991;145(9):967–969.
299. Coles FB, Hipp SS, Silberstein GS, Chen
JH. Congenital syphilis surveillance in
upstate New York, 1989-1992: implications
for prevention and clinical management. J
Infect Dis. 1995;171(3):732–735.
300. Azam et al. ***2000.
43
Disorders of Amniotic Fluid
JAN E. DICKINSON
KEY POINTS
• Amniotic fluid (AF) volume is usually well regulated in
pregnancy.
• Subjective or semiquantitative ultrasound measurement systems
are used to identify and categorise disorders of AF volume.
• Low (oligohydramnios) or high (polyhydramnios) AF volumes
are associated with increased maternal and perinatal compli-
cations.
• Obstetric management is based on the underlying cause of
the abnormal AF volume.
Introduction
The fetus exists in a fluid-filled environment which assists in pul-
monary maturation and musculoskeletal development, provides
some protection from infection and trauma, protects the umbili-
cal cord from compression and provides some nutrition. Regula-
tion of the amniotic fluid (AF) volume is not well understood, but
aberrations from normal are associated with increased perinatal
morbidity and mortality.  although important for determination of actual fluid volumes, are
Amniotic Fluid Physiology
Amniotic fluid is 98% to 99% water, with variance in chemical
composition with advancing gestation.1 In early pregnancy, AF
is a dialysate of maternal and fetal plasma, with water and solutes
traversing the fetal skin bidirectionally. By the second trimester,
when the fetal skin keratinises and becomes impermeable to water,
the AF becomes increasingly hypotonic and is derived predomi-
nantly from fetal urine and lung fluid. AF is removed mainly by
fetal swallowing and absorption into fetal blood in the chorionic
plate vessels (intramembranous pathway).2 This dynamic process
of AF regulation results in a fairly stable volume of 800 mL from
24 weeks’ gestation until near term, when there is a decline.3 The
precise mechanisms of AF regulation remain uncertain, although
the intramembranous pathway is currently believed to be the prin-
cipal regulator of AF volume.4
There are several studies assessing the change in AF volume
with increasing gestation. The earlier studies of Queenan and
colleagues (1972)5 and Brace and Wolf (1989)3 using dye-dilution
techniques demonstrated an increase in AF volume from 15 to 20
weeks’ gestation with a maximum volume at 33 to 34 weeks’ gesta-
tion. Both these authors demonstrated a progressive decrease in AF
volume from the peak until term. A decade later, Magann and col-
leagues conducted another study in 144 singleton pregnancies using
526
dye-dilution techniques to assess AF volume over gestation in an
attempt to overcome some of the methodological problems with the
earlier data.6 Using a growth curve model, Magann and colleagues
demonstrated a continued increase in AF volume across gestation,
peaking at 40 weeks’ gestation (Fig. 43.1).
Amniotic fluid provides several important functions for fetuses:
• The trophic factors found in AF (e.g., insulinlike growth factor,
granulocyte colony-stimulating factor), have been postulated
to play a role in fetal growth and development.7
• Protective support permitting fetal growth and movement
• Antimicrobial function (e.g., human β-defensins 1–4)8
• Fetal lung growth
• Fetal musculoskeletal development
• Maturation of the gastrointestinal system 
Methods for the Clinical Assessment of
Amniotic Fluid Volume
Research studies of absolute AF volume using dye dilution tech-
niques, radioactive isotopes or direct measurement at hysterotomy,
not applicable for use in the clinical practice setting. Recognising
the importance of AF in obstetric outcomes—normal, increased
or decreased—the assessment of AF volume for daily clinical
practice has centred around ultrasound using either subjective or
semiquantitative methodologies. Of the semiquantitative meth-
odologies, only amniotic fluid index (AFI) and maximum vertical
pocket (MVP) are in routine clinical practice.
Subjective Assessment of Amniotic Fluid Volume
The subjective assessment of AF volume is a visual interpreta-
tion of AF using ultrasound examination without an objective
measurement. It is unclear how frequently this is used in clinical
practice.9,10 There are limited data comparing the subjective evalu-
ation of AF volume with direct measures; however, the few pub-
lications available demonstrate a satisfactory correlation.9,11 For
experienced sonographers, the subjective impression of normal
compared with abnormal AF volume may be used but is difficult
to translate across users and time. 
Semiquantitative Ultrasound Assessment of
Amniotic Fluid Volume
There are two main semiquantitative measurement systems of AF
volume in clinical use: the AFI and the MVP (also known as the

2000
Brace and Wolf3
Magann et al.
Queenan et al.5
1000
Amniotic fluid volume (mL)
100
16 18 20 22 24
• Fig. 43.1  Normal amniotic fluid volume across gestation.6 (Reprinted with permission Magann EF, Sandlin
AT, Ounpraseuth ST. Amniotic fluid and the clinical relevance of the sonographically estimated amniotic fluid
volume. J Ultrasound Med 30:1573–1585, 2011. Stanford University Libraries Highwire Press and AIUM.)
single deepest vertical pocket). Since first suggested in 1998 by
Moore and Brace,12 there have been several studies assessing the
relationship between AF volume (both AFI and/or MVP) and dye-
determined or directly measured AF volume. Disappointingly, the
sonographic estimates of normal AF volume do not correlate consis-
tently well with the direct or dye-determined techniques (sensitivi-
ties, 71%–98%).13 For oligohydramnios, detection with ultrasound
techniques compared with dye-determination or directly measured
volumes the sensitivities are poor, ranging from 6.7% to 27%.14,15
The AFI is determined by summing four vertical quadrants
with the transducer positioned in a sagittal place perpendicular to
the floor (Fig. 43.2) and was first introduced by Phelan and col-
leagues in 1987 for term pregnancies.16 This measurement system
was subsequently expanded to include second and third trimester
pregnancies (16–42 weeks’ gestation),17 and gestation-specific nor-
mal ranges for AFI were developed. The MVP technique (Fig. 43.3)
is also widely used with a 2-cm cutoff being most widely clinically
accepted as discriminating normal from low AF.18 The ultrasound
threshold for low AF volume is generally accepted as an AFI of 5 cm
or less or a MVP of 2 cm or less because as these values have been
associated with an increased risk for adverse perinatal outcomes.19
Interestingly, the upper limit of AFI to define polyhydramnios is less
well-defined but is typically greater than 24 cm (or MVP >8 cm).19  structurally normal fetuses with intact membranes has been recently
Amniotic Fluid Volume and Perinatal
Outcome
For clinicians, the absolute relationship of true AF volume with
sonographic techniques is of lesser importance than the association
CHAPTER 43  Disorders of Amniotic Fluid 527
Max at week 34
Max at week 40
Max at week 33
26 28 30 32 34 36 38 40 42
Gestational age (wk)
of sonographic estimation of AF and adverse pregnancy outcomes.
Measurements of AF volume are routinely performed as a compo-
nent of fetal surveillance protocols throughout pregnancy. There is
a well-recognised association with fetal abnormality and extremes
of AF volume (see later discussion).
In a systematic review, Nabhan and Abdelmoula20 assessed four
randomised controlled trials in singleton pregnancies comparing
AFI and MVP as components of antepartum fetal surveillance to
prevent adverse pregnancy outcome. Use of AFI to determine oli-
gohydramnios compared with MVP was associated with an increase
in the diagnosis of oligohydramnios (relative risk [RR], 2.33; 95%
confidence interval [CI], 1.67–3.24), an increase in labour induc-
tion (RR, 2.10; 95% CI, 1.60–2.76) and an increase in caesarean
delivery for fetal compromise (RR, 1.45; 95% CI, 1.07–1.97).
Additionally, there was no difference in adverse perinatal outcomes
using either measurement technique (admission to neonatal inten-
sive care unit, cord arterial pH <7.1, Apgar score <7 at 5 minutes or
meconium). These authors concluded that MVP was the preferred
measurement method for AF volume, with AFI overestimating the
presence of oligohydramnios and leading to increased obstetric
intervention with no improvement in pregnancy outcomes.
The association of AF perturbations and pregnancy outcomes in
reviewed by Morris and colleagues.21 In their systematic review of
43 studies comparing AF volume assessments with adverse preg-
nancy outcomes in 244,493 fetuses, the authors observed:
• Strong association between oligohydramnios (variously defined)
and
• Birth weight below the 10th percentile in high-risk popula-
tions (odds ratio [OR], 6.31; 95% CI, 4.15–9.58)
528 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
Voluson
E8
Q1
Voluson
E8
Q2
• Fig. 43.2  Ultrasound evaluation of amniotic fluid using the amniotic fluid index (AFI) technique.
Voluson
E8
Q2
• Fig. 43.3 Ultrasound evaluation of amniotic fluid using the maximum
vertical pocket technique.
•  Birth weight below the 10th percentile in low-risk or
unselected populations (OR, 2.34; 95% CI, 1.76–3.09)
• Neonatal death (OR, 8.72; 95% CI, 2.43–31.26)
• Perinatal mortality in high-risk populations (OR, 11.54;
95% CI, 4.05–32.9)
• Strong association between polyhydramnios (MVP >8 cm or
AFI >25 cm) and birthweight greater than the 90th percentile
(OR, 11.41; 95% CI, 7.09–18.36)
Associations of oligohydramnios and assessments of neona-
tal morbidity were only moderate with substantial heterogeneity
between the studies. There was no association between polyhy-
dramnios and low birth weight. For all outcomes assessed, how-
ever, the prediction models for abnormalities of AF volume and
adverse outcomes were poor. In particular, although specificity was
generally good, sensitivity was low, indicating an increased risk for
an adverse outcome with an abnormal result but a normal result
did not alter outcomes. Assessment of AF volume should not be
Voluson
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Q3
Voluson
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Q4
used in isolation but rather in combination with other prognostic
features (e.g., umbilical artery Doppler) to more accurately predict
outcomes.21 
Amniotic Fluid Assessment in Multiple
Pregnancies
Multiple pregnancies have a recognised baseline increase in
adverse perinatal outcomes compared with singletons. The pres-
ence of a twin gestation adds another layer of complexity to AF
volume assessment in terms of techniques of assessment, cause
of AF variance and management. The dividing membrane in
diamniotic multiple pregnancies complicates the conduct and
reproducibility of ultrasound-based AF assessments. A recent
systematic review on the assessment of AF volume in twin preg-
nancies demonstrated that AFI and/or MVP was typically used
for ultrasound volume studies.22 The AFI and MVP performed
equivalently when compared with dye-dilution techniques, but
concerns have been raised about the reliability of AFI in twin
pregnancies.23 It has been reported that there is no significant
relationship between gestation and MVP in diamniotic twins,24
although a recent study of uncomplicated monochorionic diam-
niotic twins has demonstrated this does not hold true in this
subset.25 This may well be secondary to the predominance of
dichorionic pregnancies in the initial paper24 and the focus solely
on monochorionic pregnancies in the second.25 Most centres have
moved to using MVP for multiple pregnancies because of its ease
of use, reliability and more robust performance characteristics for
oligohydramnios24 (Fig. 43.4). In general, an MVP less than 2 cm
is used to define oligohydramnios, and an MVP greater than 8
cm to define polyhydramnios.24 For monochorionic twins, an
MVP greater than 10 cm is used to define polyhydramnios after
20 weeks’ gestation.25 
 CHAPTER 43  Disorders of Amniotic Fluid 529

M4 M3
FR 28 Hz M3 FR 28 Hz +14.4
RS R1
Z 1.7
Z 1.2 2D
2D 54%
57% C 60
P Low
C 57 Res
P Low CF
HGen 77%
-14.4
1500Hz 5
cm/s
T2 T1 WF 90Hz
Med

Dist 6.46 cm
Dist 5.80 cm
A
• Fig. 43.4 Ultrasound evaluation of amniotic fluid using the maximum FR 46 Hz
R1
M3

vertical pocket technique in a diamniotic twin pregnancy.

2D
37%
Oligohydramnios C 58
P Low
HGen
Low AF, or oligohydramnios, is usually defined as an MVP less
than 2 cm or an AFI les s than 5 cm.26 It remains of concern that
a consistent universal definition is not used, creating difficulty in
comparing studies and outcomes. The pregnancy outcome princi-
pally depends on the underlying cause of the low AF volume and
the gestation at recognition. Oligohydramnios in early midpreg-
nancy typically has a different cause to that diagnosed in the late
second or the third trimester, reflecting the different perinatal out-
comes. The three main underlying causes of oligohydramnios are:
• Premature rupture of the membranes (PPROM) B 12
• Congenital abnormalities
• Intrauterine growth restriction Voluson
The cause of periviable PPROM is poorly understood, but there E8
are several consistent risk factors associated with its occurrence,
including antepartum haemorrhage, history of preterm labour,
cervical cerclage, multiple pregnancy, cigarette smoking and previ-
ous PPROM.27 PPROM occurring in early pregnancy is typically
associated with poor perinatal outcomes, particularly if before
22 weeks’ gestation. Early studies provided an extremely dismal
prognosis with survival rates of 10% for fetuses with PPROM at 13
to 21 weeks’ gestation.28 A recent observational study of 143 cases
of periviable PPROM (16–24 weeks’ gestation) reported a 17%
survival rate to discharge rate for cases at 16 to 20 weeks’ gestation
(n = 24) and 39% for those at 20 to 24 weeks’ gestation (n = 82)
(P = .042), demonstrating some improvement in later gestations
with contemporary neonatal management.29 Factors associated C
with increased survival in periviable PPROM include gestation
at membrane rupture, latency (time period from PPROM until • Fig. 43.5  Renal causes of oligohydramnios: bilateral renal agenesis (A),
delivery) and mode of delivery. The ultrasound assessment of AFI autosomal recessive polycystic kidney disease (B) and lower urinary
was a poor predictor of survival.29 For babies born alive after peri- tract obstruction (C).
viable PPROM, respiratory morbidity (pulmonary hypoplasia,
respiratory distress syndrome and bronchopulmonary dysplasia) appearances (Fig. 43.5B) or megacystis (Fig. 43.5C) (See Chapter 33
is common. Bronchopulmonary dysplasia was reported in 29% on fetal renal abnormalities). The outcome for these fetuses tends
of survivors in a recent review.27 The occurrence of periviable to be universally dismal.
PPROM requires careful multidisciplinary counselling with realis- Fetal growth restriction may be associated with oligohydram-
tic outcome data provided and the management options of expect- nios, the cause believed to be reduction in fetal urine output sec-
ant management or pregnancy interruption clearly explained. ondary to abnormalities in placental perfusion.30 This relationship
Congenital malformations of the fetal renal tract with absence between oligohydramnios and low birth weight has recently been
of functioning renal tissue or lower urinary tract obstruction are reviewed by Morris and colleagues,21 a consistent and significant
recognised causes of second trimester oligohydramnios. These association observed between birth weight less than 2500 g or
severe fetal conditions are readily recognised by ultrasound with below the 10th percentile and low AF as measured by ultrasound.
absence of renal tissue bilaterally (Fig. 43.5A), abnormal renal (Fetal growth restriction is discussed in detail in Chapter 39.)
530 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
The presence of oligohydramnios without fetal abnormali-
ties, fetal growth restriction, intrauterine infection and in the
absence of known maternal disease is termed isolated oligohy-
dramnios. Isolated oligohydramnios in near-term or postterm
fetuses is well recognised (incidence, 0.5%–5%31) and delivery
frequently facilitated. Shrem and colleagues reviewed 12 studies
involving 36,000 women (6.7% with isolated oligohydramnios
and 93.3% with normal AF volume) to assess the relationship
of isolated oligohydramnios and adverse perinatal outcomes.31
Obstetric intervention rates were increased in pregnancies with
isolated oligohydramnios: labour induction (OR, 7.56; 95% CI,
4.58–12.48) and caesarean delivery (OR, 2.07; 95% CI, 1.77–
2.41) increased. Short-term perinatal morbidity was increased
with a higher occurrence of low Apgar scores at 5 minutes (OR,
2.01; 95% CI, 1.3–3.09) and admission to the neonatal intensive
care unit (NICU) (OR 1.47; 95% CI, 1.17–1.84). Similarly, in
another recent systematic review by Rabie and colleagues,32 for
uncomplicated pregnancies with isolated oligohydramnios, there
was an increase in meconium aspiration syndrome (RR, 2.83;
95% CI, 1.41–5.70), caesarean deliveries for fetal compromise
(RR, 2.16; 95% CI, 1.64–2.85) and admission to the NICU (RR,
1.72; 95% CI, 1.72; 95% CI, 1.20–2.47). These authors were not
able to determine an optimal delivery timing to reduce the risk for
adverse perinatal outcomes, and this is an area in need of ongoing
research. It is not clear whether some of these short-term neonatal
outcomes are secondary to the obstetric intervention of labour
induction rather than the underlying diagnosis of oligohydram-
nios in otherwise uncomplicated pregnancies. In 2009, a survey
of members of the Society for Maternal Fetal Medicine to assess
practice patterns for isolated oligohydramnios demonstrated that
only 33% of those surveyed believed labour induction would
decrease perinatal morbidity with 45% unsure and 21% disagree-
ing.33 This significant divergence of opinion in the management
of isolated oligohydramnios and the paucity of high-quality data
indicated the need for a randomised trial.  • Twin-reversed arterial perfusion sequence
Polyhydramnios
Polyhydramnios, the excess accumulation of AF, has a prevalence
of 0.2% to 1.6%34 and is typically defined by ultrasound as an
MVP of AF on ultrasound greater than 8 cm or an AFI greater
than 24 cm (Fig. 43.6).
There are four fundamental concerns after polyhydramnios is
recognised:
• The underlying cause, which typically determines the outcome
• The risk for preterm birth
• Peripartum risks of malpresentation and postpartum haemor-
rhage
• Maternal symptomatology
Cause of Polyhydramnios
The more severe the excess of AF volume, the more likely a specific
underlying cause will be found. In mild polyhydramnios (MVP
8–11 cm or AFI 25–30 cm), a cause is evident in about 17% of
cases compared with more than 90% when the polyhydramnios
is severe (MVP >15 cm or AFI >35 cm).35 Most cases of polyhy-
dramnios are mild (68%) with no cause evident.36
Recognised causes of polyhydramnios include:
• Fetal malformations
• Chromosomal: trisomy 18, trisomy 21, Pallister-Killian
• Gastrointestinal: oesophageal atresia, tracheoesophageal fis-
tula, duodenal atresia, ileal and jejunal atresia
FR 28 Hz M3
RS
2D
56%
C 58
P Low
HGen
16
Q3 11.6 cm
• Fig. 43.6 Polyhydramnios as demonstrated by the maximum vertical
pocket technique.
• Neurologic: anencephaly, hydrocephalus, akinesia syndromes
• Cardiac: Ebstein anomaly, cardiac arrhythmias
• Intrathoracic: congenital diaphragmatic hernia, hydrotho-
rax, tumours
• Skeletal dysplasias
• Renal abnormalities: mesoblastic nephroma, prenatal Bart-
ter syndrome
• Diabetes
• Type 1 and 2
• Gestational diabetes
• Fetal infections
• Cytomegalovirus
• Toxoplasmosis
• Multiple pregnancies
• Twin–twin transfusion syndrome
The varied causes of polyhydramnios require a systematic
approach to assessment and management of the pregnancy.
Most women present with increased abdominal size or tenseness,
prompting an ultrasound evaluation. Ultrasound is the corner-
stone to the management of polyhydramnios: assessment of fetal
structures with consideration to karyotyping has an important
diagnostic role. The more severe the polyhydramnios, the higher
the risk for fetal abnormality: 8% with mild polyhydramnios,
12% with moderate and 31% with severe.34 The combination of
fetal growth restriction and polyhydramnios has a well-recognised
association with chromosomal abnormalities (e.g., trisomy 18;
Fig. 43.7A) and should prompt consideration to amniocentesis.
Karyotype and genetic abnormalities are reported in 2% to 16%
of pregnancies complicated with polyhydramnios.37 Fetal gastro-
intestinal disorders frequently present with severe persistent poly-
hydramnios, although often only in the third trimester, and are
usually evident on ultrasound (Fig. 43.7B) Fetal hydrops may be
accompanied by polyhydramnios, and the investigation of this
issue is well established (see Chapter 36 on fetal hydrops). Poor
fetal movement in the presence of polyhydramnios should raise
the spectrum of neuromuscular disorders, either primary fetal
(e.g., akinesia syndromes) or secondary to maternal disease such
as myotonic dystrophy. The placenta should not be overlooked in
the ultrasound assessment of polyhydramnios with giant placental
chorioangioma a known cause of polyhydramnios (Fig. 43.7C).
Maternal hyperglycaemia is a well-recognised cause of polyhy-
dramnios; fetal macrosomia is a usual accompaniment to polyhy-
dramnios in this situation.38 Maternal diabetes accounts for 15%

Voluson
E8
A 12
Voluson
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B induction (adjusted odds ratio [aOR], 1.7; 95% CI, 1.01–2.8),42
Voluson
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C maternal morbidity that may occur in managing these pregnancies.
• Fig. 43.7  Specific causes of polyhydramnios: trisomy 18 (A), duodenal
atresia (B) and placental chorioangioma (C).
of cases of polyhydramnios and in this occurrence is strongly asso-
ciated with poor maternal glycaemic control; polyhydramnios is
rarely seen in women with well-controlled diabetes in pregnancy.38
There is an association with AF glucose, birth weight and AFI.38,39
The cause of the polyhydramnios in maternal diabetes is most likely
related to an osmotic diuresis by the fetus.39 Severe fetal anaemia
(e.g., α-thalassemia, parvovirus, red blood cell isoimmunisation)
may occasionally be associated with polyhydramnios, although
oligohydramnios (parvovirus) occurs as well and most often AF is
normal. Other more specific signs of fetal anaemia (placentomegaly,
cardiomegaly, hydrops) and elevated middle cerebral artery peak
systolic velocities are more useful to raise the suspicion.
CHAPTER 43  Disorders of Amniotic Fluid 531
FR 21 Hz M3
RS
2D
45%
C 58
P Low
HGen
• Fig. 43.8  Ultrasound-guided amnioreduction in severe polyhydramnios
(18-gauge spinal needle).
Often, no cause for the excess AF volume is evident, known as
idiopathic polyhydramnios. The precise cause is uncertain, but idio-
pathic polyhydramnios has a consistent association with increased
perinatal mortality (two- to fivefold increase) and macrosomia.40,41
The literature is inconsistent on the association of idiopathic
hydramnios and adverse perinatal outcomes, probably because the
studies are retrospective database reviews with varying definitions
and perinatal practices. Two recent retrospective cohort studies
have reported increases in adverse perinatal outcomes in pregnan-
cies complicated by idiopathic polyhydramnios, including labour
caesarean delivery (aOR, 2.6; 95% CI, 1.7–4.0),42 operative vaginal
delivery and shoulder dystocia and NICU admission (aOR, 3.71;
95% CI, 2.77–4.99).43 The risk for macrosomia appears to increase
with the severity of the polyhydramnios.43
In addition to the identification of potential causes and spe-
cific management interventions when indicated, several groups
have used amnioreduction to ameliorate maternal symptoms and
potentially prolong gestation in pregnancies complicated by severe
polyhydramnios44,45 (Fig. 43.8). The complication rate from
large-volume amnioreduction is low, and most women deliver
near term, making this a reasonable option for women symptom-
atic from severe polyhydramnios.
Maternal obstetric complications are increased in pregnancies
complicated with polyhydramnios (e.g., malpresentation, postpartum
haemorrhage), and obstetric units need to be vigilant to reduce the
 
Conclusion
Disorders of AF volume are associated with increased perinatal com-
plications, at either end of the volume spectrum. Recognition is by
antenatal ultrasound examination and the MVP appears the most reli-
able of the measurement techniques to diagnose and monitor oligo-
hydramnios and polyhydramnios. Pregnancy evaluation to establish
the underlying cause is central to developing a management and sur-
veillance protocol for women. Many cases of both oligohydramnios
and polyhydramnios are idiopathic, and intervention remains con-
troversial, with a need for prospective trials to assess if current inter-
ventional strategies actually improve obstetric and neonatal outcomes.
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References 5th and above the 95th and 97th percentiles)
predict oligohydramnios and hydramnios?
30. Moore TR. The role of amniotic fluid assess-
ment in evaluating fetal well-being. Clin Peri-
Am J Obstet Gynecol. 2004;190:164–169. natol. 2011;38:33–46.
1. Modena AB, Fieni S. Amniotic fluid dynam-
16. Phelan JP, Smith CV, Broussard P, Small M. 31. Shrem G, Nagawkar SS, Hallak M, Walfisch
ics. Acta Biomed. 2004;75(suppl 1):11–13.
Amniotic fluid volume assessment with the A. Isolated oligohydramnios at term as an
2. Brace RA. Physiology of amniotic fluid regu-
four quadrant technique at 36-42 weeks’ ges- indication for labor induction: a systematic
lation. Clin Obstet Gynecol. 1997;40(2):280–
tation. J Reprod Med. 1987;32:540–542. review and meta-analysis. Fetal Diagn Ther
289.
17. Moore TR, Cayle JE. The amniotic fluid Fetal Diagn Ther. 2016;40(3):161–173.
3. Brace RA, Wolf EJ. Normal amniotic fluid
index in normal human pregnancy. Am J 32. Rabie N, Magann E, Steelman S,
volume changes throughout pregnancy. Am J
Obstet Gynecol. 1990;162:1168–1173. Ounpraseuth S. Oligohydramnios in compli-
Obstet Gynecol. 1989;161:382–388.
18. Chamberlain PF, Manning FA, Morrison cated and uncomplicated pregnancies: a sys-
4. Robertson P, Faber JJ, Brace RA, et  al.
I, et  al. Ultrasound evaluation of amniotic tematic review and meta-analysis. Ultrasound
Responses of amniotic fluid volume and Its
fluid volume. I. The relationship of mar- Obstet Gynecol. 2017;49(4):442–449.
four major flows to lung liquid diversion and
ginal and decreased amniotic fluid volumes 33. Schwartz N, Sweeting R, Young BK. Prac-
amniotic Infusion in the ovine fetus. Reprod
to perinatal outcome. Am J Obstet Gynecol. tice patterns in the management of isolated
Sci. 2009;16(1):88–93.
1984;150:245–249. oligohydramnios: a survey of perinatologists.
5. Queenan JT, Thompson W, Whitfield CR,
19. Magann EF, Ounpraseuth S, Chauhan SP, J Matern Fetal Neonatal Med. 2009;22(4):357–
Shah SI. Amniotic fluid volumes in normal preg-
et  al. Correlation of ultrasound estimated 361.
nancies. Am J Obstet Gynecol. 1972;114(1):34–
with dye-determined or directly measured 34. Dashe JS, McIntire DD, Ramus RM,
38.
amniotic fluid volume revisited. Gynecol et  al. Hydramnios: anomaly prevalence
6. Magann EF, Bass D, Chauhan SP, et  al.
Obstet Invest. 2015;79:46–49. and sonographic detection. Obstet Gynecol.
Amniotic fluid volume in normal singleton
20. Nabhan AF, Abdelmoula YA. Amniotic fluid 2002;100(1):134–139.
pregnancies. Obstet Gynecol. 1997;90:524–
index versus single deepest vertical pocket: a 35. Hamza A, Herr D, Solomayer EF, Meyberg-
528.
meta-analysis of randomized controlled trials. Solomayer G. Polyhydramnios: causes, diag-
7. Hirai C, Ichiba H, Saito M, et  al. Trophic
Int J Gynecol Obstet. 2009;104:184–188. nosis and therapy. Geburtshilfe Frauenheilkd.
effect of multiple growth factors in amniotic
21. Morris RK, Meller CH, Tamblyn J, et  al. 2013;73(12):1241–1246.
fluid or human milk on cultured human fetal
Association and prediction of amniotic fluid 36. Harman CR. Amniotic fluid abnormalities.
small intestinal cells. J Pediatr Gastroenterol
measurements for adverse pregnancy out- Semin Perinatol. 2008;32(4):288–294.
Nutr. 2002;34(5):524–528.
come: systematic review and meta-analysis. 37. Boito S, Crovetto F, Ischia B, et al. Prenatal
8. Stock SJ, Kelly RW, Riley SC, Calder
BJOG. 2014;121:686–699. ultrasound factors and genetic disorders in
AA. Natural antimicrobial production
22. Ippolito DL, Bergstrom JE, Lutgendorf MA, pregnancies complicated by polyhydramnios.
by the amnion. Am J Obstet Gynecol.
et  al. A systematic review of amniotic fluid Prenat Diagn. 2016;36(8):726–730.
2007;196(3):255.e1–e6.
assessments in twin pregnancies. J Ultrasound 38. Vink JY, Poggi SH, Ghidini A, Spong CY.
9. Magann EF, Perry Jr KG, Chauhan SP,
Med. 2014;33(8):1353–1364. Amniotic fluid index and birth weight: is
et  al. The accuracy of ultrasound evaluation
23. Magann EF, Chauhan SP, Whitworth NS, there a relationship in diabetics with poor
of amniotic fluid volume in singleton preg-
et al. The accuracy of the summated amniotic glycemic control? Am J Obstet Gynecol.
nancies: the effect of operator experience
fluid index in evaluating amniotic fluid vol- 2006;195:848–850.
and ultrasound interpretive technique. J Clin
ume in twin pregnancies. Am J Obstet Gyne- 39. Dashe JS, Nathan L, McIntire DD, Leveno
Ultrasound. 1997;25:249–253.
col. 1997;177(5):1041–1045. KJ. Correlation between amniotic fluid glu-
10. Hallak M, Kirshon B, O’Brien Smith E,
24. Magann EF, Doherty DA, Ennen CS, et  al. cose concentration and amniotic fluid volume
et  al. Subjective ultrasonographic assessment
The ultrasound estimation of amniotic fluid in pregnancy complicated by diabetes. Am J
of amniotic fluid depth: comparison with
volume in diamniotic twin pregnancies and Obstet Gynecol. 2000;182:901–904.
the amniotic fluid index. Fetal Diagn Ther.
prediction of peripartum outcomes. Am J 40. Magann EF, Chauhan SP, Doherty DA,
1993;8:256–260.
Obstet Gynecol. 2007;196(6):570. e1–e6. et al. A review of idiopathic hydramnios and
11. Magann EF, Chauhan SP, Whitworth NS,
25. Dekoninck P, Deprest J, Lewi P, et al. Ges- pregnancy outcomes. Obstet Gynecol Surv.
et  al. Subjective versus objective evaluation
tational age-specific reference ranges for 2007;62(12):795–802.
of amniotic fluid volume of pregnancies less
amniotic fluid assessment in monochorionic 41. Taskin S, Pabuccu EG, Kanmaz AG, et  al.
than 24 weeks’ gestation: how can we be accu-
diamniotic twin pregnancies. Ultrasound Perinatal outcomes of idiopathic polyhydram-
rate? J Ultrasound Med. 2001;20:191–195.
Obstet Gynecol. 2013;41(6):649–652. nios. Interv Med Appl Sci. 2013;5(1):21–25.
12. Moore TR, Brace RA. Amniotic Fluid index
26. Moise Jr KJ. Toward consistent terminology: 42. Aviram A, Salzer L, Hiersch L, et al. Associa-
(AFI) in the Term Ovine Pregnancy: a Predict-
assessment and reporting of amniotic fluid tion of isolated polyhydramnios at or beyond
able Relationship between AFI and Amniotic
volume. Semin Perinatol. 2013;37(5):370– 34 weeks of gestation and pregnancy out-
Fluid Volume [Abstract 286]. Philadelphia:
374. come. Obstet Gynecol. 2015;125(4):825–832.
Paper presented at Annual Meeting of the
27. Waters TP, Mercer BM. The management of 43. Wiegand SL, Beamon CJ, Chescheir NC,
Society for Gynecological Investigation; 1988.
preterm premature rupture of the membranes Stamilio D. Idiopathic polyhydramnios:
13. Magann EF, Sandlin AT, Ounpraseuth ST.
near the limit of fetal viability. Am J Obstet severity and perinatal morbidity. Am J Perina-
Amniotic fluid and the clinical relevance of
Gynecol. 2009;201(3):230–240. tol. 2016;33(7):658–664.
the sonographically estimated amniotic fluid
28. Shipp TD, Bromley B, Pauker S, et  al. 44. Dickinson JE, Tjioe YY, Jude E, et al. Amnio-
volume. J Ultrasound Med. 2011;30:1573–
Outcome of singleton pregnancies with reduction in the management of polyhydram-
1585.
severe oligohydramnios in the second and nios complicating singleton pregnancies. Am J
14. Magann EF, Nolan TE, Hess LW, et al. Mea-
third trimesters. Ultrasound Obstet Gynecol. Obstet Gynecol. 2014;211(4):434.e1–e7.
surement of amniotic fluid volume: accuracy
1996;7(2):108–113. 45. Kleine RT, Bernardes LS, Carvalho MA, et al.
of ultrasonographic techniques. Am J Obstet
29. Hunter TJ, Byrnes MJ, Nathan E, et  al. Pregnancy outcomes in severe polyhydram-
Gynecol. 1992;167:1533–1537.
Factors influencing survival in pre-viable nios: no increase in risk in patients needing
15. Magann EF, Doherty DA, Chauhan SP, et al.
preterm premature rupture of membranes. amnioreduction for maternal pain or respira-
How well do the amniotic fluid index and sin-
J Matern Fetal Neonatal Med. 2012;25(9): tory distress. J Matern Fetal Neonatal Med.
gle deepest pocket indices (below the 3rd and
1755–1761. 2016;29(24):4031–4034.

531.e1
44
Multiple Pregnancy
SIEGLINDE M. MÜLLERS, FIONNUAL A MCAULIFFE AND FERGAL D. MALONE
KEY POINTS
• Multiple pregnancies are at high risk for adverse perinatal
outcome. The standard of care is early confirmation of chori-
onicity and timely referral when complications arise.
• Current available data report similar pregnancy-loss rates in
twins for both chorionic villous sampling and amniocentesis
with an excess risk for about 1% above the background risk.
• Selective intrauterine growth restriction or discordant growth
complicates approximately 20% of twins. A threshold of 18% dis-
cordance or greater should prompt referral to a fetal medicine spe-
cialist because of the increased risk for adverse perinatal outcome.
• Single intrauterine death of one twin complicates 2% to 7%
of all twins and is associated with a two- to fivefold risk for
co-twin demise or neurodevelopmental impairment in the
co-twin in monochorionic (MC) compared with dichorionic
(DC) pregnancies.
• There have been significant advances in the treatment of
twin–twin transfusion syndrome (TTTS) over the past 2
decades, and although selective fetoscopic laser ablation
remains the procedure of choice, recent evidence suggests
the Solomon technique offers the advantage of reduction in
recurrent TTTS and twin anaemia–polycythemia sequence
with comparable perinatal outcomes.
• The perinatal outcome of higher order multifetal gestations
reduced to twins has been shown to approach that of sponta-
neously conceived twins.
• Bipolar cord occlusion appears to be superior to radiofre-
quency ablation for selective feticide; however, long-term
prospective studies on neurodevelopmental outcome in
survivors is needed.
• For uncomplicated MC twins, an elective delivery between
36 and 37 weeks’ gestation is advocated, extending this to
38 weeks’ gestation for DC twins. For complicated MC twins,
an elective delivery before 36 weeks’ gestation is usually
indicated, the timing of which depends on the underlining
pathology. In uncomplicated cases, monoamniotic (MA) twins
can undergo delivery between 32 and 34 weeks’ gestation
with appropriate antenatal monitoring.
Introduction
The incidence of multifetal gestations has risen dramatically over
the past number of decades. Assisted reproductive techniques
(ARTs) and the rising trend in advanced maternal age at first birth
532
are the principal factors involved.1,2 Despite restrictions in num-
bers of embryo transfers, the twin birth rate began to rise again
from 33.7 to 33.9 per 1000 between the years 2013 and 2014.3
The incidence of higher order multifetal gestations has decreased
steadily since the peak of 193.4 per 100,000 in 1998 to 113 per
100,000 in 2014.4
These trends implicate an increasing need for specialised tertiary
referral fetal medicine units to address the increasing workload
generated with twins and multifetal pregnancies. This includes
the accurate assignment of chorionicity, prenatal screening and
optimal antenatal surveillance aimed at reducing the associated
perinatal disease burden, which extends to include the availability
of experts in fetal intervention techniques.
Perinatal Disease Burden
The risks for stillbirth and perinatal mortality in twins and peri-
natal death in triplets are approximately five, seven and nine
times those of singleton pregnancies, respectively.5,6 This is largely
attributable to the increased rate of spontaneous and iatrogenic
preterm delivery, in which multifetal gestations are 13 times more
likely to deliver less than 32 weeks’ gestation compared with sin-
gleton pregnancies.4 The increased rate of preterm delivery confers
a risk for developing cerebral palsy to be four times that of single-
tons.5,6 Withstanding the short and long-term complications of
prematurity, multiple pregnancy is associated with increased risk
for congenital anomalies, disorders of fetal growth and twin–twin
transfusion syndrome (TTTS), in addition to the increased mater-
nal complications of preeclampsia, gestational diabetes, antepar-
tum haemorrhage and the requirement for caesarean delivery.7
By applying a strategy of close antenatal surveillance and deliv-
ery at 36 to 37 weeks’ gestation for uncomplicated monochorionic
(MC) twins, extending this to 38 weeks’ gestation for dichorionic
(DC) twins, it has been suggested that perinatal morbidity can be
minimised significantly, albeit with a residual risk for 1.5% of late
IUFD in MC twins.8 The goal of antenatal surveillance and opti-
mum timing of delivery in multiple pregnancies is thus aimed at
reducing the risk for in utero demise, balanced against minimising
perinatal morbidity from prematurity. 
Chorionicity
Given that outcome in twins in largely driven by chorionicity, early
determination of chorionicity, with emphasis on identifying the
less common MC pairs, is critically important in minimising the
perinatal disease burden. Assignment of chorionicity in the first
trimester and before 14 weeks’ gestation approaches a sensitivity

and specificity of 100% and 99%, respectively.9 A DC twin preg-
nancy is determined by the presence of two placental masses and
the characteristic ‘lambda’ sign, in which the two thick chorionic
plates join. An MC twin pregnancy is determined by the presence
of a single placental mass and a thin wispy membrane, with the
‘T’ sign, which is created by a lack of intervening chorion.
After the second trimester, the lambda sign can disappear in
up to 7% of DC pregnancies because of regression of the cho-
rion frondosum10; hence, determination of chorionicity is more
challenging with advancing gestation. Discordant fetal gender
or a thick intertwin membrane may assist in the late identifica-
tion of DC pregnancies. In cases of concordant fetal gender
and delayed assignment of chorionicity, pregnancies should be
described as ‘undetermined chorionicity’, and monochorionic-
ity should be assumed unless proven otherwise.11 Ultimately,
definitive examination of the placenta after delivery should be
undertaken.  13 with a false-positive rate of 0.09% (1 of 1168). Although the
Aneuploidy Screening
Multiple pregnancies should be offered aneuploidy screening, and
the methods used are similar to those adopted in singletons.12,13
Noninvasive prenatal testing (NIPT) is increasingly being offered
as a screening option for multiple pregnancies, although larger
prospective studies are required to determine robust detection
rates for the common trisomies.
Risk for Aneuploidy Based on Zygosity
Monozygous (MZ) twins are almost always genetically identical;
thus, the age-related risk for aneuploidy for both twins will be the
same as for a singleton. On the other hand, for dizygous (DZ)
twins, these tend to be genetically distinct and should be consid-
ered as two separate fetuses when calculating the age-related risk for
aneuploidy. For example, for a DZ twin pair, the risk for at least one
twin being affected is calculated by adding the individual risk for
each fetus together (i.e. 1/100 + 1/100 = 1/50), and the risk for both
fetuses being affected is achieved by multiplying the risks together
(i.e. 1/100 × 1/100 = 1/10,000).14,15  fetus and is more likely to return a false-negative result. Although
Combined Nuchal Translucency and Serum
Screening
Serum screening alone is unreliable in multiple gestations and is
not recommended. Currently, the recommended test of choice for
aneuploidy screening in DC twin pregnancies is the first trimester
combined test.13,16 It offers an improved false-positive rate over
assessments based on nuchal translucency (NT) and maternal age.
Overall, for a 5% false-positive rate, NT alone, the first trimester
combined test and integrated tests will yield 69%, 72% and 80%
detection rates, respectively.16,17
For DC twins, a fetus-specific risk is calculated by combin-
ing the risk for each fetus on the basis of individual NT mea-
surements which is then multiplied by the likelihood ratio (LR)
derived from the serum markers. For MC twins, the NT mea-
surements are averaged and then multiplied by the LR of the
serum markers, and a ‘pregnancy-specific’ risk is obtained in this
way. A specific correction factor for pregnancy-associated pla-
cental protein A (PAPP-A) measurements (2.192 for DC twins
and 1.788 for MC twins) but not β-human chorionic gonado-
trophin (β-hCG) (which is divided by the observed corrected
multiples of the median [MoM] (2.03)) offers a more accurate
CHAPTER 44  Multiple Pregnancy 533
individual patient-specific risk.17 The limitations for the use of
the first trimester combined test in multiple pregnancies include
the fact that β-hCG and PAPP-A levels are affected by ART and
may be confounded by the presence of an unaffected fetus.18 
Noninvasive Prenatal Testing Performance in
Twin Pregnancies
In low and high-risk singleton pregnancies, NIPT is an effective
screening strategy for trisomies 21, 18 and 13.19 A limited number
of studies to date have reported promising detection rates for tri-
somy 21 in twins, with results that appear to be similar to that of
singleton pregnancies20-27 (Table 44.1). Cumulative data to date
from these studies, comprising a total of 1207 twin pregnancies
with complete outcome data, indicate a detection rate of 100%
(35 of 35) for trisomy 21, 63% (5 of 8) for trisomy 18 or trisomy
number of affected twin pregnancies is too small to draw definite
conclusions regarding overall screening performance for NIPT,
particularly for trisomies 18 and 13, results are promising for the
detection of trisomy 21.
The available studies of NIPT in twin pregnancies, how-
ever, are limited by their retrospective design,20,21,23,24 incom-
plete pregnancy follow-up data in some prospective studies23-25
and reporting of the mean rather than the lower fetal fraction
percentage.20-23,27
The performance of NIPT relies on a minimum fetal fraction
of 4%. For MZ pregnancies, the fetal fraction overall should be
similar to that of a singleton pregnancy, and in theory, a fetal frac-
tion of 4% could be anticipated to generate a test result with a
reliable aneuploidy detection rate. The issue is more complex for
DZ twins because each twin can contribute a different amount of
cell-free DNA (cfDNA), the difference being sometimes as much
as twofold.28 The real difficulty arises in the case of a DZ twin pair
discordant for aneuploidy and when the affected trisomic fetus
contributes independently less than 4% of cfDNA. In this case,
the higher fetal fraction from the disomic normal twin is likely
to mask or dilute any effect of the fetal fraction of the trisomic
it could be considered reasonable to use a threshold of 8%, it
has been proposed that the lower fetal fraction of the two fetuses
rather than the total or mean fraction is used in NIPT aneuploidy
risk assessment in twins.23,28 However as a result, a higher fail-
ure rate of cfDNA testing in twins is inevitable, and studies have
confirmed higher first and second sample failures in twins versus
singletons (twins, 5.6%–9.4% (first sample failure), 49%–50%
(second sample failure) compared with singletons: 1.7%–2.9%
(first sample failure) and 32%–37% (second sample failure)).25,26
Further consideration should be given to the association of
vanishing twin pregnancies and false-positive NIPT results.
Fetal DNA from a vanishing twin may be detectable for up to 8
weeks after demise.29 Using chromosome-counting techniques,
the presence of a vanishing twin has been reported to account
for 15% of false-positive results in one study,30 and in a further
study, one false-positive case of three cases of trisomy 21 was
due to a vanishing twin.31 The possibility of a vanishing twin
should thus be considered in the setting of test-positive result in
an ART pregnancy, particularly if there is a possibility of spon-
taneous fetal reduction in the setting of a two-embryo transfer
with triplets.
Within the studies cited, there have been a total of eight cases of
triplet pregnancies that underwent NIPT, which were all correctly
534 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE
44.1 Performance of Noninvasive Prenatal Testing in Twin Pregnancies

Gestational % FF (overall/test False- Resample/

Sample age at positive cases Detection rate positive rate sample
Study size testing (wk) when reported) (trisomy 21) (trisomy 21) failure Comments (detection rate)
Canick 25 15 (10–19) 20.2%, 19.6% 100% 0% Not reported 7/7 cases trisomy 21 (2/7
et al.20 concordant, 5/7 discor-
(2012) dant), 1/1 case trisomy 13
(FF, 7%)
Lau et al.21 12 100% 0% 1/1 trisomy 21 (discordant)
(2013)
Huang 189 19 (11–36) Not reported 100% 0% Not reported 9/9 cases trisomy 21 (9 dis-
et al.22 cordant), 1/2 cases T18a (2
(2013) discordant)
Grömminger (a) 16 15 + 4 (10 + 24% 100% 0% 0% 4/4 trisomy 21 (4 Dis)
et al.23 6 – 18 + 4)
(2014)b
(b) 40 14 + 2(9–23) 18%, 24.8% Undetermined at 0% 12.5% (5/40) 2/2 trisomy 21 (1 discordant,
time of print 1 concordant)
Del Mar Gil (a) 207 10–13 10.8% (trisomy T21 c 0% 7.2% 9/10 trisomy 21 (8 discor-
et al.24 test positive), dant, 2 concordant)
(2014)c 7% (trisomy 13 0/1 trisomy 18 (discordant),
test positive) 1/3 trisomy 13 (discordant)
(b) 68 10.6 7.4% Undetermined at 0% 13.2% 2/2 trisomy 21 (discordant),
(10–13.9) time of print 1/1 trisomy 18 (discordant)
Bevilacqua 515 13 (10–28) 8.7% Undetermined at 0% 5.6% 11/12 trisomy 21 (12 dis-
et al.25 time of print cordant), 5/5 trisomy 18 (5
(2015) discordant)
Sarno et al.26 438 11.7 (10.4– 8.0% 100% 0.25% 9.4% 8/8 trisomy 21, 3/4 trisomy
(2016 12.9) 18, 0/1 trisomy 13 (all
discordant)
Tan et al.27 565 11–28 8.9% 100% 0% 3.2% 4/4 trisomy 21 (discordant)
(2016)d

aOne false-negative case of discordant trisomy 18 in a monochorionic pregnancy.

bGrömminger et al.23: (a) retrospective study and (b) prospective study including two cases of triplets (low-risk result, all euploid); fetal DNA fraction not available in additional four cases.
cDelMar Gil et al.24: (a) retrospective study of the 10 trisomy 21 cases: 8/10: >99% risk score, 1/10 (72%), 1/10 (1:714), in 1 case of trisomy 18 and 2 cases of trisomy 13 a ‘no result’ was returned. 1
case of trisomy 18 risk score 59%; outcome not available on all low-risk pregnancies.
dTan et al.27: assisted reproductive techniques pregnancies.
FF, Fetal fraction.
  
identified as euploid.20,23,27 Further larger studies of NIPT perfor- • The lower level of fetal fraction should be used as the cutoff
mance in twins and higher order multiples are required.  when analysing results because this yields lower false-negative
results; however, higher test failure rates are to be expected
Considerations for Aneuploidy Screening in Twin when adopting this approach.
• Test failure rates are higher overall in twin compared with sin-
Pregnancies gleton pregnancies and are increased further in the setting of
• Conventional methods for aneuploidy screening in multiple ART or with a high maternal body mass index and may be due
gestations are available but underperform those in singletons. to the relatively smaller placental mass.
• ART is associated with higher false-positive screening results with • When a first sample fails to return a result, patients should
conventional methods (owing to lower PAPP-A, alpha-fetoprotein be counselled that a second sample may yield a result only in
(AFP), and unconjugated estriol (uE3) and higher β-hCG). approximately 50% of twin cases.
• There is preliminary evidence of a high detection rate and low • In the setting of repeated failed test results, conventional
false-positive rate with NIPT for trisomy 21 in twin pregnan- screening methods or invasive testing may be selected instead
cies. to avoid further delays in options for pregnancy management
• There are currently insufficient data on detection rates with into the second trimester.
NIPT for trisomies 18 and 13 in twin pregnancies to make • If there is a high suspicion of a vanishing twin, one approach
recommendations on performance. may be to delay NIPT for approximately 8 weeks after which

it is less likely that a false-positive result will be returned. Alter-
natively, patients may wish to opt for conventional screening
methods.  sis
Malformations in Multiple Pregnancies and
Invasive Prenatal Testing
Genetic Abnormalities
When aneuploidy in DC twins is suspected, it usually manifests as
one twin being aneuploid and the other euploid. In contrast, for
MC twins, aneuploidy, when present, will generally be concordant.
However, discordance of nearly all the common trisomies in MC
twins, although rare, have been described, and most cases involve
sex chromosomal abnormalities.32-35 Some very rare cases of MC
twins discordant for Di George syndrome (22q11.2 deletion syn-
drome) and trisomy 14 have also been described.36,37 Known as
‘heterokaryotypic monozygotism’, this rare phenomenon occurs
when either a normal or trisomic zygote undergoes either prezygotic
meiotic errors or postzygotic mitotic events. A recent case report
published on MC 46XX/46XY mosaicism detailed how an initial
47XXY zygote underwent postmitotic loss of the X chromosome
in some cells, and the Y chromosome in other cells such that twins,
although MZ, with discordant gender ensued.38
We recently encountered a rare case of MZ twins discordant for
trisomy 21 who both had evidence of the rare associated transient
abnormal myelopoiesis (TAM).39 TAM is a transient leukaemia
associated with trisomy 21 and mosaic trisomy 21. In this case,
the twin who was later confirmed to be mosaic trisomy 21 had
an intrauterine fetal death, but the co-twin survived without any
evidence of trisomy, even mosaicism, and the TAM resolved spon-
taneously in the neonatal period. One other case report detailed
TAM in both twins in the setting of concordance for trisomy 21.40
It is plausible the TAM resulted from vascular sharing between
the twins.  on the type of anomaly, whether it is concordant or discordant
Structural Malformations
Congenital structural malformations are up to two times more
common in twins as compared with singletons, contributing sig-
nificantly to the overall increased perinatal mortality rate in twins.
According to data from the British Colombia Health Surveillance
Registry, the incidence of congenital anomalies in twins is approx-
imately 6% and is two to five times higher in MZ compared with
DZ twins.41 When a structural anomaly is identified in a twin
pair, it is almost always discordant. Concordant structural malfor-
mations are rare in DC twins but occur in 18% to 23% of MC
twins.42,43
The types of abnormalities are broadly divided into two
groups: those involving midline or laterality defects and anoma-
lies caused by haemodynamic imbalance.44 It is worth mention-
ing that the incidence of open neural tube defects in twins is
controversial, with some studies citing increased rates of anen-
cephaly and encephalocele but not meningomyelocele45,46 and
with others quoting reduced rates of anencephaly compared
with singeltons.47
Structural malformations in monochorionic twin pregnancy.
1. Neural tube defects and facial clefts, holoprosencephaly, and
cardiac and anterior abdominal wall defects
2. Encephalomalacic brain lesions, pulmonary stenosis, renal
agenesis, limb reduction defects, aplasia cutis and intestinal
atresia
CHAPTER 44  Multiple Pregnancy 535
3. Cardiac defects, including ventricular septal defect (VSD),
lesions specific to TTTS: atrial septal defect, pulmonary steno-
Congenital heart disease (CHD) is more prevalent in MC
twins. There is some evidence to suggest CHD is more common
in twins conceived through ART.48 A retrospective series of 381
MC twins reported a prevalence of CHD of 5%, which increased
to 9% in the presence of TTTS.49 The most common anomalies
identified were VSDs (2.1%) and anomalies of the outflow tracts
(1.3%) in the general twin population followed by VSD (2.9%)
and anomalies of the great arteries (2.9%) in the TTTS group. In
contrast, DC twins may display more classic CHD such as hypo-
plastic heart syndrome and atrioventricular septal defects. 
Sonographic Evaluation in Twins
The sonographic evaluation of twins and higher order multiples is
often challenging with contemporary experience suggesting that
structural surveys are often not completed on the first attempt,
requiring further reevaluation and often at later suboptimal ges-
tational ages. Robust prospective data regarding detection rates
of CHD and other forms of structural malformations in twins
and particularly MC twins is lacking. Available data suggest that
approximately one third to half of major anomalies are detected
prenatally in singletons, with lower detection rates in twins.50 In
one series of 33 twins with anomalies, none of the 8 cardiac anom-
alies and none of the 12 minor anomalies was detected prenatally,
but 55% of other major anomalies were detected.51 
Management of Twins With Malformations
The options for management of genetic and structural anomalies
in twin pregnancies include expectant management, selective feti-
cide or termination of the entire pregnancy. Management depends
and the chorionicity of the pregnancy. We recommend karyotyp-
ing for twins with malformations; this is discussed further later in
this chapter.
Pregnancy management is generally straightforward in an MC
pregnancy concordant for an anomaly, but in the setting of MC
twins discordant for an anomaly, the subsequent management of
the pregnancy can be complex. Even if expectant management
is the preferred approach, there is still a risk to the pregnancy as
a whole, with preterm birth rates significantly increased (20%)
in addition to the background increased risk in multiple preg-
nancy.52 Caesarean delivery at earlier gestational ages and lower
birth weights are also more likely with the expectant management
of one or both twins with an anomaly.53,54 Preterm delivery may
be attributed to polyhydramnios associated with major anomalies
such as anencephaly or trisomies.54,55
When MC twins are discordant for anomalies, patients should
be made aware of the risk for possible demise of the anomalous
twin and subsequent risk for death or neurologic injury in the
surviving twin.56 This is discussed further in the section on single
twin death. 
Invasive Prenatal Diagnosis
Invasive testing presents a number of distinct challenges in twins.
Chorionic villous sampling (CVS) or amniocentesis should be
performed by an expert fetal medicine specialist. There is currently
a lack of data from prospective trials on the safety of invasive
536 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
testing in twins, and in the absence of such trials, it is not fea-
sible to accurately estimate the excess risk after invasive prena-
tal diagnostic procedures in twins. Current available data report
similar pregnancy-loss rates for both CVS and amniocentesis with
an excess risk for about 1% above the background risk.57 Rhesus
(Rh)-negative women should receive prophylaxis after invasive
procedures to prevent sensitisation.
Chorionic villous sampling. Chorionic villus sampling in mul-
tiple pregnancies performed between 10 and 14 weeks’ gestation
offers the advantage over amniocentesis in that it can be performed
at an earlier gestational age, affording earlier and safer options for
subsequent pregnancy management, including termination.
Procedure. Before the procedure, an ultrasound is always
performed to determine the chorionicity, number and position
of the embryos, viability and the presence of anomalies. The
ideal scenario is to perform both a transcervical and transab-
dominal approach if there are two separate placentas to reduce
the risk for contamination of the samples or of sampling the
same placenta twice. When the chorionicity is unclear, aspira-
tion or biopsy should be directed to the extreme ends of each
placenta or to the area nearest the respective umbilical cord
insertion sites.
Over the years, there has been controversy over the rate
of procedure-related loss in multiple pregnancies after CVS.
In a systematic review (2012) of 9 studies pregnancy-loss
rates after CVS were reported as overall pregnancy loss of
3.84% (95% confidence interval (CI), 2.48–5.47; n = 4),
pregnancy-loss rate before 20 weeks’ gestation of 2.75% (95%
CI, 1.28–4.75; n = 3) and pregnancy-loss rate before 28 weeks’
gestation of 3.44% (95% CI, 1.67–5.81; n = 3).57 No signifi-
cant differences were noted in the rate of pregnancy loss accord-
ing to the method of CVS, transabdominal or transcervical.
Cross-contamination or sampling error has been reported to be
between 0.45% and 3.17%.57 Thus, because of potential prob-
lems with contamination, some investigators suggest restrict-
ing CVS to high-risk cases such as monogenic diseases or an
aneuploidy risk for more than 1 in 50.  As in singletons, growth restriction in twins can be a conse-
Amniocentesis. Before the procedure, an ultrasound examina-
tion is performed to determine the number of fetuses, the chori-
onicity, fetal position, fetal viability, identification of the fetus(s)
when an anomaly is present, placental location and umbilical cord
location, and results are carefully mapped out with samples being
labelled according to the documented identifier information of
each twin. Under ultrasound guidance, the usual scenario is that
both sacs are sampled with separate needles; however, a single-
needle technique can also be used. For the single-needle tech-
nique, the proximal sac is sampled first, the stylet is replaced and
the needle is advanced into the second sac. The first 1 mL from
the second sac should be discarded to avoid contamination. The
advantage over the double-entry technique is that of fewer needle
insertions, but tenting of the amniotic membrane may make it
technically difficult to enter the second sac under direct visuali-
sation, and a theoretical monoamniotic (MA) pregnancy can be
created as a result. We recommend a two-needle entry technique,
in which after the first needle is inserted in the first sac and a
sample is obtained, the needle is held in place, and indigo carmine
dye is injected into the same sac before the needle is withdrawn.
The second sac is then carefully sampled, and clear amniotic fluid
should be aspirated upon entry. Methylene blue dye is no longer
recommended because it is associated with fetal risks such as fetal
demise, intestinal atresia, methaemoglobinaemia in the infant and
staining of the fetal skin.58
A recent systematic review of procedure-related loss in twins
after amniocentesis indicated the overall pregnancy loss rate
was 3.07% (95% CI, 1.83–4.61; n = 4), with pooled data from
four case-control studies showing a higher rate of pregnancy loss
(2.59% vs 1.53%) at less than 24 weeks’ gestation.57 Losses before
28 weeks’ gestation could not be accurately determined because
of the heterogeneity of the published data. The available data are
limited by a lack of prospective trials and a lack of homogene-
ity regarding definitions of procedure-related loss, and few stud-
ies included a control group or reported on loss rates based on
chorionicity. As a result, the exact loss rate after amniocentesis in
multiple pregnancies remains undetermined but is likely in excess
of 1% above the background risk. 
Complications Common to Both
Monochorionic and Dichorionic Pregnancies
Disorders of Fetal Growth in Multifetal
Gestations
Optimum fetal growth in multiple pregnancies is dependent on
adequate vascular function in a single shared placenta of a MC
pregnancy or two independent placentas in a DC pregnancy.
Twin growth velocities have been shown to taper after 32 weeks’
gestation, and given that twin estimated fetal weights (EFWs)
are often plotted using singleton growth curves, it is likely that
there is a general overestimation of intrauterine growth restric-
tion (IUGR) in twins and higher order multiples later in gesta-
tion.59 However, twin-specific growth charts are not currently
incorporated into clinical practice, and standard fetal assess-
ments of IUGR including assessment of placental function
(Doppler indices and amniotic fluid volume) are critically used
to assess fetal status in the setting of IUGR in twins rather than
assessments of EFW alone.
quence of placental dysfunction, single-gene disorders, poor
implantation site, chromosomal abnormalities, velamentous cord
insertion and single umbilical artery.60 In MC twins, the concept
of unequal placental sharing is well documented and can result
in selective IUGR (sIUGR) in one twin.61 For DC twins, it has
been suggested that alterations in implantation sites for indi-
vidual blastocysts may predispose to discordant uteroplacental
insufficiency.62
Definition of discordant growth. Discordance in fetal growth
can affect approximately 20% of twin pregnancies overall and
approximately one third of all triplet pregnancies.63,64 In MC
twins, unequal placental sharing, with severe growth discor-
dance of 25% or greater complicates approximately 20% of all
MC twins compared with only 7.6% of DC twins.59 For DC
twins, it could be anticipated that a degree of discordance in fetal
growth is likely on the basis of differing genetic potentials, pla-
cental architectures and implantation sites. Although a thresh-
old of 10% has been suggested to represent an accepted normal
physiological difference in growth potential,64 there has been a
lack of agreement as to what constitutes pathological discordant
growth, with varying definitions of a threshold ‘cutoff ’ discor-
dance (10%–30%) described.63-66 The inclusion in some studies
of major congenital fetal anomalies and cases complicated by
TTTS, in addition to a lack of clarification regarding analysis by
chorionicity, have precluded the ability to define the exact cutoff

of significant birth weight discordance associated with increased
perinatal morbidity and mortality.63
The prospective Evaluation of Sonographic Predictors of
Restricted Growth in Twins (ESPRiT) study conducted in Ireland
with complete perinatal outcome on 1001 twin pregnancies estab-
lished that the threshold for significant birth weight discordance
(i.e., that which is associated with an increase in composite perinatal
morbidity) is 18% for both DC and MC twins in the absence of
TTTS.63 Overall, the absolute risk for adverse perinatal outcome
was found to be higher in MC versus DC twins at every level of
discordance. Further studies did not find any increased risk for
neonatal morbidity or mortality in groups of discordant but appro-
priate-for-gestational-age (AGA) twins.67,68 To address this, further
analyses within the ESPRiT study determined that after adjust-
ing for gestational age at delivery, within a subgroup of 819 twins
deemed AGA, the threshold of 18% was maintained as a significant
predictor of adverse outcome. In addition, a subgroup of twins with
sIUGR was identified (in which IUGR was defined as less than the
5th centile: 11% (108 of 977), and a threshold of 18% for a dif-
ference between the IUGR and AGA twins conferred a fourfold
increased risk for adverse perinatal outcome compared with concor-
dant twins.63 Thus, although morbidity is increased in the setting of
discordance when one twin has IUGR, the risk for adverse perinatal
outcome is maintained even in the discordant AGA group when a
threshold of 18% for intertwin growth discordance is applied.
On this basis, we recommend a threshold of 18% for signifi-
cant intertwin discordance is applied to all types of twins regard-
less of chorionicity and whether or not IUGR is a feature:
1. Discordance of 18% or greater calculated as the difference in
weights between the larger and smaller of the fetuses divided by
the weight of the larger fetus:
EFW larger twin − EFW smaller twin
× 100 %
EFW larger twin
This can occur in the following scenarios:
a.  Both twins AGA but discordant in weight of 18% or
greater.
b. Both twins IUGR and discordant in weight of 18% or
greater; however, in this scenario, usually both twins are
concordant in weight.
2. sIUGR in which one twin is AGA and one twin has IUGR
when the EFW is less than the 10th centile (<5th centile has
been defined as sIUGR in some studies16)  with intermittent AREDF may be associated with an unpredict-
Ultrasound surveillance and screening for discordant
growth in twins
Prediction. The increased risk for adverse perinatal outcomes
associated with intertwin growth discordance justifies a height-
ened fetal surveillance strategy in the prenatal care of twins aimed
at the early identification of growth discordance.  tion. Severely growth discordant MC twins (≥25%) are more likely
First Trimester. Discordant fetal crown–rump lengths (CRLs)
at 11 to 14 weeks’ gestation has been associated with, but not
strongly predictive of, an increased risk for birth weight discor-
dance.69-72 Discordant assessments of CRLs in MC twin pregnan-
cies may also signal the early onset of TTTS.  Although an intertwin growth discordance of at least 18%
Second and third trimesters. Later second and third trimester
evaluation of EFW has been reported to have higher sensitivity
but lower positive predictive value (PPV) (93% and 72%, respec-
tively) when compared with intrapair abdominal circumference
(AC) difference of 20 mm (83% sensitivity and PPV) for the
CHAPTER 44  Multiple Pregnancy 537
detection of twin growth discordancy.73 In the large prospective
ESPRiT study, differences in the AC of 10% or more between 14
to 22 weeks’ gestation was found to be the most useful predictor of
composite adverse perinatal outcome, preterm delivery and birth
weight discordance greater than 18%, with the strongest correla-
tion when differences were identified between 18 and 22 weeks’
gestation.74 EFW using two or more parameters rather than AC
alone is still recommended as a screening tool for discordance in
twins. 
Doppler surveillance. Multivessel Doppler assessments follow
the same recommendations in twins as in singletons. However,
the relative contribution that Doppler assessments can make
in the identification of twin growth discordance is undetermined.
Abnormal Doppler waveforms may follow a different pattern of
progression in MC twins compared with that of DC twins, and
this is likely a factor of the intertwin vasculature in addition
to placental insufficiency. Latency of absent end diastolic flow
(AEDF) (defined as the difference between gestational age at
diagnosis of AEDF and gestational age at delivery or intrauterine
death) has been shown to be longer (54 days) in non-TTTS MC
twins compared with DC twins (30 days; P = .04) and singletons
(11 days, P = .0001).75 This may be a factor of the earlier gesta-
tion of presentation of AEDF in MC fetuses compared with both
singleton and DC fetuses and the presence of placental anasto-
moses, particularly arterio-arterial anastomoses (AAA) that likely
maintain growth, although on a lower centile.76
In MC twins, a pattern of cyclical or intermittent absent or
reversed end-diastolic flow (AREDF) has been demonstrated in
20% of growth-restricted twins.77-79 This pattern is felt to occur
because of retrograde transmission of cyclical pressure changes
from a large AA anastomosis to the umbilical artery (UA) wave-
form in the smaller MC twin, in which it manifests as fluctuating
end-diastolic flow (EDF).
A classification system for Doppler patterns in cases of sIUGR
in MC twins was proposed by Gratacos et al in 2008, and this
system has since been applied to a number of studies in which fetal
intervention in sIUGR has been undertaken.80 The Gratacos clas-
sification system for Doppler changes in the smaller twin of a MC
pair with sIUGR includes type I, positive EDF; type II, persistent
AREDF; and type III, intermittent or cyclical AREDF. Research
groups that have applied the Gratacos system to their series of
twins with sIUGR have reported varying rates of unexpected fetal
demise within groups II and III.80,81 The surprisingly high rate of
unexpected demise in one study within group III suggest sIUGR
able natural history, and such cases warrant close surveillance and
timely delivery.81 It may be that differing patterns of Doppler
waveforms exist in the setting of generalised discordance in fetal
weight, as opposed to when one twin is IUGR, and further larger
studies are required. 
Perinatal outcome with selective intrauterine growth restric-
to be delivered before 30 weeks’ gestation and have a longer neona-
tal intensive care stay (>10 days) than their DC counterparts.67,69,70
Overall, the reported incidence of IUFD in the growth-restricted
twin is reported to be between 14% and 40%.80-82
adversely affects perinatal outcome at any gestational age, lon-
ger term neurodevelopmental outcome appears to be largely
driven by gestational age at delivery. Within the ESPRiT study,
119 twins (including 24 MC pairs) with a birth weight discor-
dance of 20% or greater have been analysed to date at 24 and
538 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
42 months of age.83 Compared with the larger twin, the smaller
twin of a discordant pair significantly underperformed in assess-
ments of cognition language and motor skills (mean composite
cognitive score difference, −1.7; 95% CI, 0.3–3.1; P = .01) and
language and motor skills. However, before 33 weeks’ gestation,
gestational age at birth had a far greater impact on cognitive out-
comes than degree of birth weight discordance (mean composite
cognitive score difference, −5.8; 95% CI, 1.2–10.5; P = .008).
Neuromorbidity (determined radiologically up to 28 days of
life) has also been identified in the larger twin of a discordant
pair; the incidence of neurologic damage was reported to be as
high as 37% in the AGA-co-twin of a pair when the smaller one
had evidence of intermittent AREDF and when the risk per-
sisted even if both survived.84 As with cases of larger co-twin
demise, neuromorbidity is also thought to be attributed to ante-
natal ischemic events driven by large AAA.  presents with stigmata of imminent demise.
Management of discordant growth. The frequency of surveil-
lance for uncomplicated MC twins should include sonographic
assessments including biometry, documentation of the deepest
vertical pocket (DVP) of amniotic fluid, visibility and appearance
of the fetal bladders, and UA Doppler assessment at least every 2
weeks from 16 weeks’ gestation.13 For complicated MC pregnan-
cies, surveillance should extend to include umbilical artery and
middle cerebral artery PI and peak systolic velocities, in addition
to ductus venosus (DV) waveforms in the setting of an abnormal
UA Doppler waveform.13 The basis of this scheduling of visits is
to screen for sonographic stigmata of TTTS in MC twins and fetal
growth discordance in all twins.
For uncomplicated DC twins, surveillance has been recom-
mended at every 3 to 4 weeks from the time of the anatomical
sonogram at 18 to 22 weeks’ gestation.12,13,85 More frequent
sonography for uncomplicated DC twins has also been suggested.
Data from the prospective ESPRiT study supports this concept:
of 789 DC twin pregnancies, the detection of fetal growth restric-
tion and abnormal UA Doppler was increased from 69% to 88%
and 62% to 82%, respectively, with a 2-weekly rather than a
4-weekly ultrasonography schedule for DC twins, suggesting that
sonographic surveillance every 4 weeks in DC twins is under-
performing sonography every 2 weeks.86
Dichorionic twins. The identification of discordant fetal
growth of 18% or greater in DC twins should prompt a more
intensive fetal surveillance strategy, including a referral to a fetal
medicine specialist, to include two weekly fetal growth assess-
ments and more frequent measures of placental assessment when
absent or reversed EDF is featured as per singletons. However, the
absence of interdependent placental vasculature confers a lesser
threat to the pregnancy overall and provides a theoretical protec-
tion against neurodisability of the surviving twin in the case of
death of one twin. Decisions to delivery may therefore rest on
the gestational age at delivery where a viable weight of the smaller
twin has been achieved or the presence of superimposed maternal
complications such as preeclampsia. 
Monochorionic twins. The risk for IUFD is increased in cases
of expectantly managed IUGR in MC twins. Because of the organ-
isation of the shared placental vasculature of MC twins, the conse-
quence of the sequelae of co-twin demise or neurologic disability
in the surviving larger twin must therefore be considered. Applying
the Gratacos classification, cases of type I sIUGR (positive EDF)
can generally be managed expectantly with close surveillance to
determine timing of delivery, which has been recommended by
34 to 36 weeks’ gestation.13,80,81 In the original Gratacos series,
cases with type I sIUGR achieved a mean gestational age at
delivery of 35.5 weeks’ gestation (range, 30–38 weeks). For cases
of sIUGR with persistent AREDF in the UA (type II) or inter-
mittent AREDF (type III), iatrogenic preterm delivery is usually
indicated by 32 weeks’ gestation or sooner if there is evidence of
static growth or abnormal Doppler assessment.13,81,82
Decisions to deliver may include an iatrogenic preterm delivery
when the likelihood of IUFD of the smaller twin is considerable.
It is increasingly acknowledged that selective fetal growth restric-
tion confers an increased risk to the normally grown fetus even
if both fetuses are born alive. Management approaches must be
balanced against the risks of prematurity for the larger twin, tak-
ing into consideration also that even attaining an optimum gesta-
tional age at delivery or dual survival does not confer an absolute
certainty of neurologic protection for either fetus. Cases are fur-
ther complicated when the IUGR twin is a previable weight and
 
Intervention for selective intrauterine growth restriction.
Selective reduction may be an option to maximise the intact sur-
vival of the larger twin if demise of the smaller twin is anticipated.
Survival rates of 80% to 90% can be expected in the larger twin
after cord occlusion of the smaller twin.87,88 Alternatively, selective
fetoscopic laser ablation with the aim of salvaging the larger twin
and sometimes both is a potential option. A number of centres
have now described initial outcomes after fetal intervention using
selective fetoscopic laser ablative techniques with reports of sur-
vival rates for the larger twin between 68% and 100% but also
with survival rates for the smaller twin between 25% and 39%
(Table 44.2).89-93 The limitation with the use of this technique for
sIUGR in the absence of TTTS is that the lack of polyhydramnios
in the larger twin may render this option technically difficult, and
inadvertent prelabour premature rupture of membranes is a possi-
ble adverse outcome. Additional technicalities to consider include
the setting of an anterior placenta with large AA vessels or close
proximity of cord insertion sites.93 Although there is a lack of data
from large prospective series, both options of cord occlusion and
selective fetoscopic laser ablation are feasible. It is likely going to
continue to be challenging to establish criteria for fetal therapy in
the case of sIUGR in MC pregnancies given this largely remains
a contentious issue amongst experts. Counselling should focus on
the wishes of the parents, technical concerns of individual cases
and local expertise. 
Single Intrauterine Fetal Death
Fetuses of multiple gestations have a greater chance of dying in
utero than their singleton counterparts, and the risk for pregnancy
loss increases with increasing fetal number. Although difficult to
quantify, the overall incidence of single intrauterine fetal death
(sIUFD) in twins is reported to be 2% to 7%.94 Outcome is deter-
mined by chorionicity, with MC twins at greatest risk, and gesta-
tional age at timing of sIUFD.
Gestational age at selective intrauterine growth restriction
First trimester. A considerable proportion of women will have a
spontaneous demise of one or more fetuses in the first trimester,
referred to as the phenomenon of a ‘vanishing twin’.23,29,95 In MC
twins, death of one fetus in the first trimester will likely result in
dual pregnancy loss, although survivors have been reported.95,96
The co-twin survivor of a first trimester vanishing twin in a DC
twin pregnancy usually has a good prognosis. Overall the risk for
pregnancy loss in the first trimester is estimated to be approxi-
mately 36% for twins, 53% for triplets and 65% for quadruplets.96 
TABLE
44.2 Outcome of Selective Fetoscopic Laser Ablation for Selective Intrauterine Growth Restriction (sIUGR)
Immediate Smaller PPROM <4
Number Study GA at operative twin Larger twin Neurologic wks from GA at Perinatal
Authors Study type recruited group therapy Technique complications survival survival sequelae procedure delivery survival rate
Peeva Prospective 142 Type II 20 (15–27) Laser — 39% 68% — — 32 (24–41) 53%
et al.90 cohort sIUGRa without (55/142) (96/142) (151/284)
(2015) amnioin-
fusion
Ishii et al.91 Prospective 10 Types II and (20–25 + 6) Laser with TPTL (n = 1) 30% (3/10) 100% None 0 32 (26–38) 65% (13/20)
(2015) cohort IIIb sIUGR amnioin- (10/10) reported
fusion at 28
days
Chalouchi Retrospec- 2 Types II and 19.4 Laser with TAPS (n = 1) 30% (7/23) 74% (17/23) 1/7 sIUGR 2 (8.7%) 32.5 (31.6– 52%
et al.92 tive III sIUGR amnioin- 33.9)
(2013) fusion (n
= 23)
Gratacos Retrospec- 18 Type III 22.2 (18– Laser with 2/18 technical 6/18 (33%) 17/18 1/17 (PVL, 2/18 (11%) 32.6 64%
et al.93 tive sIUGR 26.4) amnioin- difficulties (94%)a larger (23–38)
(20080) fusion 1 larger twin twin)
demise
after laser
Quintero Retrospec- 11 sIUGR 20.3 (16.1– Laser with — 5/11 (45%) 7/11 (64%) 0/22 — 29.6 (16.3– 55%
et al.89 tive defined 23.7) amnioin- 35.4)

CHAPTER 44  Multiple Pregnancy

(2001) as EFW fusion
one twin
<10th
centile
aType II sIUGR (persistent absent or reversed absent or reversed end-diastolic flow (AREDF) of the umbilical artery).
bType III sIUGR (intermittent AREDF).
EFW, Estimated fetal weight; GA, gestational age; PPROM, premature rupture of the membranes; PVL, periventricular leukomalacia; TAPS, twin anaemia–polycythaemia sequence; TPTL, threatened preterm labour.
  

539
540 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Monochorionic n/N Dichorionic n/N

Favours Favours
monochorionic dichorionic
pregnancy pregnancy
a
Axt, 1999 0/4 0/3 (Not estimable)
Baghdadi, 2003 1/1 2/8 7.800 (0.051, infinity)
Bajoria, 2003 13/50 1/42 14.405 (1.940, 626.764)
Chelli, 2009 0/9 1/19 0.649 (0.000, 82.333)
a
Fichera, 2009 0/11 0/10 (Not estimable)
Fusi, 1990 1/8 1/8 1.000 (0.011, 89.578)
a
Gaucherand, 1994 0/6 0/2 (Not estimable)
Hagay, 1986 1/1 0/11 69.000 (0.282, infinity)
a
Ishimatsu, 1994 0/12 0/3 (Not estimable)
a
Kilby, 1994 0/7 0/13 (Not estimable)
a
Krayenbuhl, 1998 0/12 0/7 (Not estimable)
Malinowski, 2000 1/4 0/7 6.429 (0.045, infinity)
a
Malinowski, 2003 0/3 0/9 (Not estimable)
a
Petersen, 1999 0/5 0/7 (Not estimable)
Santema, 1995 2/13 0/16 7.174 (0.236, infinity)
Woo, 2000 1/13 0/1 1.800 (0.009, infinity)
Combined (random) 5.243 (1.748, 15.728)

0.001 0.01 0.1 0.2 0.5 1 2 5 10 100 1000

Odds ratio (95% confidence interval)
• Fig. 44.1  Co-twin death after single intrauterine fetal death. Cochrane Q = 0.6, I2 0% (odds ratio [OR]
4.81; 95% CI, 1.39–16.6; p <0.05). (Reproduced from Shek NW, Hillman SC, Kilby MD. Single-twin
demise: pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 28(2):249–263, 2014.)

Second trimester. In the second and third trimesters, the inci-
dence of sIUFD is reported to be between 0.5% and 2% in DC
twins, 26% in MC twins and up to 17% of triplets.12,94 The risk for
adverse outcome in the surviving twin is higher in MC com-
pared with DC pregnancies owing to the organisation of the
placental and intertwin vasculature in MC pregnancies that
can be compromised after sIUFD.97 It is still unclear exactly
which gestational age at which sIUFD in a MC pregnancy con-
fers the greatest risk for adverse sequelae in the surviving twin.
Experts generally agree that the increasing size of the vascular
anastomoses would confer a greater degree of acute circulatory
compromise in the event of the death of one MC twin later in
gestation.96,97 In a recent systematic review and meta-analysis,
MC twins were eight times more likely to have neuromorbidity
than DC twins when sIUFD occurred between 28 and 33 weeks’
gestation.94
In general, for MC twins, when one twin dies, the surviving
fetus is affected by increased risk for (i) demise, (ii) neurodisabil-
ity and multiorgan failure and (iii) increased risk for premature
delivery before 34 weeks’ gestation (iatrogenic and spontaneous).
Although rates of preterm delivery are comparable for MC and
DC pregnancies, after sIUFD, MC pregnancies have approxi-
mately five times higher risk for co-twin death (odds ratio (OR),
5.24; 95% CI, 1.75–15.7; P = .05) (Fig. 44.1).  Patterns of cerebral injury are well described in survivors of
Complications
Multiorgan injury. Multiorgan morbidity in survivors of MC
pregnancies complicated by sIUFD has been described, including
pulmonary or renal failure, hepatic or splenic infarcts, intestinal
atresia, limb reduction and aplasia cutis.98-101 It is widely accepted
that the pathophysiology of multiorgan failure is caused by acute
fetofetal transfusion and the associated ‘back-bleed’ through large
AA anastomoses with a resultant acute hypotension.96,102 There
are no robust data to support older theories of metaplastic mate-
rial from the dead fetus entering the circulation of the surviving
twin, resulting in infarction and multiorgan cystic change.103 
Neuromorbidity and neuroimaging. Although, remarkably,
most survivors do not have evidence of neurodisability, neurologic
injury is by far the most pertinent concern of parents. Cerebral
palsy has been reported in between 6% and 10% of survivors after
a single IUFD compared with survival of both twins.103-104 The
systematic review of Hillman and coworkers reported that rate
of neurodevelopmental impairment for MC compared with DC
twins after single IUFD was 26% compared with 2%, or an OR
of 4.81 (95% CI, 1.39–16.6; P < .05)94 (Fig. 44.2).
Severe cerebral injury has been identified prenatally in 8% to
26% and postnatally in up to 34% of cases of MC twins compli-
cated by sIUFD.105-107 Predictors of severe cerebral injury were
found to be advanced gestational age at single IUFD (OR, 1.14;
95% CI, 1.01–1.29) for each week of gestation; P = .03), TTTS
detected before single IUFD (OR, 5.0; 95% CI, 1.30–19.13);
P =.02) and an earlier gestation at delivery (OR, 0.83; 95% CI,
0.69–0.99 for each gestational week; P = .04).105
sIUFD, including (i) hypoxic ischaemic lesions in the white matter
may lead to multicystic encephalomalacia, porencephaly, micro-
cephaly and hydrencephaly; (ii) haemorrhagic lesions and (iii)
vascular aberrations leading to neural tube defects, limb reduc-
tion anomalies and optic nerve hypoplasia.105-114 When the IUFD
occurs before 28 weeks’ gestation, it is more likely to result in
 CHAPTER 44  Multiple Pregnancy 541

Favours
Favours dichorionic
Definition Monochorionic Dichorionic monochorionic
pregnancy
n/N n/N pregnancy
Minor delay 1/4 0/3
Axt, 1999 3.00 (0.02, infinity)
in MD
Bajoria, 1999 CP/spastic 2/21 0/33
8.59 (0.30, infinity)
Fusi, 1990 Spastic 3/6 0/6
QP/CA 13.00 (0.47, infinity)

Gaucherand, TP/ 1/5 0/2

1.67 (0.01, infinity)
1994 convulsions
Ishimatus, CP/MR 4/9 0/3 5.73 (0.20, infinity)
1994
lin, 1999 2/7 0/3 3.18 (0.07, infinity)
Saito, 1999 CP 2/14 0/10
4.20 (0.13, infinity)

Combined (random) 4.81 (1.39, 16.64)

0.01 0.1 0.2 0.5 1 2 5 10 100

Odds ratio (95% confidence interval)
• Fig. 44.2 Neuromorbidity after single intrauterine fetal death. Cochrane Q = 0.99, I2 0% (odds
ratio [OR] 4.81; 95% CI, 1.39–16.6; p <0.05). CA, Cerebral atrophy; CP, cerebral palsy; MD, motor
development; MR, mental retardation; QP, quadriplegia; TP, tetraplegia. (Reproduced from Shek NW,
Hillman SC, Kilby MD. Single-twin demise: pregnancy outcome. Best Pract Res Clin Obstet Gynaecol
28(2):249–263, 2014.)

multicystic encephalomalacia which affects cerebral white mat-
ter, and is associated with profound neurologic impairment.105
Periventricular leukomalacia and germinal matrix haemorrhage
can also occur in the second trimesters. The grey matter can be
affected when the insult happens nearer term, and in the late third
trimester, subcortical leukomalacia, basal ganglia damage or len-
ticulostriate vasculopathy may develop.108,113
Although some secondary sequelae, including ventriculomeg-
aly, porencephalic cysts, cerebral atrophy or cerebral infarcts, may
be visible on prenatal fetal ultrasound some 1 to 4 weeks after
the event, the initial fetal ultrasonogram often underestimates
the degree of cerebral injury. Diffusion-weighted magnetic reso-
nance imaging (MRI) appears to be the most sensitive modality
for examining recent ischaemic changes, followed by conventional
T2-weighted MR and fetal neurosonography.111,112
Some caution should be given to interpreting findings on imag-
ing because not all lesions translate into clinically relevant morbid-
ity. Nonetheless, MR is a useful adjunct to ultrasonography. Ideally,
a multicentre registry of such select cases in addition to large pro-
spective neuroimaging data linked with long-term school age neu-
rodevelopmental outcome in all twins would permit more accurate
assessment of the actual prevalence and significance of such lesions.  ist. Management should depend on the chorionicity and whether
Co-twin demise. The death of one fetus in an MC twin pair
considerably increases the risk for death in the co-twin either in
utero or the early neonatal period. DC twins are not without risk
for co-twin demise. In one large retrospective cohort study of the
effect of chorionicity on timing of IUFD in twin pregnancies, of
2161 twins, 86 (4%) had at least one IUFD, and 32 (1%) expe-
rienced double demise.115 MC twins carried an increased risk for
single (adjusted OR, 1.69; 95% CI, 1.04–2.75) and double demise
(adjusted OR, 2.11; 95% CI, 1.02–4.37) compared with DC twins,
with 70% of double demises occurring before 24 weeks’ gestation.
Overall second twin demise after IUFD of the first predominantly
occurred before 24 weeks’ gestation, irrespective of chorionicity.
The risk for co-twin demise for this study was found to be lower
than that of the systematic reviews of Ong and coworkers56 and by
Hillman and coworkers.94 In the 2006 review by Ong and cowork-
ers (in which MC twins were reported to have a sixfold higher risk
for co-twin demise compared with DC twins), data on chorionic-
ity and gestational age at sIUFD were incomplete in a number of
cases.56 Although the systematic review and meta-analysis of Hill-
man and coworkers concluded a fivefold increased risk for complete
pregnancy loss in MC twins, this was based on the analysis of 16
publications and limited by only 17 pregnancies complicated by
dual fetal demise, underpinning the rarity of this event.94 
Maternal risks. Reports of the risk for maternal coagulopa-
thy or maternal infection caused by the retention of a deceased
twin remain unsubstantiated. There also does not appear to be
an increased risk for maternal infection in an ongoing pregnancy
and in the presence of a retained deceased twin. There is a dearth
of information regarding the psychological trauma of the loss of
one twin in a multiple pregnancy, which is likely to be underesti-
mated, and probably attributable to the focus on the subsequent
management of the surviving twin.116 
Management. After the diagnosis of sIUFD, subsequent
management should involve referral to a fetal medicine special-
or not the sIUFD occurred before or after viability. If chorionic-
ity has not previously been performed, the pregnancy should be
assigned as MC.
Dichorionic twins. In the absence of maternal or fetal compli-
cations (e.g., IUGR in the surviving twin), DC twin pregnancies
with a history of sIUFD can be managed expectantly and delivery
anticipated at around 37 weeks’ gestation.117 Prophylactic steroids
may be indicated on the basis of gestational age and mode of deliv-
ery in the expectation of higher risk for preterm delivery and cae-
sarean section.12,64,117,118 
Monochorionic twins. The subsequent management of a MC
twin pregnancy complicated by sIUFD involves assessing the
well-being of the survivor and counselling regarding the risk for
542 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
co-twin demise or neurologic morbidity in the survivor to extend
to a later assessment of neurologic injury. The ultimate goal is to
optimise the outcome for the survivor while minimising the risks
due to prematurity. Immediate delivery of the surviving twin after
the death of her or his co-twin less than 34 weeks’ gestation is
unsubstantiated and may aggravate the risks caused by iatrogenic
prematurity.117,118
The survivor should have serial biometry and placental func-
tion testing performed every 2 to 3 weeks in addition to a com-
prehensive anatomical survey. Serial ultrasonographic assessment
of the intracranial anatomy may identify secondary fetal cranial
changes, although MRI should complement ultrasonography in
this setting. Fetal MRI is more sensitive than ultrasonography in
identifying multicystic encephalomalacia, and a fetal MRI is gen-
erally recommended 3 to 4 weeks after the initial insult to iden-
tify cavitating and atrophic lesions that will likely have evolved by
then.13,119 Although an area of practical concern, the optimum
time to schedule an MRI is after 30 weeks’ gestation to assess cor-
tical development.119,120 For patients planning to terminate on
the basis of prenatal imaging and when late termination is not
feasible, an MRI can be scheduled earlier than this. A normal MRI
is likely to provide a level of reassurance than may assist couples in
their decision making, but the limitation of prenatal imaging to
definitely rule out pathology should be acknowledged.
Serial assessment of middle cerebral artery (MCA) Doppler
is conventionally used to assess for anaemia in survivors, and
although a nonspecific sign, it has been shown to have 90% sen-
sitivity and specificity for moderate to severe fetal anaemia after
single IUFD.120,121 The primary interpretation of MCA Doppler
assessment is such that normal values are likely to exclude a major
hypovolemic event, and thus the risk for cerebral injury in sur-
vivors is likely negligible, although larger studies are required to
support this. Intrauterine ‘rescue’ transfusions in the survivor after
the death of a co-twin may not be able to prevent ischaemic brain
damage. Whether they may improve outcome in terms of overall
survival or long-term health is still unclear.122,123
Regarding the mode and timing of delivery of MC twin preg-
nancies complicated by sIUFD, in the absence of pathology and
to minimise the risk for prematurity, an elective delivery at 34
to 36 weeks’ gestation may be achieved after corticosteroids have
been administered for fetal lung maturity. The placenta is rou-
tinely sent for histopathology. Placental injection studies with a
view to outlining the intertwin vascular architecture improve the
understanding of the pathophysiology and may be attempted.118
Psychological support and bereavement counselling are recom-
mended. Discussions should also extend to feasibility of autopsy
and subsequent handling and personal arrangements for the
deceased twin thereafter.  not always denote a logical order of disease progression, although
Twin–Twin Transfusion Syndrome
Twin–twin transfusion syndrome complicates between 8% and
10% of MC twin pregnancies.124 The last decade has witnessed
considerable advances in fetal intervention with increasing overall
perinatal survival rates and favourable neurodevelopmental out-
comes. Without treatment, the condition is associated with a 73%
to 100% perinatal mortality rate and a 15% to 20% risk for brain
injury in survivors.124,125
Staging. Twin–twin transfusion syndrome is staged accord-
ing to the system developed by Quintero and coworkers in 1999,
which remains the gold standard method for assessing severity of
TTTS.126 The Eurofoetus criteria are an adaptation using a cutoff
Voluson
E8
bbb
1
• Fig. 44.3 Twin–twin transfusion syndrome (TTTS) showing the donor
twin with oligohydramnios (deepest vertical pocket, 8 mm) and the notable
polyhydramnios in the sac of the recipient. In this case, the donor (bbb)
appeared to be ‘stuck’, with an absent bladder in addition to absent end-
diastolic flow in the umbilical artery; therefore, this was a case of stage 3
TTTS.
• BOX 44.1
Stage I Mildest form with amniotic fluid discordance
Polyhydramnios with DVP >8 cm in recipient and
Oligohydramnios with DVP <2 cm with bladder still
visible in donor
Stage II Above features with lack of visible bladder in donor
Stage III Critically abnormal Dopplers in either twin: absent or
reversed EDF in donor umbilical artery or reversed
ductus venosus flow or pulsatile umbilical venous
flow in the recipient ± TTTS cardiomyopathy
(atrioventricular valvular incompetence, ventricular
hypertrophy and dysfunction)
Stage IV Ascites or frank hydrops in either recipient or donor
Stage V Single or double twin death
DVP, Deepest vertical pocket; EDW, end-diastolic flow; TTTS, twin–twin transfusion syndrome.
of 10 cm instead of 8 cm after 20 weeks’ gestation.127 There are
five stages in the Quintero system broadly based on the range of
presentations of fetal disease (Fig. 44.3). The classification is use-
ful for describing the condition in a consistent manner, but it does
early TTTS is usually associated with a better prognosis than later
disease (stages III–V)128,129 (Box 44.1). 
Early prediction of twin–twin transfusion Syndrome.
Although not necessary to secure a diagnosis of TTTS, additional
first and second trimester sonographic features have been associ-
ated with the development of the condition.
A systematic review and meta-analysis that included more than
2000 pregnancies of which 323 developed TTTS concluded mod-
est sensitivities of NT discrepancy (53%; 95% CI, 43.8%–62.7%)
and abnormal DV flow (50%, 95% CI, 33.4%–66.6%) for the
subsequent development of the condition.132
Discordant amniotic fluid in both sacs, although not meeting
the criteria for stage I, has been shown to progress to TTTS in
fewer than 15% of cases.134,135 A further study found that cases

TABLE Sonographic Findings Associated with Twin–
44.3 Twin Transfusion Syndrome
First trimester
NT >95th percentile or intertwin CRL discordance >20%130-132
Absent or reversed ductus venosus A-wave133
Second trimester
Amniotic fluid discordance (not fulfilling the criteria for stage I dis-
ease)134-137
Abdominal circumference discordance, intertwin growth discordance
>20%138
Membrane folding139
Velamentous placental cord insertion (donor), close proximity of umbili-
cal cords140,141
Hyperechogenic donor placental portion142
CRL, Crown–rump length; NT, nuchal translucency.
  
of isolated oligohydramnios in the donor progressed to TTTS and
cases of isolated polyhydramnios in the recipient progressed in in
49% (26 of 53) and 20% (6 of 20) of cases, respectively.36 Per-
sistent amniotic fluid discordance in association with AREDF in
the umbilical artery has also been associated with progression to
TTTS.137 Although not based on large prospective series, findings
of discordant amniotic fluid volume or isolated amniotic fluid vol-
ume differences in either sac in the absence of TTTS, in addition
to the markers mentioned already discussed, probably warrant
more heightened sonographic surveillance for the possibility of
the development of TTTS (Table 44.3).143  fetal echocardiographic findings in early TTTS staging classifi-
Causes
Placental architecture. The primary cause of TTTS is considered
to be the presence of intertwin vascular connections within the
placenta that are a feature of virtually all MC twins. Three types
of vascular anastomoses exist: AA, venovenous (VV) and arterio-
venous (AV). AV anastomoses are found in almost all MC twin
placentas, with AA anastomoses present in 85% to 90% and VV
anastomoses present in 15% to 20%.140,144-146
Arterioarterial and VV anastomoses are superficial vessels pro-
moting bidirectional fetal flow and are visible on the chorionic
plate. AV anastomoses in comparison with AA and VV anasto-
moses, burrow deeper into the placenta. AV anastomoses promote
unidirectional flow and consist of a chorionic artery from one
twin supplying an underlying cotyledon, with the draining cho-
rionic vein returning to the co-twin.146 Thus these anastomoses
promote a constant intertwin transfusion. If the gradient is tipped
in favour of one twin, through unidirectional AV flow, and where
bidirectional AA or VV are deficient or do not compensate, an
imbalance in blood flow occurs. The subsequent clinical response
of a volume-overloaded recipient twin with polyhydramnios and a
volume-deplete donor with oligohydramnios may ensue. Placentas
in twins complicated by TTTS are less likely to have bidirectional
compensatory AA anastomoses, with 78% of those without AA
anastomoses developing TTTS.146 Additional systemic responses
such as the renin–angiotensin system have been implicated in the
condition and might contribute to the end-organ features of renal
failure and hypertensive microangiopathy in the recipient twin
that persist beyond fetal life.147  rates between 13% and 17%.127,158-162
CHAPTER 44  Multiple Pregnancy 543
Cardiovascular changes and echocardiographic features.
Twin–twin transfusion syndrome confers an increased risk for both
structural and functional cardiac disease, in particular for the recip-
ient.148,149 The abnormal placental architecture may predispose to
abnormal cardiac formation in MC twins. The cardiac functional
changes secondary to volume overload featuring in the recipient
include cardiomegaly, biventricular hypertrophy, and in severe
cases, cardiac failure. Atrioventricular regurgitation involving the
tricuspid valve and to a lesser degree the mitral value can compli-
cate up to 71% of cases of severe TTTS.150 Overall two thirds of
recipient fetuses have evidence of diastolic dysfunction, indicated
by prolonged ventricular isovolumetric relaxation time.150 Right
ventricular outflow tract obstruction can complicate up to 10%
of recipients and is associated with significant mortality and the
requirement for neonatal balloon valvuloplasty.151 The pathogen-
esis appears to be primarily related to hypertrophy and altered
diastolic filling pressures or diastolic dysfunction across the right
ventricle, where decreased flow through the right ventricle leads
to diminished growth of the right ventricular outflow tract and
resultant varying degrees of pulmonary stenosis or atresia.
Cardiac dysfunction has also been reported in 10% to 55% of
cases of early-stage disease through more sensitive assessments of
cardiac function using myocardial performance indices.152 After
intervention, cardiac function can normalise in recipients some
4 weeks after laser therapy.153 Studies have also indicated that at
approximately 10 years after a pregnancy complicated by TTTS,
both donors and recipients demonstrated cardiac function within
the normal range.154
Given the associated cardiac functional changes in the recipi-
ent, an alternative cardiovascular scoring system has been proposed
(Table 44.4).155 Although the scoring system has not been proven
to enhance prediction of outcome, proponents of the addition of
cation recognise the potential for this to become a more refined
preoperative discriminatory strategy (Fig. 44.4). Most tertiary
referral centres perform serial echocardiography before and after
treatment of TTTS. Further prospective research into novel, more
specialised fetal cardiac functional assessments to more accurately
define myocardial deformation as an index of ventricular function
may improve outcome by predicting progression of early disease
to help determine appropriate timing of delivery.156 
Treatment. The past 2 decades have witnessed considerable
modifications in the treatment of TTTS. Before the introduc-
tion of fetoscopic laser ablation for TTTS in 1990, treatment
options were limited to either serial amnioreduction as a tem-
porary solution to minimise the risk for premature rupture of
the membranes (PPROM) and the maternal symptoms second-
ary to uterine overdistention caused by recipient polyhydram-
nios or microseptostomy aimed at equilibrating the amniotic
fluid volume.129,157,158 These treatments were palliative and did
not address the underlying cause. Furthermore, the landmark
Eurofoetus randomised controlled trial reported higher survival
rates with at least one survivor and less short-term neurodisabil-
ity after fetoscopic laser ablation versus serial amnioreduction
(76% compared with 56% and 6% compared with 14%, respec-
tively).127 Regarding serial amnioreduction, a subsequent meta-
analysis and Cochrane review reported mortality rates of up to
60% and rate of neurodisability as high as 50%.158 The current
recommended treatment of choice for TTTS is fetoscopic laser
coagulation of the vascular anastomoses, with dual survival rates
reported as ranging between 35% and 78%, and neuromorbidity
544 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
TABLE
44.4 CHOP Cardiovascular Scoring System for Twin–Twin Transfusion Syndrome
Variable Parameter
Donor Umbilical artery
Recipient Ventricular hypertrophy
Cardiac dilation
Ventricular dysfunction
Tricuspid valve regurgitation
Mitral valve regurgitation
Tricuspid valve inflow
Mitral valve inflow
Ductus venosus
Umbilical vein
Right-sided outflow tract
Pulmonary regurgitation
Reproduced from Rychik J, Tian Z, Bebbington M, et al. The twin-twin transfusion syndrome: spectrum of cardiovascular abnormality and development of a cardiovascular score to assess severity of
disease. Am J Obstet Gynecol 197(4):392.e1–e8, 2007.
CHOP, Children’s Hospital of Philadelphia.  
Fetoscopic laser surgery. The goal of fetosopic laser surgery
for TTTS is to coagulate the intertwin placental vascular anasto-
moses so as to effectively create a dichorionisation of an MC pla-
centa. The procedure can be done under local anaesthetic, avoiding
the need for regional anaesthesia for routine cases.163 Prophylactic
Fetuses with this
Finding Numeric score finding (n)
Normal 0 96 (64%)
Decreased diastolic blood flow 1 34 (23%)
Absent or reversed diastolic blood flow 2 20 (13%)
None 0 77 (51%)
Present 1 73 (49%)
None 0 78 (52%)
Mild 1 47 (31%)
>Mild 2 25 (17%)
None 0 117 (78%)
Mild 1 12 (8%)
>Mild 2 21 (14%)
None 0 97 (65%)
Mild 1 31 (21%)
>Mild 2 22 (15%)
None 0 131 (87%)
Mild 1 6 (4%)
>Mild 2 13 (9%)
Double peak 0 113 (75%)
Single peak 1 37 (25%)
Double peak 0 135 (90%)
Single peak 1 15 (10%)
All antegrade 0 114 (76%)
Absent diastolic blood flow 1 13 (9%)
Reverse diastolic blood flow 2 23 (15%)
No pulsations 0 136 (91%)
Pulsations 1 14 (9%)
Pulmonary artery > aorta 0 126 (84%)
Pulmonary artery = aorta 1 13 (9%)
Pulmonary artery < aorta 2 8 (5%)
Right ventricle outflow obstruction 3 3 (2%)
None 0 145 (97%)
Present 1 5 (3%)
antibiotics and calcium channel blockers are administered peri-
operatively. All fetal interventions should be undertaken by expe-
rienced operators.164 Under continuous ultrasound guidance, a
trochar (with a size 7- to 12-Fr cannula) is inserted into the recipi-
ent sac. The Seldinger technique using a cannula and guidewire

CV 4 CV 3 CV 2 CV 1
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Quintero Quintero Quintero Quintero
stage I stage II stage III stage IV
• Fig. 44.4 Children’s Hospital of Philadelphia (CHOP) Cardiovascular
Scoring system of twin–twin transfusion syndrome according to the Quin-
tero stage. The bar graph shows the percentage of twin pairs with a par-
ticular cardiovascular grade for each Quintero stage. Note the presence of
significant discrepancy in categorisation of severity in particular for Quin-
tero stages II and III, in which a wide variety of cardiovascular (CV) grades
are found. (Reproduced from Rychik J, Tian Z, Bebbington M, et al. The
twin-twin transfusion syndrome: spectrum of cardiovascular abnormality
and development of a cardiovascular score to assess severity of disease.
Am J Obstet Gynecol 197(4):392.e1–e8, 2007.)
has also been described.165 Depending on the gestational age at
the time of the laser procedure, a 1.3- or 2.0-mm fetoscope is used
with a straight scope and 0-degree angle in cases of a posterior pla-
centa and a 30-degree curved scope to aid optimum visualisation
of an anterior placenta. Some operators recommend the use of a
purely diagnostic larger fetoscope for the mapping process first,
switching to a smaller one with a separate channel for the laser
fibre thereafter. The vascular equator, having been mapped out
ultrasonographically before the procedure, is then directly visual-
ised in its entirety, and all visible anastomoses crossing the inter-
twin membrane are identified and coagulated using a neodymium
(Nd):YAG laser with a 400- or 600-μm laser fibre (Fig. 44.5).
After confirmation that all anastomoses are coagulated, the recipi-
ent sac is then drained under direct ultrasound guidance to a verti-
cal pocket of approximately 6 cm. A fetal karyotype is routinely
sent after patient consent. Fetal survival is typically confirmed
within 24 hours, and pregnancies are followed up according to
local protocols.
Fetoscopic laser ablation has undergone some modifications
over the years. The first procedure described consisted of coagula-
tion of all vessels crossing the membrane, but after initial reports
of high associated procedure-related loss, the procedure was sub-
sequently modified by Quintero in 1998 as the ‘selective’ laser
approach.166 The selective approach involves carefully mapping
out the vascular equator and coagulating only the vessels believed
truly to be intertwin anastomoses. This was further modified again
as the sequential selective approach, in which the vessels are coagu-
lated in a specific order aimed at minimising disruption in inter-
twin blood flow during coagulation.167 In brief, for the sequential
selective laser approach, donor AV anastomoses are first targeted
followed by recipient AV to donor, then AA anastomoses if pres-
ent (rare) and last VV anastomoses to allow for some interproce-
dure blood flow back from the recipient to the donor. Limited
CHAPTER 44  Multiple Pregnancy 545
• Fig. 44.5  Laser ablation of an arteriovenous anastomosis during a feto-
scopic procedure for treatment of twin–twin transfusion syndrome. Arter-
ies have a darker colour because of deoxygenated blood.
evidence from three nonrandomised cohort studies of 344 MC
pregnancies comparing the sequential technique (n = 224) with
the standard selective technique (n = 120) indicated improved
dual survival (75% vs 52%; P = .002, respectively) and decreased
donor and recipient demise (10% vs 34%; P = .02 and 7% vs
16%; P = .02, respectively).168 However, this approach has not
been universally accepted. 
Solomon technique. The most recent modification is known as
the ‘Solomon’ technique, whereby after the vessels are coagulated,
in essence, a ‘line’ is drawn with the laser fibre along the placen-
tal vascular equator connecting the ‘dots’ to completely separate
the placenta, at least at the chorionic surface level (Fig. 44.6).165
This procedure evolved to target even the smallest and some-
times ‘invisible’ connecting vessels because of the high percent-
age of residual vascular anastomoses reported after laser therapy,
in addition to the complication of twin anaemia–polycythemia
sequence and recurrent TTTS rates reported as 33%, 13% and
14%, respectively.169-171
Twin anaemia–polycythemia sequence (TAPS) has been
described in MC pregnancies and includes the presence of anae-
mia in the donor (defined as middle cerebral artery peak systolic
velocity (MCA-PSV) >1.5 MoMs) and polycythemia in the
recipient (MCA-PSV <0.8 or 1.0 MoMs) but without amni-
otic fluid abnormalities.171 A staging system has been proposed
(Table 44.5).171 It is a rare condition, and although it can occur
spontaneously (3%–5%), the available evidence suggests it is can
complicate up to 13% of cases of TTTS after fetoscopic laser
ablation and is believed to be a consequence of chronic intertwin
transfusion through very small placental anastomoses.169-171 The
goal of the Solomon technique is to thus target these small vessels
and prevent the development of TAPS. The available evidence of
this novel technique suggests that TAPS and recurrent TTTS are
reduced with the Solomon technique compared with the standard
laser technique.165,172,173
Regarding the Solomon technique, a randomised trial and two
retrospective studies have been described to date.165,172,173 For
546 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

A B
• Fig. 44.6  Colour-dye staining of placentas after standard treatment with selective fetoscopic laser abla-
tion (A) and using the Solomon technique (B). In A, the arrows point to the individual laser spots, but in B,
the complete vascular equator has been coagulated from one margin of the placenta to the other. (Repro-
duced from Slaghekke F, Lopriore E, Lewi L, et al. Fetoscopic laser coagulation of the vascular equator
versus selective coagulation for twin-twin transfusion syndrome: an open-label randomised controlled
trial. Lancet 383(9935):2144–2151, 2014.)

TABLE Proposed Staging System for Twin
44.5 Anaemia–Polycythemia Sequence
Antenatal stage Findings at Doppler ultrasound examination
1 MCA-PSV donor >1.5 MoM and MCA-PSV recipi-
ent <1.0 MoM without other signs of fetal
compromise
2 MCA-PSV donor >1.7 MoM and MCA-PSV recipi-
ent <0.8 MoM without other signs of fetal
compromise
3 As stage 1 or 2, with cardiac compromise of
donor, defined as critically abnormal flowa
4 Hydrops of donor
5 Intrauterine demise of one or both fetuses pre-
ceded by TAPS
aCriticallyabnormal Doppler is defined as absent or reversed end-diastolic flow in umbili-
cal artery, pulsatile flow in the umbilical vein, increased pulsatility index or reversed flow in
ductus venosus.
Reproduced from Slaghekke F, Kist WJ, Oepkes D, et al. Twin anaemia-polycythemia sequence:
diagnostic criteria, classification, perinatal management and outcome. Fetal Diagn Ther
27:181–90, 2010.
MCA-PSV, Middle cerebral artery peak systolic velocity; MoM, multiples of the median; TAPS,
twin anaemia–polycythaemia sequence.
  
the two retrospective studies, a total of 97 MC pregnancies were
treated with the Solomon technique compared with 152 treated
with the standard laser surgery. In these studies, dual survival
was significantly higher in the Solomon versus the standard laser
treated group (84.6% and 68.4% vs 46.1% and 50.5%, respec-
tively).172,173 In one of the studies, Ruano and coworkers reported
no cases of TAPS or recurrent TTTS with the Solomon technique
compared with 5.3% and 7.9% of cases treated with laser, respec-
tively. In the study by Baschat and coworkers, significantly less
TAPS (2.6% vs 4.2%; P < .05) and recurrent TTTS were reported
(3.9% vs 8.5%).172,173 In the multicentre randomised controlled
trial (RCT), 274 women were randomised to either the Solomon
technique (n = 139) or the standard laser group (n = 135), and
no significant differences were found for overall perinatal survival
(74% vs 73%, 1.04; 95% CI, 0.66–1.63), dual survival (64% vs
60%, 1.16; 95% CI, 0.71–1.89) or at least one survivor (85% vs
87%, 0.85; 95% CI, 0.71–1.89).165 However, there was a signifi-
cant reduction in TAPS in the Solomon group (3% vs 16% for
the standard group; OR, 0.16; 95% CI, 0.05–0.49) and recurrent
TTTS (1% vs 7%, 0.21; 95% CI, 0.04–0.98). 
Treatment by stage. Fetoscopic laser ablation is the recom-
mended treatment of choice for severe TTTS from 16 to 26 weeks’
gestation, and recent data suggest its feasibility at earlier and later
gestational ages in experienced hands.174,175 There is some limited
evidence to suggest feasibility for fetoscopic laser treatment for
TAPS, although it may be technically challenging in the absence
of polyhydramnios.171
Early-stage disease. Although approximately 60% of cases of
stage I TTTS may remain stable or regress, progression to a more
advanced stage can be acute and unpredictable.176 Occasionally,
stage I disease may present clinically with maternal symptoms
secondary to polyhydramnios, and this ‘tense’ polyhydramnios
results in flattening of the placenta such that fetoscopic surgery is
feasible. A number of studies have reported on outcomes for surgi-
cally managed stage I disease. Quintero and coworkers reported no
difference in outcome for cases of stage 1 disease treated by serial
amnioreduction versus fetosopic laser ablation, but higher rates
of survival with laser have been reported in other studies.177,178
Regarding cases managed expectantly versus intervention, Wagner
and coworkers reported no difference in perinatal survival rates
between the groups, but expectantly managed stage I fetuses had a
higher degree of neurodevelopmental impairment compared with
the treated group (39% vs 0%; P < .01).179
A recent systematic review by Rossi and coworkers concluded
that conservative management and laser for stage I disease had
equivalent outcomes and were superior to amnioreduction.180
However, a more recent retrospective observational study within
10 North American Fetal Therapy network (NAFTnet) centres
of 124 cases of stage 1 TTTS treated by expectant management,
serial amnioreduction or laser concluded intervention either by
amnioreduction or laser treatment was protective against fetal

loss.176 Within this study, intervention specifically with laser
was found to confer additional protection against double fetal
demise or delivery before 26 weeks’ gestation, and increased the
likelihood of delivery at 30 weeks’ gestation or later. The lack
of significance of factors at presentation (including nulliparity,
gestational age at diagnosis, recipient maximum DVP or pla-
cental location) as predictors of progression of TTTS highlights
the unmet need of a predictive tool for early-stage disease.180 A
multicentre RCT is currently underway that may provide more
robust guidance regarding the management of stage I TTTS.  cal management of TTTS recurrence.96
Advanced-stage disease. The current recommended treat-
ment for advanced-stage disease (stages II, III and IV) is fetoscopic
laser ablation. Most of the available outcome data discussed per-
tains to stage II and stage III; for example, in the Eurofoetus trial,
more than 90% of patients had stage II and stage III disease, and
there were only two patients with stage IV TTTS.127 For stage V
disease, the death of one twin carries the same risks to the co-twin
as for cases of sIUFD without TTTS, of death or neuromorbidity
of 10% to 15% and 10% to 30% respectively.56,94 Prior interven-
tion with laser ablation appears to improve neurologic outcomes
in survivors in the setting of co-twin demise.181  44.6).159 Although the improved techniques have impacted sur-
Complications. Single or dual fetal demise may occur immedi-
ately and up to several weeks after laser treatment. Single deaths
within 24 hours of the procedure were reported in 60% of cases
and within 1 week in 75% of cases.181,182 Donors are more sus-
ceptible to IUFD, possibly partly because of underlying unequal
placental sharing. Postoperative IUGR and AREDF in the UA
increases risk for donor IUFD, and this can occur even some
time after intervention. Recipient twin demise can occur with
more advanced disease in the setting of reversed DV A-wave and
hydrops and with recipient IUGR.182
Premature rupture of the membranes and preterm delivery are
inherent risks with the invasive nature of the procedure. Postpro-
cedural PPROM complicates approximately 10% to 30% of all
cases, although it is variably defined within studies.96,183 In gen-
eral, perioperative factors, including instrument choice, have not
been found to be associated with PPROM.184,185 Laser ablation
performed at an earlier gestational age (<17 weeks’ gestation) may
increase the risk for PPROM occurring shortly after surgery.174 In
terms of risk factors for preterm delivery, in addition to iatrogenic
PPROM, cervical length was found to be significantly associated
with preterm delivery.186 Placement of a cervical cerclage after
laser treatment may be offered, but data are controversial, and
randomised controlled trials are lacking.187
Immediate procedural complications include perioperative
haemorrhage, for which an amnioinfusion of saline may improve
visibility. Intermediate complications include chorioamnionitis,
abruption, sepsis and rarely (also occurring without fetoscopic
laser ablation) limb necrosis.188-190 Maternal mirror syndrome
and a case of spontaneous posterior uterine rupture have been
described.191,192 We recently had a case of TTTS associated with
gross maternal obesity, in which the patient went into cardiac
arrest shortly after regional anaesthesia and before the fetoscopic
procedure commenced, ultimately requiring an emergency hys-
terotomy.164 Overall maternal complications are estimated at
approximately 5%.193 These complications underpin the invasive
nature of the procedure, and future studies should consistently
report on all outcomes, not just perinatal survival.
Recurrence of twin–twin transfusion syndrome. Twin–twin
transfusion syndrome recurs in up to 15% of cases, and there does
not appear to be consensus in the literature regarding optimum
management of recurrence. We performed a systematic review of
CHAPTER 44  Multiple Pregnancy 547
the literature, and only a small number of publications provided
comprehensive outcome data for cases of recurrent TTTS.169 The
overall rate of neurologically intact survival was 44%. The data
were inadequate to determine the effects of secondary therapeutic
approach, placental location or gestational age on perinatal out-
come in cases of recurrent TTTS. Although limited follow-up
data suggest that recurrence is associated with significant perinatal
mortality and morbidity, further study is needed. Currently, there
are insufficient data available to guide recommendations for clini-
 
Prognosis
Perinatal outcome. Perinatal outcome has significantly
improved over the years with the evolution of the laser tech-
nique from nonselective to selective to selective sequential and
the Solomon technique. A systematic review including 34 stud-
ies and 3868 MC pregnancies concluded that there has been a
significant increase in the mean survival rates of both twins from
35% to 65%, (P = .012) and of at least one twin from 70% to
88% (P = .009) with changing interventions over 25 years (Table
vival, additional factors such as improved neonatal care, referral
pathways and the ‘learning-curve’ effect are likely to signifi-
cantly contribute to this improvement. Larger trials powered for
all possible outcome measures and complications are required.
Although Quintero stage does not always correlate with out-
come, in general, survival decreases with increasing stage. 
Neurodevelopmental outcome. Neurologic injury in either
twin remains a major cause of perinatal morbidity in TTTS, and
intervention does not appear to be fully protective against subse-
quent cerebral injury. Neuromorbidity in treated TTTS cases has
been reported to be between 6% to 18% across studies.160-162 In a
systematic review and meta-analysis of neuromorbidity after laser
treatment by Rossi and coworkers, the rate of neurologic impair-
ment was reported as 6% at birth increasing to 11% at follow
up (range, 6–48 months), of which CP complicated 40%.162 Pre-
maturity was a significant contributor, and donors and recipients
were equally affected, with single demise after laser not appearing
to confer increased neuromorbidity on survivors. It is important
to point out that neuromorbidity is inherent to the MC twinning
process, and even when controlling for TTTS, sIUFD, sIUGR
and delivery before 32 weeks’ gestation, the rate of neurodevel-
opmental impairment in uncomplicated MC twins has been
reported to be 7% and the rate of cerebral palsy to be 0.6%.194
The higher rates of severe cerebral injury with serial amniore-
duction have been attributed to a longer interval of exposure to
in utero TTTS. This is further supported by the fact that expec-
tantly managed cases of TTTS are also at high risk for long-term
neurologic impairment, suggesting a disadvantage to prolonged in
utero exposure to the condition even at a consistent mild stage.176
Although amnioreduction is no longer advocated for the treat-
ment of TTTS, results from on ongoing RCT regarding expectant
management versus treatment for stage 1 TTTS may favour inter-
vention on the basis of neurodevelopmental outcomes.
Long-term neurodevelopmental disability in children from 2 to
6 years of age, although inconsistently reported, ranges from 4%
to 11%.196-199 In addition to gestational age at delivery, increas-
ing Quintero stage appears to be associated with poorer cogni-
tive outcomes.195,196 Furthermore, the follow-up of 141 cases
randomised to the Solomon technique compared to the standard
laser approach in the trial of Slaghekke et al (2014)165 has shown
comparable 2-year neurodevelopmental outcomes.200
548 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE
44.6 Review of Survival Outcomes (Dual Survival) After Fetoscopic Laser Ablation Over the Past 25 Years
Patients Inclusion Dual survival
Reference (n) period Type of study rate (%) GA at birth (wk)c Comments
De Lia et al, 1995259 26 1988–1994 Prospective single-centre cohort 35 32.2 ± 2.8
Ville et al, 1995260 45 1992–1994 Prospective single-centre cohort 36 35.0 ± 3.8
De Lia et al, 1999261 67 1995–1998 Prospective single-centre cohort 57 30.0 ± 5.0
Hecher et al, 200 1995–1999 Prospective single-centre cohort 48 34.0 ± 2.7 Early vs late series to
2000262a,b show learning curve
effect
Quintero et al, 89 194–1999 Prospective multicentre cohort 39 32.0 ± 2.5 Nonselective laser vs
2000263a,b selective laser
Gray at al, 2006264 31 2002–2003 Prospective single-centre cohort 39 34.0 ± 4.5
Huber et al, 2006178 200 1999–2003 Prospective single-centre cohort 60 34.3 ± 2.9
Ierullo et al, 2007265 77 2002–2006 Prospective single-centre cohort 40 NA
Middeldorp et al, 100 2000–2004 Prospective single-centre cohort 58 33.0 ± 3.7
2007266
Quintero et al, 193 2003–2005 Prospective single-centre cohort 65 33.7 ± 4.0 Sequential laser vs stan-
2007167b dard selective laser
Sepulveda et al, 33 2003–2006 Prospective single-centre cohort 27 32.0 ± 3.8
2007267
Stirneman et al, 287 1999–2005 Retrospective single-centre cohort 42 32.0 ± 3.6
2008268
Cincotta et al, 100 2002–2007 Prospective single-centre cohort 66 31.0 ± 3.2
2009269
Nakata et al, 52 2002–2006 Prospective multicentre cohort 50 32.0 ± 4.2 Excluded for time-based
2009270b analysis but included
for technique-based
analysis because of
overlap
Ruano et al, 2009271 19 2006–2008 Retrospective single-centre cohort 26 32.1 ± 3.0
Chmait et al, 99 2006–2008 Prospective single-centre cohort 68 32.2 ± 4.5 Sequential laser vs stan-
2010272b dard selective laser
Meriki et al 2010273 75 2003–2008 Retrospective single-centre cohort 60 32.0 ± 2.7
Morris et al, 2010274 164 2004–2009 Prospective single-centre cohort 38 33.2 ± 1.3
Yang et al, 2010275 30 2002–2008 Retrospective single-centre cohort 60 32.0 ± 4.0
Delabaere et al276 49 2006–2008 Retrospective single-centre cohort 59 32.0 ± 2.6 Article in English
Hernandez-Andrade 35 2008–2009 Retrospective single-centre cohort 49 32.0 ± 3.7 Article in Spanish
et al 2011277
Lombardo et al 70 2000–2010 Retrospective single-centre cohort 59 32.1 ± NA
2011278
Tchirikov et al 80 2006–2011 Retrospective single-centre cohort 78 33.8 ± 3.2 Use of 1-mm optic vs
2011279 2-mm optic
Weingertner et al 100 2004–2010 Retrospective single-centre cohort 52 32.6 ± 3.8
2011280
Chang et al 2012281a 44 2005–2010 Retrospective single-centre cohort 50 30.6 ± 5.9
Liu et al 2012282 33 2003–2010 Retrospective single-centre cohort 52 31.0 ± 6.0 Excluded in technique-
based analysis
because of unclear
technique; article in
Chinese
 CHAPTER 44  Multiple Pregnancy 549

TABLE
44.6 Review of Survival Outcomes (Dual Survival) After Fetoscopic Laser Ablation Over the Past 25 Years—cont’d
Patients Inclusion Dual survival
Reference (n) period Type of study rate (%) GA at birth (wk)c Comments
Murakoshi et al 152 2002–2010 Retrospective single-centre cohort 63 33.0 ± NA
2012283
Rustico et al 2012284a 150 2004–2009 Retrospective single-centre cohort 41 32.1 ± 2.2
Sundberg et al 55 2004–2010 Retrospective single-centre cohort 35 34.8 ± 4.0
2012285
Swiatkowska- 85 2005–2010 Retrospective single-centre cohort 45 32.0 ± 2.5
Freund, 2012286
Valsky et al 2012175 334 2006–2009 Retrospective single-centre cohort 68 33.2 ± 3.0 GA at laser, 16–26 wk
vs >26 wk
Baschat et al 2013173 147 2005–2011 Retrospective single-centre cohort 60 32.6 ± 3.5 Selective laser vs Solo-
mon laser
Baud et al 2013174 325 1999–2012 Retrospective single-centre cohort 63 31.3 ± 4.0 GA at laser <16 wk vs
16–26 wk vs >26 wk
Ruano et al 2013172 102 2010–2012 Retrospective multicentre cohort 65 31.6 ± 4.4 Selective laser vs Solo-
mon laser
Slaghekke et al 272 2008–2012 Multicentre RCT 62 32.3 ± 3.3 Selective laser vs Solo-
2014165 mon laser
aThese studies described more series over different time periods and were split up in the time-based analyses.
bThese studies described comparisons between different techniques.
cFigures for gestational age (GA) are mean ± standard deviation.
NA, Not assessed; RCT, randomised controlled trial.
Reproduced from Akkermans J, Peeters SH, Klumper FJ, et al. Twenty-five years of fetoscopic laser coagulation in twin-twin transfusion syndrome: a systematic review. Fetal Diagn Ther 38(4):241–253,
2015.
  
In a recent retrospective review of 1023 cases of TTTS treated pregnancies and accounts for 1% of MC twin pregnancies.96,201 It
by laser surgery, severe prenatal cerebral lesions were reported in is a rare abnormality in which one twin has an absent, rudimen-
only 2% of all cases, and this was found to be significantly associ- tary or nonfunctioning heart (acardiac twin) and the other twin is
ated with TAPS and recurrent TTTS, further supporting the use of the normal (pump) twin. The condition is associated with adverse
the Solomon technique.200 As with cases of sIUFD, fetal MRI may perinatal outcomes for the surviving twin.
be used as an adjunct to ultrasound in the detection of ischaemic Cause. The majority of cases occur with an MC placenta,
haemorrhagic lesions as a result of TTTS, with the optimum tim- although DC cases have been described. The presence of large AA
ing 2 to 3 weeks after surgery and ideally after 28 weeks’ gestation.  between the two twins’ umbilical arteries during embryogenesis
Antenatal surveillance and timing of delivery. There are results in low pressure deoxygenated blood from the ‘‘pump twin’s’
no data regarding optimal antenatal monitoring for pregnan- umbilical artery to flow retrogradely into the perfused twin’s
cies complicated by TTTS. Weekly monitoring of UA Doppler, umbilical arteries (or artery because there is often only one) to
MCA-PSV and DV Doppler velocities should initially be under- the iliac vessels, thus perfusing the lower part of the body to a far
taken, extending this to 2-weekly assessments after 2 weeks with greater extent than the upper part.202 The result is a spectrum of
documentation of adequate fetal growth.13 A targeted fetal echo- malformations, reduction anomalies of previously existing tissues
cardiogram is recommended by a fetal medicine specialist. The and incomplete morphogenesis of tissues primarily in the upper
average gestational age at delivery for cases treated by fetoscopic body. The pump twin may become compromised and is at risk
laser surgery remains constant at 32 weeks’ gestation and does not for congestive cardiac failure because of continued growth of the
appear to be affected by technique of fetoscopic laser treatment perfused twin and the overall volume of parasitic tissue needing to
used.159 Although with the advent of the Solomon technique and be perfused by the normal heart. 
projected reduced incidence of recurrent TTTS and TAPS, more Sonographic assessment. The acardiac twin typically appears
cases may well be delivered at a later gestational age. With the as an amorphous mass with tissue oedema, deformed lower limbs
currently available data, in the absence of PPROM, a course of and rudimentary or absent upper limbs and head, with a two-ves-
corticosteroids and elective delivery between 34 and 36 weeks’ sel cord. There may be polyhydramnios in the acardiac twin’s sac,
gestation may be reasonable.13  indicating the presence of functional renal tissue.202 The pump
twin may have cardiomegaly, polyhydramnios, hydrops and pleu-
ral and pericardial effusions.203
Twin-Reversed Arterial Perfusion The TRAP sequence can be diagnosed as early as 9 weeks’ ges-
The twin-reversed arterial perfusion (TRAP) sequence, previously tation but may be mistaken for a demised twin or an anenceph-
known as acardia or acardiac twin, occurs in 1 in 35,000 to 50,000 alic fetus.204,205 In the second trimester, a history of continued
550 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
growth or ‘twitching’ of a ‘presumed dead’ twin on subsequent
scans should alert one to the diagnosis. Although cardiac pulsa-
tions are as a rule absent, rare cases of monoventricular heart with
pulsations have been reported. Definitive diagnosis can be estab-
lished by demonstrating the TRAP sequence using colour flow to
demonstrate reverse flow through the perfused twin’s aorta, and
pulsed Doppler to show the paradoxical direction of arterial flow
towards the acardiac twin.206  twins after fetal therapy, of 17 cases of TRAP sequence treated by
Perinatal complications. Polyhydramnios resulting in preterm
delivery is reported in up to 50% of these cases.203,204 The nor-
mal pump twin is also at risk for fetal hydrops (28%) and intra-
uterine demise (25%).203 Healy and coworkers (1994) reviewed
189 case reports in the literature spanning approximately 30 years
(1960–1991) and reported the overall perinatal mortality rate for
the pump twin to be 35% in twins and 45% in triplets.203 Pre-
mature delivery before 32 weeks’ gestation and the presence of
arms, ears, larynx, trachea, pancreas, kidney and small intestine in
the perfused fetus have been significantly associated with perinatal
death.203,204 Poor prognostic features such as acardiac twin: pump
twin weight or AC ratio greater than 0.7, elevated combined ven-
tricular output, elevated cardiothoracic ratio and congestive car-
diac failure, polyhydramnios, abnormal venous Dopplers or fetal
anaemia have been proposed as predictors of outcome.204,206,207 
Antenatal management. Aneuploidy has been reported in
33% of perfused twins and 9% of pump twins, and a karyotype
should be offered on this basis.203,204 Serial measurements of both
twins should be undertaken because of the association between
increased acardiac pump twin weight or AC ratio or rapid growth
in the acardiac twin and adverse outcome. Given the poor prog-
nosis associated with cardiac failure and polyhydramnios, the
occasional unanticipated demise of pump twins and the technical
difficulties in treatment at later gestations, prophylactic interven-
tion is increasingly advocated and at an early gestation age.208-211  stillborn, and more than 50% of those born alive die during the
Intervention. Selective delivery or termination of the acardiac
twin with the goal of optimising the outcome for the pump twin
have been described. There are a few case reports of successful
selective delivery of the acardiac twin by hysterotomy and subse-
quent delivery of the pump twin between 27 and 35 weeks’ gesta-
tion.212,213 A number of less invasive techniques have since been
described, including interstitial laser, fetoscopic-guided occlusion
of the acardiac twin’s cord and radiofrequency ablation (RFA).214-
218 Technical difficulties occasionally arise during external cord
procedures in the presence of short, thin or hydropic cords.
Similarly, intrafetal ablative techniques may fail when there is sig-
nificant flow or large intraabdominal vessels within the acardiac
structure. Treatment is rarely indicated after 25 weeks’ gestation
when a temporising approach with amnioreduction, and early
delivery may be preferable.
Radiofrequency ablation of intraabdominal vessels is emerg-
ing as the procedure of choice for TRAP sequence. It is rela-
tively simple compared with other cord occlusive methods and
probably safer than interstitial laser. Tines extending from the
tip of the needle anchor the ablative device in the region of the
intraumbilical vessels. One sizeable series of 29 cases reported a
survival rate with RFA between 18 and 24 weeks’ gestation in
TRAP of 86% with a mean gestational age at delivery of 34.6
weeks gestation.217 In a recent systematic review of 26 stud-
ies spanning 20 years, survival rates for pump twins were bet-
ter with intervention (ablation or cord coagulation) compared
with expectant management (OR, 2.22; 95% CI, 1.23–4.01, P =
.008).209 Furthermore ablation was favoured over cord occlusion
(pump twin survival (OR, 9.84; 95% CI, 1.56–62.00; P = .01),
with the difference being greater in the presence of one or more
poor prognostic features (OR, 8.58; 95% CI, 1.47–49.96; P =
.02). However, there were insufficient data to determine which
features should guide management. A multicentre randomised
trial comparing early versus late intervention is ongoing (TRAP
Intervention STudy: Early Versus Late Intervention for Twin
Reversed Arterial Perfusion Sequence [TRAPIST]).
Regarding long-term neurodevelopmental outcome in MC
selective reduction in one centre, of 11 pump twins who survived,
all were reported to have had a normal neurodevelopmental out-
come (range of follow-up, 1 month–5 years).214 In a further study
of follow-up of 10 survivors after cord coagulation for TRAP,
three had evidence of neurodevelopmental delay, including 1
with severe mental delay and 2 with minor delays; 2 cases were
complicated by PPROM and delivered at 28 and 29 weeks’ gesta-
tion, and the other case was delivered at 29 weeks’ gestation for
nonreassuring fetal testing.215 The authors favoured prophylactic
intervention in cases of TRAP rather than delayed intervention
on the basis of cardiac compromise of the pump twin. Long-term
larger follow-up series on neurologic outcome in surviving pump
twins is deficient. Selective termination procedures used for TRAP
sequence are described in more detail later in this chapter. 
Conjoined Twins
The precise incidence of conjoined twins is unknown but is esti-
mated at between 1 in 50,000 to 250,000 live births.219 The
underlying pathogenic mechanism may result from incomplete
division of the single blastocyst between 13 and 15 days postcon-
ception or secondary union of two separate embryonic disks.220
There is a female preponderance with about 75% of conjoined
twins being female. Approximately 40% of conjoined twins are
neonatal period.221
Conjoined twins have been classified based on the fused ana-
tomic region (with suffix ‘pagus’ meaning fixed or fastened). The
most common type of conjoined twins include thoracopagus,
xiphagus or omphalopagus, pygopagus, ischiopagus and crani-
opagus. A newer classification has been proposed on the basis
of likely three-dimensional relationships between the two fetal
body planes during early embryogenesis.221 There is a high inci-
dence of associated congenital anomalies, 60% to 70% of which
involve structural abnormalities not associated with the area of
fusion. Neural tube defects, orofacial clefts and cardiac anomalies
predominate.222
Sonographic assessment. In the first trimester, the typical picture
is that of an monoamniotic (MA) twin pregnancy with a single yolk
sac alongside two embryonic poles. The diagnosis should be made
with caution in the first trimester, and follow-up is recommended.
From 8 weeks’ gestation, fetal activity helps differentiate normal
MA from conjoined twins. Increased NT and subcutaneous oedema
may be noted in thoracopagus twins, the most common form of
conjoined twins. The fetal pole is typically bifid in appearance.
In the second trimester, the sonographic features comprise of
lack of a separating membrane and an inability to demonstrate
completely separate fetal bodies, with both heads persistently at
the same level with no change in their relative positions. In the
case of omphalopagus, the conjoined twins’ fetal presentations
may be discordant because of backward flexion of the upper spine.
There may be more than three vessels when the umbilical cord is
single. Doppler waveforms of the UA show a characteristic ‘dou-
ble-layer’ spectral pattern reflecting two separate arterial supplies
within the same umbilical cord, which is considered diagnostic of

conjoined twins.223 Prenatal MRI, particularly in the third trimes-
ter, can provide additional information in planning for delivery
and postnatal surgery. Fetal MRI recently identified significantly
reduced cerebral and placental perfusion in omphalopagus con-
joined twins compared with a singleton pregnancy.224 MRI can
be superior to ultrasonography in cases of maternal obesity or
oligohydramnios and produces three-dimensional reconstructed
images in any plane.225 Postnatal MRI is important, particularly
in craniopagus, to assess cortical fusion and in thoracopagus to
evaluate intracardiac anatomy, blood flow and ventricular wall
motion. 
Management. Aneuploidy is rare, and invasive testing is usu-
ally not recommended. The option of termination of pregnancy
should be discussed. The prognosis for the twins depends upon
the extent of fusion and the presence of separate organs. Twins
with cerebral or cardiac fusion have poor prognosis.226 Antena-
tal paediatric surgical consultation with a national centre with
expertise in conjoined twins is valued, and although the majority
of parents decide on termination, those who continue may do
so with the understanding of the need for major surgical separa-
tion and reconstruction and its associated short- and long-term
morbidity.
After defining the extent of fusion and associated anomalies,
serial scans are required to monitor fetal growth and amniotic fluid
volume. In the third trimester, polyhydramnios complicates 50%
of cases, and amnioreduction may occasionally be indicated.96
In terms of mode of delivery, vaginal delivery may be possible in
preterm cases and when neonatal survival is not anticipated, but
near-term caesarean section is necessary and might need to be clas-
sical to maximise survival and minimise trauma to viable twins.
Uterine rupture has been reported with vaginal delivery.
Postnatally, detailed MRI and computed tomography imaging
with a multidisciplinary team approach involving paediatric sur-
geons specialised in separation procedures is warranted. In addi-
tion, psychiatric, social services, physiotherapy, rehabilitation and
nursing support are necessary.
Conjoined twins fall into three management categories: (i)
inoperable cases, (ii) operable but warrant emergency separation
because of cardiac instability and (iii) planned elective separation.
Emergency separation carries a high mortality rate of 71%.
In contrast, elective separation, usually planned between 2 to 4
months of age, is safer and has a survival rate of up to 80% in
some centres.226 In a recent review of 36 sets of conjoined twins
spanning over 12 years, the majority were thoracopagus, and of 5
sets of twins who underwent surgery, 6 children survived.227  fied if looked for, allowing relatively unrestricted fetal movements.
Monoamniotic Twins
Monoamniotic twin pregnancies represent approximately 2% to
5% of all MC twins (1 in 10,000 pregnancies). It was previ-
ously held that MA twinning was associated with a high peri-
natal mortality rate of 30% to 70%, primarily because of cord
entanglement, with more recent series reporting a reduced risk
for 10% to 12%, attributed to fetal surveillance and elective
delivery.228,229 A recent review of 20 MA pregnancies diagnosed
at less than 16 weeks’ gestation that were prospectively followed
up concluded an overall survival for fetuses alive at initial scan
of 45% (18 of 40), and a further meta-analysis of 13 studies
concluded a perinatal mortality rate of only 4.5% (95% CI,
3.3%–5.8%) after 24 weeks’ gestation.229 Given that most losses
in MA twins are attributable to fetal anomalies and spontaneous
miscarriages and with a lack of RCTs of planned delivery versus
expectant management for MA twins, the merits of intensive
CHAPTER 44  Multiple Pregnancy 551
• Fig. 44.7  Colour Doppler demonstrating cord entanglement and wave-
form showing two different heart rates in a monoamniotic twin pregnancy.
fetal surveillance, inpatient monitoring and elective preterm
delivery is contentious.230
Sonographic assessment. The diagnosis can be made in the
first trimester by visualisation of two separate fetuses with no clear
dividing membrane and a single yolk sac. Visualisation of two yolk
sacs does not necessarily exclude an MA pregnancy because the
number of yolk sacs depends on the time of splitting of the germi-
nal disk. When there are two yolk sacs and no dividing membrane
before 9 weeks’ gestation, a repeat scan must be performed at a
later stage because the intertwin membrane in an MCDA twin
pregnancy may not easily be seen in early pregnancy.
The presence of a single placenta; absent intertwin membrane;
same-sex, freely moving nonstuck twins in normal amniotic
fluid volume supports the diagnosis from the second trimester.
A pathognomonic feature of MA twins is the presence of cord
entanglement, which can be demonstrated from 10 weeks’ ges-
tation onwards by insonating a common mass of cord vessels
between the two fetuses with colour-flow Doppler (Fig. 44.7).
Two distinct arterial waveform patterns with different heart rates
can be discerned in the same direction within the same-pulsed
wave sampling gate.231 
Perinatal complications. Cord knots, tightening of entangle-
ment and single or double twin deaths are the most common
complications. Cord entanglement is ubiquitous with MA twin-
ning, and cord entanglement will more often than not be identi-
Doppler waveforms in the cord may suggest tightening of cord
entanglement or knotting include a diastolic notch, elevated sys-
tolic-to-diastolic ratio or absent EDF in the umbilical artery or
umbilical venous pulsations.231,232
In a recent review of 114 MA twin sets with documented cord
entanglement, the overall survival rate was 88.6%; the perinatal
mortality rate was 11%, of which approximately two thirds died
in utero and one third at birth; 5 neonatal deaths occurred as a
result of prematurity, 2 were caused by structural anomalies and
2 were caused by cord entanglement.232 Sonographic visualisation
of cord entanglement did not improve outcome, and overall there
was no difference in mortality rates between control participants
and twins with cord entanglement. A Cochrane review in 2015
failed to identify sufficient evidence from RCTs on which to draw
strong conclusions regarding best practice.230
The presence of fetal anomalies such as anencephaly, con-
genital cardiac defects and TRAP almost doubles the perinatal
mortality rate.233 Hence, a detailed anatomy scan of both twins
552 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
is imperative. Although TTTS occurs in 15% of MCDA preg-
nancies, it is rare in MA twins (3%).234 This reflects their ubiq-
uitous AA anastomosis, which protects against the development
of TTTS. TTTS cannot be diagnosed on the basis of discordant
amniotic fluid volume in MA twins, so suggestive signs include
polyhydramnios, fetal growth discordance and discordant bladder
volumes. MA twins appear to have a lower incidence of growth
restriction than diamniotic twins, again arguably because of the
greater vascular anastomotic sharing.  adverse sequelae associated with multiple pregnancies. This proce-
Obstetric management. The main focus of antenatal manage-
ment is prevention of fetal demise and optimal timing of delivery,
and although the exact timing of delivery is contentious, elective
delivery at no later than approximately 33 weeks’ gestation has
been proposed.235
Antenatal fetal surveillance. This includes serial two weekly
scans to assess fetal growth, amniotic fluid volume and UA Doppler.
Expectant inpatient management from 25 weeks’ gestation with
daily or twice-daily cardiotocographs (CTGs) and frequent ultra-
sound monitoring has been recommended but is contentious.230
Not only is there no evidence that close fetal surveillance improves
fetal outcome, but there is significant risk for iatrogenic preterm
delivery, especially because CTGs before 28 weeks’ gestation con-
tain more fetal heart rate decelerations than accelerations.236  twins had comparable outcomes.246
Triplets and Higher Order Multiples
Perinatal outcome is significantly compromised in higher order
multiple pregnancies, primarily as a result of preterm delivery and
low birth weight. Apart from mortality, survivors have an increased
risk for long-term disabilities such as cerebral palsy, developmen-
tal delay and visual impairment. Cerebral palsy, not attributed to
prematurity, is 6 times more common in twins and 24 times more
common in triplets.237
High order multiples have considerably increased maternal
risks including gestational hypertension, diabetes, incompetent
cervix, requirement for tocolysis, bleeding and maternal mortal-
ity.238 The rate of triplets and higher order multiples has declined
with more stringent limits on the number of embryo transfers;
however, multifetal pregnancy reduction (MFPR) is not available
to all, and thus twins and higher order multiples continue to con-
fer a significant workload on many fetal medicine centres.
Triplets
Perinatal outcome by chorionicity. In triplets, outcome is
affected by chorionicity. MC and DC triplets have higher fetal
loss rates than trichorionic (TC) triplets. Complications such
as sIUGR, fetofetal transfusion syndrome and TAPS can arise.
Fetofetal transfusion syndrome has been successfully treated in
triplets, and a recent review of 126 triplets treated with fetoscopic
therapy concluded that the survival rate of at least one fetus in a
DC and MC pair was 95% and 89%, and the survival rate of all
triplets was 56% and 48%, respectively.239
It is estimated that one third of all TC triplets will be delivered
before 32 weeks’ gestation.240,241 In a recent large retrospective
cohort study of 159 pregnancies (477 neonates) consisting of 108
TC and 51 DC triplets, the perinatal mortality rate did not, how-
ever, correlate with chorionicity, and more than 94% of moth-
ers had all three infants survive to discharge.242 Pregnancies that
underwent selective reduction were excluded. DC triplets were
more likely to have very low birth weights and delivered before 30
weeks’ gestation compared with TC triplets. Neonatal mortality
rate appeared to be affected by the presence or absence of TTTS
for DC triplets, in which the 28-day survival rate was worse in DC
triplets with TTTS but was similar between DC not affected by
TTTS and TC pregnancies. There was also no difference in IUFD
based on DC or TC placentation, which is in contrast to previous
published reports, although the rate of fetofetal transfusion in this
study was less than other reports.243,244
Multifetal pregnancy reduction or nonselective fetal reduction
was developed about two decades ago in an attempt to reduce the
dure has since evolved over time, reflecting modifications in ART,
patient attitudes and practice among fetal medicine specialists.245 
Fetal Reduction
Multifetal pregnancy reduction (MFPR) is usually scheduled in
the first trimester between 11 and 13 weeks’ gestation to allow
for sufficient time for spontaneous first trimester reduction and to
facilitate the selection of fetuses with increased NT. It is typically
achieved via a transabdominal approach and targeted intracardiac
potassium chloride injection. Although transcervical approaches
have been associated with increased miscarriage risk, a large study
of 55 triplets reduced to twins compared with 78 nonreduced
Reduction of Triplets
The perinatal outcome of higher order multifetal gestations
reduced to twins has been shown to approach that of spontane-
ously conceived twins.246,247 The approach to reduction of trip-
lets to twins or singletons remains contentious. Compared with
unreduced triplets, reduced triplets from three to two fetuses in
a TC triplet pregnancy have been shown to have less prematurity
but no clear survival advantage.241,248,249 Also, a TC pregnancy
can be managed expectantly with reasonable perinatal outcomes,
considering a delivery at 32 weeks’ gestation would not confer
considerable advantage over a delivery at 34 weeks’ gestation in
the case of a reduction to twins.
It is perhaps a little more complex in the setting of a DC pair
because of the inherent risks with the pair sharing a placenta. In a
recent review of five studies and 331 triplets with a DC pair, the
miscarriage rate and severe preterm delivery rate for expectantly
managed triplets were 8.9% and 33.3%, respectively; for reduc-
tion of the MC pair, rates were 14.1% and 5.5% respectively, for
reduction of one fetus of the MC pair: 8.8% and 11.8%, respec-
tively, and for reduction of the ‘singleton’: 23.5% and 17.5%,
respectively.250 The authors suggested that if the ultimate goal is
a live-born infant, DC triplets can be managed expectantly, but
if the goal is to minimise severe preterm delivery and its atten-
dant sequelae, then fetal reduction can be considered. The wide
confidence intervals of the pooled results of this review should be
acknowledged. It is not every patient’s wish to undertake a fetal
reductive procedure in the absence of malformations or complica-
tions inherent to MC pairs, and it can be a very difficult decision
for a family to make. There are insufficient data to determine the
most appropriate method of reduction owing to a lack of RCTs
and long-term data. 
Selective Feticide
Selective feticide is usually performed at a later gestation than MFPR.
Dichorionic twins. Intracardiac potassium chloride injection
using a 22-gauge needle under ultrasound guidance is the pre-
ferred method in DC pregnancies. If the target is a structurally

anomalous fetus, then identification of that fetus is relatively
straightforward, but the identification of an aneuploid fetus of a
pair requires consistent labelling of each respective fetus from the
time of invasive testing. Pregnancy loss rates of between 4.7% and
9.6% have been reported.251,252  from 20% to 30%.256-258 Higher PPROM rates are reported for
Monochorionic twins. The indications for selective fetal reduc-
tion in MC twins include TRAP sequence, severe discordant fetal
growth restriction, discordant fetal anomalies, heterokaryotypic
chromosomal abnormality and severe TTTS in cases in which laser
therapy is not the preferred treatment. The choice of instrument
may be based on technical considerations and chorionicity. For MA
twins, direct cord occlusive techniques can be followed by cord tran-
section using laser or scissors to avoid entanglement-related demise.  Although MC twins have a considerably increased risk for peri-
Cord Occlusion
A variety of techniques have been described, aimed at occluding
the cord, including bipolar cord coagulation (BCC), laser coagula-
tion and fetoscopic ligation. These techniques require a relatively
large-diameter port (2.7–3.8 mm) into the sac of the target fetus,
and thus there is a risk for PPROM, haemorrhage and preterm
labour.249 BCC requires an operative sleeve though one large port,
and the blades completely obliterate the umbilical arteries and
vein. This technique requires sufficient amniotic fluid in the sac
for entry, and the sleeve permits fetoscopic visualisation of the
cord in addition to permitting additional manoeuvres.
Laser cord coagulation is performed using a 1.9- to 2.0-mm
fetoscope and a 400- or 600-μm Nd:YAG laser fibre; however,
as gestation advances, incomplete cessation of flow in addition
to intraoperative haemorrhage may limit the application of this
option.215 Umbilical cord ligation may be necessary at later gesta-
tions when the bipolar forceps may not be sufficient enough to
grasp a larger umbilical cord, and this can be achieved by ultra-
sound guided ligation through a single port.253
Intrafetal ablation may be used in cases of TRAP sequence,
and it involves targeting the intraabdominal umbilical or aorto-
pelvic vessels visualised on colour Doppler ultrasound and may be
superior to whole-cord techniques in this setting. Intrafetal abla-
tion can be achieved by monopolar thermocoagulation, interstitial
laser and radiofrequency energy. Interstitial laser ablation uses a
17-or 18-gauge needle under ultrasound guidance, and a 400- or
600-μm fibre is passed down the lumen to target vessels in short,
sharp bursts until colour flow ceases.254  cord entanglement and adverse outcome and the increased risk
Radiofrequency Ablation
Radiofrequency ablation has been adapted from oncology treat-
ments for use on solitary tumours. It uses high-frequency sinu-
soidal currents (400–500 Hz) to induce local ionic agitation and
thermal injury. A 17-gauge RFA probe is inserted under ultra-
sound guidance, and the needle is positioned within the fetal
abdomen; this usually requires approximately 3 minutes, though
a slower occlusive time may risk IUFD of the co-twin. It can be
carried out at an earlier gestational ages than cord occlusive tech-
niques and in the setting of oligo- or anhydramnios.
All selective reduction procedures are associated with a risk for
co-twin demise, with reports as high as 15%, although the risk is
still lower than that encountered with spontaneous IUFD of one
twin.255 A recent retrospective review of outcome of selective ter-
mination by BCC compared with RFA found that BCC was asso-
ciated with a higher rate of overall survival (85.2% vs 70.5%).256
CHAPTER 44  Multiple Pregnancy 553
The difference was attributed to an increase in the rate of co-twin
demise before 28 weeks’ gestation (59% for RFA vs 29% for
BCC), and this was thought to be due to the longer time it takes
to achieve cessation of blood flow with RFA. PPROM rates range
BCC with the larger port versus RFA and the smaller diameter
port.257 Newer techniques using microwave ablation require fur-
ther validation, although may be promising.258 
Conclusion
Multiple pregnancies remain a major concern to clinicians.
natal morbidity and mortality over their DC counterparts, DC
twins are not without risk. Although conventional aneuploidy
screening tests in twins as a rule underperform those in single-
tons, NIPT is emerging as a useful screening option for multiple
gestations with detection rates for trisomy 21 comparable to
those for singletons, albeit with higher sample failure rates. Inva-
sive prenatal testing in twins carries an additional 1% risk above
the background risk for miscarriage for both CVS and amnio-
centesis. Early determination of chorionicity is paramount for
the antenatal care of multiple pregnancies because of the inher-
ent and treatable risks associated with MC twins such as TTTS,
with invasive fetal treatments now extending to TAPS and cases
of selective IUGR. The past two decades have witnessed consid-
erable advances and modifications in the treatment for TTTS.
Fetoscopic laser ablation is the preferred treatment, the latest
modification of which is the Solomon technique, which appears
to confer the advantage of a lesser degree of recurrent TTTS
and TAPS and with at least comparable perinatal outcomes to
the standard fetoscopic approach. All in all, with intervention
for TTTS, the dual survival rate is estimated between 35% and
78%, and in survivors, normal long-term neurodevelopmental
outcome is expected in 82% to 94%.
Discordant fetal growth of 18% or greater is associated with
significant perinatal morbidity and mortality, and elective preterm
delivery is usually advocated. sIUFD in an MC pregnancy is asso-
ciated with a combined risk for co-twin demise or severe neurodis-
ability in the survivor of approximately 30%. The well-established
target elective delivery of MA twins at 32 weeks’ gestation has been
challenged owing to the lack of evidence for association between
for iatrogenic preterm delivery indicated by interpretations of
frequent inpatient CTG monitoring. Nonetheless, most experts
would not extend delivery beyond 33 weeks’ gestation.
Monochorionic and dichorionic triplet pregnancies have
higher fetal loss rates compared with TC triplet pregnancies.
MFPR is optimally carried out at 11 to 13 weeks’ gestation,
and miscarriage rates are low in experienced hands. Selective
fetal reduction is usually carried out at a later gestational age
and involves intracardiac potassium chloride for DC twins. The
options for selective feticide in MC twins include cord occlu-
sion techniques, interstitial and intrafetal options and RFA. As
with TTTS, the increasing use of fetal intervention for compli-
cated multiple pregnancies requires data on long-term outcomes
for survivors to substantiate treatment options and fully inform
patients of all eventual outcomes.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 18. Gjerris AC, Loft A, Pinborg A, et  al. First-
trimester screening markers are altered in
evaluate both fetuses: a case report. J Reprod
Med. 2016;61(3-4):167–170.
pregnancies conceived after IVF/ICSI. Ultra- 34. Reuss A, Gerlach H, Bedow W, et al. Mono-
1. Blondel B, Kaminski M. Trends in the
sound Obstet Gynecol. 2009;33(1):8–17. zygotic twins discordant for trisomy 18. Ultra-
occurrence, determinants, and conse-
19. Gil MM, Quezada MS, Revello R, et  al. sound Obstet Gynecol. 2011;38(6):727–728.
quences of multiple births. Semin Perinatol.
Analysis of cell-free DNA in maternal blood 35. Gilbert B, Yardin C, Briault S, et al. Prenatal
2002;26(4):239–249.
in screening for fetal aneuploidies: updated diagnosis of female monozygotic twins discor-
2. Fitzpatrick KE, Tuffnell D, Kurinczuk JJ, et al.
meta-analysis. Ultrasound Obstet Gynecol. dant for Turner syndrome: implications for
Pregnancy at very advanced maternal age: a
2015;45(3):249–266. prenatal genetic counselling. Prenat Diagn.
UK population-based cohort study. BJOG.
20. Canick JA, Kloza EM, Lambert-Messerlian 2002;22(8):697–702.
2017;124(7):1097–1106.
GM, et  al. DNA sequencing of maternal 36. Vincent MC, Heitz F, Tricoire J, et al. 22q11
3. Hamilton BE, Martin JA, Osterman MJ, et al.
plasma to identify Down syndrome and other deletion in DGS/VCFS monozygotic twins
Births: final data for 2014. Natl Vital Stat Rep.
trisomies in multiple gestations. Prenat Diagn. with discordant phenotypes. Genet Couns.
2015;64(12):1–64.
2012;32(8):730–734. 1999;10(1):43–49.
4. Martin JA, Hamilton BE, Ventura SJ, et  al.
21. Lau TK, Jiang F, Chan MK, et  al. Non- 37. He M, Pepperell JR, Gundogen F, et al. Mono-
Births: final data for 2009. Natl Vital Stat Rep.
invasive prenatal screening of fetal Down syn- chorionic twins discordant for mosaic trisomy
2011;60(1):1–70.
drome by maternal plasma DNA sequencing 14. Am J Genet A. 2014;164A(5):1227–1233.
5. Donovan EF, Ehrenkranz RA, Shankaran S,
in twin pregnancies. J Matern Fetal Neonatal 38. Zech NH, Wisser J, Natalucci G, et  al.
et al. Outcomes of very low birth weight twins
Med. 2013;26(4):434–437. Monochorionic-diamniotic twins discor-
cared for in the National Institute of Child
22. Xuan H, Zheng J, Chen M, et al. Non-inva- dant in gender from a naturally conceived
Health and Human Development Neonatal
sive prenatal testing of trisomies 21 and 18 pregnancy through postzygotic sex chromo-
Research Network’s intensive care units. Am J
by massively parallel sequencing of maternal some loss in a 47,XXY zygote. Prenat Diagn.
Obstet Gynecol. 1998;179:742–749.
plasma DNA in twin pregnancies. Prenat 2008;28(8):759–763.
6. Rettwitz-Volk W, Tran TM, Veldman A.
Diagn. 2014;34:335–340. 39. Butler GH, Flood K, Doyle E, et  al. Similar
Cerebral morbidity in preterm twins. J Matern
23. Grömminger S, Yagmur E, Erkan S, et  al. but different: identical pathology with differ-
Fetal Neonatal Med. 2003;13(4):218–223.
Fetal aneuploidy detection by cell-free DNA ing outcome in ‘not-so-identical’ twins. Br J
7. Conde-Agudelo A, Belizan JM, Lindmark
sequencing for multiple pregnancies and qual- Haematol. 2017;178(1):152–153.
G. Maternal morbidity and mortality associ-
ity issues with vanishing twins. J Clin Med. 40. Shimada A, Xu G, Toki T, et al. Fetal origin
ated with multiple gestations. Obstet Gynecol.
2014;3(3):679–692. of the GATA1 mutation in identical twins
2000;95:899–904.
24. Del Mar Gil M, Quezada MS, Bregant B, with transient myeloproliferative disorder and
8. Breathnach FM, Mc Auliffe FM, Geary M,
et al. Cell-free DNA analysis for trisomy risk acute megakaryoblastic leukemia accompany-
et  al. Optimum timing for planned deliv-
assessment in first-trimester twin pregnancies. ing Down syndrome. Blood. 2004;103(1):366.
ery of uncomplicated monochorionic and
Fetal Diagn Ther. 2014;35(3):204–211. 41. Schinzel AA, Smith DW, Miller JR. Monozy-
dichorionic twin pregnancies. Obstet Gynecol.
25. Bevilacqua E, Gil MM, Nicolaides KH, et al. gotic twining and structural defects. J Pediatr.
2012;119(1):50–59.
Performance of screening for aneuploidies by 1979;95(6):921–930.
9. Carroll SG, Soothill PW, Abdel-Fattah SA,
cell-free DNA analysis of maternal blood in 42. Chen CJ, Wang CJ, Yu MW, et al. Perinatal
et al. Prediction of chorionicity in twin preg-
twin pregnancies. Ultrasound Obstet Gynecol. mortality and prevalence of major congenital
nancies at 10-14 weeks of gestation. BJOG.
2015;45(1):61–66. malformations of twins in Taipei city. Acta
2002;109(2):182–186.
26. Sarno L, Revello R, Hanson E, et al. Prospec- Genet Med Gemellol (Roma). 1992;41(2-
10. Stagiannis KD, Sepulveda W, Southwell
tive first-trimester screening for trisomies by 3):197–203.
D, et  al. Ultrasonographic measurement of
cell-free DNA testing of maternal blood in 43. Cameron AH, Edwards JH, Derom R, et  al.
the dividing membrane in twin pregnancy
twin pregnancy. Ultrasound Obstet Gynecol. The value of twin surveys I the study of mal-
during the second and third trimesters: a
2016;47(6):705–711. formations. Eur J Obstet Gynecol Reprod Biol.
reproducibility study. Am J Obstet Gynecol.
27. Tan Y, Gao Y, Lin G, et  al. Noninvasive 1983;14(5):347–356.
1995;173(5):1546–1550.
prenatal testing (NIPT) in twin pregnancies 44. Little J, Bryan E. Congenital anomalies in
11. Senat MV, Quarello E, Levaillant JM, et  al.
with treatment of assisted reproductive tech- twins. Semin Perinatol. 1986;10(1):50–64.
Determining chorionicity in twin gestations:
niques (ART) in a single center. Prenat Diagn. 45. Windham GC, Bjerkedal T, Sever LE.
three-dimensional (3D) multiplanar sono-
2016;36(7):672–679. The association of twining and neural tube
graphic measurement of intra-amniotic mem-
28. Struble CA, Syngelaki A, Oliphant A, et  al. defects: studies in Los Angeles, California,
brane thickness. Ultrasound Obstet Gynecol.
Fetal fraction estimate in twin pregnancies and Norway. Acta Genet Med Gemellol (Roma).
2006;28(5):665–669.
using directed cell-free DNA analysis. Fetal 1982;31(3-4):165–172.
12. Multifetal gestations. twin, triplet and high-
Diagn Ther. 2014;35(3):199–203. 46. Doyle PE, Beral V, Botting B, et  al. Con-
order multifetal pregnancies. ACOG Pract
29. Curnow KJ, Wilkins-Haug L, Ryan A, et  al. genital malformations in twins in England
Bull. 2014;144.
Detection of triploid, molar, and vanishing and Wales. J Epidemiol Community Health.
13. Monochorionic twin pregnancy, management.
twin pregnancies by single-nucleotide poly- 1991;45(1):43–48.
RCOG Green-Top Guideline 51; 2016.
morphism-based noninvasive prenatal test. 47. Little J, Nevin NC. Congenital anomalies
14. Bush MC, Malone FD. Down syn-
Am J Obstet Gynecol. 2015;212(1):79.e1–e9. in twins in Northern Ireland II: neural tube
drome screening in twins. Clin Perinatol.
30. Futch T, Spinosa J, Bhatt S, et  al. Initial defects, 1974-1979. Acta Genet Med Gemellol
2005;32(2):373–386.
clinical laboratory experience in noninvasive (Roma). 1989;38(1-2):17–125.
15. Chasen ST, Perni SC, Kalish RB, et al. First-
prenatal testing for fetal aneuploidy from 48. Olson CK, Keppler-Noreuil KM, Romitti PA,
trimester risk assessment for trisomies 21 and
maternal plasma DNA samples. Prenat Diagn. et al. In vitro fertilisation is associated with an
18 in twin pregnancy. Am J Obstet Gynecol.
2013;33(6):569–574. increase in major birth defects. Fertil Steril.
2007;197(4):374.e1–e3.
31. Porreco RP, Garite TJ, Maurel K, et al. Non- 2005;84(5):1308–1315.
16. Audibert F, Gagnon A, Genetics Committee
invasive prenatal screening for fetal trisomies 49. Springer S, Mlczoch E, Krampl-Bettelheim
of the Society of Obstetricians and Gynaeco-
21, 18, 13 and common sex chromosome E, et  al. Congenital heart disease in mono-
logists of Canada; Prenatal Diagnosis Com-
aneuploidies from maternal blood using mas- chorionic twins with and without twin–
mittee of the Canadian College of Medical
sively parallel genomic sequencing of DNA. twin transfusion syndrome. Prenat Diagn.
Geneticists. Prenatal screening for and diagno-
Am J Obstet Gynecol. 2014;21(4):365.e1–e12. 2014;34(10):994–999.
sis of aneuploidy in twin pregnancies. J Obstet
32. Dixit A, Tanteles G, Ocraft K, et al. Monozy- 50. Simpson LL. Ultrasound in twins: dichori-
Gynaecol Can. 2011;33(7):754–767.
gotic twins discordant for trisomy 13: coun- onic and monochorionic. Semin Perinatol.
17. Spencer K, Kagan KO, Nicolaides KH.
selling and management issues. J Perinatol. 2013;37(5):348–358.
Screening for trisomy 21 in twin pregnancies
2012;32(8):639–641. 51. Allen SR, Gray LJ, Frentzen BH, et  al.
in the first trimester: an update of the impact
33. Macatangga M, De la Calle M, Torres Ultrasonographic diagnosis of congenital
of chorionicity on maternal serum markers.
ML, et  al. Monozygotic monochorionic anomalies in twins. Am J Obstet Gynecol.
Prenat Diagn. 2008;28(1):49–52.
twins discordant for trisomy 21: a reason to 1991;165:1056–1060.

553.e1
553.e2 References
52. Malone FD, Craigo SD, Chelmow D. e tal.
Outcome of twin gestations complicated
by a single anomalous fetus. Obstet Gynecol.
1996;88(1):1–5.
53. Gul A, Cebeci A, Aslan H, et  al. Perina-
tal outcomes of twin pregnancies discor-
dant for major anomalies. Fetal Diagn Ther.
2005;20(4):244–248.
54. Sebire NJ, Sepulveda W, Hughes KS, et  al.
Management of twin pregnancies discor-
dant for anencephaly. Br J Obstet Gynaecol.
1997;104(2):216–219.
55. Sebire NJ, Snijders RJ, Santiago C, et  al.
Management of twin pregnancies with
fetal trisomies. Br J Obstet Gynaecol.
1997;104(2):220–222.
56. Ong SS, Zamora J, Khan KS, et al. Prognosis
for the co-twin following single-twin death: a
systematic review. BJOG. 2006;113(9):992–
998.
57. Agarwal K, Alfirevic Z. Pregnancy loss after
chorionic villous sampling and genetic
amniocentesis in twin pregnancies: a sys-
tematic review. Ultrasound Obstet Gynecol.
2012;40(2):128–134.
58. Vink J, Wapner R, D’Alton ME. Prenatal
diagnosis in twin gestations. Semin Perinatol.
2012;36(3):169–174.
59. Smith AP, Ong S, Smith NC, et  al. A pro-
spective longitudinal study of growth velocity
in twin pregnancy. Ultrasound Obstet Gynecol.
2001;18(5):485–487.
60. Victoria A, Mora G, Arias F. Perinatal out-
come, placental pathology, and severity of dis-
cordance in monochorionic and dichorionic
twins. Obstet Gynecol. 2001;97(2):310–315.
61. Chang YL, Chang SD, Chao AS, et al. Clini-
cal outcome and placental territory ratio of
monochorionic twin pregnancies and selective
intra-uterine growth restriction with differ-
ent types of umbilical artery Doppler. Prenat
Diagn. 2009;29(3):253–256.
62. Blickstein I, Mincha S, Golman R, et al. The
Northwestern twin chorionicity study: testing
the ‘placental crowding’ hypothesis. J Perinat
Med. 2006;34(2):158–161.
63. Breathnach FM, McAuliffe FM, Geary M,
et  al. Definition of intertwin birth weight
discordance. Obstet Gynecol. 2011;118(1):94–
103.
64. Blickstein I, Kalish RB. Birthweight dis-
cordance in multiple pregnancy. Twin Res.
2003;6(6):523–526.
65. Patterson RM, Wood RC. What is birthweight
discordance? Am J Perinatol. 1990;7(3):217–
219.
66. Breathnach FM, Malone FD. Fetal growth
disorders in twin gestations. Semin Perinatol.
2012;36(3):175–181.
67. Hsieh TT, Chang TC, Chiu TH, et al. Growth
discordancy, birthweight and neonatal adverse
events in third trimester twin gestations. Gyne-
col Obstet Invest. 1994;38(1):36–40.
68. Talbot GT, Goldstein RF, Nesbitt T, et  al.
Is size discordancy an indication for deliv-
ery of preterm twins? Am J Obstet Gynecol.
1997;177(5):1050–1054.
69. Cohen SB, Elizur SE, Goldenberg M,
et  al. Outcome of twin pregnancies with
extreme weight discordancy. Am J Perinatal.
2001;18(8):427–432.
70. Kilic M, Aygun C, Kaynar-Tuncel E, et  al.
Does birth weight discordance in preterm
twins affect neonatal outcome? J Perinatol.
2006;26(5):268–272.
71. Kalish RB, Chasen ST, Gupta M, et al. First
trimester prediction of growth discordance
in twin gestations. Am J Obstet Gynecol.
2003;189(3):706–709.
72. Johansen ML, Oldenburg A, Rosthoj S, et al.
Crown-rump length discordance in the first
trimester: a predictor of adverse outcome in
twin pregnancies? Ultrasound Obstet Gynecol.
2014;43(3):277–283.
73. Hill LM, Guzick D, Chenevey P, et  al. The
sonographic assessment of twin growth discor-
dancy. Obstet Gynecol. 1994;84(4):501–504.
74. O’Connor C, Mc Auliffe FM, Breathnach
FM, et  al. Prediction of outcome in twin
pregnancy with first and second trimester
ultrasound. J Matern Fetal Neonatal Med.
2013;26(10):1030–1035.
75. Vanderheyden TM, Fichera A, Pasquini L,
et al. Increased latency of absent end-diastolic
flow in the umbilical artery of monochori-
onic twin fetuses. Ultrasound Obstet Gynecol.
2005;26(1):44–49.
76. Denbow ML, Cox P, Taylor M, et  al. Pla-
cental angioarchitecture in monochori-
onic twin pregnancies: relationship to fetal
growth, fetofetal transfusion syndrome, and
pregnancy outcome. Am J Obstet Gynecol.
2000;182(2):417–426.
77. Hecher K, Jauniaux E, Campbell S, et  al.
Artery-to-artery anastomosis in mono-
chorionic twins. Am J Obstet Gynecol.
1994;171(2):570–572.
78. Wee LY, Taylor MJ, Vanderheyden T, et  al.
Transmitted arterio-arterial anastomosis
waveforms causing cyclically intermittent
absent/reversed end-diastolic umbilical artery
flow in monochorionic twins. Placenta.
2003;24(7):772–778.
79. Gratacos E, Lewi L, Carreras E, et al. Incidence
and characteristics of umbilical artery intermit-
tent absent and/or reversed end-diastolic flow
in complicated and uncomplicated monochori-
onic twin pregnancies. Ultrasound Obstet Gyne-
col. 2004;23(5):456–460.
80. Gratacos E, Lewi L, Munoz B, et  al. A clas-
sification system for selective intrauterine
growth restriction in monochorionic twins
according to umbilical artery Doppler flow in
the smaller twin. Ultrasound Obstet Gynecol.
2007;30(1):28–34.
81. Ishii K, Murakoshi T, Takahashi Y, et al. Peri-
natal outcome of monochorionic twins with
selective intrauterine growth restriction and
different types of umbilical artery Doppler
under expectant management. Fetal Diagn
Ther. 2009;26(3):157–161.
82. Engineer N, Fisk N. Multiple pregnancy. In:
Rodeck CH, Whittle MJ, eds. Fetal Medicine:
Basic Science and Clinical Practice. London:
Churchill Livingstone; 2009.
83. Halling C, Malone FD, Breathnach FM, et al.
Neuro-developmental outcome of a large
cohort of growth discordant twins. Eur J Pedi-
atr. 2016;175(3):381–389.
84. Gratacos E, Carreras E, Becker J, et al. Preva-
lence of neurological damage in monochori-
onic twins with selective intrauterine growth
restriction and intermittent absent or reversed
end-diastolic umbilical artery flow. Ultrasound
Obstet Gynecol. 2004;24(2):159–163.
85. 
Management of multiple pregnancy. HSE Clini-
cal Practice Guideline; June 2012.
86. Corcoran S, Breathnach FM, Burke G,
et  al. Dichorionic twin ultrasound surveil-
lance: sonography every 4 weeks significantly
underperforms sonography every 2 weeks:
results of the prospective Multicenter ESPRiT
Study. Am J Obstet Gynecol. 2015;213(4):551.
e1–e5.
87.  Bebbington MW, Danzer E, Moldenhauer J,
et al. Radiofrequency ablation vs bipolar umbili-
cal cord coagulation in the management of com-
plicated monochorionic pregnancies. Ultrasound
Obstet Gynecol. 2012;40(3):319–324.
88. Lewi LGE, Gratacos E, Ortibus E, et al. Preg-
nancy and infant outcome of 80 consecutive
cord coagulations in complicated monochori-
onic multiple pregnancies. Am J Obstet Gyne-
col. 2006;194(3):782–789.
89. Quintero RA, Bornick PW, Morales WJ, et al.
Selective photocoagulation of communicat-
ing vessels in the treatment of monochorionic
twins with selective growth retardation. Am J
Obstet Gynecol. 2001;185(3):689–696.
90. Peeva G, Bower S, Orosz L, et al. Endoscopic
placental laser coagulation in monochori-
onic diamniotic twins with type II selective
fetal growth restriction. Fetal Diagn Ther.
2015;38(2):86–93.
91. Ishii K, Nakata M, Wada S, et  al. Feasibil-
ity and preliminary outcomes of fetoscopic
laser photocoagulation for monochorionic
twin gestation with selective intrauterine
growth restriction accompanied by severe
oligohydramnios. J Obstet Gynaecol Res.
2015;41(11):1732–1737.
92.  Chalouhi GE, Marangoni MA, Quibel T, et al.
Active management of selective intrauterine
growth restriction with abnormal Doppler in
monochorionic diamniotic twin pregnancies
diagnosed in the second trimester of pregnancy.
Prenat Diagn. 2013;33(2):109–115.
93. Gratacos E, Antolin E, Lewi L, et al. Mono-
chorionic twins with selective intrauterine
growth restriction and intermittent absent
or reversed end-diastolic flow (type III): fea-
sibility and perinatal outcome of fetoscopic
placental laser coagulation. Ultrasound Obstet
Gynecol. 2008;31(6):669–675.
94. Hillman SC, Morris RK, Kilby MD. Co-twin
prognosis after single fetal death: a system-
atic review and meta-analysis. Obstet Gynecol.
2011;118(4):928–940.
95. Dickey RP, Taylor SN, Lu PY, et al. Sponta-
neous reduction of multiple pregnancy: inci-
dence and effect on outcome. Am J Obstet
Gynecol. 2002;186(1):77–83.
96. D’Alton ME, Simpson LL. Syndromes in
twins. Semin Perinatol. 1995;19(5):375–386.
97. Lee YM, Wylie BJ, Simpson LL, et  al. Twin
chorionicity and the risk of stillbirth. Obstet
Gynecol. 2008;111:301–308.
98. Pharoah PO, Adi Y. Consequences of in-
utero death in a twin pregnancy. Lancet.
2000;355(9215):1597–1602.
99. Bulla M, von Lilien T, Goecke H, et  al.
Renal and cerebral necrosis in survivor after
in utero death of co-twin. Arch Gynecol.
1987;240(2):119–124.
100. Szymonowicz W, Preston H, Yu VY. The
surviving monozygotic twin. Arch Dis Child.
1986;61(5):454–458.
101. Genova L, Sueters M, vanSteenis A, et  al.
Renal failure after single fetal demise in mono-
chorionic twins: incidence and description of
a case. Fetal Diagn Ther. 2014;35(4):302–305.
102. Benirschke K. Intrauterine death of a twin:
mechanisms, implications for surviving twin,
and placental pathology. Semin Diagn Pathol.
1993;10(3):222–231.

103. Bajoria R, Wee LY, Anwar S, et al. Outcome of
twin pregnancies complicated by single intra-
uterine death in relation to vascular anatomy
of the monochorionic placenta. Hum Reprod.
1999;14(8):2124–2130.
104. Bonellie SR, Currie D, Chalmers J. Com-
parison of risk factors for cerebral palsy in
twins and singletons. Dev Med Child Neurol.
2005;47(9):587–591.
105. Griffiths PD, Sharrack S, Chan KL, et al. Fetal
brain injury in survivors of twin pregnancies
complicated by demise of one twin as assessed
by in-utero MR imaging. Prenat Diagn.
2015;35(6):583–591.
106. van Klink JM, van Steenis A, Steggerda SJ,
et  al. Single fetal demise in monochori-
onic pregnancies: incidence and patterns of
cerebral injury. Ultrasound Obstet Gynecol.
2015;45(3):294–300.
107. Simonazzi G, Segata M, Ghi T, et  al. Accu-
rate neurosonographic prediction of brain
injury in the surviving fetus after the death of
a monochorionic cotwin. Ultrasound Obstet
Gynecol. 2006;27(5):517–521.
108. Luu TM, Vohr B. Twinning on the brain:
the effect on neurodevelopmental out-
comes. Am J Med Genet C Semin Med Genet.
2009;151(2):142–147.
109. O’Donoghue K, Rutherford MA, Engineer
N, et  al. Transfusional fetal complications
after single intrauterine fetal death in mono-
chorionic multiple pregnancy are reduced but
not prevented by vascular occlusion. BJOG.
2009;116(6):804–812.
110. Spruijt M, Steggerda S, Rath M, et al. Cere-
bral injury in twin-twin transfusion syndrome
treated with fetoscopic laser surgery. Obstet
Gynecol. 2012;120(1):15–20.
111. Weisz B, Hoffmann C, Ben-Baruch S,
et  al. Early detection by diffusion-weighted
sequence magnetic resonance imaging of
severe brain lesions after fetoscopic laser coag-
ulation for twin-twin transfusion syndrome.
Ultrasound Obstet Gynecol. 2014;44(1):44–49.
112. Hoffmann C, Weisz B, Yinon Y, et  al. Dif-
fusion MRI findings in monochorionic twin
pregnancies after intrauterine fetal death. Am J
Neuroradiol. 2013;34(1):212–216.
113. Glenn OA, Norton ME, Goldstein RB,
et al. Prenatal diagnosis of polymicrogyria by
fetal magnetic resonance imaging in mono-
chorionic cotwin death. J Ultrasound Med.
2005;24(5):711–716.
114. Quarello E, Molho M, Ville Y. Incidence,
mechanisms, and patterns of fetal cerebral
lesions in twin-to-twin transfusion syndrome.
J Matern Fetal Neonatal Med. 2007;20(8):589–
597.
115. McPherson JA, Odibo AO, Shanks AL, et al.
Impact of chorionicity on risk and timing of
intrauterine fetal demise in twin pregnancies.
Am J Obstet Gynecol. 2012;207(3):190.e1–e6.
116. Bryan E. The impact of multiple preterm
births on the family. BJOG. 2003;110(suppl
20):24–28.
117. D’Alton ME, Newton ER, Cetrulo CL. Intra-
uterine fetal demise in multiple gestation. Acta
Genet Med Gemellol (Roma). 1984;33(1):43–49.
118. Shek NW, Hillman SC, Kilby MD. Single-
twin demise: pregnancy outcome. Best Pract
Res Clin Obstet Gynaecol. 2014;28(2):249–
263.
119. Levine D. Timing of MRI in pregnancy,
repeat exams, access, and physician qualifica-
tions. Semin Perinatol. 2013;37(5):340–344.
120. Bahado-Singh RO, Goncalves LF. Techniques,
terminology, and indications for MRI in preg-
nancy. Semin Perinatol. 2013;37(5):334–339.
121. Senat MV, Loizeau S, Couderc S, et  al. The
value of middle cerebral artery peak systolic
velocity in the diagnosis of fetal anemia after
intrauterine death of one monochorionic
twin. Am J Obstet Gynecol. 2003;189(5):1320–
1324.
122. Quarello E, Stirnemann J, Nassar M, et  al.
Outcome of anaemic monochorionic single
survivors following early intrauterine rescue
transfusion in cases of feto-fetal transfusion
syndrome. BJOG. 2008;115(5):595–601.
123. Tanawattanacharoen S, Taylor MJ, Letsky
EA, et  al. Intrauterine rescue transfusion in
monochorionic multiple pregnancies with
recent single intrauterine death. Prenat Diagn.
2001;21(4):274–278.
124. Lewi L, Jani J, Blickstein I, et  al. The out-
come of monochorionic diamniotic twin ges-
tations in the era of invasive fetal therapy: a
prospective cohort study. Am J Obstet Gynecol.
2008;199(5):514.e1–e8.
125. Lopriore E, van Wezel-Meijler G, Middeldorp
JM, et  al. Neurodevelopmental outcome after
laser therapy for twin-twin transfusion syn-
drome. Am J Obstet Gynecol. 2007;196(1):e20;
author reply e20–e21.
126. Quintero RA, Morales WJ, Allen MH, et al.
Staging of twin-twin transfusion syndrome.
J Perinatol. 1999;19(8 Pt 1):550–555.
127. Senat MV, Deprest J, Boulvain M, Paupe A,
Winer N, Ville Y. Endoscopic laser surgery
versus serial amnioreduction for severe twin-
twin-transfusion syndrome. N Engl J Med.
2004;351(2):136–144.
128. Taylor MJ, Govender L, Jolly M, et al. Valida-
tion of the Quintero staging system for twin-
twin transfusion syndrome. Obstet Gynecol.
2000;100(6):1257–1265.
129. Rossi AC, D’Addario V. Laser therapy and
serial amnioreduction as treatment for twin-
twin transfusion syndrome: a meta-analysis
and review of the literature. Am J Obstet Gyne-
col. 2008;198(2):147–152.
130. Linskens IH, de Mooij YM, Twisk JW,
et  al. Discordance in nuchal translucency
measurements in monochorionic diamni-
otic twins as predictor of twin-twin trans-
fusion syndrome. Twin Res Hum Genet.
2009;12(6):605–610.
131. Fratelli N, Prefumo F, Fichera A, et al. Nuchal
translucency thickness and crown rump length
discordance for the prediction of outcome in
monochorionic diamniotic pregnancies. Early
Hum Dev. 2011;87(1):27–30.
132. Stagnati V, Zanardini C, Fichera A, et al. Early
prediction of twin-to-twin transfusion syn-
drome: systematic review and meta-analysis.
Ultrasound Obstet Gynecol. 2017;49(5):573–
582.
133. Matias A, Montenegro N, Loureiro T, et  al.
Screening for twin-twin transfusion syndrome
at 11-14 weeks of pregnancy: the key role of
ductus venosus blood flow assessment. Ultra-
sound Obstet Gynecol. 2010;35(2):142–148.
134. Huber A, Diehl W, Zikulnig L, et  al. Peri-
natal outcome in monochorionic twin
pregnancies complicated by amniotic fluid
discordance without severe twin-twin trans-
fusion syndrome. Ultrasound Obstet Gynecol.
2006;27(1):48–52.
135. Hinch E, Henry A, Wilson I, et  al. Out-
comes of stage I TTTS or liquor discordant
References 553.e3
twins: a single-centre review. Prenat Diagn.
2016;36(6):507–514.
136. Kawamura H, Ishii K, Mabuchi A, et al. Peri-
natal outcome of monochorionic diamniotic
twin pregnancies complicated with isolated
amniotic fluid volume abnormality of one
twin less than 26 weeks of gestation. J Obstet
Gynaecol Res. 2016;42(12):1657–1665.
137. Hayashi S, Anami A, Ishii K, et al. Outcome
of monochorionic twin pregnancies with
moderate amniotic fluid discordance adjoin-
ing twin-twin transfusion syndrome. Prenat
Diagn. 2016;36(2):170–176.
138. Lewi L, Lewi P, Diemert A, et al. The role of
ultrasound examination in the first trimester
and at 16 weeks’ gestation to predict fetal
complications in monochorionic diamni-
otic twin pregnancies. Am J Obstet Gynecol.
2008;199(5):493.e1–e7.
139. Sebire NJ, Souka A, Skentou H, et  al. Early
prediction of severe twin-to-twin transfusion
syndrome. Hum Reprod. 2000;15(9):2008–
2010.
140. De Paepe ME, Shapiro S, Greco D, et  al.
Placental markers for twin-to-twin transfu-
sion syndrome in diamniotic-monocho-
rionic twins: a morphometric analysis of
deep artery-to-vein anastomoses. Placenta.
2010;31(4):269–276.
141. Sato Y, Ishii k, Yonetani N, Yamamoto R,
Mitsuda N. Twin-twin transfusion Syndrome
in cases with suspected close proximity of
umbilical cord insertions. Fetal Diagn Ther.
2016;40(4):306–309.
142. Kusanovic JP, Romero R, Gotsch F, et al. Dis-
cordant placental echogenicity: a novel sign
of impaired placental perfusion in twin-twin
transfusion syndrome? J Matern Fetal Neonatal
Med. 2010;23(1):103–106.
143. Simpson LL. Twin-twin transfusion syn-
drome. Am J Obstet Gynecol. 2013;208(1):3–
18.
144. Denbow ML, Cox P, Taylor M, et  al. Pla-
cental angioarchitecture in monochori-
onic twin pregnancies: relationship to fetal
growth, fetofetal transfusion syndrome, and
pregnancy outcome. Am J Obstet Gynecol.
2000;182(2):417–426.
145. Nikkels PG, Hack KE, van Gemert MJ.
Pathology of twin placentas with special atten-
tion to monochorionic twin placentas. J Clin
Pathol. 2008;61(12):1247–1253.
146. Bajoria R, Wigglesworth J, Fisk NM. Angioar-
chitecture of monochorionic placentas in rela-
tion to the twin-twin transfusion syndrome.
Am J Obstet Gynecol. 1995;172(3):856–863.
147. Galea P, Barigye O, Wee L, et al. The placenta
contributes to activation of the renin angio-
tensin system in twin-twin transfusion syn-
drome. Placenta. 2008;29(8):734–742.
148. Bahtiyar MO, Dulay AT, Weeks BP, et  al.
Prevalence of congenital heart defects in
monochorionic/diamniotic twin gestations: a
systematic literature review. J Ultrasound Med.
2007;26(11):1491–1499.
149. Lopriore E, Bokenkamp R, Rijlaarsdam
M, et  al. Congenital heart disease in twin-
twin transfusion syndrome treated with
fetosopic laser surgery. Congenit Heart Dis.
2007;2(1):38–43.
150. Barrea C, Alkazaleh F, Ryan G, et al. Prenatal
cardiovascular manifestations in the twin-to-
twin transfusion syndrome recipients and the
impact of therapeutic amnioreduction. Am J
Obstet Gynecol. 2005;192(3):892–902.
553.e4 References
151. Lougheed J, Sinclair BG, Fung Kee Fung K,
et al. Acquired right ventricular outflow tract
obstruction in the recipient twin in twin-twin
transfusion syndrome. J Am Coll Cardiol.
2001;38(5):1533–1538.
152. Michelfelder E, Gottliebson W, Border E, et al.
Early manifestations and spectrum of recipient
twin cardiomyopathy in twin-twin transfusion
syndrome: relation to Quintero stage. Ultra-
sound Obstet Gynecol. 2007;30(7):965–971.
153. Moon-Grady AJ, Rand L, Lemley B, et  al.
Effect of selective fetoscopic laser photoco-
agulation therapy for twin-twin transfusion
syndrome on pulmonary valve pathology in
recipient twins. Ultrasound Obstet Gynecol.
2011;37(1):27–33.
154. Halvorsen CP, Bilock SL, Pilo C, et al. Child-
hood cardiac function after twin-twin trans-
fusion syndrome: a 10-year follow up. Acta
Paediatr. 2009;98(9):1468–1474.
155. Rychik J, Tian Z, Bebbington M, et  al. The
twin-twin transfusion syndrome: spectrum of
cardiovascular abnormality and development
of a cardiovascular score to assess severity of
disease. Am J Obstet Gynecol. 2007;197(4):
392.e1–e8.
156. Rychik J, Zeng S, Bebbington M, et  al.
Speckle tracking-derived myocardial tissue
deformation imaging in twin-twin transfu-
sion syndrome: differences in strain and strain
rate between donor and recipient twins. Fetal
Diagn Ther. 2012;32(1-2):131–137.
157. Moise Jr KJ, Dorman K, Lamvu G, et  al. A
randomized trial of amnioreduction versus
septostomy in the treatment of twin-twin
transfusion syndrome. Am J Obstet Gynecol.
2005;193(3 Pt 1):701–707.
158. Roberts D, Gates S, Kilby M, et al. Interven-
tions for twin-twin transfusion syndrome: a
Cochrane review. Ultrasound Obstet Gynecol.
2008;31(6):701–711.
159. Akkermans J, Peeters SH, Klumper FJ,
et  al. Twenty-five years of fetoscopic laser
coagulation in twin-twin transfusion syn-
drome: a systematic review. Fetal Diagn Ther.
2015;38(4):241–253.
160. van Klink JM, Koopman HM, van Zwet EW,
et al. Cerebral injury and neurodevelopmental
impairment after amnioreduction versus laser
surgery for twin-twin transfusion syndrome:
a systematic review and meta-analysis. Fetal
Diagn Ther. 2013;33(2):81–89.
161. Spruijt M, Steggerda S, Rath M, et al. Cere-
bral injury in twin-twin transfusion syndrome
treated with fetoscopic laser surgery. Obstet
Gynecol. 2012;120(1):15–20.
162. Rossi AC, Vanderbilt D, Chmait RH. Neu-
rodevelopmental outcomes after laser surgery
for twin-twin transfusion syndrome: a system-
atic review and meta-analysis. Obstet Gynecol.
2011;118(5):1145–1150.
163. Cooley S, Walsh J, Mahony R, et al. Success-
ful fetoscopic laser coagulation for twin-twin
transfusion syndrome under local anaesthesia.
Ir Med J. 2011;104(6):187–190.
164. Müllers SM, McAuliffe FM, Kent E, et  al.
Outcome following selective fetoscopic
laser ablation for twin to twin transfusion
syndrome: an 8 year national collaborative
experience. Eur J Obstet Gynecol Reprod Biol.
2015;191:125–129.
165. Slaghekke F, Lopriore E, Lewi L, et  al.
Fetoscopic laser coagulation of the vas-
cular equator versus selective coagulation
for twin-twin transfusion syndrome: an
open-label randomised controlled trial. Lancet.
2014;383(9935):2144–2151.
166. Quintero RA, Morales WJ, Mendoza G, et al.
Selective photocoagulation of placental vessels
in twin-twin transfusion syndrome: evolution
of a surgical technique. Obstet Gynecol Surv.
1998;53(suppl 12):S97–S103.
167. Quintero RA, Ishii K, Chmait RH, et  al.
Sequential selective laser photocoagulation of
communicating vessels in twin-twin transfu-
sion syndrome. J Matern Fetal Neonatal Med.
2007;20(10):763–768.
168. Akkermans J, Peeters SH, Klumper FJ, et al.
Is the sequential laser technique for twin-twin
transfusion syndrome truly superior to the
standard selective technique? A meta-analysis.
Fetal Diagn Ther. 2015;37(4):251–258.
169. Walsh CA, McAuliffe FM. Recurrent twin–
twin transfusion syndrome after selective
fetoscopic laser photocoagulation: a system-
atic review of the literature. Ultraound Obstet
Gynecol. 2012;40(5):506–512.
170. Lopriore E, Middeldorp JM, Oepkes D, et al.
Residual anastomoses after fetoscopic laser
surgery in twin-twin transfusion syndrome:
frequency, associated risks and outcome.
Placenta. 2007;28(2-3):204–208.
171. Slaghekke F, Kist WJ, Oepkes D, et al. Twin
anemia-polycythemia sequence: diagnostic
criteria, classification, perinatal management
and outcome. Fetal Diag Ther. 2010;27(4):
181–190.
172. Ruano R, Rodo C, Peiro JL, et al. Fetoscopic
laser ablation of placental anastomoses in
twin-twin transfusion syndrome using ‘Solo-
mon technique.’ Ultrasound Obstet Gynecol.
2013;42(4):434–439.
173. Baschat AA, Barber J, Pedersen N, et al. Out-
come after fetoscopic selective laser ablation
of placental anastomoses vs equatorial laser
dichorionization for the treatment of twin–
twin transfusion syndrome. Am J Obstet Gyne-
col. 2013;209(3):234.e1–e8.
174. Baud D, Windrim R, Keunen J, et  al. Feto-
socpic laser therapy for twin-twin transfusion
syndrome before 17 and after 26 weeks’ gesta-
tion. Am J Obstet Gynecol. 2013;208(3):197.
e1–e7.
175. Valsky DV, Eixarch E, Martinez-Crespo JM,
et  al. Fetoscopic laser surgery for twin-twin
transfusion syndrome after 26 weeks of gesta-
tion. Fetal Diagn Ther. 2012;31(1):30–34.
176. Emery SP, Hasley SK, Catov JM. t al. North
American Fetal Therapy Network: interven-
tion vs expectant management for stage 1
twin-twin transfusion syndrome. Am J Obstet
Gynecol. 2016;215(3):346.e1–e7.
177. Quintero RA, Dickinson JE, Morales WJ,
et  al. Stage-based treatment of twin-twin
transfusion syndrome. Am J Obstet Gynecol.
2003;188(5):1333–1340.
178. Huber A, Diehl W, Bregenzer T, et al. Stage-
related outcome in twin-twin transfusion syn-
drome treated by fetoscopic laser coagulation.
Obstet Gynecol. 2006;108(2):333–337.
179. Wagner MM, Lopriore E, Klumper FJ, et al.
Short and long term outcome in stage 1
twin-to-twin transfusion syndrome treated
with laser surgery compared with conser-
vative management. Am J Obstet Gynecol.
2009;201(3):286.e1–e6.
180. Rossi AC, D’Addario V. Survival outcomes of
twin-twin transfusion syndrome stage I: sys-
tematic review of the literature. Am J Perinatol.
2013;30(1):5–10.
181. Cavicchioni O, Yamamoto M, Robyr R, et al.
Intrauterine fetal demise following laser treat-
ment in twin-to-twin transfusion syndrome.
BJOG. 2006;113(5):590–594.
182. Skupski DW, Luks FI, Walker M, et al. Pre-
operative predictors of death in twin-to-twin
transfusion syndrome treated with laser abla-
tion of placental anastomoses. Am J Obstet
Gynecol. 2010;203(4):388.e1–e11.
183. Diehl W, Diemert A, Hecher K. Twin-twin
transfusion syndrome: treatment and out-
come. Best Prac Res Clin Obstet Gynecol.
2014;28(2):227–238.
184. Snowise S, Mann LK, Moise Jr KJ, et  al. Pre-
term premature rupture of membranes after
fetoscopic laser surgery for twin-twin transfu-
sion syndrome. Ultrasound Obstet Gynecol.
2017;49(5):607–611.
185. Petersen SG, Gibbons KS, Luks FI, et al. The
impact of entry technique and access diameter
on Prelabour rupture of membranes following
primary fetoscopic laser treatment for twin–
twin transfusion syndrome. Fetal Diagn Ther.
2016;40(2):100–109.
186. Papanna R, Block-Abraham D, Mann L, et al.
Risk factors associated with preterm delivery
after fetoscopic laser ablation for twin-twin
transfusion syndrome. Ultrasound Obstet
Gynecol. 2014;43(1):48–53.
187. Papanna R, Habli M, Baschat AA. Cerclage
for cervical shortening at fetoscopic laser
photocoagulation in twin-twin transfusion
syndrome. Am J Obstet Gynecol. 2012;206(5):
425.e1–e7.
188. van Kempen LE, Zhao D, Steggerda SJ,
et  al. Increased risk of early-onset neona-
tal sepsis after laser surgery for twin-twin
transfusion syndrome. Twin Res Hum Genet.
2016;19(3):234–240.
189. Zhao D, Cohen D, Middeldorp JM, et al. His-
tologic chorioamnionitis and funisitis after laser
surgery for twin-twin transfusion syndrome.
Obstet Gynecol. 2016;128(2):304–312.
190. Deprest JA, Van Schoubroeck D, Van Ballaer
PP, et  al. Alternative technique for Nd:YAG
laser coagulation in twin-to-twin transfusion
syndrome with anterior placenta. Ultrasound
Obstet Gynecol. 1998;11(5):347–352, 199.
191. Chai H, Fang Q, Huang X, et  al. Prenatal
management and outcomes in mirror syn-
drome associated with twin-twin transfusion.
Prenat Diagn. 2014;34(12):1213–1218.
192. Smid MC, Waltner-Toews R, Goodnight
W. Spontaneous posterior uterine rupture in
twin-twin transfusion syndrome. AJP Rep.
2016;6(1):e68–e70.
193. Merz W, Tchatcheva K, Gembruch u, et  al.
Maternal complications of fetoscopic laser
photocoagulation (FLP) for twin-twin trans-
fusion syndrome (TTTS). J Perinat Med.
2010;38(4):439–443.
194. Ortibus E, Lopriore E, Deprest J, et  al. The
pregnancy and long-term neurodevelopmental
outcome of monochorionic diamniotic twin
gestations: a multicentre prospective cohort
study from the first trimester onward. Am J
Obstet Gynecol. 2009;200(5):494.e1–e8.
195. Lopriore E, Ortibus E, Acosta-Rojas R, et al.
Risk factors for the neurodevelopment impair-
ment in twin-twin transfusion syndrome
treated with fetoscopic laser surgery. Am J
Obstet Gynecol. 2009;113(2 pt 1):361–366.
196. Vanderbilt DL, Schrager SM, Llanes A, et al. Pre-
dictors of 2-year cognitive performance after laser

surgery for twin-twin transfusion syndrome. Am
J Obstet Gynecol. 2014;211(4):388.e1–e7.
197. Graeve P, Banek C, Stegmann-Woessner G,
et al. Neurodevelopmental outcome at 6 years
of age after intrauterine laser therapy for twin-
twin transfusion syndrome. Acta Paediatr.
2012;101(12):1200–1205.
198. McIntosh J, Meriki N, Joshi A, et  al. Long-
term neurodevelopmental outcomes pre-
school age children following laser surgery
for twin-twin transfusion syndrome. Early
Human Dev. 2014;90(12):842–887.
199. van Klink JM, Slaghekke F, Balestriero MA,
et al. Neurodevelopmental outcome at 2 years
in twin-twin transfusion syndrome survivors
randomized for the Solomon trial. Am J Obstet
Gynecol. 2016;214(1):113.e1–e7.
200. Stirnemann J, Chalouhi G, Essaoui M. Fetal
brain imaging following laser surgery in
twin-twin surgery. BJOG. 2016. https://doi.
org/10.1111/1471-0528.14162. [Epub ahead
of print].
201. Gillim DL, Hendricks CH. Holocardius;
review of the literature and case report. Obstet
Gynecol. 1953;2(6):647–653.
202. Gembruch U, Viski S, Bagamery K, et al. Twin
reversed arterial perfusion sequence in twin-to-
twin transfusion syndrome after the death of the
donor co-twin in the second trimester. Ultra-
sound Obstet Gynecol. 2001;17(2):153–156.
203. Healey MG. Acardia: predictive risk fac-
tors for the co-twin’s survival. Teratology.
1994;50(3):205–213.
204. Malone FD, D’Alton ME. Anomalies pecu-
liar to multiple gestations. Clin Perinatol.
2000;27(4):1033–1046.
205. Billah KL, Shah K, Odwin C. Ultrasonic diag-
nosis and management of acardius acephalus
twin pregnancy. Med Ultrasound. 1984;8:108.
206. Pretorius DH, Leopold GR, Moore TR, et al.
Acardiac twin: report of Doppler sonography.
J Ultrasound Med. 1988;7(7):413–416.
207. Moore TR, Gale S, Benirschke K. Perinatal
outcome of forty-nine pregnancies compli-
cated by acardiac twinning. Am J Obstet Gyne-
col. 1990;163(3):907–912.
208. Tan TY, Sepulveda W. Acardiac twin: a sys-
tematic review of minimally invasive treat-
ment modalities. Ultrasound Obstet Gynecol.
2003;22(4):409–419.
209. Mone F, Devaseelan P, Ong S. Intervention
versus a conservative approach in the manage-
ment of TRAP sequence: a systematic review.
J Perinat Med. 2016;44(6):619–629.
210. Lewi L, Valencia C, Gonzalez E, et  al. The
outcome of twin reversed arterial perfusion
sequence diagnosed in the first trimester. Am
J Obstet Gynecol. 2010;203(3):213.e1–e4.
211. Pagani G, D’Antonio F, Khalil A, et  al.
Intrafetal laser treatment for twin reversed
arterial perfusion sequence: a cohort study
and meta-analysis. Ultrasound Obstet Gynecol.
2013;42(1):6–14.
212. Robie GF, Payne Jr GG, Morgan MA. Selec-
tive delivery of an acardiac, acephalic twin.
N Engl J Med. 1989;320(8):512–513.
213. Fries MH, Goldberg JD, Golbus MS. Treat-
ment of acardiac-acephalus twin gestations
by hysterotomy and selective delivery. Obstet
Gynecol. 1992;79(4):601–604.
214. Robyr R, Yamamoto M, Ville Y. Selective feti-
cide in complicated monochorionic twin preg-
nancies using ultrasound-guided bipolar cord
coagulation. BJOG. 2005;112(10):1344–1348.
215. Lewi L, Gratacos E, Ortibus E, et  al. Preg-
nancy and infant outcome of 80 consecutive
cord coagulations in complicated monochori-
onic multiple pregnancies. Am J Obstet Gyne-
col. 2006;194(3):782–789.
216. Hecher K, Lewi L, Gratacos E, et  al. Twin
reversed arterial perfusion: fetoscopic laser
coagulation of placental anastomoses or the
umbilical cord. Ultrasound Obstet Gynecol.
2006;28(5):688–691.
217. Lee H, Wagner AJ, Sy E, et  al. Efficacy of
radiofrequency ablation for twin-reversed arte-
rial perfusion sequence. Am J Obstet Gynecol.
2007;196(5):459.e1–e4.
218. Weisz B, Peltz R, Chayen B, et  al. Tailored
management of twin reversed arterial perfu-
sion (TRAP) sequence. Ultrasound Obstet
Gynecol. 2004;23(5):451–455.
219. Hanson JW. Incidence of conjoined twinning.
Lancet. 1975;2(7947):1257.
220. Spencer R. Theoretical and analytical embry-
ology of conjoined twins: part I: embryogen-
esis. Clin Anat. 2000;13(1):36–53.
221. Machin GA. Heteropagus conjoined twins
due to fusion of two embryos. Am J Med
Genet. 1998;78(4):388–390.
222. 
The international clearinghouse for Birth
Defects Monitoring Systems. Conjoined
twins: an epidemiological study based on
312 cases. Acta Genet Med Gemellol (Roma).
1991;40(3-40):325–335.
223. Woo JS, Liang ST, Lo R. Characteristic pat-
tern of Doppler umbilical arterial velocity
waveform in conjoint twins. Gynecol Obstet
Invest. 1987;23(1):70–72.
224. Gorkem SB, Kutuk MS, Doganay S, et  al.
Decreased brain and placental perfusion in
omphalopagus conjoined twins on fetal MRI.
J Radiol Red Pract. 2016;9458540:2016.
225. McHugh K, Kiely EM, Spitz L. Imag-
ing of conjoined twins. Pediatr Radiol.
2006;36(9):899–910.
226. Spitz L. Surgery for conjoined twins. Ann R
Coll Surg Engl. 2003;85(4):230–235.
227. Brizot ML, Liao AW, Lopes LM, et al. Con-
joined twin pregnancies: experience with
36 cases from a single center. Prenat Diagn.
2011;31(12):1120–1125.
228. Rodis JF, Mc veen II PF, Egan JF, et  al.
Monoamniotic twins: improved perinatal
survival with accurate prenatal diagnosis and
antenatal fetal surveillance. Am J Obstet Gyne-
col. 1997;177(5):1046–1049.
229. Prefumo F, Fichera A, Pagani G, et al. The nat-
ural history of monoamniotic twin pregnan-
cies: a case series and systematic review of the
literature. Prenat Diagn. 2015;35(3):274–280.
230. Shub A, Walker SP. Planned early delivery
versus expectant management for mono-
amniotic twins. Cochrane Database Syst Rev.
2015;(4):CD008820.
231. Abuhamad AZ, Mari G, Copel JA, et  al.
Umbilical artery flow velocity waveforms in
monoamniotic twins with cord entanglement.
Obstet Gynecol. 1995;86(4 Pt 2):674–677.
232. Rossi AC, Prefumo F. Impact of cord entangle-
ment on perinatal outcome of monoamniotic
twins: a systematic review of the literature. Ultra-
sound Obstet Gynecol. 2013;41(2):131–135.
233. Roque H, Gillen-Goldstein J, Funai E,
et  al. Perinatal outcomes in monoamniotic
gestations. J Matern Fetal Neonatal Med.
2003;13(6):414–421.
234. van den Wijngaard JP, Umur A, Ross MG,
et al. Modelling the influence of amnionicity
References 553.e5
on the severity of twin-twin transfusion syn-
drome in monochorionic twin pregnancies.
Phys Med Biol. 2004;49(6):N57–N64.
235. Ishii K. Prenatal diagnosis and management of
monoamniotic twins. Curr Opin Obstet Gyne-
col. 2015;27(2):159–164.
236. Dodd JM, Crowther CA. Evidence-based care
of women with a multiple pregnancy. Best Pract
Res Clin Obstet Gynecol. 2005;19(1):131–153.
237. Pharoah PO, Cooke T. Cerebral palsy and
multiple births. Arch Dis Child Fetal Neonatal
Ed. 1996;75(3):F174–F177.
238. Luke B, Brown MB. Contemporary risks of
maternal morbidity and adverse outcomes
with increasing maternal age and plurality.
Fertil Steril. 2007;88(2):283–293.
239. D’Antonio F, Thilaganathan B, Toms J, et al.
Perinatal outcome after fetoscopic laser sur-
gery for twin-twin transfusion syndrome in
triplet pregnancies. BJOG. 2016;123(3):328–
336.
240. Chaveeva P, Kosinski P, Puglia D, et  al.
Trichorionic and dichorionic triplet pregnan-
cies at 10-14 weeks: outcome after embryo
reduction compared to expectant manage-
ment. Fetal Diagn Ther. 2013;34(4):199–205.
241. Papageorghiou AT, Avgidou K, Bakoulas V,
et  al. Risks of miscarriage and early preterm
birth in trichorionic triplet pregnancies with
embryo reduction versus expectant manage-
ment: new data and systematic review. Hum
Reprod. 2006;21(7):1912–1917.
242. Lopes Perdigao J, Straub H, Zhou Y, et  al.
Perinatal and obstetric outcomes of dichori-
onic vs trichorionic triplet pregnancies. Am J
Obstet Gynecol. 2016;214(5):659.e1–e5.
243. Chasen ST, Al-Kouatly HB, Ballabh P, et al.
Outcomes of dichorionic triplet pregnancies.
Am J Obstet Gynecol. 2002;186(4):765–767.
244. Adegbite AL, Ward SB, Bajoria R. Perinatal
outcome of spontaneously conceived triplet
pregnancies in relation to chorionicity. Am J
Obstet Gynecol. 2005;193(4):1463–1471.
245. Stone J, Belogolovkin V, Matho A, et al. Evolv-
ing trends in 2000 cases of multifetal pregnancy
reduction: a single-center experience. Am J
Obstet Gynecol. 2007;197(4):394.e1–e4.
246. Haas J, Hourvitz A, Dor J, et  al. Perinatal
outcome of twin pregnancies after early trans-
vaginal multifetal pregnancy reduction. Fertil
Steril. 2014;101(5):1344–1348.
247. Evans MI, Ciorica D, Britt DW, et  al.
Update on selective reduction. Prenat Diagn.
2005;25(9):807–813.
248. Abel JS, Flöck A, Berg C, et  al. Expectant
management versus multifetal pregnancy
reduction in higher order multiple pregnan-
cies containing a monochorionic pair and a
review of the literature. Arch Gynecol Obstet.
2016;294(6):1167–1173.
249. Wimalasundera RC. Selective reduction and
termination of multiple pregnancies. Semin
Fetal Neonatal Med. 2010;15(6):327–335.
250. Morlando M, Ferrara L, D’Antonio F, et  al.
Dichorionic triplet pregnancies: risk of mis-
carriage and severe preterm delivery with fetal
reduction versus expectant management. Out-
comes of a cohort study and systematic review.
BJOG. 2015;122(8):1053–1060.
251. Evans MI, Berkowitz RL, Wapner RJ, et  al.
Improvement in outcomes of multifetal reduc-
tion with increased experience. Am J Obstet
Gynecol. 2001;184(2):97–103.
252. Eddleman KA, Stone JL, Lynch L, et  al.
Selective termination of anomalous fetuses
553.e6 References
in multifetal pregnancies: two hundred
cases at a single center. Am J Obstet Gynecol.
2002;187(5):1168–1172.
253. Quintero RA, Romero R, Reich H, et  al. In
utero percutaneous umbilical cord ligation in
the management of complicated monochori-
onic multiple gestations. Ultrasound Obstet
Gynecol. 1996;8(1):16–22.
254. Jolly M, Taylor M, Rose G, et  al. Intersti-
tial laser: a new surgical technique for twin
reversed arterial perfusion sequence in early
pregnancy. BJOG. 2001;108(10):1098–1102.
255. Rossi AC, D’Addario V. Umbilical cord
occlusion for selective feticide in compli-
cated monochorionic twins: a systematic
review of the literature. Am J Obstet Gynecol.
2009;200(2):123–129.
256. Bebbington MW, Danzer E, Moldenhauer J,
et al. Radiofrequency ablation vs bipolar umbili-
cal cord coagulation in the management of
complicated monochorionic pregnancies. Ultra-
sound Obstet Gynecol. 2012;40(3):319–324.
257. van Den Bos EM, van Klink JM, Middeldorp
JM, et  al. Perinatal outcome after selective
feticide in monochorionic twin pregnancies.
Ultrasound Obstet Gynecol. 2013;41:653–658.
258. Prefumo F, Cabassa P, Fichera A, et  al. Pre-
liminary experience with microwave abla-
tion for selective feticide in monochorionic
twin pregnancies. Ultrasound Obstet Gynecol.
2013;41(4):470–471.
259. De Lia JE, Kuhlmann RS, Harstad TW, et al.
Fetoscopic laser ablation of placental vessels
in severe previable twin-twin transfusion syn-
drome. Am J Obstet Gynecol. 1995;172:1202–
1208; discussion 1208–1211.
260. Ville Y, Hyett J, Hecher K, et al. Preliminary
experience with endoscopic laser surgery
for severe twin-twin transfusion syndrome.
N Engl J Med. 1995;332:224–227.
261. De Lia JE, Kuhlmann RS, Lopez KP. Treat-
ing previable twin-twin transfusion syndrome
with fetoscopic laser surgery: outcomes fol-
lowing the learning curve. J Perinat Med.
1999;27:61–67.
262. Hecher K, Diehl W, Zikulnig L, et  al. Endo-
scopic laser coagulation of placental anastomoses
in 200 pregnancies with severe mid-trimester
twin-to-twin transfusion syndrome. Eur J Obstet
Gynecol Reprod Biol. 2000;92:135–139.
263. Quintero RA, Comas C, Bornick PW, et  al.
Selective versus non-selective laser photoco-
agulation of placental vessels in twin-to-twin
transfusion syndrome. Ultrasound Obstet
Gynecol. 2000;16:230–236.
264. Gray PH, Cincotta R, Chan FY, et al. Perina-
tal outcomes with laser surgery for twin-twin
transfusion syndrome. Twin Res Hum Genet.
2006;9:438–443. Cited as ref 178.
265. Ierullo AM, Papageorghiou AT, Bhide A,
et  al. Severe twin-twin transfusion syn-
drome: outcome after fetoscopic laser abla-
tion of the placental vascular equator. BJOG.
2007;114:689–693.
266. Middeldorp JM, Sueters M, Lopriore E, et al.
Fetoscopic laser surgery in 100 pregnancies
with severe twin-to-twin transfusion syn-
drome in the Netherlands. Fetal Diagn Ther.
2007;22:190–194. Cited as reference 167.
267. Sepulveda W, Wong AE, Dezerega V, et  al.
Endoscopic laser surgery in severe second-
trimester twin-twin transfusion syndrome: a
three-year experience from a Latin American
center. Prenat Diagn. 2007;27:1033–1038.
268. Stirnemann JJ, Nasr B, Quarello E, et  al. A
definition of selectivity in laser coagulation of
chorionic plate anastomoses in twin-to-twin
transfusion syndrome and its relationship
to perinatal outcome. Am J Obstet Gynecol.
2008;198: 62.e1–e6.
269. Cincotta RB, Gray PH, Gardener G, et  al.
Selective fetoscopic laser ablation in 100 con-
secutive pregnancies with severe twin-twin
transfusion syndrome. Aust N Z J Obstet Gyn-
aecol. 2009;49:22–27.
270. Nakata M, Murakoshi T, Sago H, et al. Modi-
fied sequential laser photocoagulation of pla-
cental communicating vessels for twin-twin
transfusion syndrome to prevent fetal demise
of the donor twin. J Obstet Gynaecol Res.
2009;35:640–647.
271. Ruano R, Brizot Mde L, Liao AW, et  al.
Selective fetoscopic laser photocoagulation
of superficial placental anastomoses for the
treatment of severe twin-twin transfusion syn-
drome. Clinics (Sao Paulo). 2009;64:91–96.
272. Chmait RH, Khan A, Benirschke K, et  al.
Perinatal survival following preferential
sequential selective laser surgery for twin-twin
transfusion syndrome. J Matern Fetal Neonatal
Med. 2010;23:10–16.
273. Meriki N, Smoleniec J, Challis D, et  al.
Immediate outcome of twin-twin transfusion
syndrome following selective laser photocoag-
ulation of communicating vessels at the NSW
Fetal Therapy Centre. Aust N Z J Obstet Gyn-
aecol. 2010;50:112–119.
274. Morris RK, Selman TJ, Kilby MD. Influences
of experience, case load and stage distribution
on outcome of endoscopic laser surgery for
TTTS - a review. Ahmed S et al. Prenatal Diag-
nosis 2010. Prenat Diagn. 2010;30:808–809.
275. Yang X, Leung TY, Ngan Kee WD, et al. Feto-
scopic laser photocoagulation in the manage-
ment of twin-twin transfusion syndrome: local
experience from Hong Kong. Hong Kong Med J.
2010;16:275–281.
276. Delabaere A, Accoceberry M, Niro J, et  al.
Favourable outcome after fetoscopic laser
surgery for twin-twin transfusion syndrome:
experience of an emerging centre. Gynecol
Obstet Fertil. 2011;39:482–485.
277. Hernandez-Andrade E, Guzman-Huerta M,
Benavides-Serralde JA, et  al. Laser ablation
of the placental vascular anastomoses for the
treatment of twin-to-twin transfusion syn-
drome. Rev Invest Clin. 2011;63:46–52.
278. Lombardo ML, Watson-Smith DJ, Muratore
CS, et al. Laser ablation of placental vessels in
twin-to-twin transfusion syndrome: a para-
digm for endoscopic fetal surgery. J Laparoen-
dosc Adv Surg Tech A. 2011;21:869–872.
279. Tchirikov M, Oshovskyy V, Steetskamp J,
et  al. Neonatal outcome using ultrathin
fetoscope for laser coagulation in twin-to-
twin-transfusion syndrome. J Perinat Med.
2011;39:725–730.
280. Weingertner AS, Kohler A, Mager C, et  al.
Fetoscopic laser coagulation in 100 consecu-
tive monochorionic pregnancies with severe
twin-to-twin transfusion syndrome. J Gynecol
Obstet Biol Reprod (Paris). 2011;40:444–451.
281. Chang YL, Chao AS, Chang SD, et al. Short-
term outcomes of fetoscopic laser surgery
for severe twin-twin transfusion syndrome
from Taiwan single center experience: dem-
onstration of learning curve effect on the
fetal outcomes. Taiwan J Obstet Gynecol.
2012;51:350–353.
282. Liu XX, Lau TK, Wang HF, et al. Fetoscopic
guided laser occlusion for twin-to-twin trans-
fusion syndrome in 33 cases. Zhonghua Fu
Chan Ke Za Zhi. 2012;47:587–591.
283. Murakoshi T, Matsushita M, Shinno T, et al.
Fetoscopic laser photocoagulation for the
treatment of twin-twin transfusion syndrome
in monochorionic twin pregnancies. Open
Med Devices J. 2012;4:4–59.
284. Rustico MA, Lanna MM, Faiola S, et al. Fetal
and maternal complications after selective
fetoscopic laser surgery for twin-to-twin trans-
fusion syndrome: a single-center experience.
Fetal Diagn Ther. 2012;31:170–178.
285. Sundberg K, Sogaard K, Jensen LN, et al. Inva-
sive treatment in complicated monochorionic
twin pregnancies: indications and outcome of
120 consecutively treated pregnancies. Acta
Obstet Gynecol Scand. 2012;91:1201–1205.
286. Swiatkowska-Freund M, Pankrac Z, et  al.
Results of laser therapy in twin-to-twin trans-
fusion syndrome: our experience. J Matern
Fetal Neonatal Med. 2012;25:1917–1920.
45
In Utero Stem Cell Transplantation
CECILIA GÖTHERSTRÖM, LILIAN WALTHER-JALLOW AND MAGNUS WESTGREN
KEY POINTS
• In utero transplantation (IUT) has the potential to cure or ame-
liorate many disorders before birth.
• Animal models for studying IUT have fundamental differences
from human models in regard to immunologic ontogeny and
placentation.
• Naturally occurring events during pregnancy result in chime-
rism in large animals and in humans and support the concept
of IUT.
• Using mesenchymal stem cells for IUT may be possible in
disorders with a functioning immune system.
• Development of advanced medicinal therapy products for
use in IUT is complicated and requires large resources and
knowledge but holds great promise for the future.
Introduction
The first successful in utero transplantation (IUT) was reported
by Touraine et  al. in 1989.1 The report was followed by a lim-
ited number of IUTs in human fetuses with severe combined
immune deficiency (SCID). Most of these reports claim partial
engraftment of the transplants.2-5 Several researchers tried a simi-
lar approach with haematopoietic stem cell (HSC) transplantation
in utero for nonimmunologic disorders; unequivocally, these cases
failed.6-11 HSC is the stem cell that give rise to all blood cells, both
of the myeloid and lymphoid lineages. The arguments for IUT, as
summarised in Table 45.1, are well known and have repeatedly
been broadcasted in different reviews on this topic for almost 3
decades.12-16 However, we are still lacking knowledge on why IUT
with HSC in fetuses with normal immunologic function do not
engraft, and the evidence supporting the arguments are, to put it
cautiously, not very strong.
A number of reviews on this topic have been published, with
extensive examination of past literature on animal work and pre-
vious human experience.12-16 Recently two excellent reviews on
IUT with HSC were published,17,18 and we will therefore in the
present review focus mainly on IUT with mesenchymal stem
cells (MSCs). MSCs are the stem cells that give rise to cell types
of the mesodermal lineage, such as osteoblasts, chondrocytes,
myocytes and adipocytes. Before embarking on such undertak-
ing, we will briefly comment on the most current experience
of IUT in animal models and whether we can learn more from
nature regarding engraftment of foreign stem cells in human
fetuses.  formed the base for the arguments for IUT, is that several research
554
What Can We Learn From Animal Models?
General problems with animal models for IUT are the funda-
mental differences among different species models and differences
between animals and humans with regard to immunologic ontog-
eny and placentation. These are crucial questions that need to be
kept in mind in the interpretation of the current literature.
Achieving engraftment in the mouse model is complicated
without a great advantage for donor cells. In 1953, Billingham and
colleagues published a pioneer report on donor specific tolerance
to skin grafts after IUT in mice.19 Three decades later, Fleischman
and colleagues reported on successful haematopoietic chimerism
after IUT that could ameliorate a genetic disorder in a mouse
model.20 These studies were directed to study stem cell biology
rather than exploring the therapeutic possibility of IUT with stem
cells. Several researchers embarked on similar studies, but when
using immunocompetent recipients, the results were poor with
regard to donor chimerism and not until immunodeficient mice
were studied was significant chimerism obtained.4,20-23 Recently,
Flake and associates have used the mouse model to extensively
explore the fetal mouse immune system.24 Their studies indicate
clonal deletion, anergy (absence of a normal immune response)
and immune tolerance after IUT. It seems that a low level of chi-
merism is associated with postnatal tolerance across full major
histocompatibility complex (MHC) barriers.25-28 Furthermore,
recent studies on IUT in mice have revealed evidence of maternal
alloimmunisation and that maternal–fetal T-cell trafficking (bidi-
rectional transfer of cells between the mother and the fetus) could
be an explanation for loss of chimerism after birth.25 Arguments
against this hypothesis have been raised by Alhajjat and colleagues,
who have pointed out that in the context of the human experience
of IUT, there has been no evidence of immunisation.29 Although
this might be true, we are not aware of any studies on maternal
immunology in conjunction with these IUTs. Shaaban’s research
group has focused on the significance of the natural killer (NK)
cells of the innate immune system and has identified a subset of
cells within the fetal liver that may pose a barrier for early engraft-
ment.29 Interestingly these naïve NK cells were recently also found
in early human tissues and in amniotic fluid.24 Kim and colleagues
have recently explored other means to enhance engraftment by
mobilising fetal HSCs by inhibition of the integrin α4β1/7.30
The most commonly used model for studies of IUT is the fetal
lamb model. After allogenic IUT with fetal liver–derived HSCs,
significant levels of chimerism have been obtained.31,32 Even
transplantations of xenogenic cells from humans have been suc-
cessful. A concern with these studies, that to a large extent has
 CHAPTER 45  In Utero Stem Cell Transplantation 555

TABLE Arguments and Evidence for In Utero assays.43 Marmosets typically show high degrees of haematopoi-
45.1 Transplantation (IUT) with Haematopoietic etic chimerism (28%–82% by peripheral blood karyotype).
Although the natural chimeras noted in dizygotic large ani-
Stem Cells (HSCs) and Mesenchymal Stem Cells
mals are highly suggestive that IUT should be successful, the most
(MSCs)a encouraging data comes from dizygotic human twins. Although
Arguments for IUT with the frequency of chimerism in dizygotic twins in these animals at
stem cells Evidence HSC Evidence MSC birth is relatively high, the level of chimerism in humans is quite
low.41 These data are consistent with placental architecture stud-
Right-to-left heart shunting that     ies showing that intertwin placental anastomosis does not occur
enhances systemic distribu-
tion of the cells
between most normal dizygotic human twins. However, more
than 30 cases of opposite-sex dizygotic twins have been reported
Small size of the fetus allows and show much higher levels of haematopoietic chimerism.39,42-46
big dose In these cases, early placental fusion and anastomosis presum-
Treat before severe conse- ably occurred. The level of chimerism in these cases is generally
quences of the disease high, and in one well-documented case, chimerism has persisted
for more than 25 years.45 Immune tolerance (as assessed by MLC
In fetal life, large-scale migra- and skin grafting) has also been demonstrated.47-49 These natu-
tion of stem cells takes place
rally occurring ‘experiments’ in large animals, including primates
Allows transplantation across     as well as humans, support the concept of IUT.
HLA barriers Another naturally occurring model is the passage of mater-
Donor-specific tolerance     nal cells across the placenta to the fetal recipient. These cells can
induction engraft, and we and others have shown that maternal cells of lym-
phoid and myeloid lineages as well as haematopoietic progeni-
Niches for exogenous stem tors are widely distributed in human second trimester fetuses.50
cells Furthermore, after birth, some cells remain and are found in the
Psychological advantages child’s lymphoid tissue.51 Later in life, maternal chimerism is very
rare, so it might be that there is some low-grade immunologic
Less expensive than postnatal
mechanism that is responsible for the loss of chimerism.52
transplantation
To summarise, induction of tolerance and engraftment
aGreen, some evidence; red, poor evidence. The darker shades depict less (red) and more of HSCs seems to be possible but is complicated to achieve in
(green) evidence. immunocompetent animal models, and it is still an open question
HLA, Human leukocyte antigen. how this will be accomplished in human fetal recipients. Several
   hurdles seem to be in play, such as the fetal immune system, the
maternal immune system and host cell competition. 
groups have tried to repeat these studies but have unequivocally
failed.31,32 Thus caution is important when evaluating these data. In Utero Transplantation
Other animal models used for studies of IUT are pigs, goats,
canines and nonhuman primates. Studies on nonhuman primates Some disorders result in irreversible damage or even perinatal
are, of course, of special value, but it has been notoriously difficult lethality, and treatment should or must be initiated before birth.
to attain engraftment.33-36 Therefore it is desirable to introduce treatment as early as possible
Therefore, although different animal models provide support before additional pathology occurs and at a time of rapid develop-
for IUT with HSCs, there are limitations in most models with ment. Additional reasons in favour for IUT include:
regard to species specific differences, and results are often diffi- 1. The right-to-left heart shunting that enhances systemic distri-
cult to comprehensively evaluate. The main question of how we bution of the cells instead of the cells being trapped in the lungs
can extrapolate from these experiments into the human situation as in childhood and adult life. Two clinical studies show that
remains.  after postnatal intravenous infusion of MSCs, the cells accu-
mulated in the lungs within 20 minutes, and after 48 hours,
Can We Learn From Nature? the cells were distributed to other organs such as the liver and
spleen.53,54 In several animal studies, MSCs were found in the
Dizygotic twin cows,37,38 goats39 and marmosets40 all provide lungs within seconds after intravenous infusion with redistri-
evidence that naturally occurring chimerism of haematopoietic bution to the liver, spleen, kidney, heart and bone marrow after
cells early during pregnancy occurs through placental blood vessel 24 to 48 hours.55-57 In contrast, after intraperitoneal IUT in
anastomosis between the two fetal circulations.39,41 It is important mouse models of osteogenesis imperfecta (OI) and muscular
to note that transmission through placental anastomosing vessels dystrophy, the transplanted human fetal MSCs were identified
is very different from IUT. With placental anastomosis, the mix- at all time points in all tissues examined, except in the lungs at
ing of fetal blood most probably occurs very early in pregnancy, birth.58,59
and the mixing of fetal blood is likely to be continuous. Dizygotic 2. The rapid growth of the fetus providing an opportunity for
twin cattle demonstrate inter twin tolerance when measured using engraftment and expansion and subsequent migration and dis-
mixed lymphocyte cultures (MLC), accept renal grafts from their tribution of the donor cells to different anatomical compart-
co-twin and have delayed rejection to skin grafts transplants.42 ments. During fetal life, naturally occurring stem cells expand
Similar evidence of intertwin tolerance has been demonstrated in and migrate to seed and populate anatomical compartments.
dizygotic twin marmosets using cytotoxic T-lymphocyte (CTL) One example is the parallel migration of HSCs and MSCs
556 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions
from the aorta-gonad-mesonephros and the yolk sac to the
liver and last to the bone marrow.60,61 These compartments
provide a potent and specialised supportive environment for
proliferation and differentiation of fetal cells, especially when a
fetal-to-fetal transplantation approach is applied.62
3. The relatively naïve fetal immune system that may permit the
development of immune tolerance towards donor cells. The
immunologic naivety in the early gestational fetus has given
rise to the concept of fetal tolerance (i.e., the inability to raise
an immunologic response against foreign antigens). During
fetal life, the developing immune system is educated to distin-
guish between autologous and foreign antigens, and if intro-
duced early enough, foreign antigens can be recognised as ‘self ’
and not be rejected.
4. The far better psychosocial situation for the mother and father
resulting from the birth of a child who has already been treated.
No comparative studies are available, but from our extensive
experience in regard to fetal therapy, we are convinced that the
parents prefer an active approach including a therapeutic strat-
egy rather than being subjected to an observational wait-and-see
attitude. These parents have often refrained from termination
and are ready to do as much as possible for their unborn child.
Thus they look at their fetus as a potential unborn patient who
should receive optimal care and management.
The possible advantages of IUT are summarised in Table 45.1.  thousands of individuals without adverse reactions.77
Pre- and Postnatal Transplantation With Fetal
Mesenchymal Stem Cells for Treatment of
Osteogenesis Imperfecta
Our research group has during the past decade been involved
in projects aiming to treat OI before and after birth. OI, often
called brittle bone disease, is a heterogeneous genetically inherited
disease with a prevalence at birth estimated to 1 in 10,000 to
20, 000.63 OI is caused by more than 1400 different dominant and
more than 150 recessive mutations.64 The most common cause
(>90%) is a dominant mutation in one of the two genes encoding
collagen type I (COL1A1 and COL1A2), which shows an autoso-
mal dominant pattern of inheritance.65 Four types of OI, types
I to IV, were originally described in 1979 by Sillence and col-
leagues based on clinical, radiologic and hereditary findings.66,67
OI is heterogeneous and range from the mild type I that may
only become evident in adulthood to the perinatally lethal type II
A/C. Type III OI is the most severe form that is compatible with
survival into adulthood. Recently, the classification was expanded
with OI types V to VIII because of distinct clinical features or
different causative gene mutations, but these types are commonly
not used in the clinical diagnosis.68 The pathology of OI develops
in fetal life, and sonographic signs of severe forms of OI can be
detected in the first trimester, for example, with increased nuchal
translucency and bony malformations.69 Later during gestation,
fetuses affected by OI type III often exhibit a typical phenotype
with sonographic signs of multiple fractures at various stages of
healing; very short, deformed long bones with bowing and angu-
lation; and sometimes reduced echogenicity.70 Conclusive prena-
tal diagnosis of OI usually requires an invasive test to obtain fetal
material for molecular analysis. The major clinical manifestations
of OI are atypical skeletal development, osteopenia, multiple
painful fractures and short stature, but individuals with OI also
have brittle teeth, hearing loss and hypermobile joints and have a
higher risk for heart valve insufficiency, aneurysms and bleeding as
a result of coagulation deficiencies throughout their lifetimes. Life
expectancy is not affected in milder OI types but may be short-
ened for those with more severe types.71 Individuals affected by
OI type III may experience hundreds of fractures in a lifetime,72
Finding suitable cell sources is one of the main challenges in
regenerative medicine. In addition to improving the dysfunctional
tissue requiring reconstruction, low immunogenicity is beneficial.
MSCs are multipotent stromal cells, originally identified in adult
bone marrow, displaying colony-forming capacity and are nonhae-
matopoietic and nonendothelial. MSCs can be easily isolated and
expanded and differentiate along the osteogenic, chondrogenic,
myogenic and adipogenic lineages.73 MSCs do not express HLA
class II antigens, are generally considered to be nonimmunogenic,
do not elicit an immune response74 and inhibit proliferating
allogeneic lymphocytes in  vitro.74,75 MSCs, when grown under
normal culture conditions, are commonly considered as safe to
transplant, and there are no reports of ectopic tissue formation or
malignant transformation.76 Because of these characteristics, they
have been tested in clinical trials for a diverse variety of disorders,
ranging from the treatment of inflammatory, autoimmune and
cardiovascular diseases and in orthopaedic applications.77 The first
MSC transplantation was performed more than 15 years ago, and
adult MSCs have been used in a diverse range of conditions in
Our research group at Karolinska Institutet were one of the
first in the world to isolate and characterise MSCs from human
fetal tissues.60,78,79 These primitive MSC types are found at a
higher frequency, with greater colony-forming capacity, have lon-
ger telomeres (cells are more long lived if the telomeres are not
shortened at each cell division) and a superior proliferative poten-
tial compared with MSC from adult sources.79-82 Furthermore,
fetal MSCs differentiate more readily into bone, muscle and oligo-
dendrocytes compared with adult MSCs.58,81,83,84 Like their adult
counterparts, fetal MSC are also nonimmunogenic.78,79
The potential of MSC transplantation for treatment of OI
was first demonstrated in mouse models of OI disease. Postna-
tal transplantation of allogeneic adult mouse MSCs resulted in
donor cell homing to the bones, where they contributed to the
osteoprogenitor population, with improvement in collagen con-
tent and bone mineralisation.85 In a later study, Panaroni and
colleagues demonstrated that IUT of allogeneic mouse adult bone
marrow transplantation in the perinatally lethal dominant BrtIIV
model of OI rescued the neonatal mouse from perinatal death
and improved the mechanical properties of bones.86 Donor cells
engrafted in haematopoietic and nonhaematopoietic tissues and
differentiated to bone cells. The donor cells synthesised up to 20%
of the total collagen type I content of bone, despite an engraft-
ment of only 2%. IUT of human fetal MSC in the oim mouse, a
naturally occurring recessive mouse model approximating to OI
type III with progressive deformities and skeletal fractures showed
that intraperitoneal injection of human fetal MSC resulted in
engraftment in the bones (∼5%). Furthermore, there were a 67%
reduction in long bone fractures and increased bone strength,
thickness and length, and normal human type I collagen could be
measured.59,87,88
Postnatal stem cell transplantation has been attempted in
a few children with severe OI. The first clinical proof-of-princi-
ple came from Horwitz and colleagues more than a decade ago
when five children with type III OI underwent transplantation
with matched adult whole bone marrow. Linear growth increased
and fracture frequency reduced despite low level (<2%) donor

osteoblast engraftment.89,90 Subsequent infusions of adult MSCs
isolated from the bone marrow of donors to the recipients showed
similar results as the bone marrow transplantation with donor cell
engraftment in the bones and an acceleration of the growth veloc-
ity.91 Data from mouse studies suggest that significant amounts of
normal collagen can be deposited by a relatively small population
of donor cells,59,86 explaining the marked improvements despite
low-level engraftment. Engraftment is likely to be higher after
IUT for the reasons discussed earlier and may therefore provide
improved phenotypic disease correction.
Encouraged by the data from preclinical and clinical studies,
we performed in 2002 the first IUT of human fetal liver–derived
MSCs in an immunocompetent fetus with OI type III (COL1A2
c.3008G>A; p.Gly1003Asp; Gly913Asp in the triple helical
domain) using cells developed by us at Karolinska Institutet with
a successful outcome.92 IUT of 6.5 × 106/kg fetal MSCs into the
umbilical vein under ultrasound guidance was performed in ges-
tational week 31. The transplantation showed promising clinical
results with long-term donor cell engraftment and site-specific dif-
ferentiation of completely HLA-mismatched MSC in the bone.
Until 8 years of age, the patient had only had one fracture and
one compression fracture per year.93 Remarkably, she continued to
grow and follow her own height and weight curve at −5 standard
deviations (SDs), until from age 6 to 8 years when it deteriorated
to −6.5 SDs. As a result and because of an increased fracture rate,
the patient was retransplanted with the same-donor MSC intrave-
nously. Over the following 2 years, she did not acquire any frac-
tures, and her linear growth and mobility improved.93
We have followed this patient for more than 15 years, and she
further received yearly infusions for 3 years with same-donor cells
in an attempt to improve her height (during years 2013–2015 at
11, 12 and 13 years of age, respectively). After each infusion, a
clinical effect was noted (reduced frequency of fractures, increased
growth and better quality of life according to the parents). A child
with an identical mutation was identified in a worldwide OI regis-
try. This child did not receive MSC transplantation before or after
birth and exhibited a very severe phenotype of OI and died as a
result of the disease at 5 months of age.93 This is the first sugges-
tion that IUT with fetal MSCs can improve severe OI in humans,
although we acknowledge that the two patients had overall differ-
ent genetic backgrounds despite identical OI-causing mutation.
Through international collaborations, we have in 2009 and
2014 performed two additional IUT with fetal liver MSCs for
treatment of OI. In the first case, we transported clinical grade
human fetal liver MSCs to National University Hospital of Sin-
gapore, where local fetal medicine experts performed an ultra-
sound-guided intrahepatic umbilical vein infusion at 31 weeks’
gestation in a fetus with a confirmed OI type IV mutation
(COL1A2 c.659G>A; p.Gly220Asp; Gly130Asp in the triple heli-
cal domain).93 The patient has received one retransplantation with
same-donor cells at 1.5 years of age with a good effect and is doing
better than expected with boosted height and reduced frequency
of fractures. In the most recent case, another international patient
with a molecularly confirmed prenatal diagnosis of OI type III
(COL1A1 c.3469G>C p.Gly1157Arg) was transplanted at Karo-
linska University Hospital in gestational week 28. The baby was
born in October 2014 and is doing better than expected.
The clinical effect after MSC transplantation in patients with
OI is not permanent and probably regular yearly or biannual
infusions are needed. This has previously been noted in the clini-
cal trial on OI using HLA-matched MSCs postnatally.91 Even
though a single IUT may not be clinically sufficient for permanent
CHAPTER 45  In Utero Stem Cell Transplantation 557
phenotype correction, an IUT approach is still justifiable because
the immunologic naïveté of the fetus may allow for the devel-
opment of immune tolerance towards the donor cells, rendering
postnatal transplantations more efficient. An early treatment of
this severe disorder is also beneficial, both for the child and for
the parents.
Building on the work carried out at Karolinska Institutet for
the past 15 years, we are now preparing an international multi-
centre EU Horizon 2020–funded clinical trial on pre- and post-
natal transplantation of fetal MSCs for ameliorating severe types
of OI (BOOSTB4.EU, grant agreement 681045). In Table 45.2,
an example of implications of a successful MSC therapy for OI
patients and health care systems is illustrated. 
Drug Development
Stem cells are, as described earlier, immature cells that can grow
into healthy cells that match the tissue that they are introduced to,
thus replacing the diseased cells and repairing the tissue. Stem cells
are promising candidates for use in cell therapy and may be effec-
tive in treating a number of to date incurable diseases, including
metabolic, diabetes, cancer, Alzheimer disease and various genetic
disorders.
Drug development as such requires resourcefulness, persever-
ance and financial strength; it takes about 15 years to develop
a new drug (Fig. 45.1), and only 1 of 10 substances that enters
clinical trials in humans reaches the market as a registered drug. To
find this single substance, between 5000 and 10,000 substances
are developed and tested in different ways, leading to research-
based pharmaceutical companies spending about 20% of revenue
on research and development. This is significantly more than most
other industrial sectors, including electronic companies and aero-
space, automobile and computer manufacturers. Since the 1980s,
pharmaceutical companies more or less doubled their spending
on research and development every 5 years, and the total cost of
developing a drug is now about €1 to €10 billion. There has been
a steady decline in the number of drugs introduced to the market,
from 70 to 100 in the 1960s to about 40 in the 1990s, and this has
led to an innovation deficit. The reasons for this deficit are several,
including extended amount of time needed for testing the drug
candidate because of increased safety demands and difficulties to
meet the regulatory requirements, leading to an increased number
and size of clinical trials to test the safety of the drug. It is also said
that the ‘low-hanging fruits’ have already been picked, meaning
that the most obvious drugs for the less complicated diseases have
already been produced and that now there are only the compli-
cated diseases left to be treated by even more advanced drugs. The
blockbusters from the early years of pharmaceutical development
are history, and the pharmaceutical industry is facing a situation
in which the demand for individualised treatment and drug regi-
mens is required.
In the era of individualised medicine and precision drugs,
advanced medicinal therapy products (ATMPs) play a promising
roll. ATMPs are medicinal products consisting of cells or tissue
which are prepared industrially or manufactured by a method
involving an industrial process. ATMPs fall into three catego-
ries: gene therapies, somatic cell therapies and tissue-engineered
products. Somatic cell therapies include the use in humans of
autologous (from the patient himself ), allogeneic (from another
human being) or xenogeneic (from animals) somatic living cells,
the biological characteristics of which have been substantially
altered as a result of their manipulation to obtain a therapeutic,
558 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE An Example of Implications of Mesenchymal Stem Cell (MSC) Therapy for Patients With Osteogenesis
45.2 Imperfecta (OI) and Health Care Systems

CURRENT TREATMENT OPTIONS FOR OI

Implications for the health
Treatment option Therapy outlook Implications for the patient care system
Bisphosphonates Limited evidence that BMD increases First available medical treatment to increase Unclear impact on QoL and costs for
Failed to demonstrate clinical benefit BMD society
(reduce pain, improved growth, Costs include recurrent inpatient stays
better functional mobility) for IV treatment
Negative impact on fracture healing
Long-term safety unclear; in adults,
possibly more fractures
Orthopaedic For example, intramedullary rods, to Physical and psychological trauma associated Indicative costs of €15,000–20,000 per
surgery correct or improve bone deformities with surgical intervention and recuperation surgical event (theatre time, staff
interventions salaries, postoperative care and
rehabilitation)a,b
Responsive Pain management, casts, corrective Lifelong exposure to painful and QoL-limiting risk Indicative costs of €10,000–€20,000
therapy due to surgery for fractures per fracture event (emergency care
fractures Severe restriction of daily function and rehabilitation)a,b
Physiotherapy Muscle strength and range of move- Scientific evidence lacking; aim is increased Costs of providing care for patients with
ment training mobility OI are unclear
POTENTIAL IMPACT OF MSC THERAPY
MSC therapy Early-stage regenerative treatment to Treating OI during fetal life or childhood Cost benefits will result from a
intervene in OI development Periodic (estimated annual or biannual) infusion reduction in fractures in treated
of MSC in an outpatient clinic with funda- patients
mental reduction in the severity of the entire
condition
aLittle
information is available in the literature describing in detail the costs associated with the care of individuals with OI. A study demonstrated that each hospital admission of an OI patient costs approxi-
mately €4000,96 recalculated with Consumer Price Index for Medical Care in the United States), and each patient is admitted at on average two times per year,96 suggesting a total average inpatient cost
of €8000. The additional costs associated with care by the parents or postdischarge medical care are not included in this table.
bSurgery including intramedullary rod placement is a pillar of treatment for OI. Data from the United Kingdom in 2005 suggest that the cost of nailing a paediatric femur fracture in an otherwise healthy
child is approximately €670097 (adjusted for 2014 prices using the Consumer Price Index for Health in the United Kingdom). No data on the cost of the procedure in patients with OI are available. However,
the additional time and care that must be taken when operating on a patient with OI, for example, to ensure appropriate anaesthesia and avoid additional fractures during surgery, and the close postopera-
tive follow-up required will at least double this cost. The same publication suggests that the costs of nonsurgical treatment for a paediatric femur fracture in an otherwise healthy child are much higher
(€9500–€18,500).
BMD, Bone mineral density; IV, intravenous; QoL, quality of life.
  

Testing and marketing

Regulatory review and approval

Clinical phase III trial

Clinical phase II trial

Clinical phase I trial

Preclinical testing

Research and development

0 2 4 6 8 10 12 14 16 Years
• Fig. 45.1  Estimated timeline for drug development. Several steps are required to ensure the safety and
efficacy of a drug before it is approved for use in humans. It may take as long as 15 years from the dis-
covery of a new drug until it reaches the patient. Drug development can be divided into phases. The first
is the preclinical phase, which might take 5 to 7 years to complete. This phase is followed by an applica-
tion to the regulatory authority, and if approved, clinical phases 1, 2 and 3 follow. The manufacturer then
files a drug application for approval. After acceptance, the regulatory authority can also request that the
manufacturer conduct additional postmarketing studies.

diagnostic or preventive effect through metabolic, pharmacologic
and immunologic means. This manipulation includes the expan-
sion or activation of autologous cell populations ex vivo, the use
of allogeneic and xenogeneic cells associated with medical devices
used ex vivo or in vivo. ATMPs have the potential to improve the
lives of patients with serious conditions in which unmet medical
needs exist, but so far, only a few cell therapy medicinal products
have received marketing approval. Developing ATMPs is, how-
ever, complex and demanding. Challenges include finding ade-
quate animal models for preclinical testing, lack of sensitive tools
to extensively characterise the product, lack of experience in the
often academic or small size pharmaceutical industry environment
in developing drugs for global commercialisation, a complicated
and sometimes difficult to interpret set of rules and regulations,
and challenges in manufacturing a highly advanced drug prod-
uct from biological sources with large in-built variation. It is thus
complicated and time consuming to perform cell therapy studies,
and it is expensive to produce the pharmaceutical—the cells.
The prospect of IUT studies in human fetuses raises impor-
tant regulatory and ethical issues and is thus to be considered one
of the most advanced types of cell therapies. A multidisciplinary
approach is needed, involving a team of experts such as doctors
with expertise in the field of the treated disease, fetal medicine
experts and manufacture and regulatory experts as well as experts
on ethical issues. Counselling is a key issue, and thus specially
trained nurses and clinicians are considered as being of utmost
importance. Current regulations state that research should not
involve greater than minimal risk, especially in early clinical stud-
ies in which no or minimal prospect of direct benefit to the fetus
or the pregnant woman is to be expected. The combination of
stem cells and IUT involves a variety of safety considerations.
First, there are risks associated with needle insertions through
the uterine wall, including spontaneous abortion and premature
birth. The prenatal infusion procedure is comparable to intrauter-
ine blood transfusions, which are routinely performed in expert
fetal therapy centres, and with a complication rate of less than 1%
to 2%.94,95 Second, manufacturing, including source control and
cell therapy product testing, must be clearly defined because cell
therapy products cannot be sterilised before administration. The
major components of source control for cell therapy production
CHAPTER 45  In Utero Stem Cell Transplantation 559
are donor testing with approved methods to prevent transmis-
sion of infection and control of all reagents and supplies that have
contact with the product to prevent introduction of infectious
agents during cell processing. Furthermore, other risks are associ-
ated with stem cell transplantation such as the potential risk for
tumour formation in the recipient. Therefore the preparation for a
clinical study involves a careful setup of the manufacturing process
as well as a clinical study protocol that has been scrutinised by
experts and authorities. An extensive ethical discussion within the
study team is also necessary.
Taken together, ATMPs using fetal human stem cells hold great
potential for curing previously untreatable diseases, but clinical
investigators and small biotech companies will need to acquire a
deep knowledge of regulatory issues and find funding for this type
of therapy for the field to expand further. 
Conclusion
Transplantation of stem cells to an unborn child is a challenging
and exciting proposition but a project full of hurdles and difficul-
ties. So far, IUT with HSCs has been associated with discouraging
results except in fetuses with inherent immunologic capacity to
mount a reaction towards the transplant. Transplantation with the
immune-privileged MSCs might be a more feasible method but
is still highly experimental. Within the next years, we will com-
mence the first trial on this concept.
In utero transplantation are associated with several difficult
ethical issues. The disease needs to be diagnosed in accurate way,
and the natural pre- and postnatal course needs to be known.
How should patients be recruited, and how should parents grant
informed consent for their unborn children? Furthermore, in
the concept lies a possibility to alter a severe phenotype to a less
diseased child. If this means that a fetus with a lethal condition
will survive in a difficult condition is indeed a very problematic
prospect. Thus, considering the complexity of these issues, there is
an obvious need for a multidisciplinary approach and a cautious
attitude.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 18. McClain LE, Flake AW. In utero stem cell
transplantation and gene therapy: recent
34. Brent L, Linch DC, Rodeck CH, et  al. On
the feasibility of inducing tolerance in man:
progress and the potential for clinical appli- a study in the cynomolgus monkey. Immunol
1. Touraine JL, Raudrant D, Royo C, et  al. In-
cation. Best Pract Res Clin Obstet Gynaecol. Lett. 1989;21(1):55–61.
utero transplantation of stem cells in bare lym-
2016;31:88–98. 35. Roodman GD, Kuehl TJ, Vandeberg JL, et al.
phocyte syndrome. Lancet. 1989;1(8651):1382.
19. Billingham RE, Brent L, Medawar PB. In utero bone marrow transplantation of fetal
2. Flake AW, Roncarolo MG, Puck JM, et  al.
Actively acquired tolerance of foreign cells. baboons with mismatched adult baboon mar-
Treatment of X-linked severe combined
Nature. 1953;172(4379):603–606. row. Blood Cells. 1991;17(2):367–375.
immunodeficiency by in utero transplanta-
20. Mintz B, Anthony K, Litwin S. Monoclonal 36. Shields LE, Gaur LK, Gough M, et  al. In
tion of paternal bone marrow. N Engl J Med.
derivation of mouse myeloid and lymphoid lin- utero hematopoietic stem cell transplantation
1996;335(24):1806–1810.
eages from totipotent hematopoietic stem cells in nonhuman primates: the role of T cells.
3. Westgren M, Ringden O, Bartmann P,
experimentally engrafted in fetal hosts. Proc Natl Stem Cells. 2003;21(3):304–314.
et  al. Prenatal T-cell reconstitution after in
Acad Sci U S A. 1984;81(24):7835–7839. 37. Jolly RD, Thompson KG, Murphy CE, et al.
utero transplantation with fetal liver cells
21. Blazar BR, Taylor PA, Vallera DA. Adult bone Enzyme replacement therapy—an experi-
in a patient with X-linked severe combined
marrow-derived pluripotent hematopoietic ment of nature in a chimeric mannosidosis
immunodeficiency. Am J Obstet Gynecol.
stem cells are engraftable when transferred in calf. Pediatr Res. 1976;10(4):219–224.
2002;187(2):475–482.
utero into moderately anemic fetal recipients. 38. Owen RD. Immunogenetic consequences of
4. Blazar BR, Taylor PA, Vallera DA. In utero
Blood. 1995;85(3):833–841. vascular anastomoses between bovine twins.
transfer of adult bone marrow cells into
22. Waldschmidt TJ, Panoskaltsis-Mortari A, Science. 1945;102(2651):400–401.
recipients with severe combined immunodefi-
McElmurry RT, et  al. Abnormal T cell- 39. Lillie FR. The theory of the free-Martin.
ciency disorder yields lymphoid progeny with
dependent B-cell responses in SCID mice Science. 1916;43(1113):611–613.
T- and B-cell functional capabilities. Blood.
receiving allogeneic bone marrow in utero. 40. Jones MZ, Cavanagh KT, Kranich R, et  al.
1995;86(11):4353–4566.
Severe combined immune deficiency. Blood. Possible beta-mannosidosis chimera. Altered
5. Wengler GS, Lanfranchi A, Frusca T, et al. In-
2002;100(13):4557–4564. expression of metabolic perturbations.
utero transplantation of parental CD34 hae-
23. Fleischman RA, Mintz B. Development J Inherit Metab Dis. 1993;16(6):1012–1023.
matopoietic progenitor cells in a patient with
of adult bone marrow stem cells in H-2- 41. Picus J, Aldrich WR, Letvin NL. A naturally
X-linked severe combined immunodeficiency
compatible and -incompatible mouse fetuses. occurring bone-marrow-chimeric primate. I.
(SCIDXI). Lancet. 1996;348(9040):1484–
J Exp Med. 1984;159(3):731–745. Integrity of its immune system. Transplantation.
1487.
24. Marquardt N, Ivarsson MA, Sundstrom E, 1985;39(3):297–303.
6. Westgren M, Ringdén O, Eik-Nes S, et al. Lack
et al. Fetal CD103+ IL-17-producing group 3 42. Howell JM, Dorling PR, Shelton JN, et  al.
of evidence of permanent engraftment after in
innate lymphoid cells represent the dominant Natural bone marrow transplantation in cat-
utero fetal stem cell transplantation in con-
lymphocyte subset in human amniotic fluid. tle with Pompe’s disease. Neuromuscul Disord.
genital hemoglobinopathies. Transplantation.
J Immunol. 2016;197(8):3069–3075. 1991;1(6):449–454.
1996;61(8):1176–1179.
25. Merianos DJ, Tiblad E, Santore MT, et  al. 43. Emery D, McCullagh P. Immunological reac-
7. Slavin S, Naparstek E, Ziegler M, et al. Clini-
Maternal alloantibodies induce a postnatal tivity between chimeric cattle twins. I. Homo-
cal application of intrauterine bone marrow
immune response that limits engraftment fol- graft reaction. Transplantation. 1980;29(1):
transplantation for treatment of genetic dis-
lowing in utero hematopoietic cell transplan- 4–9.
eases—feasibility studies. Bone Marrow Trans-
tation in mice. J Clin Invest. 2009;119(9): 44. van Dijk BA, Boomsma DI, de Man AJ. Blood
plant. 1992;9(suppl 1):189–190.
2590–2600. group chimerism in human multiple births is
8. Touraine JL, Raudrant D, Royo C, et  al. In
26. Nijagal A, Wegorzewska M, Jarvis E, et  al. not rare. Am J Med Genet. 1996;61(3):264–268.
utero transplantation of hemopoietic stem
Maternal T cells limit engraftment after in 45. Hansen HE, Niebuhr E, Lomas C. Chime-
cells in humans. Transplant Proc. 1991;23(1
utero hematopoietic cell transplantation in ric twins. T.S. and M.R. reexamined. Hum
Pt 2):1706–1708.
mice. J Clin Invest. 2011;121(2):582–592. Hered. 1984;34(2):127–130.
9. Diukman R, Golbus MS. In utero stem cell
27. Nijagal A, Wegorzewska M, Le T, et al. The 46. Vietor HE, Hamel BC, van Bree SP, et  al.
therapy. J Reprod Med. 1992;37(6):515–
maternal immune response inhibits the suc- Immunological tolerance in an HLA non-
520.
cess of in utero hematopoietic cell transplan- identical chimeric twin. Hum Immunol.
10. Touraine JL, Raudrant D, Golfier F, et  al.
tation. Chimerism. 2011;2(2):55–57. 2000;61(3):190–192.
Reappraisal of in utero stem cell transplanta-
28. Kim HB, Shaaban AF, Milner R, et al. In utero 47. Kuhl-Burmeister R, Simeoni E, Weber-
tion based on long-term results. Fetal Diagn
bone marrow transplantation induces donor- Matthiesen K, et  al. Equal distribution of
Ther. 2004;19(4):305–312.
specific tolerance by a combination of clonal congenital blood cell chimerism in dizy-
11. Monni G, Ibba RM, Zoppi MA, et  al. In
deletion and clonal anergy. J Pediatr Surg. gotic triplets after in-vitro fertilization. Hum
utero stem cell transplantation. Croat Med J.
1999;34(5):726–729; discussion 729–730. Reprod. 2000;15(5):1200–1204.
1998;39(2):220–223.
29. Alhajjat AM, Lee AE, Strong BS, et al. NK cell 48. Angela E, Robinson E, North D. A case of twin
12. Jones DR, Bui TH, Anderson EM, et  al. In
tolerance as the final endorsement of prenatal chimerism. J Med Genet. 1976;13(6):528–
utero haematopoietic stem cell transplantation:
tolerance after in utero hematopoietic cellular 530.
current perspectives and future potential. Bone
transplantation. Front Pharmacol. 2015;6:51. 49. Thomsen M, Hansen HE, Dickmeiss E.
Marrow Transplant. 1996;18(5):831–837.
30. Kim AG, Vrecenak JD, Boelig MM, et  al. MLC and CML studies in the family of a pair
13. Shields LE, Lindton B, Andrews RG, et  al.
Enhanced in utero allogeneic engraft- of HLA haploidentical chimeric twins. Scand
Fetal hematopoietic stem cell transplantation: a
ment in mice after mobilizing fetal HSCs J Immunol. 1977;6(5):523–528.
challenge for the twenty-first century. J Hema-
by alpha4beta1/7 inhibition. Blood. 2016; 50. Jonsson AM, Uzunel M, Gotherstrom C, et al.
tother Stem Cell Res. 2002;11(4):617–631.
128(20):2457–2461. Maternal microchimerism in human fetal tis-
14. Westgren M, Shields LE. In utero stem cell
31. Zanjani ED, Almeida-Porada G, Ascensao sues. Am J Obstet Gynecol. 2008;198(3):325.
transplantation in humans. Ernst Schering Res
JL, et  al. Transplantation of hematopoietic e1–e6.
Found Workshop. 2001;33:197–221.
stem cells in utero. Stem Cells. 1997;15(suppl 51. Jonsson AM, Papadogiannakis N, Granath
15. Tiblad E, Westgren M. Fetal stem-cell trans-
1):79–92; discussion 93. A, et  al. Maternal microchimerism in juve-
plantation. Best Pract Res Clin Obstet Gynae-
32. Crombleholme TM, Harrison MR, Zanjani nile tonsils and adenoids. Pediatr Res.
col. 2008;22(1):189–201.
ED. In utero transplantation of hematopoi- 2010;68(3):199–204.
16. Bui TH, Jones DR. Stem cell transplan-
etic stem cells in sheep: the role of T cells in 52. Kanold AM, Svenungsson E, Gunnarsson
tation into the fetal recipient: challenges
engraftment and graft-versus-host disease. I, et  al. A research study of the association
and prospects. Curr Opin Obstet Gynecol.
J Pediatr Surg. 1990;25(8):885–892. between maternal microchimerism and sys-
1998;10(2):105–108.
33. Shields LE, Gaur L, Delio P, et  al. Fetal temic lupus erythematosus in adults: a com-
17. Almeida-Porada G, Atala A, Porada CD. In
immune suppression as adjunctive therapy parison between patients and healthy controls
utero stem cell transplantation and gene ther-
for in utero hematopoietic stem cell trans- based on single-nucleotide polymorphism
apy: rationale, history, and recent advances
plantation in nonhuman primates. Stem Cells. using quantitative real-time PCR. PLoS One.
toward clinical application. Mol Ther Methods
2004;22(5):759–769. 2013;8(9):e74534.
Clin Dev. 2016;5:16020.

559.e1
559.e2 References
53. Gholamrezanezhad A, Mirpour S, Bagheri M,
et al. In vivo tracking of 111In-oxine labeled
mesenchymal stem cells following infusion in
patients with advanced cirrhosis. Nucl Med
Biol. 2011;38(7):961–967.
54. Erkers T, Kaipe H, Nava S, et al. Treatment
of severe chronic graft-versus-host disease
with decidual stromal cells and tracing with
(111)indium radiolabeling. Stem Cells Dev.
2015;24(2):253–263.
55. Schrepfer S, Deuse T, Reichenspurner H,
et al. Stem cell transplantation: the lung bar-
rier. Transplant Proc. 2007;39(2):573–576.
56. Gao J, Dennis JE, Muzic RF, et  al. The
dynamic in vivo distribution of bone marrow-
derived mesenchymal stem cells after infusion.
Cells Tissues Organs. 2001;169(1):12–20.
57. Niyibizi C, Wang S, Mi Z, et al. The fate of
mesenchymal stem cells transplanted into
immunocompetent neonatal mice: implica-
tions for skeletal gene therapy via stem cells.
Mol Ther. 2004;9(6):955–963.
58. Chan J, Waddington SN, O’Donoghue K,
et  al. Widespread distribution and muscle
differentiation of human fetal mesenchymal
stem cells after intrauterine transplanta-
tion in dystrophic mdx mouse. Stem Cells.
2007;25(4):875–884.
59. Guillot PV, Abass O, Bassett JH, et  al.
Intrauterine transplantation of human fetal
mesenchymal stem cells from first-trimester
blood repairs bone and reduces fractures
in osteogenesis imperfecta mice. Blood.
2008;111(3):1717–1725.
60. Campagnoli C, Roberts IA, Kumar S,
et  al. Identification of mesenchymal stem/
progenitor cells in human first- trimester
fetal blood, liver, and bone marrow. Blood.
2001;98(8):2396–2402.
61. Mendes SC, Robin C, Dzierzak E. Mes-
enchymal progenitor cells localize within
hematopoietic sites throughout ontogeny.
Development. 2005;132(5):1127–1136.
62. Taylor PA, McElmurry RT, Lees CJ, et  al.
Allogenic fetal liver cells have a distinct
competitive engraftment advantage over
adult bone marrow cells when infused into
fetal as compared with adult severe com-
bined immunodeficient recipients. Blood.
2002;99(5):1870–1872.
63. Orioli IM, Castilla EE, Barbosa-Neto JG. The
birth prevalence rates for the skeletal dyspla-
sias. J Med Genet. 1986;23(4):328–332.
64. Forlino A, Marini JC. Osteogenesis imper-
fecta: prospects for molecular therapeutics.
Mol Genet Metab. 2000;71(1-2):225–232.
65. Sillence DO, Rimoin DL. Classification
of osteogenesis imperfect. Lancet. 1978;
1(8072):1041–1042.
66. Sillence DO, Horton WA, Rimoin DL. Mor-
phologic studies in the skeletal dysplasias. Am
J Pathol. 1979;96(3):813–870.
67. Thomas IH, DiMeglio LA. Advances in
the classification and treatment of osteo-
genesis imperfecta. Curr Osteoporos Rep.
2016;14(1):1–9.
68. Van Dijk FS, Pals G, Van Rijn RR, et al. Clas-
sification of osteogenesis imperfecta revisited.
Eur J Med Genet. 2010;53(1):1–5.
69. Khalil A, Pajkrt E, Chitty LS. Early prenatal
diagnosis of skeletal anomalies. Prenat Diagn.
2011;31(1):115–124.
70. Griffin DR, Chitty LS. Prenatal diagnosis of
fetal skeletal abnormalities. In: JD, Steer PJ,
Weiner CP, Gonik B, eds. High Risk Preg-
nancy: Management Options. 4th ed. St. Louis,
MO: Elsevier Saunders; 2011:347–366.
71. Folkestad L, Hald JD, Canudas-Romo V,
et al. Mortality and causes of death in patients
with osteogenesis imperfecta: a register-based
nationwide cohort study. J Bone Miner Res.
2016;31(12):2159–2166.
72. Folkestad L, Hald JD, Ersboll AK, et  al.
Fracture rates and fracture sites in patients
with osteogenesis imperfecta: a nationwide
register-based cohort study. J Bone Miner Res.
2017;32(1):125–134.
73. Pittenger MF, Mackay AM, Beck SC,
et  al. Multilineage potential of adult
human mesenchymal stem cells. Science.
1999;284(5411):143–147.
74. Le Blanc K, Tammik L, Sundberg B, et  al.
Mesenchymal stem cells inhibit and stimulate
mixed lymphocyte cultures and mitogenic
responses independently of the major his-
tocompatibility complex. Scand J Immunol.
2003;57(1):11–20.
75. Najar M, Raicevic G, Crompot E, et al. The
immunomodulatory potential of mesen-
chymal stromal cells: a story of a regulatory
network. J Immunother. 2016;39(2):45–
59.
76. Prockop DJ, Brenner M, Fibbe WE, et  al.
Defining the risks of mesenchymal stromal
cell therapy. Cytotherapy. 2010;12(5):576–
578.
77. Squillaro T, Peluso G, Galderisi U. Clinical
trials with mesenchymal stem cells: an update.
Cell Transplant. 2016;25(5):829–848.
78. Götherström C, Ringden O, Tammik C,
et al. Immunologic properties of human fetal
mesenchymal stem cells. Am J Obstet Gynecol.
2004;190(1):239–245.
79. Götherström C, Ringden O, Westgren M,
et  al. Immunomodulatory effects of human
foetal liver-derived mesenchymal stem cells.
Bone Marrow Transplant. 2003;32(3):265–
272.
80. Guillot PV, Gotherstrom C, Chan J, et  al.
Human first-trimester fetal MSC express plu-
ripotency markers and grow faster and have
longer telomeres than adult MSC. Stem Cells.
2007;25(3):646–654.
81. Zhang ZY, Teoh SH, Chong MS, et  al.
Superior osteogenic capacity for bone tissue
engineering of fetal compared with perinatal
and adult mesenchymal stem cells. Stem Cells.
2009;27(1):126–137.
82. Götherström C, West A, Liden J, et al. Differ-
ence in gene expression between human fetal
liver and adult bone marrow mesenchymal
stem cells. Haematologica. 2005;90(8):1017–
1026.
83. Guillot PV, De Bari C, Dell’Accio F, et  al.
Comparative osteogenic transcription pro-
filing of various fetal and adult mesen-
chymal stem cell sources. Differentiation.
2008;76(9):946–957.
84. Kennea NL, Waddington SN, Chan J, et al.
Differentiation of human fetal mesenchymal
stem cells into cells with an oligodendrocyte
phenotype. Cell Cycle. 2009;8(7):1069–1079.
85. Pereira RF, O’Hara MD, Laptev AV, et  al.
Marrow stromal cells as a source of pro-
genitor cells for nonhematopoietic tissues in
transgenic mice with a phenotype of osteo-
genesis imperfecta. Proc Natl Acad Sci U S A.
1998;95(3):1142–1147.
86. Panaroni C, Gioia R, Lupi A, et  al. In
utero transplantation of adult bone mar-
row decreases perinatal lethality and rescues
the bone phenotype in the knockin murine
model for classical, dominant osteogenesis
imperfecta. Blood. 2009;114(2):459–468.
87. Jones GN, Moschidou D, Abdulrazzak H,
et al. Potential of human fetal chorionic stem
cells for the treatment of osteogenesis imper-
fecta. Stem Cells Dev. 2014;23(3):262–276.
88. Vanleene M, Saldanha Z, Cloyd KL, et  al.
Transplantation of human fetal blood stem
cells in the osteogenesis imperfecta mouse
leads to improvement in multiscale tissue
properties. Blood. 2011;117(3):1053–1060.
89. Horwitz EM, Prockop DJ, Fitzpatrick LA,
et al. Transplantability and therapeutic effects
of bone marrow-derived mesenchymal cells
in children with osteogenesis imperfecta. Nat
Med. 1999;5(3):309–313.
90. Horwitz EM, Prockop DJ, Gordon PL, et al.
Clinical responses to bone marrow transplan-
tation in children with severe osteogenesis
imperfecta. Blood. 2001;97(5):1227–1231.
91. Horwitz EM, Gordon PL, Koo WK, et al. Iso-
lated allogeneic bone marrow-derived mesen-
chymal cells engraft and stimulate growth in
children with osteogenesis imperfecta: impli-
cations for cell therapy of bone. Proc Natl
Acad Sci U S A. 2002;99(13):8932–8937.
92.  Le Blanc K, Gotherstrom C, Ringden O,
et  al. Fetal mesenchymal stem-cell engraft-
ment in bone after in utero transplantation in
a patient with severe osteogenesis imperfecta.
Transplantation. 2005;79(11):1607–1614.
93. Götherström C, Westgren M, Shaw SW,
et  al. Pre- and postnatal transplantation of
fetal mesenchymal stem cells in osteogenesis
imperfecta: a two-center experience. Stem
Cells Transl Med. 2014;3(2):255–264.
94. Tiblad E, Kublickas M, Ajne G, et al. Proce-
dure-related complications and perinatal out-
come after intrauterine transfusions in red cell
alloimmunization in Stockholm. Fetal Diagn
Ther. 2011;30(4):266–273.
95. Lindenburg IT, van Kamp IL, Oepkes D.
Intrauterine blood transfusion: current indi-
cations and associated risks. Fetal Diagn Ther.
2014;36(4):263–271.
96. Dye DE, Brameld KJ, Maxwell S, et al. The
impact of single gene and chromosomal dis-
orders on hospital admissions of children and
adolescents: a population-based study. Public
Health Genomics. 2011;14(3):153–161.
97. Gaid M, Jeer P. Cost analysis of manag-
ing paediatric femoral shaft fractures:
flexible intramedullary nailing versus non-
operative management. Acta Orthop Belg.
2006;72(2):170–175.
46
Fetal Gene Therapy
ANNA L. DAVID
KEY POINTS
• Gene therapy for fetuses may provide a therapeutic advan-
tage over postnatal treatment for certain congenital diseases.
• Three types of fetal gene therapy are available: direct ‘somatic’
fetal gene therapy, in utero transplantation of gene corrected
fetal stem cells and maternal gene therapy
• In preclinical animal models of congenital disease, direct fetal
gene therapy can bring a phenotypic cure.
• Minimally invasive ultrasound-guided injection techniques
can be used to target gene therapy to fetal organs in animal
models.
• Observed risks of fetal gene therapy include insertional muta-
genesis, germline gene transfer, vector toxicity, the fetal and
maternal immune response to the vector, and maternal and
fetal morbidity and mortality.
• Currently, direct fetal gene therapy and in utero transplanta-
tion of gene-corrected fetal stem cells remains at the experi-
mental stage.
• Maternal gene therapy is close to being translated into the
clinic.
Introduction
Gene therapy has now arrived as a major step forward in the treat-
ment of select severe genetic diseases. Already lentiviral vectors
have been used to cure severe combined immune deficiency,1
and trials of adeno-associated viral vectors show efficacy to treat
haemophilia B (factor IX deficiency).2 Many of the original bar-
riers to effective postnatal gene therapy have been overcome.
These include difficulty targeting the appropriate organ, a robust
immune response to the therapy in adults and low-level expression
of the therapeutic protein. In some diseases, gene therapy may be
most effective when it is given early in neonatal life. When con-
genital disease pathology occurs during fetal development, then
treating the fetus may be the best solution.
Preclinical studies in animal models in the last 18 years have
shown that fetal gene therapy can cure severe genetic disease.
More recently, structural anomalies in fetuses have been prevented
using a gene therapy approach.3 For some nongenetic conditions,
timely expression of a particular protein, for example, during the
last third of pregnancy, may alleviate pathology. The arrival of
noninvasive prenatal diagnosis using circulating fetal DNA in the
maternal blood allows clinicians to detect a congenital disease in
fetuses as early as 10 weeks of gestation. This gives couples time to
560
consider their options, including a possible in utero treatment to
correct the genetic disorder. 
What Is Gene Therapy?
Gene therapy uses the delivery of genetic material, generally
DNA, into cells to generate a therapeutic effect by correcting an
existing abnormality or providing cells with a new function. Vec-
tors are the vehicles that are used to carry the genetic material into
the cell. The two major classes of gene therapy methods use either
recombinant ‘genetically altered’ viruses (viral vectors) or naked
DNA or DNA complexes (nonviral methods). The genetic mate-
rial that is transferred by this type of genetic engineering is called a
transgene, and the introduction of the transgene has the potential
to change the phenotype of a patient. The transgene contains (i)
a promoter, which is a regulatory sequence that will determine
where and when the transgene is active (e.g., a liver promoter to
switch on the transgene only when it is in a liver cell); (ii) an exon,
a protein coding sequence (usually derived from the cDNA for the
protein of interest, e.g., human factor IX cDNA for treatment of
haemophilia B); and (iii) a stop sequence to turn off the transgene.
In the cell, the transgene is expressed producing a transgenic pro-
tein that has the therapeutic effect.
Somatic gene therapy treats an individual patient by insertion
of genes into non germline cells that are either outside the body
(ex  vivo) (e.g., haematopoietic stem cells grown in culture) or
in vivo (e.g., intravascular injection); pluripotential stem cells or
differentiated cells may be targeted. Fetal application of gene ther-
apy can be directly to the fetus or to fetal stem cells for autologous
transplantation (Fig. 46.1). Gene therapy can even be given to the
mother when maternal pathology affects the fetus in utero (e.g., in
the case of uteroplacental insufficiency).4 Germline gene therapy
would target oocytes or spermatocytes and potentially might eradi-
cate inherited diseases in future generations. From the earliest days
of gene therapy, however, correcting the germline was considered
to be neither scientifically nor medically justifiable, technically
unsafe and unpredictable and therefore ethically unacceptable.5 
The Potential Advantages and
Disadvantages of Fetal Gene Therapy
The advantages of fetal gene therapy over postnatal treatment are
summarised in Table 46.1. Production of clinical grade vector is
time consuming and expensive, and the small size of the fetus
could lead to increased vector biodistribution at the same vector
dose as an adult. Organs that are difficult to target after birth may
 CHAPTER 46  Fetal Gene Therapy 561

Fetal stem cell gene therapy Somatic or germ line fetal gene therapy

Ex vivo In vivo
Transduced cells injected via
IM, IP, IV routes and so on

Gene therapy vector

Transduced ex vivo
using gene therapy
vector or gene editing Fetus (or mother) injected
via IP, IV, intraamniotic,
IM routes and so on

Stem cells from fetal fluid Insertion of gene

(e.g., amniotic fluid or blood) into cell, nucleus or
genome

• Fig. 46.1  The fetus can receive gene therapy in a variety of ways. The vector can be directly applied to
the fetus or the mother by ultrasound guided injection, called somatic gene therapy. Alternatively, stem
cells can be collected from fetal fluid by ultrasound-guided sampling, gene corrected in the laboratory by
incubation with the gene therapy vector and then reintroduced back into the fetus by ultrasound guided
injection. IM, Intramuscular; IP, intraperitoneal; IV, intravascular.

TABLE
46.1 Potential Advantages and Disadvantages of Fetal Over Postnatal Gene Therapy

Advantage of Fetal Gene Therapy Disease Example or Effect

Correct disease in the fetus when pathology begins
before birth
Targets a rapidly dividing population of stem cells
that exist in the fetus
Higher vector-to-target cell ratio because the fetus is small
Target organs that are difficult to access after birth
Induce tolerance to the transgene protein product
Disadvantage of Fetal Gene Therapy
Possible increased risk for genetic modification of the
germline
Possible increased risk for insertional mutagenesis related
to particular vector types (EIAV lentivirus)
Vector toxicity
Congenital MPS
Fetal application may prevent the brain damage that occurs before birth
Provides a large population of transduced cells to produce a better therapeutic effect
Less vector needed to achieve the same therapeutic effect so more cost effective
Epidermolysis bullosa
The fetal epidermis may be targeted in utero using gene therapy.92 It undergoes remodelling by pro-
grammed cell death to be replaced postnatally by mature keratinocytes that prevent gene transfer.
Better therapeutic effect by avoiding the development of an immune response that occurs in
postnatal gene therapy
Disease Example or Effect
Gene transfer to male sheep sperm seen after first trimester injection but not second or third
trimester exposure72
Hepatocellular carcinoma in adult mice after fetal gene transfer78
Fetal ascites and death seen after injection of VSVG-pseudotyped lentivirus into fetal sheep

Maternal immune response to vector may prevent Preexisting maternal antibodies to AAV5 serotype prevented gene transfer to fetal macaques15
gene transfer to fetus
Maternal and/or fetal morbidity and mortality Intrauterine procedures could cause fetal loss and maternal and fetal morbidity

AAV5, Adeno-associated virus serotype 5; EIAV, equine infectious anaemia virus; MPS, mucopolysaccharidoses; VSVG, vesicular stomatitis virus G protein.
  

be more easily accessible during fetal life because of their develop-
mental stages or relative immaturity.6
A major obstacle to postnatal gene therapy has been the devel-
opment of an immune response against the transgenic (therapeutic)
protein or the vector itself,7 particularly when gene therapy is aim-
ing to correct a genetic disease in which complete absence of a gene
product is observed. Some individuals have preexisting antibodies to
the viral vector that will prevent long-term expression of the trans-
genic protein, limiting therapeutic efficacy and preventing repeated
vector administration. For example, preexisting neutralising anti-
bodies against adeno-associated virus (AAV) serotype 2 have been
shown to interfere with AAV2 vector-mediated factor IX (FIX) gene
562 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE Characteristics of the Ideal Vector for Fetal Gene Manipulating the vector structure and the transgene can alter
46.2 Therapy vector properties. Pseudotyping, for example, involves changing
the virus capsid (outer covering) for one of a different serotype
Characteristic Reason or of a completely different virus, thus altering its ability to infect
Highly efficient, Provide therapeutic levels of protein expres- particular cell types or organs.16 Using alternative enhancer-
regulated transgenic sion promoters can improve gene transfer to specific organs or tis-
protein expression sues. A promoter is a site on DNA to which RNA polymerase can
bind and initiate transcription, and an enhancer is a regulatory
Time and duration of Example (1): long-term transgenic protein sequence that can elevate levels of transcription from an adjacent
transgenic protein expression for a monogenic disorder
expression to suit requires protein expression to last the life-
promoter. These can be derived from the genomes of mammals,
disease time of the individual (e.g., haemophilias) viruses or other organisms and can even be manipulated to allow
Example (2): transient transgenic pro- regulatable gene expression if required.17
tein expression for a developmental Some vectors, such as lentivirus and retrovirus vectors, con-
or obstetric disorder requires protein tain integrases, an enzyme that enables the virus genetic mate-
expression at a critical window of fetal rial to be integrated into the DNA of the infected cell. This
growth (e.g., fetal growth restriction) ensures that daughter cells contain the viral genetic material
Example (3): transient expression of a after cell division. Other vectors such as adenovirus are noninte-
transgenic protein at a specific grating. Many replication-deficient lentiviruses are based on the
development time point to treat a immunodeficiency virus, and there is the theoretical possibility
structural defect (e.g., facial cleft)
of reversion to the wild type. In third- and fourth-generation
Specific vector tropism Avoid systemic gene transfer lentivirus vectors, however, the risk for in  vivo generation of
to target organ replication competent viruses is reduced by removal of the tat
Large carrying capacity Accommodate therapeutic gene and any gene. Modification of virus elements, such as mutating the inte-
of transgene insert required regulatory elements grase in lentiviral vectors, renders it incapable of integrating and
greatly reduces the risk for insertional mutagenesis.18 Clinical
No toxicity Safe for mother, fetus and future progeny grade production of vectors are tested rigorously for replication
No immunogenicity Avoid generating a fetal immune response competent viruses. For more detailed information on vectors
relevant to fetal gene therapy, the authors refer readers to other
No mutagenic properties Safe for fetus and future progeny references.19
Replication incompetent Inability to replicate itself after injection in vivo The effect of fetal exposure on vectors is important to con-
sider because many routes of fetal application require delivery into
Manufacturing process Sufficient quantities of clinical grade vector
fluid compartments such as the serum, airways or amniotic fluid
appropriate for GMP available at a reasonable cost
(AF). Human serum can inactivate retroviruses and AF inhibits
GMP, good manufacturing practice. retrovirus infection.20 Altering vector production can make them
   more robust. Lentivirus, AAV and adenovirus vectors are relatively
immune to damage.
transfer to the liver.7-9 Delivering foreign protein to the fetus can take Gene Editing. Gene editing is a process of insertion, deletion or
advantage of immune tolerance which is induced during fetal life, a replacement of DNA at a specific site in the genome of a cell which
concept that was first proposed nearly 60 years ago.10,11 Induction of is achieved in the laboratory using engineered nucleases also known
tolerance depends on the foreign protein being expressed sufficiently as molecular scissors. It is an attractive alternative approach to cor-
early in gestation, probably by 12 to 16 weeks, before the immune rect gene defects because it avoids the introduction into the cell or
system is fully developed and expression maintained at a detectable genome of the extra DNA or RNA components that viral or nonvi-
level within the fetus for presentation to the thymus at the correct ral vector approaches require. There are a number of different possi-
time. For human gestation, transgenic protein expression will need to ble strategies.21 Zinc finger nucleases (ZFNs) are artificial restriction
last at least 6 months if the vector is given early in pregnancy, which enzymes engineered to target desired DNA sequences within
limits the types of viral vectors that can be applied. Proof-of-principle complex genomes. Transcription activator–like effector nucleases
studies have shown long-term expression of proteins at therapeutic (TALENs) use DNA-recognition modules that recognise single
levels and induction of immune tolerance12 in both small13 and large base pairs. Reports of successful applications to genomic targets
animals14,15 and cured congenital disease in some animal models. are appearing at an accelerating rate.22-24 RNA-guided engineered
nucleases (RGENs) derived from the bacterial clustered regularly
Vectors for Fetal Gene Transfer interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-
associated) system are now available. CRISPR/Cas-mediated
An ideal vector for prenatal gene therapy is one that can produce genome editing has been successfully demonstrated in zebrafish and
long-term regulated and therapeutic expression of the transferred gene bacterial cells,25 giving high-precision genome editing. Gene editing
through the use of a single and efficient gene delivery method and is could be particularly attractive to target specific stem cell groups in
safe to the mother and fetus, thus allowing incorporation into clini- an in utero stem cell gene therapy (IUSCGT) approach. 
cal practice. For example, a vector carrying the β-globin gene should
deliver and express the gene only to erythroid specific cells and lineages. Selecting the Right Disease for Fetal Gene
These and other essential characteristics are described in Table 46.2.
The most commonly tested vectors in fetal gene therapy pre- Therapy
clinical studies have been adenovirus and AAV, lentivirus and As with any new therapeutic modality, the risks of fetal gene
retrovirus vectors. These and other less commonly used vector sys- therapy are not well characterised. Careful thought must be given
tems are described in Table 46.3. to decide on the right disease to select for a first-in-woman trial.
 CHAPTER 46  Fetal Gene Therapy 563

TABLE
46.3 Types of Vector and Considerations in Relation to Prenatal Gene Therapy Application
Vector DNA Efficiency Tropism Advantages Disadvantages Prenatal Considerations
Nonviral DNA No limit + Limited Low toxicity Low transduction Expression may not last through
Low immunogenicity efficiency gestation
Adenovirus 7.5 kB +++ Depends on Can grow to high titre Short-term expression Case reports of an association
serotype Highly efficient gene transfer & immunogenic with some fetal
Clinical safety and efficacy abnormalities
data long term in adults
Helper-dependent 35 kB +++ Broad Low immunogenicity, high Inefficient production
adenovirus capacity, long-term
expression in quiescent
cells
Adeno-associated 4.7 kB ++ Depends on Long-term expression Liver toxicity in adult Some subtypes associated with
virus generally sub-type Low immunogenicity trials due to anti- miscarriage
Very high titre capsid T cells. Low level integration into
Can target central nervous Risk for hepatocellular active genes so theoretical
system via systemic cancer. mutagenesis risk.
injection Androgens increase
Clinical safety and efficacy gene transfer (trans-
data long term in adults duction level higher
in males>females)
Retrovirus 10 kB + Depends on Long-term gene transfer Potential for insertional Risk for germ-line transmission
pseudotyping Clinical safety and efficacy mutagenesis. and insertional mutagenesis
data long term in neo- Infect dividing cells Virus inactivated by amniotic
nates only. fluid
Lentivirus 10 kB ++ Depends on Long-term gene transfer Potential for insertional Risk for germ-line transmission
pseudotyping Infects dividing and non- mutagenesis and insertional mutagenesis
dividing cells
Nonintegrating 10 kB ++ Depends on Insertional mutagenesis Short term expression Rapidly dividing fetal cells may
lentivirus pseudotyping unlikely result in long-term
low transgenic protein
expression

  

• BOX 46.1  Recommendations on Candidate
Diseases for Initial Application of Fetal
Gene Therapy
The disease should be associated with serious morbidity and mortality risks
for the fetus either in utero or postnatally.
There should be no effective postnatal therapy, or there is a poor outcome
using available postnatal therapies.
The treatment should correct all serious abnormalities that are associated
with the disease.
The disease must be definitively diagnosed in utero and have a well-defined
genotype–phenotype relationship.
There should be an animal model that recapitulates the human disease or
disorder for testing of in utero gene transfer.
When vectors are given directly to the fetus for correction of
genetic disease, there has been guidance given by the National
Institutes for Health Recombinant DNA Advisory Committee26
(Box 46.1).
Some of the diseases that may be suitable for fetal treatment are
listed in Table 46.4. Preclinical studies of direct fetal gene trans-
fer are encouraging. Fetal application of gene therapy in mouse
models of congenital disease such as haemophilia A27 and B,28
congenital blindness,29 Crigler-Najjar type 1 syndrome30 and
Pompe disease (glycogen storage disease type II)31 have shown
phenotypic correction of the condition. For structural anomalies,
transient transduction of the periderm via intra-amniotic delivery
of adenoviral vector encoding transforming growth factor (TGF)
β3 prevents cleft palate in a mouse model of disease.3 For obstetric
conditions that affect the fetus, maternal uterine artery injection
of adenovirus containing the vascular endothelial growth factor
(VEGF) gene improves fetal and lamb growth in growth-restricted
sheep pregnancies.32,33
The ideal fetal gene therapy would be able to effectively treat
a serious congenital disease with a single direct fetal vector injec-
tion, providing sufficient transgenic protein expression from one
injection and maintenance of this therapeutic expression for the
rest of the individual’s life. Progress in the treatment of a group of
inherited conditions, the lysosomal storage disorders, is discussed
in detail here to illustrate recent progress.
The Lysosomal Storage Diseases as Candidate Diseases. The
lysosomal storage diseases are inherited deficiencies of lysosomal
enzymes that lead to intracellular substrate accumulation. In
mucopolysaccharidosis (MPS) type VII, for example, a deficiency
of β-glucuronidase activity leads to accumulation of glycosami-
noglycans in lysosomes, resulting in an enlarged liver and spleen,
564 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE
46.4 Examples of Candidate Diseases for Fetal Gene Therapy
Disease Therapeutic Gene Product Target Cells/Organ Age at Onset Incidence Life Expectancy
Cystic fibrosis Cystic fibrosis transmem- Airway and intestinal Third trimester of 1:4000 Mid-30s
brane conductance epithelial cells pregnancy
regulator
Duchenne muscular dystrophy Dystrophin Myocytes 2 yr 1:4500 25 yr
Spinal muscular atrophy Survival motor neuron protein Motor neurons 6 mo (type 1) 1:10,000 2 yr
Haemophilia Human factor VIII or IX clotting Hepatocytes 1 yr 1:6000 Adulthood with treatment
factors
β-Thalassaemia Globin Erythrocyte <1 yr 1:2700 <20 yr in developing
precursors countries
Lysosomal storage disease (e.g., Glucocerebrosidase Hepatocytes 9.5 yr 1:9000 overall <2 yr
neuronopathic Gaucher 1:59,000
disease)
Urea cycle defects (e.g., ornithine Ornithine transcarbamylase Hepatocytes 2 days 1:30,000 overall 2 days (severe neonatal
transcarbamylase deficiency) 1:105,000 onset)
Severe combined γc Cytokine receptor Haematopoietic Birth 1:1,000,000 <6 mo if no bone marrow
immunodeficiency (X-linked SCID) precursor cells transplant
Epidermolysis bullosa (e.g., Type VII collagen Keratinocytes Birth 1:40,000 Adulthood
dystrophica)
Severe fetal growth restriction Vascular endothelial growth Trophoblast Fetus 1:500 Days
factor
  

restricted growth, developmental delay and death from cardiac
failure. The disease process starts in utero. Although rare, MPS
type VII has been a disease of choice to investigate gene therapy
because of the availability of a mouse and dog model.
Correction of the MPS phenotype theoretically requires only
low levels of the therapeutic gene product.34 Neonatal injec-
tion of a retrovirus vector in MPS VII dogs and mice transduces
hepatocytes, with uptake of the enzyme from the circulation by
other organs. The treated animals did not develop cardiac disease
or corneal clouding, and skeletal, cartilage and synovial disease
was ameliorated.35 Nonviral mediated gene transfer to the liver
of MPS I and VII mice also improved the phenotype.36 Still,
the major challenge remains to target the brain, which currently
requires multiple brain injections with accompanying risks,37,38
and immunosuppression to prevent pan-encephalitis that devel-
ops secondary to an immune response to the transgenic protein.38
Widespread correction of the pathological lesions in an MPS VII
mouse has been observed with AAV gene transfer,39 a vector that
elicits less of an immune response.
Fetal gene delivery is an alternative strategy. Injection of adeno-
virus into the cerebral ventricles of fetal mice led to widespread
and long-term gene expression throughout the brain and the spi-
nal cord.40 In the same study, delivery of a therapeutic gene to the
cerebral ventricles of fetal MPS type VII mice prevented damage
in most of the brain cells before and until 4 months after birth.
A similar study using an AAV vector had comparable results but
with longer expression.41
From a translational perspective, direct vector administration
into the fetal brain or ventricles through the fetal skull for pre-
natal gene transfer is technically difficult using minimally inva-
sive injection techniques, although this has been achieved in
nonhuman primates42 and sheep (AL David, unpublished work)
under ultrasound guidance. In contrast, ultrasound-guided access
to the human fetal circulation is commonly used for fetal blood
sampling and transfusions in clinical practice, with minimal fetal
loss rate or complication. Newer AAV vectors of serotypes 2/9
have an astonishing ability to transduce cells of the nervous system
after systemic injection in neonatal mice, cats43,44 and nonhuman
primates.45 Furthermore, intrahepatic umbilical vein injection
in fetal macaques of AAV 2/9 gives comprehensive transduction
of the central nervous system (CNS), including all areas of the
brain and retina, and the peripheral nervous system, including
the myenteric plexus.46 Systemic transduction was also achieved
using these AAV serotypes in fetal mice and macaques, particu-
larly to epithelial and muscle cells.47 A fetal gene therapy approach
using AAV therefore has the potential to treat congenital disorders
affecting a wide range of body systems, including the CNS. 
Fetal Growth Restriction as a Candidate Disease. Severe fetal
growth restriction (FGR) affects 1 in 500 pregnancies and is a
major cause of neonatal morbidity and mortality. The underlying
abnormality in many cases is placental insufficiency, whereby the
normal physiology process of trophoblast invasion that converts
the uterine spiral arteries into a high-flow large conduit for blood
fails to occur. There is no therapy to rescue poor uteroplacental
circulation or improve fetal growth. FGR is commonly diagnosed
when fetal measurements fall below the expected gestational age
charts. Abnormally increased uterine artery vascular resistance is
classically seen in midgestation.
A targeted approach to the uteroplacental circulation is needed
because intravascular infusion of sildenafil citrate, a nitric oxide
donor drops systemic blood pressure and had detrimental effects
on growth-restricted sheep fetuses.48 In the pregnant sheep,

18
p = 0.033
16
14 Marked FGR
Number of Fetuses (n)
Non-FGR
12
10
8 No fetal immune response to the vector or transgenic protein
6
4
2
0 obstetrics and neonatology.53
H + Ad.LacZ/Saline H + Ad.VEGF
• Fig. 46.2  Incidence of marked fetal growth restriction (FGR) in fetuses
of dams treated with uterine artery injection of Ad.VEGF (adenovirus vas-
cular endothelial growth factor) vector. To create FGR, singleton-bearing
adolescent ewes were fed a high (H) dietary intake from conception until
term. In midgestation, pregnant ewes were randomised into receiving a
uterine artery injection of Ad.VEGF vector, Ad.LacZ vector or saline injec-
tion at laparotomy. Fetuses were delivered by hysterotomy at 131 ± 0.2
days gestation. The mean birth weight of the fetuses of 12 contemporane-
ous control-intake ewes that demonstrated normal fetoplacental growth
was used for comparison. Marked FGR (closed bars) was defined as a
birthweight greater than 2 standard deviations (SDs) below this mean birth
weight (<4222 g for the present study). Fetuses with birthweights greater
than 4222 g were of similar weight to normally grown controls and were
therefore termed non-FGR (open bars). Proportions were compared using
Fisher’s exact test. Midgestation treatment with Ad.VEGF vector reduced
the incidence of marked FGR. (Adapted from Carr DJ, Wallace JM, Ait-
ken RP, et al. Uteroplacental adenovirus vascular endothelial growth factor
gene therapy increases fetal growth velocity in growth-restricted sheep
pregnancies. Hum Gene Ther 25:375–384, 2014.)
transient local overexpression of VEGF mediated via adeno-
virus vector injection into the uterine arteries increased uterine
artery blood flow and significantly reduced vascular contractil-
ity.49 VEGF expression was confined to the perivascular adven-
titia of the uterine arteries, together with new vessel formation,
supporting the local effect of gene transfer. These effects are long
term, lasting from midgestation (80 days) through to term (145
days).50,51 In an FGR sheep model in which uterine blood flow is
reduced by 35% in midgestation, uterine artery injection of the
same dose of Ad.VEGF significantly improved fetal growth in late
gestation,32 (Fig. 46.2) and lambs continued to thrive during the
neonatal and early postnatal period.33 The effect has been repli-
cated in FGR guinea pigs.52
For clinical translation delivery into the uterine artery could
be achieved through interventional radiology, which is sup-
ported by the Royal College of Obstetricians and Gynaecolo-
gists as a prophylactic measure before delivery in women at high
risk for postpartum haemorrhage. Although this is more inva-
sive than administering oral medication it would target vaso-
active changes to the maternal uteroplacental circulation. The
EVERREST Project (http://www.EVERREST-fp7.eu) aims
to carry out a phase I/IIa clinical trial to assess the safety and
efficacy of maternal uterine artery Ad.VEGF gene therapy for
severe early-onset FGR. The project, funded by the European
Union, involves a multinational, multidisciplinary consortium,
CHAPTER 46  Fetal Gene Therapy 565
• BOX 46.2  Practical Considerations for Clinical Fetal
Gene Therapy
Transgene expression must last a long time, from initial fetal gene transfer
into adult life for the patient.
Transgene expression must not be switched off or silenced during the
patient’s lifetime.
No preexisting maternal immunity to the vector or virus that could prevent
gene transfer to the fetus
The vector must be targeted to achieve gene transfer to the correct organ
or cells.
including experts in bioethics, fetal medicine, fetal therapy,
 
Fetal Structural Malformations as Candidate Diseases. Fetal
structural malformations are another potentially important
application of perinatal gene therapy because although they are
individually rare, collectively they affect up to 1% of all fetuses.
Congenital diaphragmatic hernia (CDH), for example, is a con-
dition in which a defect of the diaphragm results in herniation
of intraabdominal organs into the fetal chest. With surgical cor-
rection of the diaphragmatic defect, many neonates do well.
However, the compression of the fetal lungs in utero prevents
adequate growth, which results in poor lung function at birth.
To encourage lung growth in severe CDH, fetoscopic placement
of an inflatable balloon in the fetal trachea can be used to block
outflow of the tracheal fluid. An underlying lung growth defect
may contribute to the lung pathology. Gene transfer of growth
factors at a critical stage of lung development through short-
term expression has shown signs of benefit in rat and sheep mod-
els of CDH.54–56 
Practical Considerations for Clinical Fetal Gene
Therapy
There are a number of practical considerations before any fetal
gene therapy approach will be ready for the clinic (Box 46.2).
Length of Expression. The rate of mitosis in the target cell is of
considerable importance in developing fetuses when choosing a
method of gene targeting because fetal and neonatal life has one
of the most accelerated periods of growth. In the case of nonin-
tegrating vectors, organs with a high cell turnover rate will have
genetic material diluted as the organ grows and cells are lost, limit-
ing the duration and level of expression. In a long-term study of
AAV8-hFIX gene transfer to the preimmune fetal sheep, a high-
level of factor IX expression was detected 3 weeks after in utero
vector delivery, but despite the absence of neutralising antibodies,
hFIX levels dropped rapidly as the fetal liver and lamb weight
increased.14 
Vector Silencing. Vector silencing occurs when a cell prevents
the expression of a transgene after vector gene transfer. Silenc-
ing of vectors is widely acknowledged to be a drawback that
can limit their gene therapy applications and is particularly
well documented in murine leukaemia virus (MLV) vectors.57
Complete transcriptional silencing occurring shortly after
vector infection is thought to be via methylation. Progressive
silencing of an initially expressed provirus during long-term
culture or differentiation may be a particular problem in a
fetus when cells may be relatively immature.58 Removal of
566 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

TABLE
46.5 Organ-Targeted Fetal Gene Transfer
Organ to be
Targeted Current Optimal Vector Injection Routes Limiting Factors
Lung AAV Intraamniotic Large dilution effect of AF; relies on fetal BMs
Intrapulmonary Expression remains local to injection site
Intratracheal Transthoracic injection or fetoscopic tracheal delivery from midgestation
Blood Lentivirus Umbilical vein
Intraperitoneal Increased risk for germline gene transfer to peritoneal cavity when performed early in
gestation
Skeletal Muscle AAV Intramuscular
Intraperitoneal Also targets diaphragm
Hydrothoracic Technically difficult in humans
Liver AAV Intraperitoneal Rapid division of fetal liver may limit level of transduction by vectors that only minimally
Lentivirus integrate such as AAV
Intrahepatic Transduction of liver injection sites only
Brain AAV Intraventricular Technically difficult in humans
Lentivirus Umbilical vein or Relies on vector crossing the blood–brain barrier; may be gestational age dependent
intraperitoneal
Skin Lentivirus Intraamniotic Delivery early in gestation required to target deeper epidermal layers
Sensory organs Lentivirus Intraamniotic Early stage of gestation critical to reach developing ear or eye
Otocyst or retinal Technically difficult in man
injection
Gut Lentivirus Intraamniotic Large dilution effect of AF; relies on fetal BMs
Intragastric Potential limiting effect of gastric fluid on vector
Oropharyngeal Fetoscopically guided
Heart AAV Intraperitoneal
Renal Lentivirus Intraamniotic Transgenic protein expressed in kidney but produced elsewhere
? Urinary tract
Placenta Adenovirus Placenta Transduction of injection sites only
Uteroplacental Adenovirus Uterine artery
circulation

AAV, Adeno-associated virus; AF, amniotic fluid; BM, breathing movement.

  

silencing elements for example to produce self-inactivating
γ-retrovirus vectors can be used to counteract silencing. Len-
tivirus vectors are similarly affected by silencing, but because
they can infect noncycling cells and express efficiently via
multiple copy integrations, they provide more efficient gene
transfer.  lin G antibodies can cross the placental barrier and theoretically
Immune Responses to Vector and Transgenic Protein. The fetal
immune response is still a relative barrier to long-term expression
of foreign protein. In utero injection of adenovirus and AAV vec-
tors carrying the marker gene β-galactosidase in the fetal mouse
at 13 to 15 days by a variety of routes and doses resulted in the
production of low titres of neutralising antivirus and anti–β-
galactosidase antibodies.59 This primary immune response only
partially blocked transgenic protein expression after the readmin-
istration of viral vectors postnatally. However, first delivery of
the virus postnatally triggered an immune response that com-
pletely blocked transgenic protein expression after a third virus
injection.
Fetal gene transfer is still subject to immunologic barriers relat-
ing to differences in biodistribution, timing and level of trans-
genic protein expression. Designing less immunogenic vectors and
immune conditioning before gene delivery, a less desirable option,
could partially overcome the problems. Maternal immunoglobu-
prevent long-term expression. This is especially important in AAV-
mediated gene transfer in which the limiting factor seems to be
preexisting memory T-cell immunity to AAV2 in humans, who
are the only natural hosts to this virus.7 This can be circumvented
by applying AAV serotypes that humans are not naturally exposed
to, such as AAV8. 
Targeting of Vectors. Targeting vectors to organs or specific
tissues is the ultimate goal and will most likely require the use
of several combined approaches. Choosing an appropriate route
of delivery will help to direct the therapy to the right organ6
(Table 46.5).
 CHAPTER 46  Fetal Gene Therapy 567

Fetal skin

Fetal ribs

Spinal
needle

Carina
Trachea
Pulmonary artery Fetal chest

A B

Placentome

Fetal Spinal
abdomen needle

Rumen

Fetal Aorta
ribs

Fetal spine Placentome

C D
• Fig. 46.3  Ultrasound-guided delivery of gene therapy to the fetal sheep trachea and stomach. Ultra-
sonogram (A) and diagram (B) of a sheep fetus at 114 days of gestation in longitudinal section. A 20-gauge
spinal needle is inserted into the fetal thorax between the third and fourth ribs, penetrates the lung paren-
chyma and enters the fetal trachea just proximal to the carina. Ultrasonogram (C) and diagram (D) of a
sheep fetus at 61 days of gestation in transverse section. A 22-gauge spinal needle is inserted into the
fetal stomach.

Using pregnant sheep, ultrasound-guided injection techniques
from fetal medicine practice have been adapted and new methods
developed to deliver gene therapy to specific organs. For example,
ultrasound-guided delivery of adenovirus vectors into the fetal
sheep trachea (Video 46.1) results in gene expression in the fetal
airways, and injection into the fetal sheep stomach (Video 46.2)
targets gene expression to the fetal small bowel60,61 (Figs. 46.3
and 46.4). Intrapleural injection results in gene transfer to the
respiratory muscles.62 The maternal mortality rate in the pregnant
sheep is negligible, and the fetal mortality rate was between 3%
and 15% depending on the route of injection. More than 90% of
the fetal deaths were caused by iatrogenic infection, usually with
known fleece commensals. Invasive procedures such as tracheal
injection had a complication rate of 6% related to blood vessel
damage within the thorax.63 Intracardiac and umbilical vein injec-
tion had an unacceptably high procedure-related fetal mortality
rate in first trimester fetal sheep,64 and umbilical vein injection
was only reliably achieved from 70 days of gestation, equivalent to
20 weeks of gestation in humans.65 In nonhuman primates, ultra-
sound-guidance has been used to deliver gene therapy directly into
the lung parenchyma by teams in the United States.66
Alterations in receptor profiles are probably responsible for the
alternative targeting in newborn and adult mice that was seen after
adenoviral vector delivery.58 Differential cell availability through
development was after intraamniotic injection of lentiviral vec-
tors containing the GFP gene in mice. At 8 days postconception,
GFP expression was observed in tissues derived from mesoderm
and neural ectoderm, but beyond 11 days postconception,
expression was limited to epithelial cells. This expression profile
correlated closely with the cell types exposed to the AF at these
different developmental stages.67 Therefore additional steps may
be required to target vectors such as transcriptional targeting using
appropriate promoters. However, expression patterns are changing
rapidly during development as tissues mature, and the epigenetic
568 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

A B C

D E F

• Fig. 46.4  Expression of transgenic lacZ in the fetal sheep airways and gut after application of gene
therapy to the fetus. Positive X gal histochemistry (blue cells, A and F) and positive β-galactosidase immu-
nohistochemistry (brown-stained nuclei, B—E) of fetal tissues is shown. Fetuses were sampled 2 days
after ultrasound-guided injection of an adenovirus vector containing the lacZ gene. Positive lacZ expres-
sion is seen in the medium sized airways (A and C) and in the trachea (B) after delivery of the vector into
the midgestation fetal trachea. Positive lacZ expression is seen in the small bowel (D), rectum (E) and (F)
stomach after delivery to the early-gestation fetal stomach.

environment is likely to be very different from that found in the
terminally differentiated tissues of an adult. In mouse embryonic
stem cells (ESCs), for example, the type of promoter in a lentivirus
vector determined the time of transgenic protein expression dur-
ing ESC differentiation.68 Micro-RNA technology has been used
to downregulate gene expression in certain cell types such as the
Kupffer cells and hepatocytes in the liver.69,70  from rams postnatally and immunohistochemistry on their testes
Potential Adverse Consequences of Fetal Gene
Therapy
Safety is a necessary prerequisite for introducing any new therapy,
and therefore monitoring the consequences of administration of
both vector and transgene to fetuses is particularly important. The
unique features of fetal development that make it an attractive
target for gene therapy, such as its immature immune system and
rapidly dividing populations of stem cells, also mean that small
perturbations in pregnancy can have significant short- and long-
term consequences.
Effects on Fetal Growth and Development. Fetal growth and
development result from a complex interaction between the
genetic blueprint and the environment and depend on a constant,
balanced supply of nutrition provided by a healthy mother, with a
functional placenta and a well-developed fetoplacental circulation.
Viral vector-mediated gene delivery to the mother or fetus could
interfere with any stage of this sequence of events. Congenital viral
infections such as rubella and cytomegalovirus are associated with
FGR and developmental abnormalities such as sensorineural hear-
ing loss, visual impairment and cerebral palsy. Early studies com-
monly detected adenovirus DNA in the AF of women with FGR
or a fetal abnormality, but larger and more rigorous studies have
found no increased risk for complications in pregnancies infected
with adenoviruses.71 Transduction of the syncytiotrophoblast or
underlying cytotrophoblast cells might adversely affect the func-
tion of active transporter mechanisms for amino acids and lipids,
leading to growth restriction, so long-term follow up studies will
be needed after any clinical trials.  eral weeks after injection but with evidence of mild hearing loss.76
Vector Spread. Vector leakage with transduction and gene
expression in tissues other than those desired is a risk associated
with most gene delivery systems. Particularly in pregnancy, the
inadvertent transduction of the male germline has been assessed
in sheep, mice and monkeys. After intraperitoneal injection of ret-
roviral vector, polymerase chain reaction on purified sperm cells
revealed very low numbers of transduced germ cells, particularly
if vector was injected in the mid and late trimesters. The authors
estimated a transduction frequency of 1 in 6250 germ cells, which
is several orders of magnitude below the calculated frequency of
naturally occurring endogenous insertions and below the upper
tolerable limit set by the US Food and Drug Administration.72
There is only low-level spread of vector to the mother after fetal
injection in large and small animal studies of fetal gene transfer, prob-
ably limited by the placenta. The ability of adenoviruses to infect
human trophoblast cells in vitro is related to the state of trophoblast
differentiation.73 Adenovirus vectors efficiently transduce the inner
cytotrophoblast of the human placenta, but transduction of the
terminally differentiated multinucleated syncytiotrophoblast that is
exposed to the maternal blood is uncommon. This is probably due in
part to the lack of coxsackie adenovirus receptor expression on the syn-
cytiotrophoblast,74 thus rendering it resistant to maternal adenovirus
infection and limiting maternal to fetal transplacental transmission. 
Vector Toxicity. Many viral vectors are toxic at high concen-
trations, mainly through their immunogenic properties. A severe
reaction was observed in a human trial of adult gene therapy for
ornithine transcarbamylase deficiency in which in one case, a sys-
temic inflammatory response was fatal.75 The effects can be highly
species specific (e.g., vesicular stomatitis virus G protein [VSVG]
pseudotyped lentivirus), used successfully in mouse studies, caused
fetal ascites and death, even at low doses when given to the early
gestation fetal sheep (unpublished data, AL David). More subtle
signs of toxicity can be detected; for instance, VSVG-pseudotyped
HIV delivering GFP cDNA to the fetal murine cochlear resulted
in good expression in inner and outer hair cells of the cochlea sev-
 

Insertional Mutagenesis. It is postulated that the high level of
cellular proliferation in the fetus, the abundance of growth fac-
tors and the transcriptionally active state of genes associated with
the regulation of growth and differentiation might predispose to
increased risk for cancer from vectors which integrate into the host
genome. A high incidence of hepatocellular carcinoma was seen
in mice after delivery of lacZ or human factor IX cDNA using an
equine infectious anaemia virus (EIAV) vector but not after use
of an HIV vector.77 Fetuses may be uniquely sensitive to genetic
perturbations arising from integrating vector gene transfer because
EIAV has been used in adult animals for transduction of the ner-
vous system with no adverse events reported.78  animal model, and a balance is needed, taking into consideration
Undesirable Effects of Transgenic Protein Expression. Expres-
sion of a therapeutic gene at a particular stage in fetal development
may be damaging to the fetus. For example, adenovirus-mediated
expression of the cystic fibrosis transmembrane regulator (CFTR)
in normal fetal rats and mice resulted in altered lung development
and morphology.79,80 Adverse effects of ectopic or dysregulated
transgenic protein expression are unpredictable, and therefore each
gene will need to be assessed rigorously before clinical translation.  tions, such as those described by the Committee for Medicinal
Risks Arising From the Vector Delivery Procedure. Intrauterine
procedures such as those performed under ultrasound guidance
carry a finite but definitive risk for miscarriage, infection and
preterm labour. Earlier gene transfer may be beneficial because
there are profound increases in the numbers of circulating T cells
observed between 12 and 14 weeks of fetal life.81 However, appli-
cation of gene therapy before this time limits the routes that can
be safely used. In addition, fetal gene treatment during a certain
pregnancy could pose a risk to future pregnancies by affecting the
mother’s health through the effects of gene therapy or the delivery
procedure itself.  tate dehydrogenase, released into the culture medium. Placental
Combining Gene Therapy With Fetal Stem Cell
Transplantation
An alternative strategy that has been used most effectively in neo-
natal life is to combine stem cell transplantation (SCT) with gene
therapy. To treat severe combined immune deficiency, haemato-
poietic stem cells collected from the bone marrow of patients can
be gene corrected ex vivo and transplanted autologously into the
donor, resulting in complete cure of the disease.1 The UK Gene
Therapy Advisory Committee (GTAC) considered an IUSCGT
approach in its broader judgments about fetal gene therapy, find-
ing that the use of genetically modified stem cells in SCT to the
fetus was a possibility. The committee stated that ‘such ex  vivo
modification would be unlikely to carry with it any higher risk
to the germ line than the trials of postnatal somatic gene therapy
which have already been approved’.
An autologous stem cell gene transfer approach using fetal stem
cells from a number of sources within the fetus including the blood,
liver, AF and placenta could be adopted. Fetal liver or blood sampling
at an early gestational age carries a significant risk for miscarriage, but
pluripotent stem cells can be readily derived from fetal samples col-
lected at amniocentesis82 or chorionic villus sampling,83,84 procedures
that have a low fetal mortality rate. Human amniotic fluid stem (AFS)
cells can differentiate into a variety of cell types and transduced with-
out altering their characteristics.82,85 There was good fetal survival
after autologous AF mesenchymal stem cell (MSC) transplantation in
sheep using ultrasound-guided amniocentesis and subsequent intra-
peritoneal injection of selected, expanded and transduced AF MSCs
into the donor fetus. Widespread cell migration and engraftment,
particularly in the liver, heart, muscle, placenta, umbilical cord and
CHAPTER 46  Fetal Gene Therapy 569
adrenal gland, was seen.86 The functional haematopoietic potential of
transduced GFP-positive sheep AF-derived stem cells before and after
autologous IUSCGT has also been demonstrated in fetal sheep as
shown in Figure 46.5.87 The findings in a large animal model support
the concept for clinical translation to treat congenital haematopoietic
diseases in utero. 
Clinical Translation of Fetal Gene Therapy
Preclinical testing in animal models of disease will be an impor-
tant step before clinical translation is realised. There is no ideal
the gestational development of the organ to be targeted and how
that relates to the human, the type of placentation, fetal size, num-
ber and lifespan, parturition and the fetal and maternal immune
response.
Toxicology studies will be needed using animals such as the
pregnant rabbit, in which reproductive toxicology is commonly
performed, with good historical datasets. Guidelines and regula-
Products for Human Use of the European Medicines Agency, will
be considered when planning preclinical study protocols.88
The effect of gene therapy vectors on the human placenta can
be assessed in  vitro. Two models are available, cultured villous
explants or perfused whole placental cotyledons. Villi isolated
from placental lobules are cultured in net-wells submerged in
growth medium. After 1 day of culture, the syncytiotrophoblast is
routinely shed in vitro and then regenerates through the differen-
tiation of underlying cytotrophoblast cells 2 days later.89 Cellular
integrity and apoptosis can be assessed using markers, such as lac-
perfusion preserves cellular and tissue architecture whilst allowing
a dual fetal- and maternal-side haemodynamic compartment to be
maintained. Movement of substances such as adenovirus vector,
applied to the maternal or fetal side of the placenta, can be studied
in the opposite side of the placenta using this model, over a 5- to
9-hour time period after delivery of the placenta.90
Phase I Trials and Ethical Considerations. Phase I human trials
face hurdles because of difficulties in testing pregnant women in
whom toxicologic studies are usually contraindicated. Thus, when
human application becomes possible, extensive unbiased parental
counselling and informed consent are paramount because of the
uncertainties about the efficacy and long-term safety of prenatal
gene therapy, which may not become evident until much later
in the individual’s life. This can be difficult when the decision to
participate in a fetal gene therapy trial will occur close to the time
of prenatal diagnosis of the condition. Because the risks involve
the mother, fetus and possibly future progeny, parents will also
be required to consent their offspring and themselves to lifelong
follow-up. One criticism levelled at fetal gene therapy is a belief
that couples pregnant with an affected child would be unlikely
to proceed with prenatal therapy and would opt for a termina-
tion instead. This concern is not solely applicable to perinatal gene
therapy, however, but also can be raised for any fetal treatment
such as fetal surgery and in utero SCT.
We recently evaluated the ethical and social acceptability of a
proposed clinical trial using maternal uterine artery VEGF gene
therapy to treat severe early-onset FGR in pregnant women. The
literature review on the ethics and legality of experimental treat-
ments in pregnant women concluded that there were no ethical or
legal objections to the intervention or to a trial of this interven-
tion. Issues that were identified from the literature helped develop
570 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

AF

1. Frozen AF CD34+ cells

2. Fresh AF CD34+ cell
3. Adult BM CD34+ cells Secondary
transplantation
Primary transplantation of mice BM cells

BM

1. Fresh AF CD34+ cells

In utero autologous
transplantation of fresh Lamb delivery and Lamb
transduced AF CD34+ cells follow-up 6 months BM cells
into peritoneal cavity

5
X16A X16B X17A X17B X18A
4.5
X18B M3 M4 M5 Control
GFP+ cells in the blood (%)

4
3.5
3
2.5
2
1.5
1
0.5
0
Before Day 0 1 wk 2 wk 4 wk 8 wk 10 wk 12 wk 14 wk 16 wk 20 wk 24 wk
B IUT
• Fig. 46.5  In utero autologous transplantation of fresh transduced amniotic fluid (AF) CD34+ cells into the
peritoneal cavity of fetal sheep. A, Diagram showing the experimental setup. Transduced sheep eGFP1
CD34+ selected fresh or frozen amniotic fluid (AF) and adult bone marrow (BM) cells were transplanted into
immunocompromised NOD-SCID-γ (NSG) mice (primary and secondary xenogeneic transplantation). Green
fluorescent protein (GFP)–positive cells were detected in the haematopoietic organs and peripheral blood of
NSG mice primary and secondary recipients 3 months later demonstrating their haematopoietic potential. 
In a separate set of experiments, CD34+ cells were selected by fluoresence activated cell sorting
(FACS) analysis of AF collected by amniocentesis in first trimester fetal sheep. CD34+AF cells were trans-
duced overnight in suspension culture with an HIV lentivirus containing the GFP transgene. Transduced
sheep GFP + CD34 + fresh AF cells were then injected into the donor sheep fetuses (in utero autologous
transplantation) that were subsequently delivered and followed for up with serial blood sampling. At 6
months of postnatal age, BM from these primary sheep recipients was then used to perform xenogeneic
secondary transplantation into NSG mice. Donor sheep cells were detected in in the haematopoietic
organs and peripheral blood of these secondary NSG recipients showing haematopoietic reconstitution.
B, Engraftment in the peripheral blood after in utero transplantation of autologous sheep CD34 + GFP +
AF cells. All five born lambs showed GFP + cells in the peripheral blood at birth, and all three survivors
revealed persistent levels of engraftment of around 2% up to the last sampling point at 6 months of age.
Negative control: peripheral blood from uninjected sheep. M1, M2 and M3: the three ewes showed nega-
tivity for GFP signal. IUT, In utero transplantation. (From Shaw SWS, Blundell MP, Pipino C, et al. Sheep
CD34+ amniotic fluid cells have hematopoietic potential and engraft after autologous in utero transplanta-
tion. Stem Cells. 2015;33(1):122–132.)

interview guides for semistructured, qualitative interview, carried
out in four European countries with 34 key stakeholders (disabil-
ity groups, professional bodies and patient support groups) and 24
women or couples who had experienced a pregnancy affected by
severe early-onset FGR. Overall, respondents viewed the proposed
trial in positive terms. Women were generally interested in partici-
pating in clinical trials that conferred a potential benefit to their
unborn children. The risk for disability of the premature child was
a concern but not considered a major stumbling block for mater-
nal VEGF gene therapy.91 

Conclusion
Fetal gene therapy offers the potential for clinicians not only to
diagnose but also to treat inherited genetic disease, structural
disease in fetuses and maternal obstetric conditions that affect
fetuses. Fetal application may prove better than neonatal or adult
gene therapy for treatment of early-onset genetic disorders such
as CNS and liver disorders. Gene transfer to developing fetuses
targets rapidly expanding stem cell populations that are inacces-
sible after birth. Integrating vector systems give permanent gene
transfer. In animal models of congenital disease, the functionally
CHAPTER 46  Fetal Gene Therapy 571
immature fetal immune system does not respond to the product
of the introduced gene, and therefore immune tolerance can be
induced. For the treatment to be acceptable, it must be safe for
both the mother and fetus and preferably avoid germline trans-
mission. Currently, fetal gene therapy remains an experimental
procedure, but it is rapidly moving into the clinic with the poten-
tial of a first-in-woman study within the next 2 years to treat fetal
growth restriction.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 17. Guo ZS, Li Q, Bartlett DL, et al. Gene trans-
fer: the challenge of regulated gene expres-
34. Sands MS, Davidson BL. Gene therapy
for lysosomal storage diseases. Mol Ther.
sion. Trends Mol Med. 2008;14(9):410–418. 2006;13(5):839–849.
1. Hacein-Bey-Abina S, Pai S-Y, Gaspar HB,
18. Philpott NJ, Thrasher AJ. Use of nonintegrat- 35. Mango RL, Xu L, Sands MS, et al. Neonatal
et  al. A modified γ-retrovirus vector for
ing lentiviral vectors for gene therapy. Hum retroviral vector-mediated hepatic gene ther-
X-linked severe combined immunodefi-
Gene Ther. 2007;18(6):483–489. apy reduces bone, joint, and cartilage disease
ciency. N Engl J Med. 2014;371(15):1407–
19. Shangaris P, David AL. Perinatal gene therapy. in mucopolysaccharidosis VII mice and dogs.
1417.
In: Fauza DO, Bani M, eds. Fetal Stem Cells and Mol Genet Metab. 2004;82(1):4–19.
2. Nathwani AC, Reiss UM, Tuddenham EGD,
Regenerative Medicine Principles and Transla- 36. Aronovich EL, Bell JB, Belur LR, et al. Pro-
et al. Long-term safety and efficacy of factor
tional Strategies. Berlin: Springer Science; 2016. longed expression of a lysosomal enzyme in
IX gene therapy in hemophilia B. N Engl J
20. Douar AM, Themis M, Sandig V, et al. Effect mouse liver after Sleeping Beauty transposon-
Med. 2014;371(21):1994–2004.
of amniotic fluid on cationic lipid mediated mediated gene delivery: implications for non-
3. Wu C, Endo M, Yang BH, et al. Intra-amni-
transfection and retroviral infection. Gene viral gene therapy of mucopolysaccharidoses.
otic transient transduction of the periderm
Ther. 1996;3(9):789–796. J Gene Med. 2007;9(5):403–415.
with a viral vector encoding TGFβ3 prevents
21. Kim H, Kim J-S. A guide to genome engi- 37. Berges BK, Yellayi S, Karolewski BA, et  al.
cleft palate in Tgfβ3−/− mouse embryos. Mol
neering with programmable nucleases. Nat Widespread correction of lysosomal storage
Ther. 2012;21(1):8–17.
Rev Genet. 2014;15(5):321–334. in the mucopolysaccharidosis type VII mouse
4. Spencer RN, Carr DJ, David AL. Treatment
22. Beumer KJ, Trautman JK, Christian M, et al. brain with a herpes simplex virus type I vec-
of poor placentation and the prevention of
Comparing zinc finger nucleases and tran- tor expressing beta-glucuronidase. Mol Ther.
associated adverse outcomes—what does the
scription activator-like effector nucleases for 2006;13(5):859–869.
future hold? Prenat Diagn. 2014;4(7):677–
gene targeting in Drosophila. G3 (Bethesda). 38. Ciron C, Desmaris N, Colle MA, et al. Gene
684.
2013;3(10):1717–1725. therapy of the brain in the dog model of Hurl-
5. Clothier CM. Report of the Committee on the
23. Bedell VM, Wang Y, Campbell JM, er’s syndrome. Ann Neurol. 2006;60(2):204–
Ethics of Gene Therapy, London: Her Majesty’s
et  al. In  vivo genome editing using a 213.
Stationery Office (HMSO) Publications; 1992.
high-efficiency TALEN system. Nature. 39. Cearley CN, Wolfe JH. A single injection of
6. Mehta V, Abi Nader K, Waddington S,
2012;491(7422):114–118. an adeno-associated virus vector into nuclei
David AL. Organ targeted prenatal gene
24. Xiao A, Wang Z, Hu Y, et  al. Chromo- with divergent connections results in wide-
therapy–how far are we? Prenat Diagn.
somal deletions and inversions mediated spread vector distribution in the brain and
2011;31(7):720–734.
by TALENs and CRISPR/Cas in zebrafish. global correction of a neurogenetic disease.
7. Manno CS, Pierce GF, Arruda VR, et al. Suc-
Nucleic Acids Res. 2013;41(14):e141. J Neurosci. 2007;27(37):9928–9940.
cessful transduction of liver in hemophilia
25. Gaj T, Gersbach CA, Barbas CF. ZFN, 40. Shen JS, Meng XL, Yokoo T, et  al. Wide-
by AAV-Factor IX and limitations imposed
TALEN, and CRISPR/Cas-based methods spread and highly persistent gene transfer to
by the host immune response. Nat Med.
for genome engineering. Trends Biotechnol. the CNS by retrovirus vector in utero: impli-
2006;12(3):342–347.
2013;31(7):397–405. cation for gene therapy to Krabbe disease.
8. Nathwani AC, Tuddenham EGD,
26.  U. National Institutes of Health. Recom- J Gene Med. 2005;7(5):540–551.
Rangarajan S, et  al. Adenovirus-associated
binant DNA Advisory Committee. Prena- 41. Karolewski BA, Wolfe JH. Genetic correc-
virus vector–mediated gene transfer in hemo-
tal gene transfer: scientific, medical, and tion of the fetal brain increases the lifespan
philia B. N Engl J Med. 2011;365(25):2357–
ethical issues: a report of the Recombinant of mice with the severe multisystemic disease
2365.
DNA Advisory Committee. Hum Gene Ther. mucopolysaccharidosis type VII. Mol Ther.
9. Nathwani AC, Gray JT, Ng CYC, et al. Self-
2000;11(8):1211–1229. 2006;14(1):14–24.
complementary adeno-associated virus vec-
27. Lipshutz GS, Sarkar R, Flebbe-Rehwaldt L, 42. Tarantal AF, Chu F, O’Brien WD,
tors containing a novel liver-specific human
et  al. Short-term correction of factor VIII Hendrickx AG. Sonographic heat generation
factor IX expression cassette enable highly
deficiency in a murine model of hemophilia in  vivo in the gravid long-tailed macaque
efficient transduction of murine and nonhu-
A after delivery of adenovirus murine fac- (Macaca fascicularis). J Ultrasound Med.
man primate liver. Blood. 2006;107(7):2653–
tor VIII in utero. Proc Natl Acad Sci U S A. 1993;12(5):285–295.
2661.
1999;96(23):13324–13329. 43. Foust KD, Nurre E, Montgomery CL, et al.
10. Billingham RE, Brent L. Acquired tolerance
28. Waddington SN, Nivsarkar MS, Mistry AR, Intravascular AAV9 preferentially targets neo-
of foreign cells in newborn animals. Proc R Soc
et  al. Permanent phenotypic correction of natal neurons and adult astrocytes. Nat Bio-
Lond B Biol Sci. 1956;146(922):78–90.
hemophilia B in immunocompetent mice by technol. 2009;27(1):59–65.
11. Billingham RE, Brent L, Medawar PB.
prenatal gene therapy. Blood. 2004;104:2714– 44. Duque S, Joussemet B, Riviere C, et al. Intra-
Actively acquired tolerance of foreign cells.
2721. venous administration of self-complementary
Nature. 1953;172(4379):603–606.
29. Dejneka NS, Surace EM, Aleman TS, et al. In AAV9 enables transgene delivery to adult motor
12. Waddington SN, Buckley SM, David AL,
utero gene therapy rescues vision in a murine neurons. Mol Ther. 2009;17(7):1187–1196.
et al. Fetal gene transfer. Curr Opin Mol Ther.
model of congenital blindness. Mol Ther. 45. Bevan AK, Duque S, Foust KD, et  al. Sys-
2007;9(5):432–438.
2004;9(2):182–188. temic gene delivery in large species for tar-
13. Waddington SN, Kennea NL, Buckley SMK,
30. Seppen J, van der Rijt R, Looije N, et  al. geting spinal cord, brain, and peripheral
et  al. Fetal and neonatal gene therapy: ben-
Long-term correction of bilirubin UDP- tissues for pediatric disorders. Mol Ther.
efits and pitfalls. Gene Ther. 2004;11(suppl
glucuronyltransferase deficiency in rats by 2001;19(11):1971–1980.
1):S92–S97.
in utero lentiviral gene transfer. Mol Ther. 46. Mattar C, Waddington S, Biswas A, et al. Sys-
14. David AL, McIntosh J, Peebles DM, et  al.
2003;8(4):593–599. temic delivery of scAAV9 in fetal macaques
Recombinant adeno-associated virus-mediated
31. Rucker M, Fraites TJ, Porvasnik SL, et  al. facilitates neuronal transduction of the central
in utero gene transfer gives therapeutic trans-
Rescue of enzyme deficiency in embryonic and peripheral nervous systems. Gene Ther.
gene expression in the sheep. Hum Gene Ther.
diaphragm in a mouse model of metabolic 2013;20:69–83.
2011;22:419–426.
myopathy: Pompe disease. Development. 47. Mattar CN, Wong AMS, Hoefer K, et  al.
15. Mattar CNZ, Nathwani AC, Wadding-
2004;131:3007–3019. Systemic gene delivery following intravenous
ton SN, et  al. Stable human FIX expres-
32. Carr DJ, Wallace JM, Aitken RP, et al. Utero- administration of AAV9 to fetal and neonatal
sion after 0.9G intrauterine gene transfer
placental adenovirus vascular endothelial mice and late-gestation nonhuman primates.
of self-complementary adeno-associated
growth factor gene therapy increases fetal FASEB J. 2015;29(9):3876–3888.
viral vector 5 and 8 in macaques. Mol Ther.
growth velocity in growth-restricted sheep 48. Miller SL, Loose JM, Jenkin G, Wallace EM.
2011;19(11):1950–1960.
pregnancies. Hum Gene Ther. 2014;25:375– The effects of sildenafil citrate (Viagra) on
16. Kobinger GP, Weiner DJ, Yu QC, Wil-
384. uterine blood flow and well being in the intra-
son JM. Filovirus-pseudotyped lentiviral
33. Carr DJ, Wallace JM, Aitken R, et  al. Peri- uterine growth-restricted fetus. Am J Obstet
vector can efficiently and stably transduce
and postnatal effects of prenatal adenoviral Gynecol. 2009;200(1):102.e1–e7.
airway epithelia in  vivo. Nat Biotechnol.
VEGF gene therapy in growth-restricted 49. David AL, Torondel B, Zachary I, et al. Local
2001;19(3):225–230.
sheep. Biol Reprod. 2016;94(6):142. delivery of VEGF adenovirus to the uterine

571.e1
571.e2 References
artery increases vasorelaxation and uterine
blood flow in the pregnant sheep. Gene Ther.
2008;15:1344–1350.
50. Mehta V, Abi-Nader K, Peebles D, et  al.
Long-term increase in uterine blood flow is
achieved by local overexpression of VEGF-
A(165) in the uterine arteries of pregnant
sheep. Gene Ther. 2012;19(9):925–935.
51. Mehta V, Abi-Nader KN, Shangaris P, et al.
Local over-expression of VEGF-DΔNΔCin
the uterine arteries of pregnant sheep results
in long-term changes in uterine artery
contractility and angiogenesis. PLoS One.
2014;9(6):e100021.
52. Swanson AM, Rossi CA, Ofir K, et al. Mater-
nal therapy with Ad.VEGF-A165 increases
fetal weight at term in a guinea pig model
of fetal growth restriction. Hum Gene Ther.
2016;27(12):997–1007.
53. Gancberg D, Hoeveler A, Draghia-Akli R.
Gene therapy and gene transfer projects of
the 7th Framework Programme for Research
and Technological Development of the
European Union. Hum Gene Ther Clin Dev.
2015;26:77.
54. Toelen J, Carlon M, Claus F, et al. The fetal
respiratory system as target for antenatal ther-
apy. Facts, views. Vis ObGyn. 2011;3(1):22–
35.
55. Larson JE, Cohen JC. Improvement of pul-
monary hypoplasia associated with congenital
diaphragmatic hernia by in utero CFTR gene
therapy. Am J Physiol Lung Cell Mol Physiol.
2006;291(1):L4–L10.
56. Saada J, Oudrhiri N, Bonnard A, et al. Com-
bining keratinocyte growth factor transfection
into the airways and tracheal occlusion in a
fetal sheep model of congenital diaphragmatic
hernia. J Gene Med. 2010;12(5):413–422.
57. Ellis J. Silencing and variegation of gamma-
retrovirus and lentivirus vectors. Hum Gene
Ther. 2005;16(11):1241–1246.
58. Huard J, Lochmuller H, Acsadi G, et al. Dif-
ferential short-term transduction efficiency
of adult versus newborn mouse tissues by
adenoviral recombinants. Exp Mol Pathol.
1995;62(2):131–143.
59. Jerebtsova M, Batshaw ML, Ye X. Humoral
immune response to recombinant adenovi-
rus and adeno-associated virus after in utero
administration of viral vectors in mice. Pedi-
atr Res. 2002;52(1):95–104.
60. Peebles D, Gregory LG, David A, et al. Wide-
spread and efficient marker gene expression
in the airway epithelia of fetal sheep after
minimally invasive tracheal application of
recombinant adenovirus in utero. Gene Ther.
2004;11(1):70–78.
61. David AL, Peebles DM, Gregory L, et  al.
Clinically applicable procedure for gene
delivery to fetal gut by ultrasound-guided
gastric injection: toward prenatal prevention
of early-onset intestinal diseases. Hum Gene
Ther. 2006;17(7):767–779.
62. Weisz B, David AL, Gregory LG, et al. Target-
ing the respiratory muscles of fetal sheep for
prenatal gene therapy for Duchenne muscular
dystrophy. Am J Obstet Gynecol. 2005;193(3
Pt 2):1105–1109.
63. David AL, Weisz B, Gregory L, et al. Ultrasound-
guided injection and occlusion of the trachea
in fetal sheep. Ultrasound Obstet Gynecol.
2006;28:82–88.
64. David A, Peebles D, Miah M, et al. Ultrasound-
guided delivery of viral vectors encoding the
beta-galactosidase and human factor IX genes
to early gestation fetal sheep in utero. Hum
Gene Ther. 2002;364:353–364.
65. Coutelle C, Themis M, Waddington SN, et al.
Gene therapy progress and prospects: fetal
gene therapy—first proofs of concept—some
adverse effects. Gene Ther. 2005;12:1601–1607.
66. Tarantal AF, Lee CI, Ekert JE, et al. Lentiviral
vector gene transfer into fetal rhesus monkeys
(Macaca mulatta): lung-targeting approaches.
Mol Ther. 2001;4(6):614–621.
67. Endo M, Henriques-Coelho T, Zoltick PW,
et al. The developmental stage determines the
distribution and duration of gene expression
after early intra-amniotic gene transfer using
lentiviral vectors. Gene Ther. 2010;17(1):61–
71.
68. Hong S, Hwang D-Y, Yoon S, et  al. Func-
tional analysis of various promoters in len-
tiviral vectors at different stages of in  vitro
differentiation of mouse embryonic stem
cells. Mol Ther. 2007;15(9):1630–1639.
69. Brown BD, Gentner B, Cantore A, et  al.
Endogenous microRNA can be broadly
exploited to regulate transgene expression
according to tissue, lineage and differentiation
state. Nat Biotechnol. 2007;25(12):1457–1467.
70. Brown BD, Cantore A, Annoni A, et  al. A
microRNA-regulated lentiviral vector medi-
ates stable correction of hemophilia B mice.
Blood. 2007;110(13):4144–4152.
71. Wenstrom KD, Andrews WW, Bowles NE,
et  al. Intrauterine viral infection at the time
of second trimester genetic amniocentesis.
Obstet Gynecol. 1998;92(3):420–424.
72. Park PJ, Colletti E, Ozturk F, et  al. Factors
determining the risk of inadvertent retrovi-
ral transduction of male germ cells after in
utero gene transfer in sheep. Hum Gene Ther.
2009;20(3):201–215.
73. MacCalman CD, Furth EE, Omigbodun A,
et al. Transduction of human trophoblast cells
by recombinant adenoviruses is differentiation
dependent. Biol Reprod. 1996;54:682–691.
74. Koi H, Zhang J, Makrigiannakis A, et  al.
Differential expression of the coxsackievirus
and adenovirus receptor regulates adenovi-
rus infection of the placenta. Biol Reprod.
2001;64(3):1001–1009.
75. Wilson JM. Lessons learned from the gene
therapy trial for ornithine transcarbamylase
deficiency. Mol Genet Metab. 2009;96(4):
151–157.
76. Bedrosian JC, Gratton MA, Brigande JV,
et al. In vivo delivery of recombinant viruses
to the fetal murine cochlea: transduction char-
acteristics and long-term effects on auditory
function. Mol Ther. 2006;14(3):328–335.
77. Themis M, Waddington SN, Schmidt M,
et  al. Oncogenesis following delivery of
a nonprimate lentiviral gene therapy vec-
tor to fetal and neonatal mice. Mol Ther.
2005;12(4):763–771.
78. Wong LF, Goodhead L, Prat C, et al.
Lentivirus-mediated gene transfer to the central
nervous system: therapeutic and research appli-
cations. Hum Gene Ther. 2006;17(1):1–9.
79. Morrow SL, Larson JE, Nelson S, et al. Modi-
fication of development by the CFTR gene
in utero. Mol Genet Metab. 1998;65(3):203–
212.
80. Larson JE, Delcarpio JB, Farberman MM,
et  al. CFTR modulates lung secretory cell
proliferation and differentiation. Am J Physiol
Lung Cell Mol Physiol. 2000;279:L333–L341.
81. Pahal GS, Jauniaux E, Kinnon C, et al. Normal
development of human fetal hematopoiesis
between eight and seventeen weeks’ gestation.
Am J Obstet Gynecol. 2000;183(4):1029–1034.
82. De Coppi P, Bartsch G, Siddiqui MM,
et  al. Isolation of amniotic stem cell lines
with potential for therapy. Nat Biotechnol.
2007;25:100–106.
83. Portmann-Lanz CB, Schoeberlein A, Huber
A, et al. Placental mesenchymal stem cells as
potential autologous graft for pre- and peri-
natal neuroregeneration. Am J Obstet Gynecol.
2006;194(3):664–673.
84. Antoniadou E, David AL. Placental stem cells.
Best Pract Re Clin Obst Gynaecol. 2016;31:
13–29.
85. Bollini S, Pozzobon M, Nobles M, et  al.
In vitro and in vivo cardiomyogenic differen-
tiation of amniotic fluid stem cells. Stem Cell
Rev Reports. 2011;7(2):364–380.
86. Steven Shaw SW, Bollini S, Nader KA, et al.
Autologous transplantation of amniotic fluid-
derived mesenchymal stem cells into sheep
fetuses. Cell Transplant. 2011;20:1015–1031.
87. Shaw SWS, Blundell MP, Pipino C, et  al.
Sheep CD34+ amniotic fluid cells have hema-
topoietic potential and engraft after autolo-
gous in utero transplantation. Stem Cells.
2015;33(1):122–132.
88.  European Medicines Agency. Guideline on
the Quality, Non-clinical and Clinical Aspects
of Gene Therapy Medicinal Products. London:
European Medicines Agency; 2015.
89. Crocker IP, Tansinda DM, Baker PN. Altered
cell kinetics is cultured placental villous
explants in pregnancies complicated by pre-
eclampsia and intrauterine growth restriction.
J Pathol. 2004;204(1):11–18.
90. Brownbill P, Desforges M, Sebire N, et  al.
Human placental ex  vivo studies to support
an adenovirus-mediated vascular endothelial
growth factor (VEGF) gene medicine for the
treatment of severe early onset fetal growth
restriction. Hum Gene Ther. 2014;25(11):
A60.
91. Sheppard MK, David AL, Spencer R, et  al.
Ethics and ethical evaluation of a proposed
clinical trial with maternal uterine artery
vascular endothelial growth factor gene ther-
apy to treat severe early onset fetal growth
restriction in pregnant. Hum Gene Ther.
2014;25:A98.
92. Endo M, Zoltick PW, Radu A, et  al. Early
intra-amniotic gene transfer using lentiviral
vector improves skin blistering phenotype in a
murine model of Herlitz junctional epidermol-
ysis bullosa. Gene Ther. 2012;19(5):561–569.
47
The Developmental Origins of Health
and Disease
SCOTT W. WHITE, SARA L. HILLMAN AND JOHN P. NEWNHAM
KEY POINTS
• Influences during early life have substantial impact upon
adult health and disease.
• This concept is most commonly known as the developmental
origins of health and disease.
• Environmental, genetic and epigenetic factors, as well as
interactions among these factors, underlie this association.
• Modification of these factors in early development has the po-
tential to influence an individual’s health across the life course.
Introduction
An association between the early life environment and health
in later life has been known at least since the time of Hip-
pocrates. Contemporary research in this field grew following
on from the seminal work of David Barker and colleagues in
England in the 1980s. Barker found that the geographical pat-
tern of adult ischaemic heart disease mortality matched that
of infant mortality several decades earlier.1 The most common
cause of infant mortality at the time was low birth weight, lead-
ing to the ‘Barker hypothesis’ that exposure to an intrauterine
environment leading to poor fetal growth caused metabolic
changes that persisted into adulthood and increased the risk
for ischaemic heart disease.2
This initial work was then validated across several popula-
tions and continuing refinement led to the coining of the term
‘fetal origins of adult disease’.3,4 Further work extended this
theory, and it became evident that early life impacts upon later
life disease were not limited to pregnancy but also the maternal
preconception environment and early postnatal life. Gluckman
and Hanson5 first described the DOHaD theory to better incor-
porate these influences outside of the fetal period upon adult
health.
Much scientific research now focuses on DOHaD across a
wide range of fields from both clinical and basic science arenas,
and complex analysis of associations has led to new frontiers in
572
statistics.6 The implications of the research extend now to the
development of public health policy and global recommendations
about pregnancy and childhood care.7 A large current thrust of
the research focuses upon identifying the underlying biology of
DOHaD in the hope that this might identify novel approaches
to the prevention of the associated adult diseases. It is now clear
that complex gene–environment interactions lie at the heart of
this process.8,9 
History of the Developmental Origins of
Health and Disease Theory
Ancient
Historical works from the time of Hippocrates link the health of a
woman during pregnancy, the condition of the child at birth and
the growth of the infant to well-being in adulthood. Attributed
to Hippocrates, Airs, Waters and Places describes the association
between an adverse environment during pregnancy and the ongo-
ing health of the offspring, noting that:
In the first place women who happen to be with child, and whose
accouchement should take place in springs . . . have feeble and
sickly children, so that they either die presently or are tender, feeble,
and sickly, if they live. 
Kermack and Forsdahl
During the 20th century, a link between early life and adult
health was proposed by several researchers.10 As early as 1934,
reductions in all-cause mortality in Europe were thought to be
related to improved childhood conditions.11 Forsdahl12 later
recognised an association between infant mortality rates and
subsequent ischaemic heart disease decades later, insightfully
hypothesising that poor early life nutrition may result in later
life susceptibility to a mismatched environment of nutritional
plenty. 
 CHAPTER 47  The Developmental Origins of Health and Disease 573

Hazardratio of ischaemic 1.6 2.6

Hazardratio of ischaemic
1.4 2.2
heart disease death

heart disease death

1.2 1.8

1.0 1.4

0.8 1.0

0.6 0.6
5 6 7 8 9 10 17 19 21 23 25 27
A Birth weight (lb) B Weight at 1 year (lb)

• Fig. 47.1 Relationship between early life growth and adult cardiovascular disease. A, Relationship
between birth weight and death from ischaemic heart disease. B, Relationship between weight at 1 year
and death from ischaemic heart disease. (Data from Barker DJ. Childhood causes of adult diseases. Arch
Dis Child 63(7):867–869, 1988.)

The Barker Hypothesis Mechanisms of the Developmental Origins

It was not for another decade that, with the recognition in 1986
by Barker and colleagues1 of an association between low birth
weight and adult cardiovascular disease, this area began to receive
substantial research interest. In his analysis of trends in mortality
across geographical regions of England and Wales, Barker found
that the areas with the highest mortality rates due to ischaemic
heart disease were the same as those that had the highest rates
of infant mortality decades earlier. The most common cause of
infant death at the time was ‘low birth weight’, and the ‘Barker
hypothesis’ was formulated, suggesting that events contributing
to low birth weight also contributed to the development of car-
diovascular disease in adulthood (Fig 47.1).13 Reflection upon
previous research and the continued work of Barker and others
developed this theory by confirming these associations across vari-
ous cohorts.2,12,14–17  mental impact upon gene expression and phenotype.21-23
The Fetal Origins of Adult Disease
Continuing work demonstrated associations with an increas-
ing number of adult diseases. This led to the proposal in 1992
of the ‘fetal origins of adult disease’ theory, which expanded
upon the Barker hypothesis to acknowledge that impacts
upon the fetus had the potential to affect a variety of adult
diseases.18,19  heart is complete relatively early in gestation, late insults may alter
The Developmental Origins of Health and
Disease
The field continued to grow as evidence mounted across vari-
ous populations, species and diseases. By early in the new mil-
lennium, it was clear that adult outcomes were related to both
specific diseases and overall well-being and were associated not
only with the fetal environment but also with those of the pre-
conception period and early childhood. To better acknowledge
these factors, Gluckman and Hanson5 coined the phrase ‘devel-
opmental origins of health and disease’ as the field is now most
commonly known.  represent subtle changes resulting from developmental disruption
of Health and Disease
One principle underlying the DOHaD phenomenon is that of
‘developmental plasticity’, the notion that a range of phenotypes
can result from the same genotype as a result of altered environmen-
tal exposures during an organism’s development.20 In the human
and in the DOHaD realm, this plasticity manifests as altered dis-
ease risk in later life. The association with low birth weight does
not necessarily reflect a causal role of abnormal fetal growth in the
development of disease but, rather, abnormal fetal growth is but
one measure of an abnormal intrauterine environment, exposure
to which induces phenotypic variation leading to increased disease
risk. Developmental plasticity is a well-recognised phenomenon in
nonhuman species, with substantial animal evidence of the environ-
Plasticity is time dependent and can only take place during criti-
cal periods of organogenesis. For example, brain development takes
place over a longer period than cardiac development, and environ-
mental impacts only result in changes to cardiac structure when
present during earlier development compared with those which
may produce neurologic changes.24 Periods of plasticity may vary
between the structural and functional development of an organ,
however. For example, although structural development of the fetal
terminal cardiac myocyte differentiation, leading to more subtle
functional changes. The physiological regulation of metabolism and
inflammation is relatively late to develop and is therefore subject to
environmental influences for much of the early life period and more
susceptible to perturbations resulting in later disease.
Gluckman and Hanson20 describe an important distinction
in this observation, that between developmental adaptation and
developmental disruption. Not all plasticity results in advanta-
geous changes for the developing organism, and in some cases,
changes are the result of environmental damage to normal devel-
opmental processes, as in teratogenesis. It is important to con-
sider that deleterious effects of environment on phenotype may
574 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

18
1
17

0 16 Body mass
24 index at
26 15 11 years
28 (kg/m2)
30
Ponderal index at birth (kg/m3)
• Fig. 47.2  Catch-up growth is associated with increased cardiovascular mortality. Relationship between
ponderal index at birth, body mass index at age 11 years and hazard ratio of death from cardiovascular
disease. (Data from Forsen T, Eriksson JG, Tuomilehto J, et  al. Growth in utero and during childhood
among women who develop coronary heart disease: longitudinal study. BMJ 319(7222):1403–1407,
1999.)

rather than an adaptation to better suit an environment. Experi-
mental evidence must take this into account; however, develop-
mental plasticity which occurs at a certain point may confer an
advantage at one point but a change in environment may render
that adaptation harmful, especially if it occurs after the window of
plasticity, preventing a reversal of the phenotypic expression. This
trade-off is the basis of the thrifty phenotype hypothesis.
Developmental ‘Trade-Off’
Waddington described ‘homeorhesis’ in 1957 (cited by Gluckman
and Hanson20) as long-term change to a phenotypic developmental
trajectory as a response to prolonged exposure to an adverse environ-
ment, as opposed to a short-term change in response to a brief envi-
ronmental exposure that has no extended consequences. A trade-off
exists when a change has immediate advantage but later disadvantage.
This is best illustrated in humans by two observations. First, children
born after experiencing fetal growth restriction (FGR) who have
slower growth trajectories postnatally enter puberty earlier than their
normally grown counterparts.25 A trade-off exists that allows survival
under poor nutrition but results in poor postnatal growth. Earlier
reproductive development confers an evolutionary advantage by
allowing the individual to reproduce even under poor developmental
conditions. Second, growth-restricted fetuses may undergo ‘rescue by
preterm birth’26 in which the initiation of preterm delivery is a trade-
off which allows the fetus to escape the hostile intrauterine environ-
ment and stillbirth but exposes it to the challenges of prematurity.  cally to modulate the effect of the abnormal intrauterine growth on
Predictive Adaptive Responses
Gluckman and Hanson5 described a different form of devel-
opmental plasticity in which the developing organism makes
phenotypic adaptations better suited to the predicted adult envi-
ronment, anticipated from signals within the intrauterine envi-
ronment. These ‘predictive adaptive responses’ (PARs) do not
confer benefit to the developing organism but, rather, better equip
the organism for adult life. The association with adult survival
advantage requires the prediction of the adult environment to be
accurate.27 If an inappropriate PAR is made, this may manifest
as altered disease risk, as in the metabolic disease associated with
FGR. Accurate predictions of adult environment may be impaired
by disordered maternal–placental–fetal signalling in the case of
maternal–placental disease. Here, the fetus undernourished by a
dysfunctional placenta prepares (by becoming insulin and leptin
resistant, among other adaptations) for an extrauterine life of
nutritional paucity but, when born into a mismatched environ-
ment of caloric plenty, develops metabolic disease. 
Postnatal ‘Catch-up’ Growth
Prader and colleagues28 coined the term ‘catch-up growth’ as a
description of increased growth velocity in small children after the
treatment of a disease limiting their growth, suggesting that this was a
healthy phenomenon. However, in the context of a growth-restricted
fetus, growth trajectories during infancy were found epidemiologi-
 CHAPTER 47  The Developmental Origins of Health and Disease 575

Fetal genotype

Fetal insulin resistance Adult insulin resistance

Disordered IGF-1 Metabolically unsuited Metabolically suited

signalling constant food supply to periods of
undernutrition

Slow fetal growth Obesity

Low birth weight Type 2 diabetes Health

Association

• Fig. 47.3  The thrifty genotype hypothesis. IGF-1, Insulin-like growth factor-1. (Adapted from Neel JV. Dia-
betes mellitus: a ‘thrifty’ genotype rendered detrimental by ‘progress’? Am J Hum Genet 14:353–362, 1962.)

adult disease, with the highest risk in children who gained the most
weight in childhood (Fig 47.2).29,30 These associations were repli-
cated experimentally, with rapid postnatal growth associated with an
increase in adult disease.20 Furthermore, adult disease can be induced
by accelerating infant growth even in those of normal birth weight,
suggesting that the period in early life during which developmental
plasticity acts extends beyond birth into infancy. Given this postnatal
plasticity, there may be potential to introduce postnatal interventions
to modulate the risk for adult disease.  association between severe growth restriction and adult disease, it
The ‘Thrifty Genotype’ Hypothesis
The thrifty genotype hypothesis, initially proposed as a purely
genetic explanation for regional variation in insulin resistance, sug-
gested that certain populations had evolutionarily selected genetic
insulin resistance, which allowed them to cope better with periods
of nutritional deficit (Fig 47.3). Neel31 suggested that this insulin
resistance predisposed these populations to type 2 diabetes in the
presence of modern-day nutritional abundance. A modification
to this hypothesis was proposed in the early days of the DOHaD
field whereby the genes thought responsible for the insulin resis-
tance could also explain the observed changes in birth weight. This
was certainly biologically valid, given the known interplay between
insulin and insulin-like growth factor-1 (IGF-1) and the role of
IGF-1 in the regulation of fetal growth. Genetic polymorphisms
must play a role in DOHaD, but they are not enough alone to
describe all of the epidemiologic evidence, in particular the con-
sistency of associations across ethnic populations and the speed
with which DOHaD manifests in socioeconomically transitioning
populations.20 Moreover, the array of evidence for ‘developmental
programming’ induced by environmental change highlights the
importance of a place for environment in any complete theory.32  individuals. Neonates with HNF1β MODY mutations have been
The ‘Thrifty Phenotype’ Hypothesis
Hales and Barker,33 in response to the inadequacies of the thrifty
genotype hypothesis, proposed the thrifty phenotype hypothesis as
one of the first theories to explain the underlying physiology of their
observed associations between the fetal environment and adult disease
(Fig 47.4). They proposed a mechanism by which a fetus exposed to
intrauterine undernutrition grew more slowly, resulting in a trajectory
to small growth, and developed insulin resistance and other changes
appropriate to a nutritionally poor environment. A developmental
mismatch then occurred in later life in the presence of nutritional
abundance, where the fetal adaptations increased risk for adult disease.
This theory, however, also does not fully explain the observa-
tions from epidemiology. In particular, although it explains the
remains uncertain if it can account for observed changes in disease
risk across the range of normal growth. For example, why should an
individual with a birth weight on the 30th centile have an increased
risk compared with one on the 70th centile, as the early epidemi-
ologic data suggested, when both are clearly ‘normally’ grown at
birth?2,20 Regardless, it is increasingly clear that the relationships
between birth weight and subsequent disease risk extends across the
birth weight spectrum rather than being confined to the extremes.
Furthermore, this hypothesis does not explain observed changes in
adult systems that provide no apparent fetal survival advantage. 
The Fetal Insulin Hypothesis
Hattersley and Tooke34 proposed the fetal insulin hypothesis along
similar lines to the thrifty genotype hypothesis, suggesting that
genetically determined insulin resistance could explain both FGR
and later glucose intolerance. Evidence for this hypothesis arose
through the study of monogenic conditions where a single defect
in a gene results in disease. Maturity-onset diabetes of the young
(MODY) is a rare form monogenic diabetes characterised by single
mutations in a range of individual genes. MODY-causing gene vari-
ants have been directly implicated in lower birth weight for affected
shown to be up to 900 g lighter than negative control participants.34
Further support for a genetic role in fetal growth has come
from genome-wide association studies of birth weight, which have
demonstrated an association between a specific allelic variant in
the ADCY5 gene and reduced fetal growth.35 This variant had pre-
viously been demonstrated to be associated with an increased risk
for adult type 2 diabetes mellitus (T2DM).36 
576 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Maternal malnutrition

Other maternal or
placental
abnormalities

Fetal malnutrition

↓ β-Cell mass or islet function

↓ Fetal growth

Infant malnutrition

↓ Adult β-cell function

Obesity
Insulin resistance
Age

Other organ Type 2

dysfunction diabetes Hypertension

Metabolic syndrome

• Fig. 47.4  Diagrammatic representation of the thrifty phenotype hypothesis as it applies to type 2 diabe-
tes and the metabolic syndrome. (Adapted from Hales CN, Barker DJ. The thrifty phenotype hypothesis.
Br Med Bull 60:5–20, 2001.)

Gene–Environment Interactions environment resulted in the phenotype of disease. This confirmed,

Neither of the ‘thrifty’ hypotheses fully explains that which we
observe epidemiologically and experimentally. Clearly, genotype is
important because not all individuals of low birth weight go on to
develop adult disease. Similarly, environment also plays a role given
the rapid changes observed during short periods of famine37 and
with rapid population socioeconomic transformation.38 Wells39 also
suggests that the two hypotheses are not mutually exclusive, with
genetics possibly explaining the long-term changes in entire popu-
lations and environment explaining acute changes in individuals.
In defence of the importance of environment underlying the
DOHaD phenomenon, Hales and Barker40 acknowledged the
role of genetics in every human disease. Indeed, they conceded
that malaria and tuberculosis, among the most environmental of
diseases, still affected individuals differently depending on their
genetic susceptibility. However, they maintained, environmen-
tal exposure to the pathogen was the key factor in disease and
compared this to gene–environment interactions in DOHaD. By
contrast, Eriksson et al.41 elegantly demonstrated the importance
of gene–environment interactions underlying DOHaD in dem-
onstrating an association with the combination of PPARG gene
polymorphisms and low birth weight with adult T2DM. In this
study, neither the polymorphisms nor low birth weight alone
were associated with T2DM, but the combination of gene and
at least for this example, the combined roles for genetics and envi-
ronment underlying DOHaD phenomena. 
Genomic Variation and Epigenetics
The two main areas of genetic interest in the DOHaD field are
genomic variation and epigenetics. The genes that make up our
genetic code vary in size from a few hundred DNA bases to more
than 2 million. Each autosomal gene is represented by two copies,
or alleles, one inherited from each parent. Although most alleles are
the same, about 1% vary among people (genetic polymorphism).
Alleles vary through small difference in their DNA sequence.
The major allele refers to the one most commonly displayed in
the population. These small genetic differences can contribute to
each person’s unique features. In their rare forms, as described for
monogenic diabetes, they can be disease causing. Genomic poly-
morphism may affect gene function in two main ways. First, DNA
sequence variation in coding regions of genes can result in amino
acid substitutions, which can change the structure and function
of the protein products of the gene. Second, and probably more
commonly, polymorphisms within noncoding regions of DNA,
such as transcription factor binding sites (and other regulating
regions), may affect the expression of proteins, with more quanti-
tative than qualitative effects on the protein product of the gene.
 CHAPTER 47  The Developmental Origins of Health and Disease
Genomic variation is clearly important in DOHaD. Several
studies have demonstrated associations between genetic poly-
morphisms, abnormal fetal growth and adult disease.42-44 The
genetic regulation of both fetal growth and adult metabolic dis-
ease is complex, involving multiple genes, and it is evident that
the genetic mechanisms underlying their association will also be
complex, with multiple variations across multiple genes each mak-
ing a small contribution to the phenotypic outcome.
Epigenetic phenomena, on the other hand, do not alter the
genomic DNA sequence but involve changes to the structure and
function of the DNA complex. Epigenetic mechanisms enable
developing organisms to produce disparate cellular phenotypes
from the same genotype.45 These modifications take the form
of DNA methylation, chromatin remodelling, covalent modi-
fications to histones, parental imprinting and X-chromosome
inactivation.46
Epigenetic events may explain the relationship between an
individual’s genetic background, the environment, aging and dis-
ease. Although a DNA sequence always remains the same, cells
in a specific tissue have the ability to vary their epigenetic state
and hence gene expression through their life course. It is therefore
likely that these mechanisms are important in DOHaD and that
they interact with each other.47
Traditionally, genomic variation and epigenetics have been
seen as competing theories of DOHaD mechanisms, but in
reality, the two are probably interdependent. For example,
DNA methylation is controlled by the genes encoding the
DNA methyltransferase enzymes, and evidence links poly-
morphisms in these genes with altered DNA methylation pat-
terns in endometrial,48 pancreatic,49 gastric50 and colorectal51
cancers.
Technological advances in epigenomic sequencing have
provided a better understanding of the complex interplay
between genotype and epigenotype and have more firmly
established that genetic polymorphisms can affect meth-
ylation. For example, the gene SERPINA3 (Serpin peptidase
inhibitor clade A member 3) encodes a protease inhibitor
involved in a wide range of biological processes. It has been
found to be upregulated in human placental diseases (specifi-
cally growth restriction) in association with hypomethylation
of a regulating part of the gene in the presence of a specific
genetic variant.52
This interaction between methylation and genotype which
could lead to alteration of gene expression and therefore disease
provides a novel insight into how communicating genetic and epi-
genetic mechanisms could be involved in placental disorders such
as FGR.  were associated together in a single functional network centred
Imprinted Genes. Genomic imprinting is a process through
which the expression of a gene is dependent on the sex of the
parent from which it was inherited.53 Unlike the majority of
genes in which both alleles are expressed in imprinted genes,
only one allele is expressed. If the allele inherited from the father
is imprinted, it is thereby silenced (paternally imprinting), and
only the allele from the mother is expressed. If the allele from
the mother is imprinted, then only the allele from the father is
expressed. Imprinted genes are highly conserved and expressed
in the placenta. These genes are effectively driven by epigenetic
regulation theorised to be controlled by differential methyla-
tion of the relevant genes.53 It has been hypothesised that spo-
radic loss of imprinting induced through methylation change
could occur in human placentas and contribute to abnormal
placental development and consequentially decreased fetal
577
growth.54 Within the placenta, regulation of imprinted gene
expression appears to be less stable than in the fetus itself. This
allows the placenta to better adapt to changing physiological
environments but with potential adverse consequences such as
poor growth.55,56
The paternally expressed insulin-like growth factor 2
(IGF2) gene is imprinted and is a major modulator of both
placental and fetal growth.57 IGF2 is regulated by a differen-
tially methylated binding region known as imprinting con-
trol region 1 (ICR1).58,59 In growth-restricted placentas, the
ICR1 region has been found to be significantly less meth-
ylated with resultant reduced expression of IGF2 compared
with normal grown placentas. Decreased placental methyla-
tion at the IGF2 imprinting control region is associated with
intrauterine growth restriction but not preeclampsia60,61 and
complete loss of placental IGF2 expression is associated with
FGR in mice.62 
Epigenetics and Other Interactions. Maternal diet is one of
the ways in which the supply of nutrients vital to DNA meth-
ylation may be affected. Famine provides an extreme example
of disruption to the supply of nutrients essential in the meth-
ylation pathway. Individuals exposed to famine in utero have
been shown to have lower birth weights,37 and furthermore,
exposure to famine during any stage of gestation was associ-
ated with glucose intolerance later in life.63 Individuals born
during the Dutch famine born 60 years ago were later found
to have less DNA methylation of the imprinted IGF2 gene
compared with their famine-unexposed same-sex siblings.64
This observation supports the hypothesis that exposure to
adverse environmental factors in utero could permanently alter
epigenetic marks, which might have a bearing in adult disease
risk. 
DNA Methylation in Placental Diseases. DNA hypomethyl-
ation at gene enhancer regions has been identified between pla-
centas from pregnancies affected by pre-eclampsia and control
participants.65 In a small cohort of term pregnancies affected by
FGR, methylation differences were found in the pathway involv-
ing hepatocyte nuclear factor 4α (HNF4A) gene.66 Because
HNF4A is a candidate gene for T2DM, this observation opens up
the possibility that differential methylation of genes important in
diabetes might also play a role in fetal growth. 
Animal Models of DNA Methylation in Fetal Growth Restric-
tion. In an animal model of FGR, cytosine methylation in pan-
creatic islet cells was differentially methylated at approximately
1400 loci in male rats at 7 weeks of age, before they went on to
develop diabetes. Of the top 53 genes, almost half of those tested
on a collection of important metabolic and cellular regulators.67
The majority of changes occurred in evolutionarily conserved
DNA sequences with some loci in proximity to genes manifesting
changes in gene expression which were enriched near genes regu-
lating vascularisation, proliferation and insulin secretion.68
Furthermore, in growth-restricted rodent offspring, histone
modifications in association with DNA methylation differences
have been identified in the promoter region of the gene PDX1,
eventuating in permanent gene silencing and a diabetic pheno-
type.69 In health, PDX1 regulates pancreatic β-cell differentiation,
but when silenced, T2DM eventuates. This study offered a new
insight in epigenetic mechanisms linking growth restriction to
diabetes development.70
Given the ability of sequence variation in genes influencing
epigenetic changes to affect phenotypic outcome, it is likely that
578 SE C T I O N 8     Diagnosis and Management of Other Fetal Conditions

Early complementary
Breastfeeding Formula feeding
feeding (<4 months)

– Energy + Energy – Energy

Macronutrient
– Protein + Protein – Protein
intake
+ Fat – Fat + Fat

– Leptin ? Leptin
+ Leptin + Ghrelin ? Ghrelin
Hormones – Ghrelin ? Adiponectin ? Adiponectin
+ Adiponectin + Insulin + Insulin
+ IGF-1 + IGF-1

+ Bifidobacteria + Clostridium
Intestinal + Enterococcus
+ Lactobacillus + Enterobacteria
microflora + Bacteroides
+ Staphylococcus + Bacteroides

+ Maternal education, SES, age

+ Maternal obesity
Socioenvironment + Acceptance of vegetables
+ Food neophobia +/– Maternal diet quality
and behaviour + Infant control of feeding
+ Maternal control of feeding
+ Self-regulation

Slower weight gain More rapid weight gain

Lower adiposity Greater adiposity

– +
Paediatric obesity

• Fig. 47.5  Pathways linking infant nutrition and later obesity. IGF-1, Insulin-like growth factor-1; SES,
socioeconomic status. (From Thompson AL. Developmental origins of obesity: early feeding environ-
ments, infant growth, and the intestinal microbiome. Am J Hum Biol 24(3):350–360, 2012.)

sequence variation will underlie some of the association seen between
epigenetics and DOHaD, with an individual’s susceptibility to epi-
genetic changes being controlled to some extent by their genotype.  tion, the prolonged fasting associated with reduced overnight
Infant Nutrition
Breastfeeding appears to be protective against the development of
adult obesity.71,72 This protection is dose responsive, with greater
benefit in those breastfed for longer durations73 and those exclusively
breastfed without formula supplementation.74 The precise underly-
ing mechanism by which infant nutrition modifies obesity risk is
difficult to elucidate given the multiple biological, environmental,
genetic, epigenetic and social contexts.75 There are likely to be signifi-
cant contributions from all of these factors (Fig 47.5). So far there is
evidence to demonstrate an impact of early nutrition upon metabolic
programming,76 upon epigenetic modification within the IGF axis77
and upon hormonal pathways including insulin and adiponectin.78  change in insulin level during the window of developmental plastic-
Behavioural Modifications. In high-resource settings, breast-
feeding tends to be more common among those from more
affluent socioeconomic backgrounds,79 raising the possibility of
confounding of associations by the higher rates of healthy lifestyle
habits in these groups.80 There are, however, significant differences
in the pattern of feeding between breast- and formula-fed infants
which may modulate long-term health. Formula-fed infants take
larger volumes of slightly more energy-dense milk at less frequent
intervals than breastfed infants.81. Breastfed infants reduce their
milk intake when solid foods are introduced, but formula-fed
infants do not, resulting in an overall higher energy intake. These
patterns are associated with more rapid postnatal weight gain82
and a greater accumulation of adipocytes and fat mass.83 In addi-
feeding in formula-fed infants favours relative insulin resistance,
which may become a programmed feature of an individual’s later
metabolism. Breastfed infants are more able to determine their
own nutritional intake, but it is unclear whether this behavioural
aspect of nutrition is maintained beyond breastfeeding.84 
Nutritional Composition. There are substantial micro- and
macronutrient differences between breast and formula milk which
have the potential to lead to altered hormonal responses and meta-
bolic programming.84 For example, cow’s milk formula is signifi-
cantly higher in protein than human breast milk, stimulating higher
insulin and IGF-1 in formula-fed infants.85 IGF-axis stimulation
drives more rapid postnatal weight gain, and this increase in weight
persists after withdrawal of the relatively high protein diet.86 This
ity may persist into later life when hyperinsulinaemia is associated
with increased obesity among other adverse metabolic consequences.
Also, in contrast to formula milk, breast milk composition changes
among feeds and across the duration of breastfeeding. It is likely that
this reflects differing nutritional requirements of infants at various
times, which are not addressed by the constant composition of for-
mula milk.
Fat content is significantly different between breast milk and cow’s
milk formula, both quantitatively and qualitatively. An infant diet of
relatively low fat-to-carbohydrate ratio, as found in formula milk, is
associated with a higher rate of adult obesity in animal models.87
 CHAPTER 47  The Developmental Origins of Health and Disease
Aside from the nutritional differences, breast milk and cow’s
milk formula differ substantially in their nonnutrient composi-
tions, with breast milk containing various hormonal mediators of
nutrition and metabolism such as leptin, ghrelin and adiponectin.
These nonnutritional components may determine programming of
metabolism and account for the differences in weight gain and adi-
posity distribution seen with various patterns of infant feeding.75
Prebiotic substances such as human milk–oligosaccharides and
immunomodulatory substances such as long-chain polyunsaturated
fatty acids also play a role in early metabolic health and are widely
different between human and bovine milk.  bacterium and Lactobacillus spp., the predominant genera in
Growth Acceleration. Singhal and Lucas88 proposed the
‘growth acceleration hypothesis’ whereby the more rapid
growth observed in formula-fed infants compared with exclu-
sively breastfed infants results in metabolic programming of
insulin resistance, dyslipidaemia, hypertension and obesity.
Apparently, small changes in weight gain in a very short period
of postnatal life were associated with substantial changes in
endothelial function of similar magnitude to type 1 diabetes
and smoking. Differences in infant nutrition change the tim-
ing of the adiposity rebound in childhood, which is associated
with an altered risk for later obesity.89  health over her or his life course. This association is influenced by
Intestinal Microbiome. The bacterial flora of the gastrointesti-
nal tract has gained much attention in recent times and may play a
significant role in the developmental origins of metabolic disease.
The gut microbiome is established early in postnatal life, deter-
mined by maternal factors such as adiposity, neonatal hygiene and
early feeding, and is then relatively stable during infancy. A major
source of infant intestinal bacteria is the maternal intestinal flora,
which may be modified in association with complications of preg-
nancy such as obesity, excessive weight gain and diabetes.90,91
579
Perturbations in the infant gut microbiome, in particular
reduction in Bifidobacterium spp. and replacement by coagu-
lase-positive staphylococci, may precede the development of
obesity in childhood.92 This is thought to relate to the influ-
ence that gut bacteria has upon the metabolism of complex
indigestible molecules into simple sugars and short-chain fatty
acids with an impact upon the growth, adiposity and immu-
nologic development of the host.75,93,94 Infant nutrition is a
major determinant of the gut microflora, which vary signifi-
cantly between breast- and formula-fed infants, with Bifido-
the former and Clostridium, Bacteroides Enterobacteria and
Enterococcus spp. more common in the latter. This variation
may explain some of the association between breastfeeding and
obesity reduction. 
Conclusion
The expansion of knowledge in the DOHaD over 3 decades has
contributed greatly to the recognition of the importance of mater-
nal and neonatal health in the determination of an individual’s
complex environmental, genetic and epigenetic factors as well as
the interactions among these factors. The phenomenon of devel-
opmental plasticity provides not only an explanation for the asso-
ciation between early life events and later disease but potentially a
window of opportunity to intervene to alter the trajectory toward
later health or disease.
Access the complete reference list online at ExpertConsult.com.
Self-assessment questions available at ExpertConsult.com.
References 20. Gluckman PD, Hanson MA. The conceptual
basis for DOHaD. In: Gluckman PD, Han-
38. Prentice AM, Moore SE. Early programming
of adult diseases in resource poor countries.
son MA, eds. Developmental Origins of Health Arch Dis Child. 2005;90(4):429–432.
1. Barker DJ, Osmond C. Infant mortal-
and Disease. Cambridge, UK: Cambridge Uni- 39. Wells JC. The thrifty phenotype hypothesis:
ity, childhood nutrition, and ischaemic
versity Press; 2006:33–50. thrifty offspring or thrifty mother? J Theor
heart disease in England and Wales. Lancet.
21. Clabaut C, Herrel A, Sanger TJ, et al. Devel- Biol. 2003;221(1):143–161.
1986;1(8489):1077–1081.
opment of beak polymorphism in the African 40. Hales CN, Barker DJ. The thrifty phenotype
2. Barker DJ. Childhood causes of adult dis-
seedcracker, Pyrenestes ostrinus. Evol Dev. hypothesis. Br Med Bull. 2001;60:5–20.
eases. Arch Dis Child. 1988;63(7):867–869.
2009;11(6):636–646. 41. Eriksson JG, Lindi V, Uusitupa M, et al. The
3. Newnham JP. The developmental origins of
22. Scoville AG, Pfrender ME. Phenotypic plas- effects of the Pro12Ala polymorphism of the
health and disease (DOHaD)—why it is so
ticity facilitates recurrent rapid adaptation to peroxisome proliferator-activated receptor-
important to those who work in fetal medicine.
introduced predators. Proc Natl Acad Sci U S gamma2 gene on insulin sensitivity and insu-
Ultrasound Obstet Gynecol. 2007;29(2):121–
A. 2010;107(9):4260–4263. lin metabolism interact with size at birth.
123.
23.  West-Eberhard MJ. Phenotypic accommo- Diabetes. 2002;51(7):2321–2324.
4. Barker DJ. The fetal origins of diseases of old
dation: adaptive innovation due to develop- 42. White SW, Ang QW, Warrington NM, et al.
age. Eur J Clin Nut. 1992;46(suppl 3):S3–S9.
mental plasticity. J Exp Zool B Mol Dev Evol. Single Nucleotide Polymorphisms Within the
5. Gluckman PD, Hanson MA. Living with the
2005;304(6):610–618. Leptin and Leptin Receptor Genes are Associated
past: evolution, development, and patterns of
24. Hoverman JT, Relyea RA. How flexible is with Fetal Growth Trajectories and Adolescent Car-
disease. Science. 2004;305(5691):1733–1736.
phenotypic plasticity? Developmental win- diovascular Disease Precursors. Santiago, Chile:
6. Ohashi J, Tokunaga K. The power of genome-
dows for trait induction and reversal. Ecology. 6th World Congress on the Developmental
wide association studies of complex dis-
2007;88(3):693–705. Origins of Health and Disease; 2009.
ease genes: statistical limitations of indirect
25. Da Silva P, Aitken RP, Rhind SM, et al. Influ- 43. Sorensen K, Aksglaede L, Petersen JH, et al.
approaches using SNP markers. J Hum Genet.
ence of placentally mediated fetal growth The exon 3 deleted growth hormone recep-
2001;46(8):478–482.
restriction on the onset of puberty in male and tor gene is associated with small birth size and
7. Solomons NW. Developmental origins of
female lambs. Reproduction. 2001;122(3):375– early pubertal onset in healthy boys. J Clin
health and disease: concepts, caveats, and
383. Endocrinol Metab. 2010;95(6):2819–2826.
consequences for public health nutrition.
26. Froen JF, Pinar H, Norwitz ER. Genetic epi- 44. Bueno AC, Espineira AR, Fernandes-Rosa
Nutr Rev. 2009;67(suppl 1):S12–S16.
demiologic studies of preterm birth: studies of FL, et al. Adiponectin: serum levels, promoter
8. Roberts CT. Review: complicated interac-
disease or of ‘rescue by birth’?. Am J Obstet polymorphism, and associations with birth
tions between genes and the environment
Gynecol. 2007;197(4):438–489; author reply size and cardiometabolic outcome in young
in placentation, pregnancy outcome and
489. adults born large for gestational age. Eur J
long term health. Placenta. 2010;31(suppl):
27. Bateson P. Fetal experience and good adult Endocrinol. 2010;162(1):53–60.
S47–S53.
design. Int J Epidemiol. 2001;30(5):928–934. 45. Jirtle RL, Skinner MK. Environmental epig-
9. Bruce KD, Hanson MA. The developmental
28. Prader A, Tanner JM, von HG. Catch-up enomics and disease susceptibility. Nat Rev
origins, mechanisms, and implications of met-
growth following illness or starvation. An Genet. 2007;8(4):253–262.
abolic syndrome. J Nutr. 2010;140(3):648–
example of developmental canalization in 46. Whitelaw E, Garrick D. Epigenetic mecha-
652.
man. J Pediatr. 1963;62:646–659. nisms. In: Gluckman PD, Hanson MA, eds.
10. McMillen IC, Robinson JS. Developmental
29. Eriksson JG, Forsen T, Tuomilehto J, et  al. Developmental Origins of Health and Disease.
origins of the metabolic syndrome: predic-
Catch-up growth in childhood and death Cambridge, UK: Cambridge University Press;
tion, plasticity, and programming. Physiol
from coronary heart disease: longitudinal 2006:62–74.
Rev. 2005;85(2):571–633.
study. BMJ. 1999;318(7181):427–431. 47. Gluckman PD, Hanson MA, Buklijas T, et al.
11. Kermack WO, McKendrick AG, McKinlay
30. Forsen T, Eriksson JG, Tuomilehto J, et  al. Epigenetic mechanisms that underpin meta-
PL. Death-rates in Great Britain and Sweden.
Growth in utero and during childhood among bolic and cardiovascular diseases. Nat Rev
Some general regularities and their signifi-
women who develop coronary heart disease: lon- Endocrinol. 2009;5(7):401–408.
cance. Lancet. 1934;223(5770):698–703.
gitudinal study. BMJ. 1999;319(7222):1403– 48. Han J, Hankinson SE, De Vivo I. Polymor-
12. Forsdahl A. Are poor living conditions in
1407. phisms in O6-methylguanine DNA meth-
childhood and adolescence an important risk
31. Neel JV. Diabetes mellitus: a ‘thrifty’ geno- yltransferase and endometrial cancer risk.
factor for arteriosclerotic heart disease? Br J
type rendered detrimental by ‘progress’? Am J Carcinogenesis. 2006;27(11):2281–2285.
Prev Soc Med. 1977;31(2):91–95.
Hum Genet. 1962;14:353–362. 49. Jiao L, Bondy ML, Hassan MM, et  al.
13. Barker DJ. The origins of the develop-
32. Warner MJ, Ozanne SE. Mechanisms involved Selected polymorphisms of DNA repair genes
mental origins theory. J Intern Med. 2007;
in the developmental programming of adult- and risk of pancreatic cancer. Cancer Detect
261(5):412–417.
hood disease. Biochem J. 2010;427(3):333– Prev. 2006;30(3):284–291.
14. Barker DJ, Osmond C. Inequalities in health
347. 50. Fan H, Liu D, Qiu X, et al. A functional poly-
in Britain: specific explanations in three
33. Hales CN, Barker DJ. Type 2 (non-insulin- morphism in the DNA methyltransferase-3A
Lancashire towns. Br Med J (Clin Res Ed).
dependent) diabetes mellitus: the thrifty pheno- promoter modifies the susceptibility in gas-
1987;294(6574):749–752.
type hypothesis. Diabetologia. 1992;35(7):595– tric cancer but not in esophageal carcinoma.
15. Barker DJ, Osmond C. Death rates from
601. BMC Med. 2010;8:12.
stroke in England and Wales predicted from
34. Hattersley AT, Tooke JE. The fetal insu- 51. de Vogel S, Wouters KA, Gottschalk RW,
past maternal mortality. Br Med J (Clin Res
lin hypothesis: an alternative explanation et al. Genetic variants of methyl metabolizing
Ed). 1987;295(6590):83–86.
of the association of low birthweight with enzymes and epigenetic regulators: associa-
16. Notkola V, Punsar S, Karvonen MJ, et  al.
diabetes and vascular disease. Lancet. 1999; tions with promoter CpG island hypermeth-
Socio-economic conditions in childhood and
353(9166):1789–1792. ylation in colorectal cancer. Cancer Epidemiol
mortality and morbidity caused by coronary
35. Freathy RM, Mook-Kanamori DO, Sovio U, Biomarkers Prev. 2009;18(11):3086–3096.
heart disease in adulthood in rural Finland.
et  al. Variants in ADCY5 and near CCNL1 52. Chelbi ST, Wilson ML, Veillard AC, et  al.
Soc Sci Med. 1985;21(5):517–523.
are associated with fetal growth and birth Genetic and epigenetic mechanisms collabo-
17. Barker DJ, Osmond C. Childhood respira-
weight. Nat Genet. 2010;42(5):430–435. rate to control SERPINA3 expression and its
tory infection and adult chronic bronchitis in
36. Dupuis J, Langenberg C, Prokopenko I, et al. association with placental diseases. Hum Mol
England and Wales. Br Med J (Clin Res Ed).
New genetic loci implicated in fasting glucose Genet. 2012;21(9):1968–1978.
1986;293(6557):1271–1275.
homeostasis and their impact on type 2 diabe- 53. Haycock PC, Ramsay M. Exposure of mouse
18. Barker DJ, Martyn CN. The maternal and
tes risk. Nat Genet. 2010;42(2):105–116. embryos to ethanol during preimplantation
fetal origins of cardiovascular disease. J Epide-
37. Lumey LH. Decreased birthweights in infants development: effect on DNA methylation
miol Community Health. 1992;46(1):8–11.
after maternal in utero exposure to the Dutch in the h19 imprinting control region. Biol
19. Barker DJ. Fetal growth and adult disease. Br
famine of 1944-1945. Paediatr Perinat Epide- Reprod. 2009;81(4):618–627.
J Obstet Gynaecol. 1992;99(4):275–276.
miol. 1992;6(2):240–253.

579.e1
579.e2 References
54. Maccani MA, Marsit CJ. Epigenetics
in the placenta. Am J Reprod Immunol.
2009;62(2):78–89.
55. Novakovic B, Yuen RK, Gordon L, et al. Evi-
dence for widespread changes in promoter
methylation profile in human placenta in
response to increasing gestational age and envi-
ronmental/stochastic factors. BMC Genomics.
2011;12:529.
56. Nelissen EC, van Montfoort AP, Dumoulin
JC, et al. Epigenetics and the placenta. Hum
Reprod Update. 2011;17(3):397–417.
57. Constancia M, Hemberger M, Hughes J,
et  al. Placental-specific IGF-II is a major
modulator of placental and fetal growth.
Nature. 2002;417(6892):945–948.
58. Murrell A, Heeson S, Reik W. Interaction
between differentially methylated regions par-
titions the imprinted genes Igf2 and H19 into
parent-specific chromatin loops. Nat Genet.
2004;36(8):889–893.
59. Engel N, West AG, Felsenfeld G, et al. Antag-
onism between DNA hypermethylation and
enhancer-blocking activity at the H19 DMD
is uncovered by CpG mutations. Nat Genet.
2004;36(8):883–888.
60. Bourque DK, Avila L, Penaherrera M, et  al.
Decreased placental methylation at the H19/
IGF2 imprinting control region is associ-
ated with normotensive intrauterine growth
restriction but not preeclampsia. Placenta.
2010;31(3):197–202.
61. McMinn J, Wei M, Schupf N, et al. Unbal-
anced placental expression of imprinted genes
in human intrauterine growth restriction.
Placenta. 2006;27(6-7):540–549.
62. Fowden AL, Sibley C, Reik W, et  al.
Imprinted genes, placental development and
fetal growth. Horm Res. 2006;65(suppl 3):50–
58.
63. Roseboom T, de Rooij S, Painter R. The
Dutch famine and its long-term conse-
quences for adult health. Early Hum Dev.
2006;82(8):485–491.
64. Heijmans BT, Tobi EW, Stein AD, et al. Per-
sistent epigenetic differences associated with
prenatal exposure to famine in humans. Proc
Natl Acad Sci U S A. 2008;105(44):17046–
17069.
65. Blair JD, Yuen RK, Lim BK, et  al. Wide-
spread DNA hypomethylation at gene
enhancer regions in placentas associated with
early-onset pre-eclampsia. Mol Hum Reprod.
2013;19(10):697–708.
66. Einstein F, Thompson RF, Bhagat TD, et al.
Cytosine methylation dysregulation in neo-
nates following intrauterine growth restric-
tion. PLoS One. 2010;5(1):e8887.
67. Thompson RF, Fazzari MJ, Niu H, et  al.
Experimental intrauterine growth restriction
induces alterations in DNA methylation and
gene expression in pancreatic islets of rats.
J Biol Chem. 2010;285(20):15111–15118.
68. Stoffers DA, Desai BM, DeLeon DD, et  al.
Neonatal exendin-4 prevents the develop-
ment of diabetes in the intrauterine growth
retarded rat. Diabetes. 2003;52(3):734–740.
69. Park JH, Stoffers DA, Nicholls RD, et  al.
Development of type 2 diabetes following
intrauterine growth retardation in rats is asso-
ciated with progressive epigenetic silencing of
Pdx1. J Clin Invest. 2008;118(6):2316–2324.
70. Brissova M, Blaha M, Spear C, et al. Reduced
PDX-1 expression impairs islet response
to insulin resistance and worsens glucose
homeostasis. Am Physiol Endocrinol Metab.
2005;288(4):E707–E714.
71. Gillman MW, Rifas-Shiman SL, Camargo Jr
CA, et al. Risk of overweight among adoles-
cents who were breastfed as infants. JAMA.
2001;285(19):2461–246.
72. Owen CG, Martin RM, Whincup PH,
et  al. Effect of infant feeding on the risk of
obesity across the life course: a quantita-
tive review of published evidence. Pediatrics.
2005;115(5):1367–1377.
73. Harder T, Bergmann R, Kallischnigg G,
et  al. Duration of breastfeeding and risk of
overweight: a meta-analysis. Am J Epidemiol.
2005;162(5):397–403.
74. Arenz S, Ruckerl R, Koletzko B, et al. Breast-
feeding and childhood obesity—a system-
atic review. Int J Obes Relat Metab Disord.
2004;28(10):1247–1256.
75. Thompson AL. Developmental origins of
obesity: early feeding environments, infant
growth, and the intestinal microbiome. Am J
Hum Biol. 2012;24(3):350–360.
76. Anzman SL, Rollins BY, Birch LL. Paren-
tal influence on children’s early eating envi-
ronments and obesity risk: implications
for prevention. Int J Obes (Lond). 2010;
34(7):1116–1124.
77. Waterland RA, Lin JR, Smith CA, et al. Post-
weaning diet affects genomic imprinting at
the insulin-like growth factor 2 (Igf2) locus.
Hum Mol Genet. 2006;15(5):705–716.
78. de Zegher F, Sebastiani G, Diaz M, et al. Breast-
feeding vs formula-feeding for infants born
small-for-gestational-age: divergent effects on
fat mass and on circulating IGF-I and high-
molecular-weight adiponectin in late infancy.
J Clin Endocrinol Metab. 2013;98(3):1242–1247.
79. Thulier D, Mercer J. Variables associated with
breastfeeding duration. J Obstet Gynecol Neo-
natal Nurs. 2009;38(3):259–268.
80. Weyers S, Dragano N, Richter M, et al. How
does socio economic position link to health
behaviour? Sociological pathways and per-
spectives for health promotion. Glob Health
Promot. 2010;17(2):25–33.
81. Sievers E, Oldigs HD, Santer R, et al. Feeding
patterns in breast-fed and formula-fed infants.
Ann Nutr Metab. 2002;46(6):243–248.
82. Mihrshahi S, Battistutta D, Magarey A, et al.
Determinants of rapid weight gain during
infancy: baseline results from the NOURISH
randomised controlled trial. BMC Pediatr.
2011;11:99.
83. Harvey NC, Poole JR, Javaid MK, et al. Paren-
tal determinants of neonatal body composition.
J Clin Endocrinol Metab. 2007;92(2):523–526.
84. Singhal A, Lanigan J. Breastfeeding, early
growth and later obesity. Obesity Rev. 2007;
8(suppl 1):51–54.
85. Rzehak P, Grote V, Lattka E, et al. Associa-
tions of IGF-1 gene variants and milk protein
intake with IGF-I concentrations in infants
at age 6 months—results from a random-
ized clinical trial. Growth Horm IGF Res.
2013;23(5):149–158.
86. Michaelsen KF, Greer FR. Protein needs early
in life and long-term health. Am J Clin Nutr.
2014;99(3):718S–22S.
87. Shahkhalili Y, Mace K, Moulin J, et al. The
fat:carbohydrate energy ratio of the wean-
ing diet programs later susceptibility to obe-
sity in male Sprague Dawley rats. J Nutr.
2011;141(1):81–86.
88. Singhal A, Lucas A. Early origins of cardiovas-
cular disease: is there a unifying hypothesis?
Lancet. 2004;363(9421):1642–1645.
89. Oddy WH. Infant feeding and obesity risk in
the child. Breastfeed Rev. 2012;20(2):7–12.
90. Collado MC, Isolauri E, Laitinen K, et al. Dis-
tinct composition of gut microbiota during
pregnancy in overweight and normal-weight
women. Am J Clin Nutr. 2008;88(4):894–899.
91. Prince AL, Antony KM, Ma J, et al. The micro-
biome and development: a mother’s perspec-
tive. Semin Reprod Med. 2014;32(1):14–22.
92. Kalliomaki M, Collado MC, Salminen S, et al.
Early differences in fecal microbiota composi-
tion in children may predict overweight. Am J
Clin Nutr. 2008;87(3):534–538.
93. Saavedra JM, Dattilo AM. Early development
of intestinal microbiota: implications for
future health. Gastroenterol Clin North Am.
2012;41(4):717–731.
94. Nauta AJ, Ben Amor K, Knol J, et al. Rele-
vance of pre- and postnatal nutrition to devel-
opment and interplay between the microbiota
and metabolic and immune systems. Am J
Clin Nutr. 2013;98(2):586S–593S.
48
Pharmacokinetics and
Pharmacodynamics
LARA S. LEMON, RAMAN VENKATARAMANAN AND STEVE N. CARITIS
KEY POINTS
• P hysiological changes associated with pregnancy have a
profound effect on how medications are handled in pregnant
women.
• Most medications taken by pregnant women reach the fetus
to varying degrees. The fetus is in part protected by placental
efflux transporters and placental drug-metabolising enzymes.
The fetus can also metabolise some drugs but to a minor
degree.
• Drugs reaching the fetus can be teratogenic or toxic, but the
vast majority of pharmacologic agents are neither teratogenic
nor toxic. Most drugs are inadequately studied in pregnancy,
limiting the usefulness of the US Food and Drug Administra-
tion classification of medications.
Current Status of Pharmacotherapy in
Pregnancy
Medication use in pregnancy is on the rise. A majority of pregnant
women in the United States use at least one over-the-counter or
prescription medication during pregnancy; the average number
of medications taken by pregnant women in a survey in 2006 to
20081 was four. Over the past decade, women have become older
and heavier at the time of conception.2 Consequently, the num-
ber of women entering pregnancy already receiving treatment for
chronic hypertension, diabetes, asthma and other chronic condi-
tions has also increased. Pregnant women may also receive medi-
cations for a gestation-related condition such as preeclampsia,
gestational diabetes or preterm labour. Substance use disorder also
leads to additional drug exposure in certain pregnant women.
Medication use in pregnancy is innately complicated because
the medication may affect not only the mother but may also affect
fetal organogenesis, fetal organ function and maturation. Thus
fetal risk and benefit must also be considered when prescribing
medications for a pregnant woman. This is particularly impor-
tant during the first trimester, during fetal organogenesis. During
this sensitive period, about 50% of women are exposed to at least
one medication.1 Prescription medications commonly used in the
first trimester include progestins; antibiotics; and medications for
hypertension, asthma, diabetes, nausea and vomiting of pregnancy
(i.e., promethazine, ondansetron) and thyroid disorders, among
others.3 Medications taken during pregnancy after the period of
organogenesis may also impact fetal organ function or maturation.
Common medications include labetalol for hypertensive disor-
ders, oral hypoglycaemic agents such as glyburide and metformin
for gestational diabetes, magnesium sulphate, betamethasone and
nifedipine and indomethacin for preterm labour.
Most medications used by pregnant women are used ‘off label’,
meaning that the medication is Food and Drug Administration
(FDA) approved but not specifically for pregnant women. In fact,
very few drugs commonly used in pregnancy are FDA approved
for use in this population. Pharmacokinetic and safety informa-
tion is required for all drugs before FDA approval, but this infor-
mation is virtually nonexistent for medications used in pregnancy.
Of the 172 newly approved drugs from 2000 to 2010, there were
insufficient data to assess teratogenicity in 97.7% of the drugs,
and 73.3% of the newly approved drugs provided no data regard-
ing the medication use in pregnancy.4 Despite this dearth of data,
pregnant women are being prescribed medications not approved
by the FDA for use in pregnancy. In fact, from 1996 to 2000,
only 2.4% of women received a prescription medication that was
rated Category A (i.e., Controlled studies in women fail to dem-
onstrate a risk to the fetus in the first trimester. Risk of fetal harm
appears remote.); nearly 50% were rated Category C-X.5 Extend-
ing from this problem, clinicians have little to no pharmacokinetic
and pharmacodynamic data by which to adapt dosing regimens
specific to pregnancy. It is common practice to apply pharmacoki-
netic data obtained from men and nonpregnant women to deter-
mine dosing during pregnancy despite the dramatic physiologic
changes that occur during pregnancy that might affect dosing.
In June 2015, the FDA released new labelling requirements to
be used in pregnancy. These new requirements are titled: the Pre-
scription & Lactation Labeling Rule (PLLR). This labelling replaces
the standard Category Rankings A, B, C, D and X. The prior cat-
egories were deemed oversimplistic and were often inappropriately
interpreted as a grading system. The newly implemented PLLR is
composed of three sections: (8.1) Pregnancy, (8.2) Lactation, (8.3)
Females and Males of Reproductive Potential. The new ‘Females
and Males of Reproductive Potential’ section provides information
relating to contraception, pregnancy testing and infertility.
The ‘Pregnancy’ and ‘Lactation’ sections each contain a Risk Sum-
mary, Clinical Considerations and Data. Taken together, this infor-
mation is provided to assist clinicians in making an informed decision
about medication use in pregnancy and provides more information
581
582 SE C T I O N 9     Interface of Maternal, Fetal and Neonatal Medicine
on both the risks and benefits than previous labelling. The new rule
also mandates that if a pregnancy exposure registry exists, it must be
listed in the ‘Pregnancy’ section. Such registries facilitate epidemio-
logic studies of medication use in this vulnerable population.
Several factors contribute to the paucity of pharmacokinetic
and pharmacodynamic data in pregnancy. A major barrier is the
enormous liability concerns that discourage pharmaceutical com-
panies from studying pregnant women. When studying medica-
tions in pregnancy, the risk posed to both the mother and the fetus
must be considered. This additional liability is often enough to
dissuade pharmaceutical companies from performing such stud-
ies. The relatively small market for many pregnancy medications
is another factor discouraging involvement of the pharmaceutical
industry. Pregnant women account for only 4% of the female US
population, according to the Centers for Disease Control and Pre-
vention (CDC), and pregnancy lasts only 40 weeks. Therefore the
potential revenue to be gained by these companies for studying
medications in this population is relatively small. Big pharma is
also aware that women with chronic conditions frequently con-
tinue their medication regimens throughout pregnancy regardless
of the lack of pregnancy-specific pharmacological data. There is
little incentive for pharmaceutical companies to study drugs dur-
ing pregnancy and assume potential litigation risk because the
pregnant woman’s care provider often continues to provide the
medication if the clinical circumstances demand that the medica-
tion be used despite a lack of precise pharmacologic information.
Further complicating research in this population is the need for
long-term follow-up for childhood outcomes. To fully quantify
risk for outcomes such as attention deficit hyperactive disorder,
follow-up must be adequate. In addition to long lengths of follow-
up, large sample sizes are required to document associations with
rare birth defects. For example, congenital heart defects (the most
common birth defect diagnosed in the United States) occur at a
rate of about 1% with an estimated 40,000 affected births each
year in the United States,6,7 according to the CDC. To demon-
strate an increased risk resulting from medication exposure, inves-
tigators may need thousands of exposed pregnant women.  it is 100% bioavailable. This is also true for most drugs that are
Pregnancy Altered Drug Behaviour:
Pharmacokinetics
Pharmacokinetics is the study of what the body does to a drug;
this includes absorption, distribution, metabolism and elimina-
tion (ADME). Each aspect of pharmacokinetics can influence the
exposure of the patient to the drug. In practice, the area under the
plasma concentration × time curve (AUC) or the trough concen-
trations are commonly used as surrogate markers to measure drug
exposure (Fig. 48.1).
Throughout pregnancy, extraordinary anatomic and physi-
ologic changes occur in the mother. These adaptations impact
most maternal organ systems and collectively produce an environ-
ment that optimises fetal growth and development. These changes
dramatically affect both the pharmacokinetics and pharmacody-
namics of a drug. The physiologic adaptations occurring during
pregnancy are generally gradual but may change from trimester to
trimester. For example, blood pressure decreases early in gestation
(lowest point at 20–24 weeks) but typically returns to normal lev-
els by delivery. Others changes persist throughout pregnancy, such
as the decrease in plasma proteins.8
We will now describe each component of pharmacokinet-
ics (ADME) and discuss how pregnancy can alter each of these
Absorption
ADME
Metabolism Distribution
Excretion
• Fig. 48.1  Pharmacokinetic absorption, distribution, metabolism and
elimination (ADME) components.
processes. This list is not complete but aims to give readers a per-
spective on how influential these physiologic changes can be on
drug exposure to mother and the fetus.
Absorption
Absorption is defined as the movement of drug into the systemic
circulation from the site of administration. Bioavailability is the
per cent of the administered drug that reaches the systemic circu-
lation in intact form. When a drug is administered intravenously,
administered intramuscularly, subcutaneously and by inhala-
tion. However, when a drug is administered orally, intraperito-
neally, dermally or rectally, the drug is often not fully absorbed
or bioavailable. Orally administered medications have the great-
est variability in absorption and bioavailability because they are
affected by multiple factors such as gastric acidity, gastric transit
time, permeability from the gastrointestinal tract, involvement of
uptake and efflux transporters, metabolism by intestinal enzymes
and hepatic enzymes after entering the portal circulation. Whereas
metabolism occurs primarily in the liver, enzymes in extrahepatic
tissues such as in the gastrointestinal tract, lungs, the gut wall
and gut flora can also contribute to metabolism of certain drugs.9
Metabolism is influenced by the motility of the gut, enzymatic
activity in the gut and the impact of transporters.
This ‘first-pass’ effect dramatically reduces the amount of drug that
reaches the systemic circulation. Therefore the term ‘bioavailability’
is commonly used to describe the extent to which a drug is systemi-
cally available.10 Drugs with low bioavailability require a higher oral
dose to reach plasma concentrations equivalent to that observed after
intravenous administration. Variations in oral bioavailability of drugs
have direct clinical consequences. Drugs with low bioavailability typi-
cally will have large intersubject variation in drug exposure that may
translate into variable clinical effects and potentially side effects.
Numerous maternal changes in pregnancy impact absorption
of oral medications. In a nonpregnant woman, gastric acidity is
 CHAPTER 48  Pharmacokinetics and Pharmacodynamics
between a pH of 1 and 3. Gastric acidity is reduced in pregnancy,
resulting from both decreased production of gastric acid along with
increased mucous secretion. Gastric acidity influences ionisation of
drugs and thereby absorption. Both weak acids (e.g., aspirin) and
Per cent increase above nonpregnant
weak bases (e.g., caffeine) diffuse more easily in unionized form; the
pKa of a drug is the pH at which a drug will be 50% ionized and
50% unionized. Weak acids are more likely to be unionized when
the pH of the stomach is lower than pKa of the drug; conversely
for weak bases the pH must be higher (i.e., more basic) to remain
in unionized form. When the pH is increased in pregnancy, weak
acids such as aspirin will have decreased absorption. Conversely,
these changes in acidity can increase the absorption of weak bases
such as caffeine because they are more likely to be unionized in this
environment and more readily cross the membrane.
Pregnant women also commonly experience nausea and vomit-
ing in pregnancy, particularly in the first trimester. More than 80%
of pregnant women develop this condition.11 Nausea and vomit-
ing impact drug absorption through many ways. First, nausea and
vomiting can limit a woman’s ability to ingest oral medications.
In severe cases, known as hyperemesis gravidarum, women may
require intravenous treatment.11 In addition, a drug’s absorption
may vary according to whether it is administered during the fed
or fasted state. Nausea and vomiting can impact the woman’s diet,
which ultimately affects drug absorption. Medications typically
used to treat these conditions, such as antacids, act by decreas-
ing gastric acidity, again ultimately affecting a drug’s absorption.
Beyond that, antacids are also known to adsorb some drugs and
impact their absorption.
In regards to first-pass metabolism, pregnancy can either
increase or decrease the activity of drug-metabolising enzymes
located in the gut. Contact of the drug with these enzymes is
also increased because progesterone, a smooth muscle relaxant,
decreases gut transit time by an estimated 30% to 50% in preg-
nancy.8 This increased time for interaction allows for more gut
metabolism to occur. Finally, portal venous flow is increased dur-
ing pregnancy,12 which may facilitate absorption13 and increase
drug delivery to the liver.  (by 40%–50%), plasma volume (by 40%–50%) and red blood
Distribution
Distribution of a drug is defined as the reversible transfer of drug
from one location to another within the body after it reaches the
systemic circulation. The apparent volume of distribution (Vd)
is a theoretical volume into which a drug is distributed at a con-
centration similar to the plasma concentration. The Vd relates the
plasma concentration and the amount of drug in the body at the
corresponding time point. The Vd is used to measure the extent
of a drug’s distribution within the body. A drug with a low Vd is
predominantly within the vascular system. A drug with a large Vd
is predominantly in the extravascular space and is highly bound
to the tissues.
The distribution of a drug is a function of both the innate prop-
erties of the drug and certain physiologic and pathologic states of
pregnancy. The distribution of a drug is primarily determined by
four physical properties of the drug in addition to blood perfusion
of the tissue: (i) plasma protein binding, (ii) lipid solubility, (iii)
vascular permeability and (iv) tissue binding. Plasma protein bind-
ing defines the extent to which the drug is protein bound in the
plasma. The lower the protein binding, the greater the drug distri-
bution outside the vascular compartment. Similarly, an increase in
plasma protein binding leads to a smaller Vd because most drug
will remain within the intravascular space. A drug that does not
583
100
Plasma volume
Blood volume
90
RBC mass
80
70
Delivery
60
50
40
30
20
10
0 4 8 12 16 20 24 28 32 36 40 42
Weeks of pregnancy
• Fig. 48.2  Plasma volume, blood volume and red blood cell (RBC) mass
changes that occur in pregnancy by gestational week. (With permission
from Gabbe SG, Niebyl JR, et al. Maternal physiology. In: Obstetrics: Nor-
mal and Problem Pregnancies. 7th ed. Philadelphia: Elsevier; 2016:38–63.)
bind to any proteins in the body will have a Vd similar to that of
total body water. In women, total body water accounts for an esti-
mated 50% of their weight compared with 60% in men and 70%
in infants. Lipid solubility also affects drug distribution; drugs that
have high lipid solubility (i.e., are nonpolar) typically have greater
distribution and Vd because they can partition into other tissues
such as fat. If a drug is highly bound to the tissues, it will result
in a very high Vd. When a drug has a large VD, this can cause an
apparent plasma ‘dilutional effect’; more of the drug may reach
the target site or tissue and lead to a lower plasma concentrations.
Pregnant women experience an increase in total blood volume
cell volume (by 10%–15%). These changes begin around 6 to 8
weeks in gestation with their most dramatic effects occurring at 32
weeks and then typically return to prepregnancy values by 6 weeks
postpartum8 (Fig. 48.2).
In addition to these changes in blood volume, extracellular
fluid and total body water are also increased in pregnancy by 6.5
to 8.5 L.14 As these volumes are increasing, total fat also rises
as women gain weight in pregnancy with most women gaining
beyond the recommendations of the Institute of Medicine.2,15
Beyond these volume and fat changes, pregnancy introduces an
entirely new compartment available for drug distribution: the
fetus and placenta. All of these pregnancy changes may potentially
increase the apparent Vd for drugs.
Protein binding is also profoundly impacted by pregnancy. The
primary protein involved in drug binding is albumin. Albumin
is decreased by an estimated 13% by the end of a normal preg-
nancy.13,16 With lower concentrations of plasma proteins, less
drug is bound, resulting in an increased concentration of free drug
for high clearance drugs. A greater amount of free drug impacts
drug distribution as well as elimination. This is a particularly
important consideration for drugs such as phenytoin which are
highly protein bound and active in their free form. When changes
in protein levels and binding are not considered, pregnant women
may be exposed to drastically higher levels of the medication as
gestation progresses. This increased exposure can result in both an
584 SE C T I O N 9     Interface of Maternal, Fetal and Neonatal Medicine

CYP1A2

CYP2A6

CYP2B6
CYP3A4/5

CYP2C8

CYP2C9 CYP2J2

CYP2E1

CYP2C19
CYP2D6
• Fig. 48.3  Contribution of various hepatic CYP P450 enzymes to drug metabolism. (Adapted from
Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression,
enzyme activities, and impact of genetic variation. Pharmacol Ther 138:103–141, 2013.)

exaggerated intended effect and potential side effects and toxici-
ties. Many drugs, such as midazolam, digoxin and phenytoin, are
affected by these alterations in albumin.8,17
Finally, alterations in regional blood occurring in pregnancy
affect distribution of drugs. Pregnant women have 30% to 50%
increased cardiac output that impacts regional distribution
throughout the body.18 Blood flow to the uterus is increased more
than 10-fold (from 5 0mL/min prepregnancy to 500 mL/min at
term). Blood flow is also increased to the skin, breasts and kidneys.
These can impact drug distribution.  affected by pregnancy and its associated hormones.14,23 DMEs
Metabolism
Metabolism of a drug is the biochemical modification of a chemi-
cal through enzymatic systems. In general, metabolism converts
a drug into an inactive and readily excretable form. However, for
prodrugs (which are less common) metabolism is necessary to
convert the drug from an inactive to an active form.13
The enzymes responsible for metabolism are typically divided
into two groups: phase I enzymes and phase II enzymes. The
majority of drugs will go through at least phase I metabolism.
Phase I enzymes, cytochrome P450 (CYP) enzymes and flavin
mono-oxygenases, are primarily located in the liver but are also
present in smaller amounts in the gut, kidney and placenta. These
substrate specific drug-metabolising enzymes (DMEs) act through
oxidation, reduction and hydrolysis.8 Phase I enzymes that metab-
olise drugs fall into three major families: CYP1, CYP2 and CYP3.
Of the vast number of DMEs and 38 drug-metabolising isoen-
zymes of CYP450,19 only a small proportion is responsible for the
majority of drug metabolism. Many drugs on the market today
are metabolised by CYP1A2, CYP2C9, CYP2D6 and CYP3A4/5.
Together hepatic CYP450 enzymes account for greater than 80%
of all hepatic enzyme activity20 (Fig. 48.3).
Phase II DMEs are also located primarily in the liver and act by
conjugating the drug substrate into more polar compounds with
sulphate, glucuronic acid, glutathione or glycine. This process is
much less common than phase I metabolism. Phase II DMEs
include UDP glucosyltransferases (UGTs), glutathione transfer-
ases (GSTs), sulfotransferases (SULTs) and N-acetyltransferases
(NATs).
Activity and expression of DMEs varies by race,20 age, gen-
der and genetics.21,22 Activity is further influenced by concur-
rent medications taken by the patient if they share a DME. The
activity of these enzymes is also dramatically and differentially
that have increased activity during pregnancy include CYP3A,
CYP2D6, CYP2C9, CYP2E1 and UGT1A1. Conversely, activity
of CYP1A2, CYP2C19, NAT2 and CBR1 is decreased in preg-
nancy13,14,23 (Table 48.1).
The effect of pregnancy on drug metabolism extends beyond
those related to DMEs. There is also a delay in gut transit time
(as mentioned in the Absorption section), which allows for more
interaction between xenobiotics and gut metabolic enzymes. In
addition to the liver and gut, both placental and fetal hepatic
enzymes can also play a role in drug metabolism. Finally, oestro-
gen and progesterone, two pregnancy hormones, individually and
in combination have major effects on DMEs.24-26
Any increase in the metabolism of a drug in pregnancy results
in less exposure to the parent compound but increases exposure
to the metabolite. Conversely, any reduction in metabolism may
increase exposure to the parent drug but reduce exposure to the drug
metabolite. These changes with a fixed dosage can translate to sub-
or supratherapeutic drug concentrations. A well-documented com-
pound demonstrating this effect is caffeine. Because of a decrease in
CYP1A2 activity throughout pregnancy, the DME responsible for
the breakdown of caffeine, women experience a 50% reduction in
caffeine metabolism in pregnancy. This directly translates to increased
exposure and a half-life increasing from 3.4 to 8.3 hours.23,27 Refer-
ring to phase II DMEs, the anticonvulsant lamotrigine is metabo-
lised primarily by UGT1A4, which has markedly increased activity
 CHAPTER 48  Pharmacokinetics and Pharmacodynamics 585

TABLE Altered Enzymatic Activity Induced by Clinically, increases in elimination allow the woman’s body
48.1 Pregnancy to eliminate both the parent drug and metabolite more quickly.
This increase in clearance often results in less drug exposure. Con-
Pregnancy-Induced Potential Substrates in versely, decreasing elimination leads to accumulation of the drug.
Enzyme Change Obstetrics A prime example of the impact of elimination changes is seen with
CYP3A4 Increased Glyburide, nifedipine and
the administration of lithium in pregnancy. This drug is indicated
indinavir for treatment of bipolar disorder. Because of the dramatic increase
in glomerular filtration rate, the clearance of this renally elimi-
CYP2D6 Increased Metoprolol, dextromethorphan, nated drug is doubled by the third trimester.17,31 Subsequently,
paroxetine, duloxetine, the dose of lithium must be increased, often significantly, by the
fluoxetine and citalopram end of pregnancy to maintain adequate therapeutic levels.8 
CYP2C9 Increased Glyburide, NSAIDs, phenytoin
and fluoxetine Pharmacodynamics
CYP2C19 Decreased Glyburide, citalopram, diazepam,
omeprazole, pantoprazole Whereas pharmacokinetics is the study of what the body does
and propranolol to a drug, pharmacodynamics is the study of what a drug does
to the body. Pharmacodynamics includes both the biochemical
CYP1A2 Decreased Theophylline, clozapine, olan- and physiologic effects of the drug and its mechanism of action.
zapine, ondansetron and Because of the changes in pharmacokinetics described earlier,
cyclobenzaprine
changes in the pharmacodynamic effect often follows.
UGT1A4 Increased Lamotrigine To establish proper dosing of a drug, pharmacodynamics stud-
ies are required to relate the plasma or tissue concentration to the
UGT1A1/9 Increased Acetaminophen
clinical effect. Without such studies, the dose may be excessive and
NAT2 Decreased Caffeine cause side effects, or conversely, the dose may be inadequate and
lack effect. Often when the dose is insufficient, it is mistaken as a
NSAID, Nonsteroidal antiinflammatory drug.
drug failure or less than optimal response. In pregnancy, women
With permission from Feghali M, Venkataramanan R, Caritis S. Pharmacokinetics of drugs in
pregnancy. Semin Perinatol 39(7):512–519, 2015.
often have reduced exposure to medications (lower AUCs) result-
ing from higher clearance (see Elimination). When this occurs,
   the reduced exposure may lead to an inadequate effect. Examples
of this phenomenon include oseltamivir, metformin and gly-
in pregnancy.28,29 Therefore to maintain therapeutic levels and sei- buride. Conversely, a higher exposure (AUC) to certain medica-
zure control, women receiving this treatment for epilepsy may require tions because of decreased metabolism or elimination will result in
increased doses up to 250% as pregnancy progresses.28,29 excessive high plasma concentrations and may be associated with
Because pregnancy-related metabolic changes impact many unwanted side effects. Ideally, exposure should be equated with
drugs (e.g., metoprolol, methadone, sertraline, nifedipine, glipi- desired endpoint through a pharmacodynamics study. This is the
zide), changes in dosing may be needed for a large number of only way to appropriately adjust the dose.
medications. It is crucial that physicians treating pregnant women An example of titrating to effect in practice is the dosing of
address these variations in metabolism and adjust doses accord- insulin in women with diabetes. Doses are increased to lower blood
ingly. This is a difficult task when the drug is subject to multiple sugars to the accepted norms. For many medications, dosing is not
metabolic pathways. In some cases, a medication will be metab- adjusted, but rather, a fixed dose is administered without regard
olised by two enzymes with opposite effects on the activity of to maternal characteristics or response. Generally, this approach is
DMEs in pregnancy. In these circumstances, it is important to be used when the target response is not easily measured except over
aware of the changes occurring in metabolism and to monitor the