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EMERY AND RIMOIN’S
PRINCIPLES AND PRACTICE
OF MEDICAL GENETICS AND
GENOMICS
This page intentionally left blank
     
EMERY AND RIMOIN’S
PRINCIPLES AND PRACTICE
OF MEDICAL GENETICS AND
GENOMICS
Perinatal and Reproductive Genetics
Seventh Edition

Edited by
Reed E. Pyeritz
Perelman School of Medicine at the University of Pennsylvania,
Philadelphia, PA, United States

Bruce R. Korf
University of Alabama at Birmingham, Birmingham, AL, United States

Wayne W. Grody
UCLA School of Medicine, Los Angeles, CA, United States
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 CONTENTS

List of Contributors ix 3.3 Chorionic Villus Sampling 43

Preface to the Seventh Edition of Emery and Rimoin’s 3.4 Fetal Blood Sampling 49
Principles and Practice of Medical Genetics and 3.5 Fetal Skin and Tissue Biopsy
Genomics xi Procedures 50
Preface to Perinatal and Reproductive Genetics xiii Acknowledgments 51
References 51
Introduction to Perinatal Disorders and
1  4 Neonatal Screening 57
Reproductive Genetics 1 Inderneel Sahai and Richard W. Erbe
Susan J. Gross 4.1 Introduction 57
1.1 Introduction 1 4.2 Historical Aspects 58
1.2 Imaging During Pregnancy—A First 4.3 Components of Screening Programs 59
Look 1 4.4 Potential Problems in Newborn
1.3 Prenatal Diagnostics—Confirming Screening 65
Genetic Disorders 3 4.5 Disorders and Conditions Detected by
1.4 Prenatal Screening for Genetic Newborn Blood Screening: Inborn Errors
Disorders—Aneuploidy and Single of Metabolism 67
Gene 4 4.6 Other Congenital Disorders and
1.5 The End of the Beginning and What Lies Conditions Detected by Newborn Blood
Ahead 5 Screening 77
1.6 Conclusion 6 4.7 Issues and Concerns in Screening 79
References 6 References 80
Further Reading 8 Further Reading 86
Web Resources 86
2 Prenatal Screening for Neural Tube Defects and
Aneuploidy 9 5 Hypogonadotropic and Hypergonadotropic
Robert G. Best Hypogonadism in Females: Disorders of
2.1 Introduction 9 Reproductive Ducts 87
2.2 Prenatal Screening for Birth Defects 10 Joe Leigh Simpson
2.3 Risk Determination and Thresholds 19 5.1 Kallmann Syndrome and Idiopathic
2.4 Modalities of Testing for NTD and Down (Congenital) Hypogonadotropic
Syndrome 21 Hypogonadism 87
2.5 Follow-up to Positive Screens—NTD 22 5.2 Hypergonadotropic Hypogonadism:
2.6 Maintaining and Monitoring Screening Historical Overview and Evolution of
Performance 24 Scientific Approach 91
2.7 Keeping Screening in Perspective 25 5.3 Mechanism of Action for Genes Causing
2.8 Summary 26 Hypergonadotropic Hypogonadism 95
References 26 5.4 Related Gynecological Disorders Causing
Infertility 102
3 Techniques for Prenatal Diagnosis 35
5.5 Structural Anomalies of the Uterus and
Lee P. Shulman, Jeffrey S. Dungan and Andrew F. Wagner
Vagina 106
3.1 Introduction 35
References 110
3.2 Amniocentesis 36

v
vi CONTENTS

6 
Genetics of Male Infertility 121 Acknowledgments 228
Csilla Krausz, Viktoria Rosta, Ronald S. Swerdloff and References 228
Christina Wang 10 Noninvasive Prenatal Testing and Noninvasive
6.1 Male Infertility—Introduction 121 Prenatal Screening 235
6.2 Chromosome Anomalies 123 Charles M. Strom
6.3 Gene Defects Involved in Endocrine 10.1 Precision in Screening Tests 235
Forms of Infertility 128 10.2 Fetal Fraction 239
6.4 Monogenic Defects of Male Infertility 135 10.3 Sex Chromosome Aneuploidies and
6.5 Syndromic Monogenic Defects 138 Gender Determination 240
6.6 Conclusion 139 10.4 Segmental Aneuploidies 240
References 140 10.5 Triploidies and Haploidies 241
10.6 Mendelian Disorders in NIPS 241
7 The Genetics of Disorders Affecting the Premature 10.7 Gender Determination 241
Newborn 149 10.8 Multiple Pregnancies and Vanishing
Aaron R. Prosnitz, Jeffrey R. Gruen and Vineet Bhandari Twins 241
7.1 Introduction 149 10.9 Confined Placental Mosaicism 242
7.2 Respiratory Distress Syndrome 150 10.10 Maternal Factors 242
7.3 Bronchopulmonary Dysplasia 157 10.11 Inappropriate Use of NIPS 243
7.4 Patent Ductus Arteriosus 162 10.12 NIPT Paternity Testing 244
7.5 Intraventricular Hemorrhage 164 10.13 Noninvasive Whole Genome Fetal
7.6 Retinopathy of Prematurity 168 Sequencing 245
7.7 Necrotizing Enterocolitis 171 10.14 Conclusion 245
References 175 References 245
8 Fetal Loss 187 Further Reading 248
Rhona Schreck, John Paul Govindavari and John Williams III 11 Preimplantation Genetic Testing 249
8.1 Background 187 Svetlana A. Yatsenko and Aleksandar Rajkovic
8.2 Definition of Terms 187 11.1 Introduction 249
8.3 Early Pregnancy Loss 188 11.2 Milestones in PGT 250
8.4 Late Pregnancy Loss 202 11.3 Indications for Preimplantation Genetic
8.5 Evaluation and Management of Recurrent Testing 251
Abortion 204 11.4 Technical Approaches 253
8.6 Conclusions 205 11.5 Testing and Analysis of Embryonic
References 205 Nuclear DNA 254
Further Reading 215 11.6 Embryo Testing for Monogenic
Conditions (PGT-M) 254
9 Preeclampsia 217
11.7 PGT-M for Mitochondrial
Anthony R. Gregg
Conditions 256
9.1 The Preeclampsia Phenotype 217
11.8 Preimplantation Genetic Testing
9.2 Preeclampsia Is a Quantitative Trait
for Structural Chromosome
Disorder 218
Rearrangements 256
9.3 Preeclampsia and the Placenta 219
11.9 Preimplantation Genetic Testing for
9.4 Preeclampsia Biomarkers in Clinical
Aneuploidy 258
Use 224
11.10 Interpretation of PGT Results and
9.5 Preeclampsia Management and Future
Clinical Dilemmas 259
Health 225
11.11 PGT-A: Mosaicism 262
9.6 Genetic Basis of Preeclampsia 226
11.12 Advantages and Limitations of PGT 262
9.7 Preeclampsia and Animal Models 228
 CONTENTS vii

11.13 Prenatal Follow-Up and Confirmatory 12.6 Introduction of Expanded Carrier

Testing 263 Screening into Clinical Practice 285
11.14 Genetic Counseling 264 12.7 Process of Carrier Screening 286
11.15 Future Technological Advances in ART 12.8 Pretest Counseling 286
and PGT 265 12.9 Interpretation of Molecular
11.16 Regulatory Policies, Ethical Findings 287
Considerations, and Challenges in 12.10 Reproductive Options for Carrier
PGT 267 Couples Identified During
References 268 Pregnancy 290
12.11 Reproductive Options for
12 Expanded Carrier Screening 281
Carrier Couples Identified Before
Ronald J. Wapner, Katherine Johansen Taber, Gabriel Lazarin
Pregnancy 290
and James D. Goldberg
12.12 Posttest Counseling of Pregnant Carrier
12.1 Introduction 281
Couples 290
12.2 History of Reproductive Carrier
12.13 Preimplantation Genetic Testing for
Screening 281
Carrier Couples 291
12.3 Expanding Carrier Screening: One Gene
12.14 Use of PGT-M for Identifying Potential
at a Time 283
HLA Donor Embryos for Affected
12.4 Introduction of Expanded Carrier
Siblings 292
Screening Panels 284
12.15 Conclusions 292
12.5 Changes in Technology from Genotyping
References 292
to Sequencing Drive Carrier Screening
Performance Improvements 285 Index 295
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 LIST OF CONTRIBUTORS
Robert G. Best
Department of Biomedical Sciences, University
of South Carolina School of Medicine Greenville,
Greenville, SC, United States
Vineet Bhandari
Department of Pediatrics, Cooper Medical School of
Rowan University, The Children’s Regional Hospital at
Cooper, Camden, NJ, United States
Jeffrey S. Dungan
Department of Obstetrics and Gynecology, Division
of Clinical Genetics, Feinberg School of Medicine of
Northwestern University, Chicago, IL, United States
Richard W. Erbe
Departments of Pediatrics and Medicine, University
at Buffalo, Division of Genetics, Oishei Children’s
Hospital, Buffalo, NY, United States
James D. Goldberg
Department of Obstetrics and Gynecology, Vagelos
College of Physicians and Surgeons, Columbia
University Irving Medical Center, New York, NY,
United States; Myriad Women’s Health, South San
Francisco, CA, United States
John Paul Govindavari
Division of Molecular Pathology, Medical Genetics &
Pathology and Laboratory Medicine, Cedars-Sinai
Medical Center, Los Angeles, CA, United States
Anthony R. Gregg
Department of Obstetrics and Gynecology, PRISMA
Health, Columbia, SC, United States
Susan J. Gross
Department of Genetics and Genomic Sciences, Icahn
School of Medicine at Mount Sinai, New York, NY,
United States; Cradle Genomics, San Diego, CA,
United States
Jeffrey R. Gruen
Departments of Pediatrics, Genetics and Investigative
Medicine, Yale University School of Medicine, Yale
Child Health Research Center, New Haven, CT, United
States
Csilla Krausz
Department of Experimental and Clinical Biomedical
Sciences “Mario Serio”, University of Florence,
Florence, Italy
Gabriel Lazarin
Department of Obstetrics and Gynecology,
Vagelos College of Physicians and Surgeons,
Columbia University Irving Medical Center,
New York, NY, United States; Myriad Women’s Health,
South San Francisco, CA, United States
Aaron R. Prosnitz
Sanger Heart and Vascular Institute, Levine Children’s
Hospital at Atrium Health, Charlotte, NC, United States
Aleksandar Rajkovic
Department of Pathology, University of California
San Francisco, San Francisco, CA, United States;
Department of Obstetrics, Gynecology and
Reproductive Sciences, University of California San
Francisco, San Francisco, CA, United States;
Institute of Human Genetics, University of California
San Francisco, San Francisco, CA, United States
Viktoria Rosta
Department of Experimental and Clinical Biomedical
Sciences “Mario Serio”, University of Florence,
Florence, Italy
Inderneel Sahai
Department of Pediatrics, University of Massachusetts
Medical School, New England Newborn Screening
Program, Worcester, MA, United States
ix
x LIST OF CONTRIBUTORS
Rhona Schreck
Division of Molecular Pathology, Medical Genetics &
Pathology and Laboratory Medicine, Cedars-Sinai
Medical Center, Los Angeles, CA, United States
Lee P. Shulman
Department of Obstetrics and Gynecology, Division
of Clinical Genetics, Feinberg School of Medicine of
Northwestern University, Chicago, IL, United States
Joe Leigh Simpson
Departments of Human and Molecular Genetics and of
Obstetrics and Gynecology, Herbert Wertheim College
of Medicine, Florida International University Miami,
FL, United States
Charles M. Strom
UCLA School of Dentistry, Center for Head and Neck
Oncology, Los Angeles, CA, United States
Ronald S. Swerdloff
Division of Endocrinology, Department of Medicine,
Harbor-UCLA Medical Center and The Lundquist
Institute, Torrance, CA, United States
Katherine Johansen Taber
Department of Obstetrics and Gynecology, Vagelos
College of Physicians and Surgeons, Columbia
University Irving Medical Center, New York, NY,
United States; Myriad Women’s Health, South San
Francisco, CA, United States
Andrew F. Wagner
Department of Obstetrics and Gynecology, Division
of Clinical Genetics, Feinberg School of Medicine of
Northwestern University, Chicago, IL, United States
Christina Wang
Division of Endocrinology, Department of Medicine,
Harbor-UCLA Medical Center and The Lundquist
Institute, Torrance, CA, United States
Ronald J. Wapner
Department of Obstetrics and Gynecology, Vagelos
College of Physicians and Surgeons, Columbia
University Irving Medical Center, New York, NY,
United States
John Williams III
Division of Maternal-Fetal Medicine, Department
of Obstetrics and Gynecology, Cedars-Sinai Medical
Center, Los Angeles, CA, United States
Svetlana A. Yatsenko
Department of Pathology, University of Pittsburgh
School of Medicine, Pittsburgh, PA, United States;
Department of Obstetrics, Gynecology and
Reproductive Sciences, University of Pittsburgh School
of Medicine, Pittsburgh, PA, United States;
Magee-Womens Research Institute, Pittsburgh, PA,
United States; Department of Human Genetics,
Graduate School of Public Health, University of
Pittsburgh, Pittsburgh, PA, United States
 P R E FAC E TO T H E S E V E N T H
­E D I T I O N O F E M E R Y A N D
­R I M O I N ’ S P R I N C I P L E S A N D
PRACTICE OF MEDICAL
GENETICS AND GENOMICS
The first edition of Emery and Rimoin’s Principles and
Practice of Medical Genetics appeared in 1983. This was
several years prior to the start of the Human Genome
Project in the early days of molecular genetic testing,
a time when linkage analysis was often performed for
diagnostic purposes. Medical genetics was not yet a rec-
ognized medical specialty in the United States, or any-
where else in the world. Therapy was mostly limited to
a number of biochemical genetic conditions, and the
underlying pathophysiology of most genetic disorders
was unknown. The first edition was nevertheless pub-
lished in two volumes, reflecting the fact that genetics
was relevant to all areas of medical practice.
Thirty-five years later we are publishing the seventh
edition of Principles and Practice of Medical Genetics and
Genomics. Adding “genomics” to the title recognizes the
pivotal role of genomic approaches in medicine, with
the human genome sequence now in hand and exome/
genome-level diagnostic sequencing becoming increas-
ingly commonplace. Thousands of genetic disorders
have been matched with the underlying genes, often
illuminating pathophysiological mechanisms and in
some cases enabling targeted therapies. Genetic testing
is becoming increasingly incorporated into specialty
medical care, though applications of adequate family
history, genetic risk assessment, and pharmacogenetic
testing are only gradually being integrated into routine
medical practice. Sadly, this is the first edition of the
book to be produced without the guidance of one of the
founding coeditors, Dr. David Rimoin, who passed away
just as the previous edition went to press.
The seventh edition incorporates two major changes
from previous editions. The first is publication of the
text in 11 separate volumes. Over the years, the book
had grown from two to three massive volumes, until
the electronic version was introduced in the previous
edition. The decision to split the book into multiple
smaller volumes represents an attempt to divide the con-
tent into smaller, more accessible units. Most of these
are organized around a unifying theme, for the most
part based on specific body systems. This may make the
book more useful to specialists who are interested in the
application of medical genetics to their area but do not
wish to invest in a larger volume that covers all areas
of medicine. It also reflects our recognition that genetic
concepts and determinants now underpin all medical
specialties and subspecialties. The second change might
seem on the surface to be a regressive one in today’s
high-tech world—the publication of the 11 volumes
in print rather than strictly electronic form. However,
feedback from our readers, as well as the experience of
the editors, indicated that access to the web version via a
password-protected site was cumbersome, and printing
a smaller volume with two-page summaries was not use-
ful. We have therefore returned to a full print version,
although an eBook is available for those who prefer an
electronic version.
One might ask whether there is a need for a compre-
hensive text in an era of instantaneous Internet searches
for virtually any information, including authoritative
open sources such as Online Mendelian Inheritance in
Man and GeneReviews. We recognize the value of these
and other online resources, but believe that there is still
a place for the long-form prose approach of a textbook.
Here the authors have the opportunity to tell the story of
their area of medical genetics and genomics, including
in-depth background about pathophysiology, as well as
giving practical advice for medical practice. The willing-
ness of our authors to embrace this approach indicates
that there is still enthusiasm for a textbook on medical
genetics; we will appreciate feedback from our readers
as well.
xi
xii PREFACE TO THE SEVENTH EDITION OF EMERY AND RIMOIN’S PRINCIPLES

The realities of editing an 11-volume set have become
obvious to the three of us as editors. We are grateful to
our authors, many of whom have contributed to mul-
tiple past volumes, including some who have updated
their contributions from the first or second editions.
We are also indebted to staff from Elsevier, particu-
larly Peter Linsley and Pat Gonzalez, who have worked
patiently with us in the conception and production of
this large project. Finally, we thank our families, who
have indulged our occasional disappearances into writ-
ing and editing. As always, we look forward to feedback
from our readers, as this has played a critical role in
shaping the evolution of Principles and Practice of Med-
ical Genetics and Genomics in the face of the exponen-
tial changes that have occurred in the landscape of our
discipline.
 P R E FAC E TO P E R I N A T A L A N D
REPRODUCTIVE GENETICS
Mention the term “genetics” to most laypeople and
they will think first of “inheritance,” the transmission
of inborn traits from one generation to the next. In the
case of Homo sapiens, this process involves sexual repro-
duction via gametogenesis, fertilization, embryonic and
fetal development during gestation, followed by labor,
delivery, and the immediate newborn period. These
processes in aggregate comprise the perinatal period,
and the myriad ways in which any of these steps can
go wrong constitute the content of this volume. In that
sense, this volume represents the quintessential aspect
of genetics for many people.
This volume boasts state-of-the-art updates of key
chapters in previous editions dealing with prenatal
diagnosis, infertility, newborn screening, fetal loss, and
other critical topics. In addition, several new chapters
not present in the previous editions have been intro-
duced, reflecting the latest advances in molecular and
bioinformatic technology to enable such impressive
applications as noninvasive prenatal screening, preim-
plantation genetic testing, and highly expanded carrier
screening by next-generation DNA sequencing.
As with all such technological advances, ethical and
legal dilemmas often come to light, and the authors in
this volume do not shy away from discussion of those,
either. Some of the ethical/legal challenges are specific
to the particular techniques and their respective intel-
lectual property, while others are overarching across
the entire field of maternal–fetal medicine and genetics.
Included in that latter category are restrictions on access
to needed reproductive services, due either to inequities
in health insurance coverage for expensive procedures
or to politically motivated intrusions into reproductive
decision-making, such as legislative obstacles to preg-
nancy termination after specific (sometimes very early)
gestational ages or even for specific fetal diagnoses (such
as Down syndrome).
It is hoped that this volume will address the most
current needs of medical geneticists, genetic counsel-
ors, obstetricians, and all other healthcare profession-
als interested in this most fundamental area of clinical
genetics and patient care.
xiii
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Disorders and Reproductive
1Department
1.1 INTRODUCTION
There are multiple time points that one could point to
as signifying the “start” of the field of perinatal genetics.
Certainly, the concept of heritable disease appears early
in human history. The Talmud, an ancient legal text
compiled around 500 CE, rules against circumcising an
infant if his older brothers had died from uncontrolled
bleeding following their circumcisions—likely the first
description and management of an X-linked disorder,
specifically hemophilia (BT Yevamot 64b).
While clinical genetics as a focus area dedicated to
the management and understanding of heritable dis-
orders advanced considerably during the middle of
the last century, reproductive genetics remained hin-
dered by the very limited ability to evaluate the fetus
in any meaningful way. Fetal dysmorphology and even
the simplest metabolic or genetic tests were simply
not possible. Thus, the real breakthrough in the field
of reproductive genetics would have to wait for tech-
nological advances that would overcome these limita-
tions. This chapter will highlight key fetal imaging and
diagnostic and screening milestones that have brought
us to where we can now “see” what had been hidden
for millennia. Having achieved the ability to assess
the fetus, the future holds much promise but there
remain some important concerns that still need to be
addressed.
Emery and Rimoin’s Principles and Practice of Medical Genetics and Genomics. https://doi.org/10.1016/B978-0-12-815236-2.00001-1
Copyright © 2022 Elsevier Inc. All rights reserved.
1
Introduction to Perinatal
Genetics
Susan J. Gross1,2
of Genetics and Genomic Sciences, Icahn School of Medicine at
Mount Sinai, New York, NY, United States,
2Cradle Genomics, San Diego, CA, United States
1.2 IMAGING DURING
PREGNANCY—A FIRST LOOK
1.2.1 Radiography
While ultrasound is often the first technology that usu-
ally comes to mind when thinking of obstetrical imag-
ing, radiography in pregnancy was first reported in 1923
[1]. Abdominal X-rays to assess adequacy of a mater-
nal pelvis for delivery was first described in 1944 and,
despite concerns regarding teratogenic effects, remained
in use or under study until relatively recently [2]. This
modality can assess fetal osseous structures and there-
fore identify important birth defects, including single
gene disorders (e.g., skeletal dysplasias) as well as mul-
tifactorial anomalies (e.g., anencephaly). However, radi-
ography specifically for obstetrical reasons is usually
avoided due to concerns regarding potential teratoge-
nicity and limited clinical utility.
1.2.2 Ultrasound Imaging
There is no dispute among reproductive geneticists
that ultrasound was one of the breakout technologies
that changed the field forever. Based on the work of
Christian Doppler and other physicists in the 19th and
early 20th century, the first known medical applica-
tion was reported to the public in 1949 to detect gall-
stones, based on different echo signals from reflected
sound waves that were recorded on an oscilloscope
1
2 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
screen [3]. The next major technological step, and
what many consider the actual beginning of medical
ultrasound, begins with the report of 2D ultrasound in
the 1950s for breast anatomy and neck, although the
patient was immersed in water to overcome the artifi-
cial echoes that would otherwise be generated. Direct
skin “contact” 2D ultrasound arrived a few years later,
thanks to the Scottish Obstetrician Ian Donald and his
engineering colleague Tom Brown [4]. Reproductive
sonographers and geneticists will usually point to the
seminal paper by Donald, Brown, and gynecologist
John MacVicar [5] that described their findings related
to abdominal masses, which included not only ovar-
ian cancer but also the first ultrasound image of a fetal
head. While a sonographer with experience would be
able to make sense of these images, the resolution was
less than optimal. Furthermore, these images were gen-
erated over time and were “static” and were not actu-
ally captured in “real time.” Nevertheless, as described
by Dr. Stuart Campbell, a pioneer in the field in his
own right, the publication of this paper signaled that
“the starting gun had been fired and the ultrasound
race had begun” [3].
One cannot overstate the role of ultrasound in the
field of perinatal genetics. Below is a brief overview
as the technology has continued to advance from the
“snowstorm” images in the 1950s to current 3D (images
that add depth) and 4D (incorporates time, allowing for
assessment of movement) technologies.
Pregnancy Dating: Ultrasound dating, rather than
date of first menstrual period, has become the standard
of care for dating pregnancies. Precise dating is import-
ant for many reasons but is critical when trying to deter-
mine if a fetus is small for gestational age when working
up a potential genetic issue. Likewise, an erroneous ges-
tational age can result in false-positive or false-negative
fetal aneuploidy or neural tube defect screening test
results.
Fetal Dysmorphology: It is standard of care to offer
women routine fetal anatomy scanning during the first
half of pregnancy [6]. In many centers, this detailed
scan occurs between 18 and 20 weeks. However, as
the technology continues to improve, a detailed sono-
gram, including fetal echocardiogram, can be obtained
in the first trimester [7]. Abnormal fetal anatomy on
ultrasound exam remains one of the major reasons for
referral to centers with expertise in fetal medicine and
prenatal genetics.
Fetal Aneuploidy Screening: Standard screening for
fetal aneuploidy is still used as a frontline method in
many parts of the world and incorporates ultrasound
along with protein markers to determine risk for tri-
somy 21 and other chromosomal anomalies. Nuchal
translucency refers to a fluid-filled space normally seen
behind the fetal neck on ultrasound performed in the
first trimester of pregnancy. A measurement that is
enlarged relative to gestational age is associated with
Down syndrome, as well as other genetic disorders such
as Noonan syndrome [8] and skeletal dysplasias [9].
Some centers look for “soft markers” that are not consid-
ered structural anomalies, but do confer increased risk
for Down syndrome, such as increased renal pyelectasis
found on second trimester sonogram [10].
Ultrasound-Guided Diagnostic Procedures: Ultra-
sound was not initially used routinely to direct fetal
diagnostic procedures. Amniocentesis was available in
the 1960s, but the quality and availability of ultrasound
technology was still quite limited. Fetal injury from the
needle was a significant risk that was discussed with a
patient deciding whether to undergo a procedure. How-
ever, with the incorporation of ultrasound into prena-
tal care, use of this technology for needle guidance has
become standard of care. Perfumo and Jauniaux [11] in
their review point out the pivotal role of this technology
as “it is only the use of ultrasound-guided amniocentesis
in the 1980s that made it a very safe procedure during
the first half of pregnancy.” While amniocentesis in the
early days was possible without ultrasound guidance,
procedures such as chorionic villus sampling (CVS) of
the placenta or cordocentesis (also known as percuta-
neous umbilical blood sampling) would not have been
possible.
Counseling and Surgical Aid: 3D ultrasound that pro-
vides depth to the fetal image has provided geneticists
with a helpful tool when counseling patients for certain
fetal anomalies. For example, the surface rendering pro-
vides a clearer image of certain anomalies, especially
cleft lip and palate. In addition to defining the extent of
the finding for diagnostic purposes, having this more
recognizable image can help with patient counseling as
well as help the cleft lip and palate interdisciplinary care
team prepare [12].
1.2.3 MR Imaging
While CT imaging is sometimes used for maternal rea-
sons, due to increased risk for fetal radiation exposure,
 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
its use is limited prenatally. However, MRI can be an
important adjunct to prenatal ultrasound and has
demonstrated good sensitivity for fetal CNS malforma-
tions [13]. While neither ultrasound nor MRI is asso-
ciated with fetal risk, it is still recommended that this
technology be used judiciously, when the results could
provide medical benefit [14].
1.3 PRENATAL DIAGNOSTICS—
CONFIRMING GENETIC DISORDERS
Currently, amniocentesis and CVS remain the mainstays
when it comes to confirming genetic disease during the
prenatal period. In the past, cordocentesis was used
more frequently for genetic diagnoses. For example,
TAR syndrome that was suspected on prenatal ultra-
sound would be confirmed based on thrombocytopenia
and anemia observed in fetal cord blood analysis [15].
However, molecular diagnosis can now be made using
cells derived from an amniocentesis or CVS sample,
which is considered a safer alternative [16].
1.3.1 Amniocentesis
Amniocentesis was first described in the 1800s when
fluid was removed to treat polyhydramnios [17]. How-
ever, although used for other reasons over the inter-
vening years, it was really not until the 1950s that the
procedure became a part of obstetric care when it was
demonstrated that spectrophotometric analysis of bili-
rubin in the third trimester could be used to diagnose
and manage Rh disease [18]. This was quickly followed
by genetic diagnosis of sex using Barr body analysis [19].
However, the big hurdle that needed to be overcome
was the ability to culture the cells from the amniotic
fluid. With that achievement, prenatal diagnostics could
begin in earnest in the 1960s with a seminal publication
that described fetal chromosomal analysis [20]. 1968
saw the first reports of fetal Down syndrome as well
as galactosemia diagnoses [21,22]. Multiple case series
quickly followed, and amniocentesis has remained the
cornerstone for genetic screening confirmation and
diagnosis to the present day.
1.3.2 CVS
A limitation of traditional amniocentesis has remained
its timing during pregnancy. It is a second trimester
test, generally offered after 15 weeks gestation. Early
amniocentesis was proposed as a solution. However, a
3
randomized controlled trial demonstrated an increased
risk for talipes equinovarus in the early amniocentesis
group [23]. CVS, which entails sampling the placenta
either through an abdominal or transvaginal approach,
proved to be a first trimester diagnostic alternative. First
performed in 1983 [24], the technique has become well
established. While there is a small risk of false results due
to placental mosaicism, the chromosomal complement of
the placental cells used for this procedure closely mirrors
that of the fetal cells obtained through amniocentesis.
1.3.3 Preimplantation Genetic Testing
Preimplantation genetic testing has become more
widely available within IVF programs and is performed
prior to embryo transfer, following conception. Usually,
a biopsy is performed at the blastocyst stage, allowing
5 to 10 cells to be removed for further genetic testing.
The goal is to identify unaffected embryos for transfer
[25] and consequently avoid issues related to potential
termination of pregnancy.
1.3.4 Cytogenetic and Molecular Techniques
Used for Prenatal Diagnosis
Once fetal or placental cells could be retrieved, the
evolution of prenatal diagnosis tracked with available
cytogenetic and molecular technologies that were con-
currently available. The first paper documenting 46
chromosomes in humans was not published until 1956
[26]. In the early days of amniocentesis, G-banding was
not available and would not become part of cytogenetic
practice until the 1970s. The next major milestone was
the addition of molecular approaches to chromosomal
analysis. FISH probes allowed for the identification of
microdeletions that could not be seen using standard
karyotyping alone. Currently, microarrays are consid-
ered standard of care for prenatal diagnosis in the United
States, especially in the setting of fetal anomalies or still-
birth. If no structural anomalies are seen, conventional
karyotype and microarray should be discussed with the
patient [27]. Nor is prenatal exome sequencing still con-
sidered experimental. In the presence of fetal anomalies
or a single major anomaly suggestive of a genetic disor-
der where microarray is negative or unavailable, exome
sequencing becomes an option, similar to the postnatal
setting [28].
Noninvasive prenatal diagnosis is the next technolog-
ical phase that is garnering a lot of activity and attention.
It holds out the promise of removing the risk for fetal loss
4 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
that is associated with amniocentesis or CVS. While the
risk is low, 0.1%–0.3% in expert hands [29] and may not
even confer excess risk especially if the fetus is not anom-
alous [30], many women prefer to avoid invasive testing
if possible. The initial avenues explored were the isola-
tion of trophoblasts from the endocervical canal [31] and
fetal cells from the maternal circulation [32]. The focus
is on the separation and extraction of these cells, as once
isolated, current molecular sequencing techniques and
various analytic approaches become possible. Isolation of
intact fetal cells has now largely been superceded by direct
sequencing of cell-free fetal DNA, as discussed below.
1.4 PRENATAL SCREENING FOR GENETIC
DISORDERS—ANEUPLOIDY AND SINGLE
GENE
1.4.1 Fetal Aneuploidy Screening
It is notable that even during the 1960s and 1970s, when
amniocentesis was the only genetic testing option, women
were still involved in a screening program. Amniocente-
sis was not universally available and therefore age alone
was the clinical feature, absent any personal or family
risk, used to determine who would be offered a diagnostic
procedure. The age cut-off at 35 was used based on a few
factors including resource allocation and the “balance” of
1/200 risk of fetal loss versus 1/200 risk of any fetal chro-
mosomal anomaly at that maternal age. However, the
medical community always appreciated that despite the
increased risk in this older maternal age group, most chil-
dren with Down syndrome are born to women less than
35. Even in patients with affected offspring, the risk is
still only a few percentage points at most. Therefore, con-
ceptually, whether we are looking at the first “AFP only”
single marker aneuploidy screening test, standard first tri-
mester screening or the latest cell-free DNA noninvasive
prenatal screening (NIPS) approach, they all came about
to help refine the initial “age alone” risk algorithm [33].
NIPS has dramatically changed the landscape with pos-
itive predictive values (PPVs) that are several times bet-
ter than standard first trimester screening that combines
first trimester ultrasound NT and biomarkers (45.5% vs.
4.2% for trisomy 21% and 40.0% vs. 8.3% for trisomy 18)
[34]. However, despite this major leap forward in test
performance, NIPS has not been without controversy.
Additional disorders have been added with poor PPVs
and varied clinical utility, such as rare aneuploidies and
microdeletion syndromes [35]. Most problematic is an
ongoing confusion regarding the difference between a
screening test that can only provide a risk assessment
versus a true diagnostic test. In response, leading profes-
sional organizations have created open access calculator
tools to help healthcare professionals provide accurate
information to patients regarding PPVs and negative pre-
dictive values (NSGC PQF NIPT Calculator https://ww-
w.perinatalquality.org/Vendors/NSGC/NIPT/). It is also
worth noting that the entire fetal genome has already
been sequenced [36] using shotgun sequencing of mater-
nal plasma DNA. The approach is not practical for broad
clinical testing at this time, but it demonstrates that non-
invasive fetal sequencing can already be performed with
currently available technologies.
1.4.2 Carrier Screening for Genetic Disorders
Even prior to molecular diagnostics, fetal risk assess-
ment for Mendelian disorders was possible. A good
pedigree analysis could provide valuable information in
the case of a woman with a family history of Duchenne
Muscular Dystrophy or a previous child with cystic
fibrosis. The population-based Tay Sachs screening pro-
gram was successfully executed using maternal enzyme
analysis and was the first multi-disease panel as some
of the programs also screened for familial hypercholes-
terolemia using cholesterol levels. Hemoglobin electro-
phoresis and a simple MCV are considered the first-line
screening tests for hemoglobinopathies [37].
However, there is no doubt that molecular technol-
ogies, in particular next-generation sequencing (NGS),
have altered the carrier screening landscape. The cur-
rent approach is to sequence the mother and if a patho-
genic or likely pathogenic variant is identified, then the
father of the baby also undergoes genetic testing in the
case of an autosomal recessive disorder. While carrier
screening is on one hand a diagnostic for the mother
(if a pathogenic cystic fibrosis variant is found, she is
indeed a carrier), the term “screening” is used because
the purpose of the test is to assess the risk to fetus. The
benefits of NGS technology are manifold, including the
ability to test for more disorders in a highly precise and
efficient way. However, the larger the panels, the more
likely a patient will receive a “positive” screen result. As
more variants will be found in genes associated with
increasingly rare disorders, the odds that the other par-
ent will likewise have a pathogenic variant in that same
gene become more unlikely. Thus, there is significant
 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
concern that larger panels will result in downstream
anxiety and costs but will not necessarily provide useful
information specific to the current pregnancy. Similar to
aneuploidy screening, single gene variant detection has
already been reported using cell-free DNA in maternal
plasma [38] using droplet PCR. Other approaches have
also been reported [39,40]. A clinical test is already on
the market for select de novo and paternally inherited
variants, although it is not considered to be sufficiently
validated to be incorporated into standard of care [41].
1.5 THE END OF THE BEGINNING AND
WHAT LIES AHEAD
From a broad perspective, the above survey of prenatal
genetics tells us that we have attained what would have
seemed like a far-off achievement only a few decades
ago. We already have the technology to interrogate the
fetal genome during pregnancy and the preimplantation
period. Treatments will become available and newer
diagnostic methodologies seem poised to fulfill the
promise of noninvasive testing. There remains much to
be done with respect to scalability and cost reduction;
however, technological advances will continue and one
can expect within a few years to see prenatal diagnostics
move forward on all fronts as well, opening the door to
true precision medicine prior to delivery. While there
is much to celebrate, the same questions that have con-
cerned the specialty in the past have not diminished and
perhaps take on more urgency as our ability to finally
access the fetal genome has arrived.
1.5.1 We Can Do It, but Should We Do It?
There has always been the push and pull between our
ability to “do more” to benefit patients versus primum
non nocere—first do no harm. Thoughtful clinicians
and leaders in the field addressed this problem even
when screening panels were still just a few disorders in
size [42]. Andermann et al. [43], in a WHO bulletin,
applied the well-known Wilson and Jungner principles
of screening criteria to the genomic era. Many of the key
concepts still hold, including the “North Star” of clinical
utility. Even if a screening test works consistently well in
the laboratory and can even detect disorders of interest
in the clinical setting, should it be offered if there is no
demonstrable positive impact on outcomes? Certainly,
there are challenges as often specific genetic disorders
tend to be rare, and large broad-based research studies
5
comparable to diabetes or coronary heart disease may
not be feasible. Some screening and even diagnostic tests
may require more “shared decision-making” approaches
in the future. However, there is still the need for rigor-
ous analytic, clinical validity and ultimately clinical
utility studies if testing is to be provided to millions of
women worldwide who are or seek to become pregnant.
Professional bodies have tried to address the question
with an approach that does not necessarily provide a
defined panel of diseases, but rather seeks to specify
characteristics of disorders that may warrant screening,
for example, whether the condition could result in sig-
nificant disability or knowledge of the condition could
enhance delivery planning. Conversely, guidance also
can address what disorders should be excluded, such as
adult-onset disorders or high allele frequency variants
but low penetrance such as MTHFR [44]. Others have
looked closely at allele frequency and the identification
of carrier couples rather than just one parent. Assessing
only 40 genes with carrier rates >1.0% would identify a
substantial number of panethnic carrier couples, while
the addition of genes with lower carrier rates followed
the principle of “diminishing returns” [45]. Genome
sequencing will ensure that this conversation regard-
ing prenatal test expansion will become more, not less,
important in the future.
1.5.2 Women’s Autonomy
Related to the above discussion of what prenatal tests
should be offered is the question of who gets to decide.
For example, Canadian guidelines recommend invasive
prenatal diagnosis be offered to women at high risk [46],
while in the United States, all women have the option of
screening versus diagnostic testing [47]. Some authors
have approached the issue of women’s autonomy via the
lens of informed consent and the “routinization” of pre-
natal testing, such that women are making decisions but
based on limited knowledge. In addition, “[s]upport for
access to prenatal genetic tests and abortion services and
advocacy for robust informed consent processes grow
out of the same ethical commitment to respect for auton-
omy” [48]. Other authors have noted that historically,
the focus has been on the risk for fetal loss following
invasive testing. Rather, an autonomy-based approach
would help women identify what risk most concerns
them personally. For some it may indeed be the risk of
fetal loss but for other women, it may be the risk of hav-
ing a child with a significant genetic abnormality [49].
6 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
1.6 CONCLUSION
Advances in reproductive genetics have been inextri-
cably tied to the technologic achievements that have
greatly accelerated over the past decade. Not only can
we image the fetus, but we can sequence its genome
using both invasive and noninvasive approaches which
can conceivably lead to treatment and cures. However,
the same concerns are still present, no different than
decades ago when the field of reproductive genetics
was just beginning. In his landmark paper on the first
prenatal diagnosis of an inborn error of metabolism
(galactosemia) using amniocentesis, the author ends
his abstract with the following “… until consider-
ably more experience is gained with these techniques,
these procedures should be considered experimental
in nature” [21]. Despite ethical concerns, science and
medicine will continue to move forward with the goal
of enhancing the well-being of individuals and com-
munities. Further detail regarding the past milestones
and future promise of reproductive genetics will be
found in the pages of this volume. Nevertheless, while
celebrating the many achievements that have improved
the health of mothers and families, it is worth reflecting
on a key moment in Dr. Charles Epstein’s Presidential
Address to the American Society of Human Genetics
in 1996 [50]. The address was meaningful for many
reasons, not the least of which was the fact that Dr.
Epstein, a recognized expert in the genetics of Down
syndrome, had recently survived injuries caused by an
explosion set by the “Unabomber,” an individual who
was on a violent campaign to stop technology. Toward
the end of his presentation, and in very compassionate
terms, Dr. Epstein made the case that medical genet-
icists have a responsibility to educate and inform the
public about what medical geneticists actually do.
However, most important and especially relevant to
those in the field of prenatal genetics, he recommended
that the medical genetic community “continue to be –
and, if anything, become more – involved in the social
and ethical debate that increasingly surrounds every-
thing that we do.”
REFERENCES
[1] Benson CB, Doubilet PM. The history of imaging in ob-
stetrics. Radiology 2014;273(2 Suppl. l):S92–110. https://
doi.org/10.1148/radiol.14140238.
[2] Macones GA, Chang JJ, Stamilio DM, Odibo AO,
Wang J, Cahill AG. Prediction of cesarean delivery
using the fetal-pelvic index. Am J Obstet Gynecol
2013;209(5):431.e1–4318. https://doi.org/10.1016/j.
ajog.2013.06.026.
[3] Campbell S. A short history of sonography in obstetrics
and gynaecology. Facts Views Vis Obgyn 2013;5(3):213–
29.
[4] Zagzebski J. WE-G-211-03: development of ultrasound
imaging equipment. Med Phys 2012;39(6Part28):3964.
https://doi.org/10.1118/1.4736177.
[5] Donald I, MacVicar J, Brown TG. Investigation of
abdominal masses by pulsed ultrasound. Lancet
1958;1(7032):1188–95. https://doi.org/10.1016/s0140-
6736(58)91905-6.
[6] Committee on practice Bulletins—Obstetrics and the
American Institute of Ultrasound in Medicine. Practice
bulletin No. 175: ultrasound in pregnancy. Obstet
Gynecol 2016;128(6):e241–56. https://doi.org/10.1097/
AOG.0000000000001815.
[7] Minnella GP, Crupano FM, Syngelaki A, Zidere V,
Akolekar R, Nicolaides KH. Diagnosis of major heart
defects by routine first-trimester ultrasound examina-
tion: association with increased nuchal translucency,
tricuspid regurgitation and abnormal flow in ductus
venosus. Ultrasound Obstet Gynecol 2020;55(5):637–44.
https://doi.org/10.1002/uog.21956.
[8] Ali MM, Chasen ST, Norton ME. Testing for Noonan
syndrome after increased nuchal translucency. Prenat
Diagn 2017;37(8):750–3. https://doi.org/10.1002/
pd.5076.
[9] Syngelaki A, Hammami A, Bower S, Zidere V, Akolekar
R, Nicolaides KH. Diagnosis of fetal non-chromosomal
abnormalities on routine ultrasound examination at
11–13 weeks’ gestation. Ultrasound Obstet Gynecol
2019;54(4):468–76. https://doi.org/10.1002/uog.20844.
[10] Nyberg DA, Souter VL, El-Bastawissi A, Young S, Luth-
hardt F, Luthy DA. Isolated sonographic markers for de-
tection of fetal Down syndrome in the second trimester
of pregnancy. J Ultrasound Med 2001;20(10):1053–63.
https://doi.org/10.7863/jum.2001.20.10.1053.
[11] Prefumo F, Jauniaux E. Amniocentesis for fetal karyo-
typing: the end of an era? BJOG 2016;123(1):99. https://
doi.org/10.1111/1471-0528.13497.
[12] Carlson DE. The ultrasound evaluation of cleft lip and
palate–a clear winner for 3D. Ultrasound Obstet Gyne-
col 2000;16(4):299–301. https://doi.org/10.1046/j.1469-
0705.2000.00323.x.
[13] Manganaro L, Bernardo S, Antonelli A, Vinci V, Saldari
M, Catalano C. Fetal MRI of the central nervous system:
state-of-the-art. Eur J Radiol 2017;93:273–83. https://
doi.org/10.1016/j.ejrad.2017.06.004.
 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
[14] Committee on Obstetric Practice. Committee opin-
ion no. 723: guidelines for diagnostic imaging during
pregnancy and lactation [published correction ap-
pears in Obstet Gynecol. 2018 Sep;132(3):786]. Obstet
Gynecol 2017b;130(4):e210–6. https://doi.org/10.1097/
AOG.0000000000002355.
[15] Donnenfeld AE, Wiseman B, Lavi E, Weiner S. Pre-
natal diagnosis of thrombocytopenia absent radius
syndrome by ultrasound and cordocentesis. Prenat
Diagn 1990;10(1):29–35. https://doi.org/10.1002/
pd.1970100106.
[16] Society for Maternal-Fetal Medicine (SMFM), Gandhi
M, Rac MWF, McKinney J. Radial ray malformation.
Am J Obstet Gynecol 2019;221(6):B16–8. https://doi.
org/10.1016/j.ajog.2019.09.024.
[17] Elias S, Simpson JL. Amniocentesis. In: Milunsky A, editor.
Genetic disorders and the fetus. Boston, MA: Springer;
1986. https://doi.org/10.1007/978-1-4684-5155-9_2.
[18] Bevis DC. The antenatal prediction of haemolytic
disease of the newborn. Lancet 1952;1(6704):395–8.
https://doi.org/10.1016/s0140-6736(52)90006-8.
[19] Fuchs F, Riis P. Antenatal sex determination. Nature
1956;177(4503):330. https://doi.org/10.1038/177330a0.
[20] Steele MW, Breg Jr WR. Chromosome analysis of
human amniotic-fluid cells. Lancet 1966;1(7434):383–5.
https://doi.org/10.1016/s0140-6736(66)91387-0.
[21] Nadler HL. Antenatal detection of hereditary disorders.
Pediatrics 1968;42(6):912–8.
[22] Valenti C, Schutta EJ, Kehaty T. Prenatal diagnosis of
Down’s syndrome. Lancet 1968;2(7561):220. https://doi.
org/10.1016/s0140-6736(68)92656-1.
[23] CEMAT Trial: randomised trial to assess safety and fetal
outcome of early and midtrimester amniocentesis. The
Canadian Early and Mid-trimester Amniocentesis Trial
(CEMAT) Group. Lancet 1998;351(9098):242–7.
[24] Brambati B, Simoni G. Diagnosis of fetal trisomy 21 in
first trimester. Lancet 1983;1(8324):586.
[25] Preimplantation genetic testing: ACOG com-
mittee opinion, number 799. Obstet Gynecol
2020;135(3):e133–7. https://doi.org/10.1097/
AOG.0000000000003714.
[26] Tjio JH, Levan A. The chromosome number of man.
Hereditas 1956;42:1–6.
[27] Society for Maternal-Fetal Medicine (SMFM), Elec-
tronic address: pubs@smfm.org, Dugoff L, Norton
ME, Kuller JA. The use of chromosomal microarray for
prenatal diagnosis [published correction appears in Am
J Obstet Gynecol. 2017 Feb;216(2):180]. Am J Obstet
Gynecol 2016;215(4):B2–9. https://doi.org/10.1016/j.
ajog.2016.07.016.
[28] International Society for Prenatal Diagnosis; Society for
Maternal and Fetal Medicine; Perinatal Quality Foun-
7
dation. Joint position statement from the International
Society for Prenatal Diagnosis (ISPD), the Society for Ma-
ternal Fetal Medicine (SMFM), and the Perinatal Quality
Foundation (PQF) on the use of genome-wide sequenc-
ing for fetal diagnosis. Prenat Diagn 2018;38(1):6–9.
https://doi.org/10.1002/pd.5195. https://www.perinatal-
quality.org/Vendors/NSGC/NIPT/.
[29] American College of Obstetricians and Gynecologists’
Committee on Practice Bulletins—Obstetrics, Commit-
tee on Genetics, Society for Maternal–Fetal Medicine.
Practice bulletin no. 162: prenatal diagnostic testing for
genetic disorders. Obstet Gynecol 2016;127(5):e108–22.
https://doi.org/10.1097/AOG.0000000000001405.
[30] Salomon LJ, Sotiriadis A, Wulff CB, Odibo A, Akolekar
R. Risk of miscarriage following amniocentesis or
chorionic villus sampling: systematic review of literature
and updated meta-analysis. Ultrasound Obstet Gynecol
2019;54(4):442–51. https://doi.org/10.1002/uog.20353.
[31] Jain CV, Kadam L, van Dijk M, et al. Fetal genome pro-
filing at 5 weeks of gestation after noninvasive isolation
of trophoblast cells from the endocervical canal. Sci
Transl Med 2016;8(363):363re4. https://doi.org/10.1126/
scitranslmed.aah4661.
[32] Beaudet AL. Using fetal cells for prenatal diagnosis: history
and recent progress. Am J Med Genet C Semin Med Genet
2016;172(2):123–7. https://doi.org/10.1002/ajmg.c.31487.
[33] Gross S, Cuckle H. Prenatal screening and diagnosis–An
introduction. Am J Med Genet C Semin Med Genet
2007;145C(1):1–4. https://doi.org/10.1002/ajmg.c.30122.
[34] Bianchi DW, Parker RL, Wentworth J, et al. DNA se-
quencing versus standard prenatal aneuploidy screen-
ing. N Engl J Med 2014;370(9):799–808. https://doi.
org/10.1056/NEJMoa1311037.
[35] Gregg AR, Skotko BG, Benkendorf JL, et al. Noninvasive
prenatal screening for fetal aneuploidy, 2016 update: a
position statement of the American College of Medical
Genetics and Genomics. Genet Med 2016;18(10):1056–
65. https://doi.org/10.1038/gim.2016.97.
[36] Fan HC, Gu W, Wang J, Blumenfeld YJ, El-Sayed
YY, Quake SR. Non-invasive prenatal measurement
of the fetal genome [published correction appears
in Nature. 2012 Sep 13;489(7415):326]. Nature
2012;487(7407):320–4. https://doi.org/10.1038/na-
ture11251.
[37] Committee on Genetics. Committee opinion no.
691: carrier screening for genetic conditions. Obstet
Gynecol 2017;129(3):e41–55. https://doi.org/10.1097/
AOG.0000000000001952.
[38] Camunas-Soler J, Lee H, Hudgins L, et al. Noninvasive
prenatal diagnosis of single-gene disorders by use of
droplet digital PCR. Clin Chem 2018;64(2):336–45.
https://doi.org/10.1373/clinchem.2017.278101.
8 CHAPTER 1 Introduction to Perinatal Disorders and Reproductive Genetics
[39] Ren Y, Zhao J, Li R, et al. Noninvasive prenatal test for
FGFR3-related skeletal dysplasia based on next-gener-
ation sequencing and plasma cell-free DNA: test per-
formance analysis and feasibility exploration. Prenat
Diagn 2018;38(11):821–8. https://doi.org/10.1002/
pd.5334.
[40] Yang X, Ye Y, Fan D, et al. Non-invasive prenatal
diagnosis of thalassemia through multiplex PCR,
target capture and next-generation sequencing. Mol
Med Rep 2020;22(2):1547–57. https://doi.org/10.3892/
mmr.2020.11234.
[41] ACOG practice advisory: cell-free DNA to screen
for single-gene disorders. February 2019. Available
from: https://www.acog.org/clinical/clinical-guid-
ance/practice-advisory/articles/2019/02/cell-free-dna-to
-screen-for-single-gene-disorders. [Accessed 17 August
2020].
[42] Grody WW, Cutting GR, Watson MS. The Cystic
Fibrosis mutation “arms race”: when less is more.
Genet Med 2007;9(11):739–44. https://doi.org/10.1097/
gim.0b013e318159a331.
[43] Andermann A, Blancquaert I, Beauchamp S, Déry V.
Revisiting Wilson and Jungner in the genomic age: a
review of screening criteria over the past 40 years. Bull
World Health Organ 2008;86(4):317–9. https://doi.
org/10.2471/blt.07.050112.
[44] 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 Obstetricians and Gynecologists, National Society
of Genetic Counselors, Perinatal Quality Founda-
tion, and Society for Maternal-Fetal Medicine. Obstet
Gynecol 2015;125(3):653–62. https://doi.org/10.1097/
AOG.0000000000000666.
[45] Guo MH, Gregg AR. Estimating yields of prenatal car-
rier screening and implications for design of expanded
carrier screening panels. Genet Med 2019;21(9):1940–7.
https://doi.org/10.1038/s41436-019-0472-7.
[46] Audibert F, De Bie I, Johnson JA, et al. No. 348-Joint
SOGC-CCMG guideline: update on prenatal screen-
ing for fetal aneuploidy, fetal anomalies, and adverse
pregnancy outcomes [published correction appears
in J Obstet Gynaecol Can. 2018 Aug;40(8):1109]. J
Obstet Gynaecol Can 2017;39(9):805–17. https://doi.
org/10.1016/j.jogc.2017.01.032.
[47] APA Rose NC, Kaimal AJ, Dugoff L, Norton ME,
American College of Obstetricians and Gynecologists’
Committee on Practice Bulletins—Obstetrics, Commit-
tee on Genetics Society for Maternal-Fetal Medicine.
Screening for fetal chromosomal abnormalities. Obstet
Gynecol 2020;136.
[48] Johnston J, Farrell RM, Parens E. Supporting wom-
en’s autonomy in prenatal testing. N Engl J Med
2017;377(6):505–7. https://doi.org/10.1056/NE-
JMp1703425.
[49] Chervenak FA, McCullough LB, Sharma G, Davis J,
Gross S. Enhancing patient autonomy with risk assess-
ment and invasive diagnosis: an ethical solution to a
clinical challenge. Am J Obstet Gynecol 2008;199(1):19.
e1–94. https://doi.org/10.1016/j.ajog.2008.02.021.
[50] Epstein CJ. 1996 ASHG presidential address. Toward the
21st century. Am J Hum Genet 1997;60(1):1–9.
FURTHER READING
Brock DJ, Bolton AE, Monaghan JM. Prenatal diagnosis of
anencephaly through maternal serum-alphafetoprotein
measurement. Lancet 1973;2(7835):923–4. https://doi.
org/10.1016/s0140-6736(73)92592-0.
Committee on Obstetric Practice, the American Institute of
Ultrasound in Medicine, and the Society for Maternal-Fe-
tal Medicine. Committee opinion no 700: methods for es-
timating the due date. Obstet Gynecol 2017;129(5):e150–
4. https://doi.org/10.1097/AOG.0000000000002046.
Cuckle H, Maymon R. Development of prenatal screening–A
historical overview. Semin Perinatol 2016;40(1):12–22.
https://doi.org/10.1053/j.semperi.2015.11.003.
Hobbins JC. Overview of imaging in pregnancy: history to
the present, including economic impact. Semin Perina-
tol 2013;37(5):290–1. https://doi.org/10.1053/j.sem-
peri.2013.06.002.
National Society of Genetic Counselors and Perinatal Quality
Foundation: NIPT/cell free DNA screening predictive
value calculator. [Accessed 17 August 2020].
Scott F, Chan FY. Assessment of the clinical usefulness of
the “Queenan” chart versus the “Liley” chart in predict-
ing severity of rhesus iso-immunization. Prenat Diagn
1998;18(11):1143–8.

Prenatal Screening for Neural
Tube Defects and Aneuploidy*
Department of Biomedical Sciences, University of South Carolina School of Medicine Greenville,
2.1 INTRODUCTION
Population-based prenatal screening using biomark-
ers began in the 1980s following successful pilot stud-
ies that demonstrated that open neural tube defects
(ONTDs) including spina bifida and anencephaly could
be effectively identified by studying the presence of
alpha-fetoprotein (AFP) in the maternal circulation in
the second trimester of pregnancy [1]. Soon thereafter,
it was discovered that AFP levels were statistically lower
in pregnancies affected with Down syndrome (DS),
and this observation was exploited to provide the first
maternal serum biomarker screening for aneuploidy
[2]. Clinical screening for these two conditions is based
on successful characterization of the different distribu-
tions of these biomarkers between the population as
a whole and the subset of pregnancies affected by the
condition for which screening is seen as valuable. This
approach has led to the discovery of dozens of other
biomarkers for which affected pregnancies exhibit dif-
ferences from the underlying population that can be
exploited for screening purposes alone, or in combina-
tion with other biomarkers. This is not only limited to
protein markers such as AFP but also smaller peptides
[3,4], steroid hormones [5], nucleic acids [6], fetal mor-
phometric measurements [7–10], and other attributes
of the maternal–fetal environment [11,12]. Although
neural tube defects (NTDs) and DS remain at the center
* This article is a revision of the previous edition article by
Amelia L.M. Sutton and Joseph R. Biggio, pp 1–23. © 2013,
Elsevier Ltd. All rights reserved.
Emery and Rimoin’s Principles and Practice of Medical Genetics and Genomics. https://doi.org/10.1016/B978-0-12-815236-2.00011-4
Copyright © 2022 Elsevier Inc. All rights reserved.
2
Robert G. Best
Greenville, SC, United States
of prenatal screening, the methodology extends well
beyond these two conditions.
Biomarker concentrations during pregnancy tend to
be dynamic, so it is generally necessary to standardize
measurements relative to gestational age by comparing
a patient’s measured value against the median value in
the overall pregnant population for that specific point
in pregnancy. A risk-based approach that is shared
among most of the conditions for which clinical prena-
tal screening is offered uses Bayesian probability mod-
ifications constructed from the statistical distributions
of the biomarkers to modify a priori risks from the
population to calculate a patient-specific risk for each
condition of interest. This construct has led to dramatic
improvements in the ability to detect DS prenatally and
has opened an approach to identify other birth defects
and potentially serious adverse pregnancy conditions
(e.g., preeclampsia, low birth weight, etc.).
This chapter is intended to highlight the general
approach to prenatal screening currently in use in clin-
ical settings with a central focus on ONTD and DS and
secondary focus on other aneuploidies and the great
variety of other conditions that can be identified in the
context of prenatal screening. This chapter begins with
an historical perspective and ends with a reminder of
the clinical public health context of prenatal screening.
The introduction of cell-free fetal DNA (cfDNA) as a
mode for screening has dramatically influenced pre-
natal screening for aneuploidies since its introduction
in practice in 2012 (see Chapter 10); however, multiple
marker testing remains as a vital component of prenatal
screening in the United States and around the world.
9
10 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
2.2 PRENATAL SCREENING FOR BIRTH
DEFECTS
Screening tests are designed to identify the potential for
health disorders from among an otherwise healthy pop-
ulation. Screening differs from diagnostic testing in that
false positives and negatives are expected and are incor-
porated into the schema. Prenatal screening focuses pri-
marily on the risk of adverse health conditions of the
fetus that are both serious and common. Current stan-
dards for healthcare screening advanced by the World
Health Organization based on iterative improvements of
earlier criteria proposed by Wilson and Junger [13,14]
require that the screening test responds to a recognized
need for a defined target population, reflects scientific
evidence that the screening program is effective, is
designed to be equitable across the entire target popula-
tion, that the benefits outweigh any harms, and that the
program integrates education, testing, clinical services,
and program management [13].
Research around the Health Belief Model exploring
the motivation of patients to accept available testing
identifies the patient’s own perceptions of susceptibility,
severity, benefits, and barriers as critically important,
conditioned on beliefs of self-efficacy (i.e., an ability to
take effective action) [15,16]. Decisions to participate are
also affected by a variety of modifying factors (e.g., race/
ethnicity, age, education, etc.) and internal or external
cues that trigger action (e.g., receiving information from
trusted sources). Thus, the mere availability of a test or
demonstration that screening is possible is not sufficient
in terms of public policy nor patient demand. Two pre-
natal conditions that seem to fully meet all criteria for
screening are ONTD and DS. In addition to these two
conditions, there are several other conditions for which
information arises while testing for ONTD or DS that
bear sufficient clinical utility to merit inclusion in the
overall screening program.
2.2.1 Neural Tube Defects
NTDs are among the most common of the serious birth
defects in the population. These are major structural
developmental defects affecting the central nervous sys-
tem that arise from an error in the maturation of the
neural tube early in pregnancy, between 14- and 28-days
postfertilization (4–6 weeks by menstrual dating).
During this 2-week period, the embryonic tissues that
give rise to the spine and brain begin with a relatively
flat geometry, develop a transverse fold that deepens
into a groove along the axis of development, that ulti-
mately circularizes to give rise to a tubular structure as
the leading edges of the neural fold begin to touch and
connect with each other [17,18], providing the founda-
tional structures for the brain and spine. Failure of the
neural tube to close completely results in a disruption of
these central structures of the nervous system. Although
failure to close can result in a complete failure of the for-
mation of the neural tube and all resulting structures
downstream (complete dysraphism), most commonly
the errors are confined to incomplete closure at one end
or the other. When the failure involves the caudal end,
the developmental failure results in an opening along
the spine (spina bifida), whereas failure at the cephalic
end results in a dramatic disruption of the primary
structures of the brain and cranial vault (encephalocele,
anencephaly). These two anomalies are almost equally
common and account for approximately 90% of all
NTDs [19].
Morbidity and mortality are variable depending upon
the size, location, and fine structure of the defect. Spina
bifida is typically associated with paralysis or weakness of
the lower structures of the body but the extent is highly
variable and ranges from a lack of clinical impairment to
fetal or neonatal death [18]. Anencephaly is considered to
be uniformly fatal with death early in the postnatal period
for babies that survive to term [20].
When NTDs are covered by skin or other membranes,
they are considered to be closed defects. Most often, NTD
lesions are not covered with skin, and are therefore con-
sidered to be open defects. This is an important distinc-
tion because the mechanism that leads to differences in
AFP concentrations between the affected and unaffected
populations is limited to open defects. Only 15%–20% of
spina bifida cases are closed defects but, in general, the
prognosis is more favorable [21,22], whereas most cases
of anencephaly are open [23]. Biochemical screening is
therefore restricted to open NTD because the open lesion
is directly related to the increased release of fetal protein
into maternal circulation. It is not the intent of this chap-
ter to fully characterize the range of NTDs and their vari-
ous clinical presentations.
Most commonly, NTDs occur without other struc-
tural anomalies unrelated to the development of the
neural tube and are considered to be isolated or non-
syndromic. Their occurrence is estimated to be 7/10,000
live births in the general population of the United
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
States [24] with notable differences in birth prevalence
around the world [25] and variability related to race,
geographical location, and the availability of folic acid
in the diet [26]. Isolated NTDs are genetically complex
traits with a heritability of approximately 60% [27,28]
with many genes associated and few genes having
been identified that clearly demonstrate major effects
[29]. Like other complex traits, recurrence is increased
when there are affected first-degree relatives [30] at a
rate of approximately the square root of the population
birth prevalence and less so for more distant affected
relatives [28]. A number of environmental factors have
been identified that influence the development of the
neural tube including folic acid, folate antimetabolites,
and type I diabetes [31–33]. Since the great majority of
NTDs occur in the absence of a positive family history,
prenatal identification is largely dependent upon gen-
eral population screening through AFP or ultrasound
examination.
NTDs can also appear in syndromic forms asso-
ciated with structural defects unrelated to the neu-
ral tube. Recurrence risks for syndromic NTDs are
highly variable and are dependent on the etiologic
mechanisms. For example, Meckel–Gruber syndrome
is a rare disorder with a birth prevalence of 2.6 per
100,000, inherited in a single-gene autosomal recessive
pattern and is associated primarily with encephalo-
cele [34]. In contrast, complete or partial aneuploidy
may also involve disruption of the neural tube during
development, with recurrence risks dependent on
­ sonalities are frequently described as affectionate and
the mechanism through which the chromosomal
­imbalance arose. Most syndromic forms of NTDs are
relatively rare and are therefore challenging to study
for recurrence and the degree to which environmental
factors might be involved. While it is not the intent of
this chapter to address the fine points of the occurrence
and distribution of all forms of NTDs, it is important
to recognize differences in recurrence risks as a limit-
ing factor in the estimation of patient-specific risk cal-
culations in screening. Biomarker screening is effective
for any ONTD independent of its causal mechanism.
2.2.2 Down Syndrome and Aneuploidies in
Pregnancy
Studies in the 1970s showed that chromosomal abnor-
malities affect approximately 1 in 160 live births [35].
A more recent European study of second trimester
amniocenteses demonstrated that this incidence may
11
be higher when minor alterations such as mosaicism
are included [36]. The majority of chromosome abnor-
malities are sex chromosome alterations involving
extra copies of the X or Y chromosomes, monosomy X
or autosomal trisomies involving chromosomes 21, 18,
or 13. Approximately 1 in 700–800 children are born
with trisomy 21 (DS), 1 in 6000 are born with trisomy
18 (Edwards syndrome), and 1 in 10,000 with trisomy
13 (Patau syndrome) [37,38]. Most autosomal trisomies
are caused by nondisjunction during maternal meiosis,
a process that is more frequent with advancing mater-
nal age [39].
2.2.2.1 Down Syndrome
DS is a complex clinical phenotype that results from
trisomy of part or all of chromosome 21. DS is the most
common autosomal aneuploidy occurring in humans,
with a current birth prevalence of approximately 1:700
live births [38] and higher birth frequency among
older mothers. People with DS typically have an IQ
in the mildly to moderately low range with character-
istic facial features that may include epicanthal folds,
upward slanting palpebral fissures, flattened facial pro-
file, short neck and small ears, hypotonia, hyperflex-
ibility, single transverse palmar creases, and a variety
of other benign or mild features [40]. Individuals with
DS are susceptible to duodenal atresia, Hirschsprung
disease, patent ductus arteriosus, early-onset Alzhei-
mer disease, and acute leukemia [41,42]. Their per-
pleasant albeit somewhat complex [43]. The combina-
tion of a relatively high birth prevalence, complexity
of the clinical phenotype, older maternal age at birth,
and relatively long life expectancy no doubt contribute
to the high level of interest in prenatal screening and
diagnosis.
2.2.2.2 Trisomy 18
Another autosomal aneuploidy, trisomy 18 (Edwards
syndrome), demonstrates an altered biomarker profile
compared with unaffected pregnancies and is there-
fore also detectable in multiple marker screening [44].
Edwards syndrome has a significantly higher mor-
bidity and mortality compared with DS, is subject to
higher rates of spontaneous abortion, and is far less
common [45–47]. Because of its rarity and differences
in severity and life expectancy, the public health ratio-
nale for trisomy 18 screening is considerably weaker
12 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
than for DS and likely would not justify screening on
its own. Inasmuch as the markers that are employed
for trisomy 21 may also be informative for trisomy
18, this screening can be included without undertak-
ing any additional testing. As with all rare conditions,
the positive predictive value (PPV) is relatively low.
Selecting the risk cutoff for screen positives to keep
specificity high serves to reduce unnecessary diagnos-
tic testing.
2.2.2.3 Other Chromosome Abnormalities
There are several other chromosome abnormalities that
occur during pregnancy. A third autosomal aneuploidy
that sometimes survives to term is trisomy 13 (Patau
syndrome) [48]. This is more severe and significantly
less prevalent than either trisomy 18 or trisomy 21 [46].
While autosomal monosomies are uniformly lethal
prenatally, several other autosomal trisomies are known
to occur, but do not survive to term except in a mosaic
state. There is a very large number of partial aneuso-
mies that each are extremely rare, with highly variable
phenotypes that occasionally are live born, as well as
lethal triploidies and tetraploidies that include whole
extra sets of chromosomes [49]. Each in this group of
chromosomal disorders shares the properties of severe
phenotypes, low probability of survival, and low birth
prevalence such that screening would not meet the basic
features of disorders for which population screening
would be merited. Finally, there are quite a number of
sex chromosome aneuploidies including Turner syn-
drome (monosomy X) [50], which has a relatively mild
phenotype but a low chance of survival to term and birth
prevalence of 1 per 10,000; Klinefelter syndrome (male
with an extra X chromosome)—a syndrome involving
infertility and some comparatively mild phenotypic fea-
tures affecting 1 per 1000 live born males [51]; trisomy
X female and disomy Y males with similar birth prev-
alence to Klinefelter that demonstrate clinical features
sufficiently unremarkable as to not be considered to
constitute a physical syndrome [52,53]; and others with
multiple extra copies of the X and/or Y chromosome
[54,55]. While there might be some value and interest
in identifying one or more of these chromosome abnor-
malities prenatally, the costs would generally be consid-
ered to outweigh the benefits under most circumstances.
These might be considered to be off-target secondary
findings discovered during clinical screening for DS,
however.
2.2.2.4 Aneuploidy and Spontaneous Fetal Loss
Pregnancies affected by aneuploidy have a greater risk of
spontaneous abortion than unaffected pregnancies. True
estimates of this rate are difficult to ascertain as a propor-
tion of affected pregnancies that are detected by prenatal
screening programs will be terminated and some may
occur so early in development that the woman does not
recognize them as a pregnancy loss. A large-scale study
showed that 43% of pregnancies with DS detected by the
first trimester chorionic villus sampling (CVS) and 23%
of pregnancies with DS detected by the second trimester
amniocenteses will end in miscarriage or stillbirth [56].
Fetal loss rates in pregnancies affected by trisomy 13 or
18 are even higher with 49% of pregnancies diagnosed
with trisomy 13 in the first trimester and 42% diagnosed
with trisomy 13 in the second trimester will end in mis-
carriage or stillbirth. 72% of pregnancies diagnosed with
trisomy 18 in the first trimester and 65% of pregnancies
diagnosed with trisomy 18 in the second trimester will
end in miscarriage or stillbirth [57]. This information
is vital for counseling women regarding prognoses for
their affected pregnancies. Accurate determination of
fetal loss influences the a priori and posterior risk esti-
mates for the autosomal trisomies and is relevant to the
patient’s decision-making from the standpoint of the
value of screening and diagnosis under the Health Belief
Model.
2.2.3 Maternal Age as a Marker for
Aneuploidy
One of the earliest and most primitive forms of screen-
ing is to consider maternal age at the expected date of
delivery as a means of estimating risk for DS and other
aneuploidies. This is a simple, cost-free approach to
identifying pregnancies that merit consideration for
diagnostic testing to identify chromosome abnormali-
ties.
The association between advanced maternal age and
an increased risk of DS was first described in the 1930s
[58]. A multitude of subsequent studies have confirmed
this association and defined its magnitude. The risk of
DS and other aneuploidies increases with maternal age,
so that a 40-year-old woman has a more than 13-fold
higher risk of having a pregnancy affected by DS than
does a 20-year-old woman [59]. Nevertheless, in terms
of raw numbers, far more autosomal trisomies occur
to younger women who are not considered to be of
advanced maternal age.
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy 13

The molecular basis for the association between
maternal age and aneuploidy is an increased rate of mei-
otic nondisjunction in aging oocytes [39]. Oocytes are
suspended in the dictyotene stage of prophase I from
the time they are formed during fetal development until
they are fertilized in adulthood. During this protracted
time in prophase, the chromosomes are kept aligned on
the equatorial plate by chiasmata, the sites of recombi-
nation. In most cases of maternal nondisjunction, it is
thought that aging causes deterioration of the chiasmata
and subsequent misalignment of sister chromatids. This
results in missegregation of chromosome pairs to the
daughter oocytes [39].
2.2.4 AFP as a Biomarker of Fetal
Development in Maternal Circulation
2.2.4.1 AFP in Unaffected Pregnancies
AFP is a protein produced almost exclusively by the
fetus, and it is therefore mostly contained in the fetal cir-
culation. During the optimal gestational period for NTD
screening (16–18 weeks of gestation), AFP is also pres-
ent in amniotic fluid, but at approximately a 1000-fold
lower concentration than in fetal circulation, with entry
into the amniotic fluid primarily through the kidneys.
With rare exception, AFP is not produced in the healthy
adult [60], so the vast majority of AFP in maternal cir-
culation is fetal in origin, passing through the fetal kid-
neys into the amniotic fluid, and then through the fetal
membranes into maternal circulation [61]. Maternal
serum AFP (msAFP) appears at dramatically lower con-
centrations than amniotic fluid AFP (afAFP), demon-
strating approximately an additional 1000-fold gradient
comparing maternal circulation to amniotic fluid (Fig.
2.1). Thus, there is nearly a one-million-fold difference
between the fetal serum and maternal serum during
the second trimester. Throughout pregnancy, median
msAFP levels change predictably in unaffected pregnan-
cies according to gestational age [62,63].
Because of the gestational age dependence of AFP
in amniotic fluid and maternal serum, AFP levels are
normalized by first establishing median levels of AFP
for each week of gestation measured in International
Units (IU) per mL for amniotic fluid or mIU per mL
for msAFP. Alternatively, labs may express concentra-
tions in micrograms or nanograms per mL (afAFP and
msAFP, respectively). The patient-specific values are
then calculated as multiple of the median (MoM) by
dividing the patient’s measured AFP concentration by

Figure 2.1 Mean concentrations of alpha-fetoprotein (AFP) in maternal serum, amniotic fluid, and fetal serum
at various stages of pregnancy. (From Haddow, JE. prenatal screening for open neural tube defects, Down’s
syndrome, and other major fetal disorders. Semin. Perinatol. 1990;14:488–503, with permission.)
14 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
Figure 2.2 Distribution of maternal serum alpha-fetoprotein (AFP) (in MoM) in anencephalic, open spina
bifida, and unaffected pregnancies.
the appropriate median value. The distribution of val-
ues across all unaffected pregnancies is centered at 1.00
MoM, approximating a log-Gaussian distribution [64].
2.2.4.2 AFP in Pregnancies Affected with Neural
Tube Defects
The basis of msAFP as a screening test for ONTD is that
AFP in the amniotic fluid attains higher concentrations
when the fetus has an open defect because the protein
can pass directly into the amniotic fluid without having
to clear through the kidneys. This results in dramati-
cally higher levels in maternal circulation in pregnan-
cies affected with ONTDs. Maternal serum AFP values,
expressed in MoMs on a log10 scale approximate a Gauss-
ian distribution for the unaffected, open spina bifida and
anencephaly populations (Fig. 2.2). Because these values
are normally distributed, each distribution can be fully
described by the mean and variance or standard devia-
tion. A screening cutoff of 2.0–2.5 MoM is typically used
to discriminate pregnancies that are screen positive for
ONTD and which merit diagnostic testing or further
evaluation. Direct testing of afAFP levels requires an
invasive procedure (amniocentesis) and provides a diag-
nostic test for ONTDs when paired with acetylcholines-
terase (AChE) to eliminate rare false positives [65].
2.2.4.3 AFP in Pregnancies Affected with Down
Syndrome
The availability of population data on AFP levels during
pregnancy led to the discovery that msAFP values are
statistically lower when the fetus has DS/trisomy 21 [66].
Prior to the discovery of AFP as a potential biomarker
for DS, maternal age served as the only prenatal popula-
tion screening test. As a test for DS, maternal age is com-
bined with msAFP to identify pregnancies that merit
diagnostic testing or other follow-up [67]. Although the
sensitivity and specificity of msAFP plus maternal age
as a screening test is relatively poor, its adoption as a
screening test represented a major advancement for pre-
natal screening in that women of any age could benefit
from testing. While it is well known that the frequency
of DS is indeed positively correlated with increased
maternal age, it has been less well appreciated that most
DS is not identified when age alone is used as the pri-
mary screening test. Further study of other potential
biomarkers during pregnancy led to the discovery of
more than a dozen potential markers for DS and a wide
range of other birth defects, the details of which are pro-
vided below. AFP remains the only marker in clinical
use to screen for ONTD to date, however.
2.2.5 Prenatal Screening—Primary Focus on
NTD and Down Syndrome
Placing the many prenatal screening options into per-
spective requires one to step back and take stock of the
evolution of screening. Initially, screening was cen-
tered on AFP for NTD identification with calculation
of patient-specific risks for DS as a secondary benefit.
While AFP is a relatively poor prenatal marker for DS,
it was in essence free information obtained during NTD
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
screening that could be combined with maternal age
to identify DS in younger women who would be com-
pletely missed by age-only screening.
Over time, the availability of serum samples from a
large prenatal population led to the identification of doz-
ens of potential biomarkers for not only DS but an array
of other fetal anomalies and maternal conditions as well.
In a short period of time, the field shifted from identifica-
tion of a single disorder (i.e., NTD) in a narrow range of
gestational ages under established principles for screen-
ing [14] into a discovery phase for both common and
rare conditions. This was paralleled by similar changes in
newborn screening which began with just phenylketon-
uria screening and progressed to routine public health
screening of 30 or so conditions with the advent of tan-
dem mass spectroscopy and other emerging technolo-
gies [68,69]. In addition to serum peptides and hormonal
biomarkers, discovery of nonbiochemical markers such
as ultrasonic biometric measurements that could be
incorporated into screening was also proliferating.
In making sense of the many options for screening
there are two views one might consider: 1) the scientific
question of what combination of markers yields optimal
detection of DS and other disorders vs. 2) a pragmatic
view which takes into account the cost, performance,
and availability of reagents, the relative public interest
in and public health significance of different condi-
tions, the cost of diagnostic follow-up, and other sys-
tems issues such as the natural cadence of prenatal care
visits and data management challenges. The approach
one takes might lead to very different conclusions. For
example, there is extensive research on the many differ-
ent forms of hCG that appear in blood and urine at dif-
ferent points in pregnancy. Many (or all) of these forms
of hCG likely reflect the same underlying differences
between euploid and trisomy 21 fetuses and the ultimate
choice by any given laboratory of which one to include
in screening might well depend more on practical con-
siderations than on the small differences in performance
of any given marker. In addition, the protocol for follow-
ing up on screen-positive cases, such as the emergence
of cfDNA as a potentially efficacious and noninvasive
alternative to invasive procedures with significant iatro-
genic risks, will also matter [70]. Since the question of
prenatal screening arises in the context of clinical care,
this chapter places the central focus on the detection of
NTD and DS using the most pragmatic, common, and
available approaches.
15
The set of variables that should guide which screen-
ing approach to use would include which reagents and
testing platforms are readily available, common iden-
tifiable conditions for which interventions are feasible
and desired by the patient population, the cost of avail-
able reagents, options for resolving positive screen tests,
availability of ultrasound, and practical considerations
around sample collection and transport.
2.2.6 Biochemical Markers for Down
Syndrome and Other Conditions
2.2.6.1 In Serum
The availability of large numbers of blood samples col-
lected for the purpose of NTD screening during the
second trimester created the conditions necessary for
biomarker discovery for DS and other disorders. As DS
screening became routine and the availability of first
trimester diagnostic testing expanded during the 1980
and 1990s, study of biomarkers in maternal circulation
during the first trimester began to be explored to per-
mit a wider range of pregnancy management options for
patients. Obtaining blood samples is a routine compo-
nent of prenatal care, and most markers that are useful in
prenatal screening are available through serum testing.
In the first trimester of pregnancy, various forms of
hCG are altered in DS, the most studied of which are
free-β hCG and intact hCG. In addition, pregnancy-as-
sociated plasma protein A (PAPP-A) has been identified
as an effective biomarker in early pregnancy [71]. Serum
levels of hCG are generally elevated in DS compared with
euploid pregnancies, and there is an interesting correla-
tion between gestational age for different forms of hCG.
After 13 weeks, hCG is a slightly more effective marker
than free-β hCG, perhaps because of sample stability
[72]. From 11 to 13 weeks of gestation, free-β is the bet-
ter marker, and prior to 11 weeks, free-β discriminates
between DS and unaffected pregnancies, whereas hCG
does not. In contrast, PAPP-A levels are decreased in
DS [73]. A preponderance of the published work in first
trimester screening involved PAPP-A and free-β hCG
and the vast majority of that work combined these bio-
markers with maternal age and measurement of nuchal
translucency (NT) [74]. Other markers that have been
utilized in the first trimester include invasive tropho-
blast antigen (ITA) which is itself a hyperglycosylated
form of hCG (↑) [75], inhibin (↑) [76], AFP (↓) [77], uE3
(↓) [78], disintegrin and metalloproteinase domain-con-
taining protein 12 (ADAM 12) (↑) [79], placental growth
16 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
factor (PlGF) (↓) [80], growth hormone–binding pro-
tein (↑) [81], human placental lactogen (hPL) (↑) [82],
and placental protein 13 (PP13) (↓) [80]. Two studies
by Alldred and colleagues systematically reviewed the
available literature for age plus first trimester biochemi-
cal tests [83] and biochemical tests combined with ultra-
sound [74] for readers with an interest in greater detail
on the wide array of combinations of markers that have
been studied. Three findings of note from systematic
review of biochemical markers include the observations
that screening performance was notably higher when
PAPP-A was combined with free-β hCG (plus maternal
age) in a double marker test compared with AFP and
free-β hCG (plus maternal age); that triple tests were
associated with higher sensitivity but were not statisti-
cally better than the double test with PAPP-A and free-β
hCG; and that higher-order combinations showed simi-
lar patterns to the double and triple tests during the first
trimester with marginal gains using more markers.
In the second trimester, we continue to see various
forms of hCG showing elevated levels in DS. Biomark-
ers studied included AFP (↓) [66], uE3 (↓) [5], free-β (↑)
[84], total hCG (↑) [85], free-α hCG (↑) [3], inhibin A
(↑) [86], pregnancy-specific β 1-glycoproteins (SP1) (↑)
[87], CA125 (↓) [88], PAPP-A (↓) [89], PlGF (↑) [90],
and ProMBP (↓) [91]. A systematic review of the 12
best and most thoroughly evaluated strategies with sec-
ond trimester tests alone and in combinations revealed
detection rates between 41% (AFP only plus maternal
age) and 84% (a 5-biomarker test plus maternal age) at a
fixed false-positive rate (FPR) of 5% [92,93].
2.2.6.2 Blood Spots
Blood spots offer an alternative approach to sample col-
lection for some serum markers. Sample collection is
somewhat less invasive (finger prick vs. phlebotomy)
and perhaps more convenient when blood is not already
being drawn for other purposes as it does not require a
phlebotomist. Analyte stability may be higher or lower
depending on the analyte and the conditions under
which samples are shipped and stored prior to testing.
Palomaki et al. compared screening performance for
hCG and PAPP-A in blood spots and fresh maternal
serum samples and found higher variance in weight-ad-
justed MoM values for both hCG and PAPP-A using
blood spot collection and projected a somewhat lower
screening performance for DS based on those differences
[94]. Analyte stability in fresh serum may be subject to
higher variation under suboptimal collection and trans-
portation conditions, however, and certain analytes such
as free-β hCG may be more stable when collected as
blood spots as a general rule as has been suggested [95].
2.2.6.3 Urine
Maternal biochemical markers may also be evaluated
from urine samples collected during the first or second
trimester. Advantages to urine as a sample type are like
those found with blood spots. Different forms of hCG,
including β-core fragment, hyperglycosylated hCG
(which is also called ITA), and intact hCG, are found
in urine as is estriol. Alldred et al. reviewed 19 studies
involving 18,013 subjects with 527 true cases of DS com-
paring the performance of urinary markers alone and
in combinations in first trimester pregnancies without
consideration of maternal age for four forms of hCG
(total hCG, free-β hCG, β-core fragment, and hyper-
glycosylated hCG/ITA); in the second trimester with-
out consideration of maternal age for estriol, total hCG,
free-β hCG, β-core fragment, hyperglycosylated hCG/
ITA, and gonadotropins; and in the second trimester
with consideration of maternal age for estriol, β-core
fragment, free-β hCG, and hyperglycosylated hCG/
ITA [96]. While hyperglycosylated hCG alone performs
quite well as a biomarker for DS, the combination of
hyperglycosylated hCG, β-core fragment, and maternal
age performed the best with an estimated detection rate
of 92% with specificity set at 95%. The highest perfor-
mance noted came from a single study of second tri-
mester AFP and β-core fragment to estriol ratio with
consideration of maternal age that yielded a 90% sensi-
tivity with FPR of 5% [96].
2.2.7 Reagents and Platforms in Clinical
Perspective
The scientific literature that has been established around
the wide range of biomarkers in their many different
forms throughout the course of pregnancy describes
a broad array of potential approaches toward prenatal
screening. In clinical practice, however, laboratories are
constrained by the specific set of assays that are available
for clinical use. Biomarkers for which assays are avail-
able for use only in other countries or that are limited
to research use only applications are not candidates
for clinical use by any given laboratory. In addition,
instrumentation is itself expensive, and specific plat-
forms all offer only a limited choice of assays, and this
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy 17

further constrains the set of tests that a laboratory may
reasonably offer. Once a reagent platform is established
in a laboratory, there is a form of technological lock-in
that occurs that limits the addition of new markers to
the screening menu, particularly for smaller laborato-
ries similar to what has been seen with other emerging
technologies [97,98]. In extreme cases, a specific analyte
might be so valuable in the context of screening that
it must be included even when the added cost is rela-
tively high, but for most markers, there is only a small
incremental gain in performance for adding any specific
marker. As an example, adding free-α hCG or substitut-
ing free-β hCG for a different form of hCG might easily
be passed over by a laboratory if reagents are expensive,
hard to acquire, pose sample collection challenges, or
exist only on a platform not available to the laboratory.
2.2.8 Nonbiochemical Markers from
Ultrasound
As prenatal ultrasound has become iteratively more refined
throughout the 1980s and beyond, reliable identification
and measurement of fetal structures and biophysical fea-
tures associated with pregnancy came to be studied in nor-
mal pregnancies as well as pregnancies associated with DS
and other disorders. Two ultrasound features of particular
note are NT and nasal bone.
2.2.8.1 Nuchal Translucency
NT is the term used to describe a collection of fluid
behind the fetal neck recognized during the first tri-
mester (Fig. 2.3). While all fetuses have some degree
of measurable NT, fetuses with trisomy 21, as well as
other chromosome abnormalities, have measurements
that are two- to threefold larger than that of unaffected
fetuses [99], making it a powerful marker for estima-
tion of the risk of fetal aneuploidy. In addition to its
role in aneuploidy screening, an increased NT has also
been identified in a multitude of single-gene disorders
and structural malformations (e.g., congenital cardiac
defects, renal malformations, neuromuscular abnor-
malities) [100,101]. For this reason, any fetus with an
NT ≥ 3–3.5 mm should have a targeted ultrasound

Figure 2.3 Nuchal translucency measurement in the first trimester. Nuchal translucency (NT) within average
range: (A) fetal prone position and (B) Fetal supine position. Increased NT: (C) prone and (D) supine.
18 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy

evaluation for careful assessment of anatomy. NT as

the sole criteria for fetal aneuploidy screening detects
approximately 70%–75% of fetuses with trisomy 21,
and when combined with the maternal age–associated
a priori risk, a detection rate in excess of 80% is achiev-
able [102–106]. NT measurements are independent of
maternal serum levels of hCG and PAPP-A; therefore,
these markers can be combined into a single screening
test.

2.2.8.2 Nasal Bone

A high proportion of aneuploidy fetuses demonstrate
hypoplasia or failure to visualize the nasal bone in the
first trimester (Fig. 2.4). Interrogation of the fetal facial
profile in the first trimester is an extension of the phys-
ical examination performed in the neonatal period,
in which a flattened nasal bridge and profile are often
noted in aneuploidy infants, especially those with tri-
somy 21 [107]. Although dependent somewhat on eth-
nicity, up to 75% of fetuses with trisomy 21 will have
nonvisualization of the nasal bone in the first trimester
compared to fewer than 1% of euploid fetuses [8,107].
By adding the nasal bone to other first trimester screen-
ing, including hCG, PAPP-A, NT, and FHR, this strategy
decreased the FPR for trisomy 13, 18, 21, and Turner
syndrome and further increased the detection rate for
trisomy 21 [8,108].

2.2.8.3 Other Ultrasound Markers for Aneuploidy

Sufficient literature has been accumulated on the follow-
ing ultrasound endpoints for them to be included in a
systematic analysis of literature with respect to prenatal
screening [74]: NT [109], nasal bone [8], ductus venous
Doppler [110], maxillary bone length [111], fetal heart
rate [112], aberrant right subclavian artery [113], fron-
tomaxillary facial angle [114], presence of mitral gap
[115], tricuspid regurgitation and blood flow [116],
and iliac angle 90 degrees [117]. This systematic review
included study of 1,604,040 pregnancies, 8454 of which
were affected with DS, and included 152 publications
describing 126 different studies with consideration of
maternal age, first trimester biochemical markers, and
ultrasound features alone or in various combinations.
Comparison of 10 of the most well-studied screen-
ing strategies demonstrated superior performance of
a combined NT, PAPP-A, and free-β hCG with mater-
nal age compared with ultrasound markers alone with
a detection rate of 90% at a FPR of 5% without the
Figure 2.4 Middle sagittal image of fetus in supine position for
assessment of fetal nasal bone. (A) Nasal bone present—note
the echogenic area (arrow) below the skin echo to give appear-
ance of an equal sign. (B) Absent nasal bone.
inclusion of nasal bone. Maternal age plus ultrasound
without inclusion of biochemical markers demonstrated
a lower detection rate (71%) as did study of maternal
age plus biochemical markers alone (87%). Nasal bone
alone showed a detection rate of 49% at an FPR of 1%.
Because of the tradeoff between detection rate and FPR,
direct comparison of different screening approaches is
often complicated by the use (or observation) of differ-
ent FPRs. The highest performing combination at a 5%
FPR was found in a single study using the combination
of NT, fetal heart rate, ductus, free-β hCG, and PAPP-A
which demonstrated a 97% DR [74]. While ultrasound
is labor-intensive and requires careful standardization
to maintain consistency between different operators, it
is noninvasive and its inclusion in prenatal screening
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
alongside biochemical markers is effective. Among the
ultrasound markers, the inclusion of NT is well-docu-
mented as being effective and may be enhanced by the
inclusion of one or two other ultrasound markers. There
is a large body of evidence supporting the combination
of NT, PAPP-A, and free-β hCG with maternal age as an
approach to screening for DS in the first trimester [74].
2.3 RISK DETERMINATION AND
THRESHOLDS
Screening tests differ from diagnostic tests in that there
are false positives and false negatives that arise during
the testing process. In the case of prenatal screening for
NTDs and DS, this arises from the overlapping distri-
bution of screening markers alone or in combination
between the affected and unaffected populations. In
most cases, it is possible to increase the detection rate
by tolerating a higher FPR and vice versa. Choosing the
optimum tradeoff between detection and false positives
might take into consideration historical norms (e.g., risk
of DS to a 35-year-old woman), a population norm (e.g.,
increase above the general population risk), the magni-
tude and nature of iatrogenic risks of diagnostic testing,
or cost. Patient perceptions of risk are also important
and are not typically uniform across the population.
For example, a couple experiencing their third or fourth
pregnancy may not perceive the iatrogenic risk of mis-
carriage the same as a couple who has struggled for years
to attain pregnancy for the first time. Consequently, an
accurate determination of patient-specific risk from a
screening test may be of great value both to the patient
and to the provider. In the case of NTDs where only a
single marker is utilized in screening, risk computation
is generally not central to decision-making. Here it is the
relative AFP value, rather than risk that is most often
used to determine the threshold for a positive screen
test. In DS screening and other aneuploidies, where
multiple markers are included in the screening protocol,
risk offers a simple reference point to define a positive
screen test that relates to historical standards for inva-
sive testing.
Fetal development is of course a highly dynamic
process and it is hardly surprising that the biochem-
ical markers that are used in screening as well as the
presence, size, and shape of fetal structures that are
observed with ultrasound change throughout the preg-
nancy. A fetal marker that is produced by the liver that
19
enters the amniotic fluid through the fetal kidneys and
then diffuses across the fetal membranes and tissues to
enter the maternal circulation will be affected by the
size of the liver and kidneys, the timing of gene expres-
sion during pregnancy, and the surface area and mass
of the extraembryonic tissues (amnion, chorion, pla-
centa, etc.). Consequently, the expected values of most
biomarkers in affected and unaffected pregnancies are
gestational age-dependent. For any given biomarker, it
is therefore vital to know the expected distributions for
both the affected and unaffected populations across the
range of gestational ages for which screening is offered.
Many or most biomarkers approximate (or can be statis-
tically manipulated to approximate) a Gaussian distri-
bution by establishing the median values of the marker
in unaffected pregnancies at different gestational ages to
serve as a reference value. Each measured patient value
is divided by the median for the specific gestational
age at the time the sample was taken to establish a unit
referred to as a MoM [64]. The expected value within
the unaffected population is 1.0 MoM by definition for
every gestational age, and thus the challenge of chang-
ing expectations for each biomarker by gestational age is
addressed. There are just two variables required to fully
describe a Gaussian distribution: the mean and variance
(or standard deviation). In the unaffected population,
the mean value is fixed at 1.0 MoM across all gestational
ages, whereas the variance may change with gestational
age. This will be true for each biomarker that is quan-
tified for screening; the mean will be 1.0 MoM and the
variance needs to be established or verified for each ges-
tational age.
Each pregnancy population for which screening is
offered must also be characterized in terms of mean and
variance. The most useful markers for screening will
be those whose distributions overlap the least with the
unaffected population. This would be a combination of
the greatest magnitude of the difference in means com-
pared with the unaffected population combined with
the lowest values of variance.
2.3.1 Computation of Risk
The simplest case for computation occurs when there is
a single biomarker used for screening, such as AFP for
NTD screening. The determination of risk is based on
a Bayesian calculation in which an a priori risk estab-
lished for the pregnancy population under study is
modified by a likelihood ratio (LR) calculated from the
20 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
Figure 2.5 (A) Distribution of maternal serum alpha-fetoprotein (AFP) (in MoM) in unaffected and Down
syndrome pregnancies. (B) Estimation of the likelihood of an affected pregnancy associated with a specific
AFP level.
relative distances from the baseline of the affected and
unaffected distribution (Fig. 2.5). This is easy enough to
visualize when there is a single marker as it represents
the length of the line for the affected population at some
measured value divided by the length of the line for the
unaffected population. A value greater than 1.0 rep-
resents an increased risk, and a value less than 1.0 indi-
cates a decreased risk. The single point at which the two
distributions cross represents a singular value at which
the risk is unchanged and is therefore the same as the
population risk (Fig. 2.5).
When there are multiple markers in use, the calcu-
lation is made in essentially the same way, but there is
some added complexity. Again, the calculation begins
with the a priori risk from the population. Here, how-
ever, there are correlation coefficients that must be deter-
mined between the various markers that are included in
the screening test. These must be established for both the
affected and unaffected populations for every combina-
tion of markers included in the test. In the case of multi-
ple markers, the LR is the equivalent of the Mahalanobis
distance of the multivariate Gaussian distribution at the
measured values of the various analytes [118].
2.3.2 A Priori Risks and Distribution Limits
Care must be taken to ensure that the a priori risk is as
accurate as possible. It is also vital to establish the range
of measured values for which the Gaussian distributions
are stable and to address the way extreme values will be
handled as the LR may behave erratically in the tails of
the distributions. In the case of NTD, a priori risks may
be significantly different based on family history, type I
diabetes, and race.
In the case of DS and other aneuploidies, a priori risks
typically consider the age of the mother (or the age of the
egg donor in the case of in vitro fertilization) and family
history. Women with a prior aneuploid pregnancy carry
an increased risk of recurrence in subsequent pregnan-
cies. For DS, the relative risk of recurrence among all
women is approximately double [119,120]. The risk is
higher in women who carry the first affected pregnancy
before age 35 with a relative risk of 3.5 [119]. The risk of
recurrence for trisomy 18 is more than triple, whereas
the relative risk of recurrence for trisomy 13 is nearly
10-fold [119]. Women with any offspring with trisomy
are at increased risk for a different trisomy in a subse-
quent pregnancy [119,120]. These recurrences can be
explained by possible gonadal mosaicism, in cases of
the homotrisomy recurrence, or by increased rates of
meiotic error in women with homo- or heterotrisomy
recurrences. These numbers may be used to alter the a
priori risk when counseling women with a prior affected
pregnancy and allow better risk estimation than the tra-
ditional 1% risk estimate provided to most women after
a prior trisomic conception.
In practice, risk algorithms for clinical screening may
use published estimates of the population parameters
for the means, standard deviations, and correlation coef-
ficients for each of the markers and marker combina-
tions, but larger screening programs may use in-house
population parameters if they are confident that they
do not have ascertainment bias that might arise from
only being able to positively identify detected cases, for
example [121–124].
Several maternal variables are known to affect a pri-
ori risk estimates and/or the relative distributions of
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
different biomarkers used in screening, and corrections
may be made for each variable individually to provide
more accurate patient-specific risk estimates. Correc-
tions may be made in the use of different median values
for different populations, adjustments in the calculation
of the MoM values, use of different population param-
eters in the risk algorithm, or adjustment of the a priori
risk estimates. This would include age [57,125], family
history [126], gestational age [2,127–129], method for
gestational age dating [130], use of assisted reproductive
technologies [2,131,132], twins [133,134], race/ethnicity
[135,136], maternal weight [137–139], maternal insulin
use [132,140–142], and cigarette smoking [143–145].
2.4 MODALITIES OF TESTING FOR NTD
AND DOWN SYNDROME
There remains just one effective second trimester
marker in clinical use for NTD screening and conse-
quently a single modality for serum screening for NTD.
In contrast, there are far too many different combina-
tions of all possible markers for DS screening to merit
evaluation of all of them or the establishment of clini-
cal screening operations for most of the modalities that
have been piloted. As detection rates have increased
over time with the addition of new screening markers,
there is a diminishing return on the inclusion of addi-
tional markers to detect residual cases, and that process
seems to be approaching its limit. In addition, with the
discovery of the comparatively higher sensitivity and
specificity of circulating cfDNA as a means of directly
sampling fetal chromosome copy number, and with the
availability of excellent systematic reviews of serum and
ultrasound biomarker performance, it seems likely that
the biochemical and ultrasound modalities will settle
into a smaller number of well-studied protocols for DS
screening for the foreseeable future.
There are now four primary modalities that are com-
mon in DS and other aneuploidy screening. These include
serum screening using second trimester biochemical
markers [146], first trimester biochemical markers with
or without ultrasound [147], integrated screening using
first and second trimester biochemical markers with or
without ultrasound evaluation in the second trimester
[148], and contingent screening—similar to integrated
screening with risk determination in the first trimes-
ter and a decision for either further screening or diag-
nostic testing based upon the calculated first trimester
21
risk [149]. In addition, there remains testing that is not
based on serum screening including blood spot–based
screening in the first trimester and urine-based screen-
ing as described below. The optimal strategy will differ
depending upon the population being tested and the rel-
ative availability of medical technologies, expertise, and
laboratory reagents.
2.4.1 Second Trimester Biochemical Screening
for Down Syndrome and Trisomy 18
The majority of second trimester biochemical screening
is conducted between 15 and 22 weeks of gestation, and
is based on modification of a maternal age–based a pri-
ori risk estimate using a Bayesian calculation with the
LR determined from a multivariate gaussian distribution
of four maternal serum markers: AFP, UE3, DIA, and
some form of hCG [150]. The term quad screen gener-
ally refers to this set of markers. Using this method and
setting the screen-positive risk threshold at 1:270 risk
for DS at term, the detection rate is approximately 80%
with a corresponding FPR of 5%. Prior to the discov-
ery and successful piloting of DIA, the test was referred
to as triple screening and had slightly lower sensitivity
(∼61%) with a 5% FPR. Other markers can be substi-
tuted, added, or removed from the panel provided that
the distribution parameters for maternal serum within
the unaffected and DS populations are known [93].
Trisomy 18 risk can also be estimated from the same
measurement of markers (including maternal age),
although DIA is not informative in the trisomy 18 risk
calculation. The detection rate for trisomy 18 using a
screen-positive risk threshold of 1:100 is similar to that
of trisomy 21 at ∼78% with a small incremental FPR
of 0.2%. Interestingly, some additional cases of DS are
identified in pregnancies that screen negative for DS but
positive for trisomy 18 [151].
2.4.2 First Trimester Biochemical Screening
with or Without Ultrasound for Aneuploidy
Detection of DS in the first trimester is typically done
during weeks 10–13 of pregnancy (menstrual dating)
with measurement of PAPP-A and the free-β form of
hCG obtained from maternal serum or dried blood
spots. When combined with ultrasound measurement
of NT, the detection rate for DS is 87% at an FPR of 5%
[74]. By comparison, screening in the first trimester
with biochemistry only was found to have a detection
rate of 68% with an associated FPR of 5% [83].
22 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
Focusing on the combined detection rate for trisomy
18, trisomy 13, monosomy X (45,X), and triploidy using
the AFP, PAPP-A and free-β hCG combined with NT or
cystic hygroma identification was found to be 78% with
an associated FPR of 6%. Limiting screening to trisomy
18 and trisomy 13 using a protocol by Spencer et al.,
the estimated combined detection rate was 95% with an
associated FPR of 0.3% [152].
2.4.3 Integrated First and Second Trimester
Screening
While there initially were logistical issues that complicated
the clinical application of integrating first trimester bio-
chemistry (with or without ultrasound) with second tri-
mester biochemistry testing, this approach has since been
successfully employed in screening in many settings. In
this protocol, maternal age is used to establish the a priori
risk for DS, PAPP-A is measured from maternal serum
or blood spots in the first trimester, and the analytes AFP,
hCG, UE3, and DIA are measured from maternal serum
in the second trimester. Ultrasound measurement of NT
may also be added from the first trimester. In the serum
integrated test where only the biochemistry results are
included in screening, the detection rate is 87% with an
associated FPR of 5%. In the full integrated test where NT
measurement is included, the DR increases to 95% with
an associated FPR of 5% [74,123].
2.4.4 Contingent Testing
In the contingent model, the same overall markers are
used as with the integrated test; however, free-β hCG
is performed in the first trimester. The screen-positive
threshold is set such that the top 0.5% of women for DS
risk is identified as being screen positive based on the
first trimester results alone resulting in an initial DR of
66%. The remaining women with risks for DS ≤ 1:2000
based on first trimester results are reported out as screen
negative with no additional testing. Women whose risks
are >1:2000 then complete second trimester testing. The
overall detection rates between the combined test and
the contingent test are roughly equal; however, the FPR
for the combined test is 2.15% versus a slightly higher
FPR of 2.4% for the contingent test. Compared with
the combined test, however, there is earlier diagnosis
or resolution to screen-negative status for the 0.5% of
women with the highest risks. In addition, contingent
testing results in lower testing costs for serum markers
and reduced anxiety for the 78% or so of women who
do not have to wait for second trimester testing to be
completed [153].
2.5 FOLLOW-UP TO POSITIVE SCREENS—
NTD
2.5.1 Biochemical Analysis from Amniotic
Fluid
Prior to the development of high-resolution ultrasound,
patients with screen-positive NTD tests were offered
amniocentesis for biochemical diagnostic testing using
AFP. In the amniotic fluid, AFP levels are approxi-
mately 1000 times as concentrated as in the maternal
serum. Median values of afAFP change over time, and
the determination of MoMs is a stabile unit of measure
from 14 to 23 weeks of gestation. There is comparatively
little overlap between the affected and unaffected pop-
ulations for NTDs, and a threshold of 2.0 MoM offers
100% detection with very few false-positive tests. A sec-
ond protein, AChE, is useful in diagnosis of open NTDs
in amniotic fluid samples. AChE is normally absent in
the amniotic fluid unless there is an open neural lesion.
Evaluation for AChE allows for the elimination of the
small number of false-positive afAFPs based on the 2.0
MoM threshold [65].
2.5.2 Prenatal Ultrasound in NTD Screening
As ultrasound equipment and techniques have become
refined over time, positive identification of spina bifida
and anencephaly can often be achieved by ultrasound in
the second trimester.
In addition to the important role that ultrasound
plays in accurate dating and appropriate interpretation
of amniotic fluid and maternal serum AFP levels, ultra-
sound screening for major fetal anomalies in the second
trimester has become a routine part of prenatal care in
many countries [154]. Detailed evaluation of most fetal
anatomy is best accomplished at 18–20 weeks of ges-
tation as major anomalies can be diagnosed, and time
allowed for the option of pregnancy termination. While
many anomalies can be visualized earlier, this time also
allows for better evaluation of the cardiac anatomy than
is possible at earlier gestational ages. The detection rates
of NTDs by ultrasound vary considerably with the qual-
ity of the equipment, the fetal position, the maternal
habitus, and the experience of the operator. In expert
hands, ultrasonography alone has 97% sensitivity and
100% specificity in the detection of NTDs [155].
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
Figure 2.6 (A) Sagittal and (B) coronal ultrasound images of a
midtrimester fetus with anencephaly. Note the absence of the
cranium above the level of the orbits and the presence of only
amorphous cerebral tissue.
Anencephaly results from failed closure of the
cephalic portion of the neural tube and disrupted cranial
development resulting in exencephaly, the precursor of
anencephaly. Ultrasound findings include decreased
crown-rump length, absent calvaria, extruding lobu-
lated cerebral tissue (exencephaly) or absent neural tis-
sue, abnormal head shape with the eyes delineating the
upper portion of the fetal face in the coronal plane, and,
later in gestation, polyhydramnios due to impaired fetal
swallowing (Fig. 2.6) [156]. Given the marked abnor-
malities evident on ultrasound, the detection rates for
anencephaly approach 100% [157]. As ossification of the
23
Figure 2.7 Axial view of fetal head demonstrating findings
associated with a neural tube defect. The normal contour of
the articulation of the frontal bones is altered so that there is
an overlap and scalloping to generate the “lemon sign.” In the
posterior fossa, the cerebellum is inferiorly displaced and elon-
gated consistent with a Type II Arnold–Chiari malformation giv-
ing the appearance of the “banana sign.”
skull is not visualized until after 12 weeks of gestation,
care should be taken in diagnosing anencephaly before
this time. The abnormal contour of the head and amor-
phous appearing cerebral tissue are often visible in the
first trimester.
Open spina bifida is detected by both cranial and spi-
nal abnormalities on ultrasound. The two major cranial
anomalies, the so-called “lemon” and “banana” signs, are
typically easily recognized [158]. The lemon sign describes
the flattening or concavity of the frontal bones of the skull
visible in the transverse plane (Fig. 2.7). Decreased intra-
spinal pressure with concomitant downward displace-
ment of the brain and decreased intracranial pressure is
thought to cause this abnormality [159]. This theory is
supported by the fact that the lemon sign is visible in 98%
of fetuses with open spina bifida before 24 weeks but in
only 13% after 24 weeks when ossification of the cranium
has progressed [160]. The banana sign signifies the cau-
dal displacement of the cerebellum with alignment of the
cerebellar hemispheres as part of type II Arnold–Chiari
malformation (Fig. 2.7). In contrast to the lemon sign,
this abnormality persists throughout gestation although
visualization with ultrasound becomes more difficult in
the third trimester [156].
Approximately 99% of fetuses with open spina bifida
lesions exhibit at least one cranial finding on ultrasound
24 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
[161]. Both lemon and banana (or absent cerebellum)
signs were observed in 97% of fetuses with spina bifida,
ventriculomegaly in 75%, cisterna magna obliteration
in 68%, and diminished biparietal diameter in 61% of
fetuses. These findings, including an effaced cisterna
magna, a small posterior fossa, and a small cerebellum,
are strongly (>90%) associated with spina bifida [162].
This study found that ventriculomegaly and the lemon
sign were less frequently associated with spina bifida
(81% and 53%, respectively). As nearly all fetuses with
closed spina bifida will have normal cranial anatomy,
this may be used to differentiate between open and
closed lesions [163].
Sonographic identification of the area of spinal dys-
raphism is often more difficult than detecting cranial
abnormalities in spina bifida. The length of the spine
must be examined in the axial, coronal, and sagittal
planes [156]. The axial view is most useful for visual-
ization of all three ossification centers and will show
splaying of the posterior lamina in fetuses with spina
bifida [164]. If the protruding sac can be visualized,
the thickness of the sac wall may indicate if the defect
is open or closed [164]. Additionally, the approximate
level of the lesion can be identified, which can provide
some prognostic data regarding future neurological
function [165]. Three-dimensional ultrasound has gar-
nered recent attention as an adjuvant imaging tool for
fetal anatomy [166,167]. This is a particularly useful tool
in imaging the fetal spine as its entirety cannot be visu-
alized in a single plane. Furthermore, three-dimensional
ultrasound is less dependent on the technical skills of
the operator and may uncover some spinal anomalies
that would otherwise go unrecognized [168].
More recent studies have examined first trimester
sonographic markers of spina bifida. At 11–13 weeks,
the nasal bone is visualized, and the NT is measured to
evaluate for aneuploidy. In the same midsagittal view,
the fourth cerebral ventricle can be visualized as an
intracranial translucency. Preliminary studies indicate
that absence of intracranial translucency is a specific
marker of spina bifida [169,170] but this screen is only
50% sensitive for the anomaly [170]. This has not been
evaluated in large-scale trials.
2.5.3 Follow-up to Positive Screens—Down
Syndrome and Other Aneuploidies
There are three primary modes of follow-up for pregnan-
cies that screen positive for DS and other chromosomal
abnormalities; two are diagnostic, and a third and
newer approach involves the use of a nucleic acid–based
screening test.
2.5.3.1 Diagnostic Testing
Diagnostic testing can be performed for nearly all chro-
mosome abnormalities through direct study of embry-
onic or extra-embryonic cells obtained either through
amniocentesis or CVS. Extensive reviews of these proce-
dures are available elsewhere [171,172]. Amniocentesis
has the advantages of being slightly more accurate and
safer, while CVS offers the distinct advantage of allow-
ing testing at an earlier gestational age. Amniocentesis
is typically performed between 15 and 22 weeks of ges-
tation and is therefore not an option for screen-positive
diagnostic follow-up in the first trimester. CVS, however,
may be performed safely as early as 9 weeks of gestation.
In terms of accuracy of the testing, CVS obtains cells
that are all extra-embryonic in origin and may identify
false-positive chromosome abnormalities that are not
found in the fetus. By contrast, amniocentesis obtains
amniocytes and fibroblasts from within the gestational
sac which are not subject to false-positive findings.
2.5.3.2 Reflex Screening via Cell-free DNA
In recent years, circulating cfDNA has been developed
and refined as a noninvasive procedure for screening for
fetal chromosome abnormalities [6,173,174]. Screening
with cfDNA has been shown to have a very high sensi-
tivity and negative predictive value (NPV) which makes
it a highly useful test in following up on screen-positive
cases based on serum markers. While it might seem
counterintuitive to use a second screening test to follow
up on the original screen-positive test, in practice it has
performed exceptionally well [175].
2.6 MAINTAINING AND MONITORING
SCREENING PERFORMANCE
Laboratories that offer clinical testing must fully meet
the prevailing standards for clinical practice in their
communities in the preanalytic, analytic, and pos-
tanalytic phases of testing. This includes a need to
establish their ability to solicit and reliably collect and
track relevant patient information, establish gesta-
tional age of the fetus, collect and transport samples,
validate assays for each analyte, establish norms for
each marker across the full range of gestational ages
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
for which screening is offered, reliably measure each
of the analytes in use, demonstrate the consistency of
measurement for each analyte over time and across
different lots of reagents, demonstrate consistent
performance against peer laboratories through pro-
ficiency testing schemes, calibrate instrumentation,
and perform epidemiological monitoring over time.
The reader is referred to published professional prac-
tice guidelines from professional societies that are
updated periodically to reflect the evolving standards
for clinical practice [65,72].
2.6.1 Measures of Screening Performance
Screening tests differ from diagnostic tests in that
the distributions of affected and unaffected individ-
uals overlap. Consequently, there are false-positive
and false-negative tests. There are four primary mea-
sures of screening efficacy that help the practitioner
and patient understand the reliability of the screen-
ing results: sensitivity (or detection rate), specificity,
PPV, and NPV.
Sensitivity and specificity are measures of the reli-
ability of the screening test relative to the correct
identification of affected and unaffected individuals,
respectively, within the population. These two mea-
sures are independent of the frequency of the disorder
in the population. Sensitivity answers the question,
“what is the likelihood that the test will correctly detect
an affected case?”, while specificity answers the ques-
tion, “what is the chance that an unaffected fetus will
be correctly identified through screening?” The FPR is
often used to convey the same information as specific-
ity. FPR is (1—specificity) and expresses the expected
proportion of unaffected pregnancies that are identified
as screen positive. Sensitivity and specificity exist in a
dynamic tension such that one can be improved at the
expense of the other.
The second pair of measures, the PPV and NPV, are
measures of reliability with respect to test outcomes—
the extent to which a positive or negative test can be
trusted. PPV is highly sensitive to birth prevalence with
rare disorders showing lower PPV values given fixed
values for sensitivity and specificity. PPV answers the
question, “given that I have a positive screen test, what
is the likelihood that the fetus is truly affected?” Con-
versely, NPV answers the question, “given that I have a
negative screen test, what is the likelihood that the fetus
is truly unaffected?”
25
2.6.2 Detection Rate and Specificity
Tradeoff in Multiple Marker Screening
Comparisons
One of the challenges in making comparisons between
studies with different markers relates to the tradeoff
between sensitivity and specificity because there are
a very large number of possible combinations. One
common approach to simplify analysis in the prenatal
screening literature is to make comparisons of detec-
tion rates when FPR is held constant (often at 5%).
Alternatively, one can show the entire receiver oper-
ating characteristic (ROC) curve of each of the dif-
ferent tests to illustrate this tradeoff. ROC curves plot
detection rate on the y-axis against FPR or (1—speci-
ficity) on the x-axis. A superior test lies above and to
the left on the ROC curve of a comparison test, and a
perfectly uninformative test is represented by the 45
degrees diagonal line. In theory, evaluation of the area
under the curve (AUC) for ROC curves would permit
comparison of different screening strategies; however,
study of AUC in this context has not proven to be effec-
tive [176,177].
2.7 KEEPING SCREENING IN PERSPECTIVE
NTDs and DS are among the most common serious
birth defects in human populations, and many patients
are cognizant of their seriousness and frequency.
­Consequently, it is common for patients to want to
undergo screening for these disorders. This is consistent
with published guidelines surrounding medical screen-
ing that generally confine medical screening to disorders
that are relatively common, have some serious health
consequence, and for which there is some actionable
strategy related to treatment or prevention. Rarer disor-
ders, even if they are serious and preventable, generally
do not merit screening because false-positive tests tend
to dominate over true positives for uncommon disor-
ders. However, markers that are already being measured
for ONTD and DS may be suitable for interpretation of
other disorders (e.g., Edwards syndrome—trisomy 18)
that would not merit the cost and implementation of a
screening test of their own. Rarer conditions or condi-
tions associated with a lower morbidity may be passed
over altogether in the interest of containing cost and
avoiding unnecessary testing associated with false posi-
tives that result from the relatively low PPV seen in rare
conditions.
26 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
2.8 SUMMARY
The discovery in the late 1990s that cfDNA can be reli­
ably identified in maternal blood and the subsequent
development of methodologies for directly identifying
chromosomal material associated with DS and other
chromosomal disorders has dramatically changed the
practice of prenatal screening for DS and other aneuploi­
dies [6,175,178]. The very low iatrogenic risk associated
with cfDNA screening as a reflex test for screen-positive
serum screening, displacing amniocentesis or CVS [70],
may influence the strategy regarding the establishment
of decision thresholds for serum screening such that
lower initial specificity might be tolerated as a means of
improving overall detections rates.
Prenatal screening for NTD and DS for women of
all ages with a combination of biochemical and sono-
graphic analysis has supplanted maternal age as the
dominant standard of care in the United States and in
many other countries. Advances in biomarker screen-
ing and ultrasound technology have improved detec-
tion rates and decreased FPRs, dramatically reducing
the use of invasive diagnostic procedures. The more
recent introduction of testing for cfDNA in maternal
circulation introduced additional tools for prenatal
identification of aneuploidies and single gene disorders;
however, the population utilization of serum screening
augmented with ultrasound measurements has contin-
ued with only a modest change in utilization rates. Even
as newer methods have taken hold and older approaches
have stabilized and simplified, the fundamental strategy
of focusing prenatal screening on common and serious
disorders over which patients may be empowered to
exercise some measure of control continues as a sound
strategy to meet public demand with increasing bene-
fits to younger mothers and women without known risk
factors.
REFERENCES
[1] Crandall BF, Lebherz TB, Schroth PC, Matsumoto
M. Alpha-fetoprotein concentrations in mater-
nal serum: relation to race and body weight. Clin
Chem 1983;29(3):531–3. https://doi.org/10.1093/
clinchem/29.3.531. PMID: 6186415.
[2] Cuckle HS, Wald NJ, Lindenbaum RH. Maternal
serum alpha-fetoprotein measurement: a screening
test for Down syndrome. Lancet 1984;1(8383):926–9.
https://doi.org/10.1016/s0140-6736(84)92389-4.
[3] Bogart MH, Pandian MR, Jones OW. Abnormal
maternal serum chorionic gonadotropin levels in preg-
nancies with fetal chromosome abnormalities. Prenat
Diagn 1987. https://doi.org/10.1002/pd.1970070904.
[4] Hallahan TW, Krantz DA, Tului L, Alberti E, Bu-
chanan PD, Orlandi F, Klein V, Larsen JW, Macri
JN. Comparison of urinary free beta (hCG) and
beta-core (hCG) in prenatal screening for chromo-
somal abnormalities. Prenat Diagn 1998. https://doi.
org/10.1002/(SICI)1097-0223(199809)18:9<893::AID-
PD362>3.0.CO;2-E.
[5] Canick JA, Knight GJ, Palomak GE, Haddow JE, Cuck-
le HS, Wald NJ. Low second trimester maternal serum
unconjugated oestriol in pregnancies with Down’s syn-
drome. BJOG An Int J Obstet Gynaecol 1988. https://
doi.org/10.1111/j.1471-0528.1988.tb06601.x.
[6] Dennis Lo YM, Corbetta N, Chamberlain PF, Rai V,
Sargent IL, Redman CWG, Wainscoat JS. Presence of
fetal DNA in maternal plasma and serum. Lancet 1997.
https://doi.org/10.1016/S0140-6736(97)02174-0.
[7] Benacerraf BR, Gelman R, Frigoletto FD. Sonographic
identification of second-trimester fetuses with down’s
syndrome. N Engl J Med 1987. https://doi.org/10.1056/
nejm198711263172203.
[8] Cicero S, Avgidou K, Rembouskos G, Kagan KO,
Nicolaides KH. Nasal bone in first-trimester screening
for trisomy 21. Am J Obstet Gynecol 2006. https://doi.
org/10.1016/j.ajog.2005.12.057.
[9] Dicke JM, Gray DL, Songster GS, Crane JP. Fetal
biometry as a screening tool for the detection of
chromosomally abnormal pregnancies. Obstet Gynecol
1989.
[10] Lockwood C, Benacerraf B, Krinsky A, Blakemore K,
Belanger K, Mahoney M, Hobbins J. A sonograph-
ic screening method for Down syndrome. Am J
Obstet Gynecol 1987. https://doi.org/10.1016/S0002-
9378(87)80059-5.
[11] Kagan KO, Valencia C, Livanos P, Wright D, Nico-
laides KH. Tricuspid regurgitation in screening for
trisomies 21, 18 and 13 and Turner syndrome at 11
+ 0 to 13 + 6 weeks of gestation. Ultrasound Obstet
Gynecol 2009b. https://doi.org/10.1002/uog.6264.
[12] Liao AW, Snijders R, Geerts L, Spencer K, Nicolaides
PKH. Fetal heart rate in chromosomally abnormal
fetuses. Ultrasound Obstet Gynecol 2000. https://doi.
org/10.1046/j.1469-0705.2000.00292.x.
[13] Sturdy S, Miller F, Hogarth S, Armstrong N,
Chakraborty P, Cressman C, Dobrow M, Flitcroft
K, Grossman D, Harris R, Hoebee B, Holloway K,
Kinsinger L, Krag M, Loblova O, Lowy I, Mackie A,
Marshall J, O’Hallahan J, Zappa M. Half a century of
Wilson & Jungner: reflections on the governance of
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
population screening [version 2; peer review: 3 ap-
proved]. Wellcome Open Res 2020;5(158). https://doi.
org/10.12688/wellcomeopenres.16057.2.
[14] Wilson JMG, Jungner G, Organization WH. In:
­Wilson JMG, Jungner G, editors. Principles and
­practice of screening for disease. World Health Orga-
nization; 1968.
[15] Janz NK, Becker MH. The health belief model: a
decade later. Health Educ Behav 1984. https://doi.
org/10.1177/109019818401100101.
[16] Rosenstock IM. Historical origins of the health
belief model. Health Educ Behav 1974. https://doi.
org/10.1177/109019817400200403.
[17] Copp AJ, Greene NDE. Genetics and development
of neural tube defects. J Pathol 2010. https://doi.
org/10.1002/path.2643.
[18] Northrup H, Volcik KA. Spina bifida and other neural
tube defects. Curr Probl Pediatr 2000. https://doi.
org/10.1067/mpp.2000.112052.
[19] Detrait ER, George TM, Etchevers HC, Gilbert JR,
Vekemans M, Speer MC. Human neural tube defects:
developmental biology, epidemiology, and genetics.
Neurotoxicol Teratol 2005. https://doi.org/10.1016/j.
ntt.2004.12.007.
[20] Baird PA, Sadovnick AD. Survival in infants
with anencephaly. Clin Pediatr 1984. https://doi.
org/10.1177/000992288402300505.
[21] Ghi T, Dall’asta A, Pilu G, Contro E, De Musso F,
Frusca T. Neural tube defects. In: Obstetric imaging:
fetal diagnosis and care. 2nd ed. 2017. https://doi.
org/10.1016/B978-0-323-44548-1.00041-3.
[22] Milani HJF, Barreto de EQS, Chau LH, To NH, Moron
AF, Meagher S, Da Silva Costa F, Araujo Júnior E.
Prenatal diagnosis of closed spina bifida: multicenter
case series and review of the literature. J Matern Fetal
Neonatal Med 2020. https://doi.org/10.1080/14767058.
2018.1500543.
[23] Gardener G, Rodeck C. Prenatal screening and diagno-
sis of neural tube defects. Prenat Diagn 2006.
[24] Williams J, Mai CT, Mulinare J, Isenburg J, Flood TJ,
Ethen M, Frohnert B, Kirby RS, Centers for Disease
Control and Prevention. Updated estimates of neural
tube defects prevented by mandatory folic Acid fortifi-
cation - United States, 1995–2011. MMWR 2015.
[25] Zaganjor I, Sekkarie A, Tsang BL, Williams J, Razzaghi
H, Mulinare J, Sniezek JE, Cannon MJ, Rosenthal
J. Describing the prevalence of neural tube defects
worldwide: a systematic literature review. PloS One
2016. https://doi.org/10.1371/journal.pone.0151586.
[26] Prevention of neural tube defects: results of the
medical research council vitamin study. Lancet 1991.
https://doi.org/10.1016/0140-6736(91)90133-A.
27
[27] Bassuk AG, Kibar Z. Genetic basis of neural tube
defects. Semin Pediatr Neurol 2009. https://doi.
org/10.1016/j.spen.2009.06.001.
[28] Neumann PE, Frankel WN, Letts VA, Coffin JM, Copp
AJ, Bernfield M. Multifactorial inheritance of neural
tube defects: localization of the major gene and recog-
nition of modifiers in ct mutant mice. Nat Genet 1994.
https://doi.org/10.1038/ng0494-357.
[29] Greene NDE, Stanier P, Copp AJ. Genetics of human
neural tube defects. Hum Mol Genet 2009. https://doi.
org/10.1093/hmg/ddp347.
[30] Seller MJ. Recurrence risks for neural tube defects in
a genetic counselling clinic population. J Med Genet
1981. https://doi.org/10.1136/jmg.18.4.245.
[31] Hall JG, Friedman JM, Kenna BA, Popkin J, Jawanda
M, Arnold W. Clinical, genetic, and epidemiological
factors in neural tube defects. Am J Hum Genet 1988.
[32] Hernández-Díaz S, Werler MM, Walker AM, Mitchell
AA. Neural tube defects in relation to use of folic acid
antagonists during pregnancy. Am J Epidemiol 2001.
https://doi.org/10.1093/aje/153.10.961.
[33] Mcmahon DM, Liu J, Zhang H, Torres ME, Best RG.
Maternal obesity, folate intake, and neural tube defects
in offspring. Birth Defects Res A Clin Mol Teratol
2013. https://doi.org/10.1002/bdra.23113.
[34] Louis-Jacques AF, Odibo AO, Bradshaw RJ. Meck-
el-gruber syndrome. In: Obstetric imaging: fetal diag-
nosis and care. 2nd ed. 2017. https://doi.org/10.1016/
B978-0-323-44548-1.00133-9.
[35] Hook EB, Hamerton JL. Analyses of data on rates of
cytogenetic disorders in live births. Am J Hum Genet
1978.
[36] Forabosco A, Percesepe A, Santucci S. Incidence of
non-age-dependent chromosomal abnormalities: a
population-based study on 88965 amniocenteses.
Eur J Hum Genet 2009. https://doi.org/10.1038/
ejhg.2008.265.
[37] Hook EB, Waller DK. Prenatal screening for neural
tube defects and chromosome abnormalities: the
complexities of program evaluation. Epidemiology
1995;6(1):1–4.
[38] Mai CT, Isenburg JL, Canfield MA, Meyer RE, Correa
A, Alverson CJ, Lupo PJ, Riehle-Colarusso T, Cho
SJ, Aggarwal D, Kirby RS. National population-based
estimates for major birth defects, 2010–2014. Birth
Defects Res 2019. https://doi.org/10.1002/bdr2.1589.
[39] Hassold T, Hunt P. To err (meiotically) is human: the
genesis of human aneuploidy. Nat Rev Genet 2001.
https://doi.org/10.1038/35066065.
[40] Korenberg JR, Chen XN, Schipper R, Sun Z, Gonsky
R, Gerwehr S, Carpenter N, Daumer C, Dignan P,
Disteche C, et al. Down syndrome phenotypes: the
28 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
consequences of chromosomal imbalance. Proc Natl
Acad Sci USA 1994;91(11):4997–5001. https://doi.
org/10.1073/pnas.91.11.4997.
[41] Antonarakis SE, Skotko BG, Rafii MS, Strydom A,
Pape SE, Bianchi DW, Sherman SL, Reeves RH.
Down syndrome. Nat Rev Dis Prim 2020. https://doi.
org/10.1038/s41572-019-0143-7.
[42] Hobson-Rohrer WL, Samson-Fang L. Down syn-
drome. Pediatr Rev 2013. https://doi.org/10.1542/
pir.34-12-573.
[43] Fidler DJ. The emergence of a syndrome-specific
personality profile in young children with Down
syndrome. Downs Syndr Res Pract 2006. https://doi.
org/10.3104/reprints.305.
[44] Staples AJ, Robertson EF, Ranieri E, Ryall RG, Haan
EA. A maternal serum screen for trisomy 18: an exten-
sion of maternal serum screening for down syndrome.
Am J Hum Genet 1991.
[45] Cereda A, Carey JC. The trisomy 18 syndrome. Or-
phanet J Rare Dis 2012. https://doi.org/10.1186/1750-
1172-7-81.
[46] Crider KS, Olney RS, Cragan JD. Trisomies 13 and 18:
population prevalences, characteristics, and prenatal
diagnosis, metropolitan Atlanta, 1994–2003. Am J Med
Genet 2008. https://doi.org/10.1002/ajmg.a.32200.
[47] Gaw SL, Platt LD. Trisomy 18. In: Obstetric imaging:
fetal diagnosis and care. 2nd ed. 2017. https://doi.
org/10.1016/B978-0-323-44548-1.00150-9.
[48] Wyllie JP, Wright MJ, Burn J, Hunter S. Natural
history of trisomy 13. Arch Dis Child 1994. https://doi.
org/10.1136/adc.71.4.343.
[49] Robinson WP, McFadden DE, Stephenson MD. The ori-
gin of abnormalities in recurrent aneuploidy/polyploidy.
Am J Hum Genet 2001. https://doi.org/10.1086/324468.
[50] Sas TCJ, van Alfen-van der Velden JAEM, De Muinck
Keizer-Schrama SMPF. Turner syndrome. Encycl
Endocr Dis 2018. https://doi.org/10.1016/B978-0-12-
801238-3.04146-5.
[51] Davis SM, Ross JL. Klinefelter syndrome. Encycl
Endocr Dis 2018. https://doi.org/10.1016/B978-0-12-
801238-3.66137-8.
[52] Kim IW, Khadilkar AC, Ko EY, Sabanegh ES. 47,XYY
syndrome and male infertility. Rev Urol 2013.
[53] Tartaglia NR, Howell S, Sutherland A, Wilson R,
­Wilson L. A review of trisomy X (47,XXX). Or-
phanet J Rare Dis 2010. https://doi.org/10.1186/1750-
1172-5-8.
[54] Linden MG, Bender BG, Robinson A. Sex
­chromosome tetrasomy and pentasomy. Pediatrics
1995;96(4 Pt 1):672–82. PMID: 7567329.
[55] Tartaglia N, Ayari N, Howell S, D’Epagnier C, Zeitler
P. 48,XXYY, 48,XXXY and 49,XXXXY syndromes: not
just variants of Klinefelter syndrome. Acta Paediatr
Int J Paediatr 2011. https://doi.org/10.1111/j.1651-
2227.2011.02235.x.
[56] Morris JK, Wald NJ, Watt HC. Fetal loss in Down
syndrome pregnancies. Prenat Diagn 1999. https://doi.
org/10.1002/(SICI)1097-0223(199902)19:2<142::AID-
PD486>3.0.CO;2-7.
[57] Savva GM, Walker K, Morris JK. The maternal
age-specific live birth prevalence of trisomies 13 and
18 compared to trisomy 21 (Down syndrome). Prenat
Diagn 2010. https://doi.org/10.1002/pd.2403.
[58] Penrose LS. The relative effects of paternal and ma-
ternal age in mongolism. J Genet 1933. https://doi.
org/10.1007/BF02984413.
[59] Snijders RJM, Holzgreve W, Cuckle H, Nicolaides
KH. Maternal age‐specific risks for trisomies at 9—14
weeks’ gestation. Prenat Diagn 1994. https://doi.
org/10.1002/pd.1970140706.
[60] Ball D, Rose E, Alpert E. Alpha-fetoprotein levels
in normal adults. Am J Med Sci 1992. https://doi.
org/10.1097/00000441-199203000-00004.
[61] Mizejewski GJ. Levels of alpha-fetoprotein during
pregnancy and early infancy in normal and dis-
ease States. Obstet Gynecol Surv 2003. https://doi.
org/10.1097/01.OGX.0000099770.97668.18.
[62] Bredaki FE, Sciorio C, Wright A, Wright D, Nicolaides
KH. Serum alpha-fetoprotein in the three trimes-
ters of ­pregnancy: effects of maternal characteristics
and ­medical history. Ultrasound Obstet Gynecol
2015;46(1):34–41. https://doi.org/10.1002/uog.14809.
Epub 2015 May 25. PMID: 25652769.
[63] Haddow JE. Prenatal screening for open neural tube
defects, Down’s syndrome, and other major fetal disor-
ders. Semin Perinatol 1990.
[64] Nicholas Wald HC. Antenatal screening and diagnosis;
Chapter 18. In: Elwood JH, Elwood JM, Little J, edi-
tors. Epidemiology and control of neural tube defects.
Oxford University Press; 1992.
[65] Palomaki GE, Bupp C, Gregg AR, Norton ME, Ogles-
bee D, Best RG. Laboratory screening and diagnosis
of open neural tube defects, 2019 revision: a technical
standard of the American College of Medical Genetics
and Genomics (ACMG). Genet Med 2020. https://doi.
org/10.1038/s41436-019-0681-0.
[66] Merkatz IR, Nitowsky HM, Macri JN, Johnson WE.
An association between low maternal serum α-feto-
protein and fetal chromosomal abnormalities. Am J
Obstet Gynecol 1984. https://doi.org/10.1016/0002-
9378(84)90530-1.
[67] DiMaio MS, Baumgarten A, Greenstein RM, Saal HM,
Mahoney MJ. Screening for fetal Down’s syndrome
in pregnancy by measuring maternal serum alpha-fe-
toprotein levels. N Engl J Med 1987. https://doi.
org/10.1056/nejm198708063170603.
 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
[68] Impact of expanded newborn screening—United
States, 2006. J Am Med Assoc 2008. https://doi.
org/10.1001/jama.300.19.2242.
[69] Waisbren SE, Albers S, Amato S, Ampola M, Brewster
TG, Demmer L, Eaton RB, Greenstein R, Korson
M, Larson C, Marsden D, Msall M, Naylor EW,
Pueschel S, Seashore M, Shih VE, Levy HL. Effect
of expanded newborn screening for biochemical
genetic disorders on child outcomes and parental
stress. J Am Med ­Assoc 2003. https://doi.org/10.1001/
jama.290.19.2564.
[70] Wald NJ, Huttly WJ, Bestwick JP, Old R, Morris JK,
Cheng R, Aquilina J, Peregrine E, Roberts D, Alfirevic
Z. Prenatal reflex DNA screening for trisomies 21,
18, and 13. Genet Med 2018. https://doi.org/10.1038/
gim.2017.188.
[71] Spencer K. Aneuploidy screening in the first trimes-
ter. Am J Med GenetC 2007. https://doi.org/10.1002/
ajmg.c.30119.
[72] Palomaki GE, Lee JES, Canick JA, McDowell GA,
Donnenfeld AE. Technical standards and guidelines:
prenatal screening for Down syndrome that includes
first-trimester biochemistry and/or ultrasound mea-
surements. Genet Med 2009. https://doi.org/10.1097/
GIM.0b013e3181ad5246.
[73] Brambati B, Tului L, Bonacchi I, Shrimanker K, Suzuki
Y, Grudzinskas JG. Serum PAPP‐A and free β‐hCG
are first‐trimester screening markers for down
­syndrome. Prenat Diagn 1994;14(11):1043–7. https://
doi.org/10.1002/pd.1970141106. PMID: 7533285.
[74] Alldred SK, Takwoingi Y, Guo B, Pennant M, Deeks JJ,
Neilson JP, Alfirevic Z. First trimester ultrasound tests
alone or in combination with first trimester serum
tests for Down’s syndrome screening. Cochrane Data-
base Syst Rev 2017. https://doi.org/10.1002/14651858.
CD012600.
[75] Cole LA, Shahabi S, Oz UA, Bahado-Singh RO,
Mahoney MJ. Hyperglycosylated human chorionic
gonadotropin (invasive trophoblast antigen) immu-
noassay: a new basis for gestational Down syndrome
screening. Clin Chem 1999. https://doi.org/10.1093/
clinchem/45.12.2109.
[76] Wald NJ, Kennard A, Hackshaw AK. First trimes-
ter serum screening for Down’s syndrome. Prenat
Diagn 1995;15(13):1227–40. https://doi.org/10.1002/
pd.1970151305. Erratum in: Prenat Diagn 1996
Apr;16(4):387. PMID: 7533285.
[77] Nebiolo L, Ozturk M, Brambati B, Miller S, Wands
J, Milunsky A. First‐trimester maternal serum
alpha‐fetoprotein and human chorionic gonado-
tropin screening for chromosome defects. Prenat
Diagn 1990;10(9):575–81. https://doi.org/10.1002/
pd.1970100905. PMID: 1702539.
29
[78] Biagiotti R, Cariati E, Brizzi L, D’Agata A. Maternal se-
rum screening for Down’s syndrome in the first trimes-
ter of pregnancy. BJOG An Int J Obstet Gynaecol 1995.
https://doi.org/10.1111/j.1471-0528.1995.tb11407.x.
[79] Baviera G, Chimicata S, De Domenico R, Granese
R, Carbone C, Dugo N, D’Anna R. First- and sec-
ond-trimester ADAM12s in down syndrome
screening. Clin Chem 2010. https://doi.org/10.1373/
clinchem.2009.139816.
[80] Cowans NJ, Spencer K, Meiri H. First-trimester mater-
nal placental protein 13 levels in pregnancies resulting
in adverse outcomes. Prenat Diagn 2008. https://doi.
org/10.1002/pd.1921.
[81] Donalson K, Turner S, Morrison L, Liitti P, Nilsson
C, Cuckle H. Maternal serum placental growth factor
and α-fetoprotein testing in first trimester screening
for Down syndrome. Prenat Diagn 2013. https://doi.
org/10.1002/pd.4087.
[82] Christiansen M, Sørensen TL, Nørgaard-Pedersen B.
Human placental lactogen is a first-trimester maternal
serum marker of Down syndrome. Prenat Diagn 2007.
https://doi.org/10.1002/pd.1600.
[83] Alldred SK, Takwoingi Y, Guo B, Pennant M, Deeks
JJ, Neilson JP, Alfirevic Z. First trimester serum tests
for Down’s syndrome screening. Cochrane Database
Syst Rev 2015. https://doi.org/10.1002/14651858.
CD011975.
[84] Orlandi F, Rossi C, Allegra A, Krantz D, Hallahan T,
Orlandi E, Macri J. First trimester screening with free
β-hCG, PAPP-A and nuchal translucency in preg-
nancies conceived with assisted reproduction. Prenat
Diagn 2002. https://doi.org/10.1002/pd.390.
[85] Gjerris AC, Loft A, Pinborg A, Christiansen M, Tabor
A. First-trimester screening markers are altered in
pregnancies conceived after IVF/ICSI. Ultrasound
Obstet Gynecol 2009. https://doi.org/10.1002/
uog.6254.
[86] Lambert-Messerlian GM. Second trimester levels of ma-
ternal serum inhibin A, total inhibin., a precursor, and
activin in Down’s syndrome pregnancy. J Med Screen
1996. https://doi.org/10.1177/096914139600300202.
[87] Qin QP, Christiansen M, Nguyen TH, Sørensen S,
Larsen SO, Nørgaard-Pedersen B. Schwangerschafts-
protein 1 (SP1) as a maternal serum marker for Down
syndrome in the first and second trimesters. Prenat
Diagn 1997. https://doi.org/10.1002/(SICI)1097-
0223(199702)17:2<101::AID-PD4>3.0.CO;2-H.
[88] Spencer K, Muller F, Aitken DA. Amniotic fluid
and maternal serum levels of CA125 in pregnancies
affected by Down syndrome: a re-evaluation of the
role of CA125 in Down syndrome screening. Prenat
Diagn 1997. https://doi.org/10.1002/(SICI)1097-
0223(199708)17:8<701::AID-PD135>3.0.CO;2-F.
30 CHAPTER 2 Prenatal Screening for Neural Tube Defects and Aneuploidy
[89] Watanabe H, Hamada H, Yamada N, Ogura T, Yasuoka
MO, Okuno S, Fujiki Y, Sohda S, Kubo T. Second-tri-
mester maternal pregnancy-associated plasma protein
A and inhibin A levels in fetal trisomies. Fetal Diagn
Ther 2002. https://doi.org/10.1159/000048026.
[90] Su YN, Hsu JJ, Lee CN, Cheng WF, Kung CCS, Hsieh
FJ. Raised maternal serum placenta growth factor con-
centration during the second trimester is associated
with Down syndrome. Prenat Diagn 2002. https://doi.
org/10.1002/pd.218.
[91] Christiansen M, Oxvig C, Wagner JM, Qin QP,
Nguyen TH, Overgaard MT, Larsen SO, Sot-
trup-Jensen L, Gleich GJ, Nørgaard-Pedersen B. The
proform of eosinophil major basic protein: a new
maternal serum marker for Down syndrome. Prenat
Diagn 1999. https://doi.org/10.1002/(SICI)1097-
0223(199910)19:10<905::AID-PD658>3.0.CO;2-Q.
[92] Alldred SK, Deeks JJ, Guo B, Neilson JP, Alfirevic Z.
Second trimester serum tests for Down’s Syndrome
screening. Cochrane Database Syst Rev 2012. https://
doi.org/10.1002/14651858.cd009925.
[93] Alldred SKK, Takwoingi Y, Guo B, Pennant M, Deeks
JJ, Neilson JP, Alfirevic Z, Guo B, Neilson JP, Alfirevic
Z. Second trimester serum tests for Down’s syndrome
screening (review). Cochrane Database Syst Rev 2015c.
[94] Palomaki GE, Neveux LM, Knight GJ, Haddow JE,
Lee JE. Estimating first-trimester combined screen-
ing performance for Down syndrome in dried blood
spots versus fresh sera. Genet Med 2007. https://doi.
org/10.1097/GIM.0b013e31809861a9.
[95] Spencer K, Macri JN, Carpenter P, Anderson R,
Krantz DA. Stability of intact chorionic gonadotro-
pin (hCG) in serum, liquid whole blood, and dried
whole-blood filter-paper spots: impact on screening
for Down syndrome by measurement of free β-hCG
subunit. Clin Chem 1993. https://doi.org/10.1093/
clinchem/39.6.1064.
[96] Alldred SK, Guo B, Takwoingi Y, Pennant M, Wisniews-
ki S, Deeks JJ, Neilson JP, Alfirevic Z. Urine tests for
Down’s syndrome screening. Cochrane Database Syst
Rev 2015. https://doi.org/10.1002/14651858.CD011984.
[97] Best R, Khushf G. The social conditions for nano-
medicine: disruption, systems, and lock-in. J Law
Med Ethics 2006. https://doi.org/10.1111/j.1748-
720X.2006.00093.x.
[98] Liebowitz SJ, Margolis SE. Path dependence, lock-
in, and history. J Law Econ Organ 1995. https://doi.
org/10.2139/ssrn.1706450.
[99] Nicolaides KH, Azar G, Byrne D, Mansur C, Marks K.
Fetal nuchal translucency: ultrasound screening for
chromosomal defects in first trimester of pregnancy. Br
Med J 1992. https://doi.org/10.1136/bmj.304.6831.867.
[100] Souka AP, Snijders RJM, Novakov A, Soares W, Nico-
laides KH. Defects and syndromes in chromosomally
normal fetuses with increased nuchal translucency
thickness at 10–14 weeks of gestation. Ultrasound
Obstet Gynecol 1998. https://doi.org/10.1046/j.1469-
0705.1998.11060391.x.
[101] Souka AP, Von Kaisenberg CS, Hyett JA, Sonek JD,
Nicolaides KH. Increased nuchal translucency with
normal karyotype. Am J Obstet Gynecol 2005. https://
doi.org/10.1016/j.ajog.2004.12.093.
[102] Chasen ST, Sharma G, Kalish RB, Chervenak FA.
First-trimester screening for aneuploidy with fetal nu-
chal translucency in a United States population. Ultra-
sound Obstet Gynecol 2003. https://doi.org/10.1002/
uog.174.
[103] Gasiorek-Wiens A, Tercanli S, Kozlowski P, Kossak-
iewicz A, Minderer S, Meyberg H, Kamin G, Germer
U, Bielicki M, Hackelöer BJ, Sarlay D, Kuhn P, Klapp J,
Bahlmann F, Pruggmayer M, Schneider KTM, Seefried
W, Fritzer E, Von Kaisenberg CS. Screening for triso-
my 21 by fetal nuchal translucency and maternal age: a
multicenter project in Germany, Austria and Switzer-
land. Ultrasound Obstet Gynecol 2001. https://doi.
org/10.1046/j.0960-7692.2001.00604.x.
[104] Nicolaides KH. Nuchal translucency and other
first-trimester sonographic markers of chromosomal
abnormalities. Am J Obstet Gynecol 2004;191(1):45–
67. https://doi.org/10.1016/j.ajog.2004.03.090. PMID:
15295343.
[105] Snijders RJM, Noble P, Sebire N, Souka A, ­Nicolaides
KH. UK multicentre project on assessment of risk of
trisomy 21 by maternal age and fetal nuchal-trans-
lucency thickness at 10–14 weeks of gestation.
Lancet 1998. https://doi.org/10.1016/S0140-
6736(97)11280-6.
[106] Zoppi MA, Ibba RM, Floris M, Monni G. Fetal nuchal
translucency screening in 12 495 pregnancies in
Sardinia. Ultrasound Obstet Gynecol 2001. https://doi.
org/10.1046/j.0960-7692.2001.00583.x.
[107] Sonek JD, Cicero S, Neiger R, Nicolaides KH. Nasal
bone assessment in prenatal screening for trisomy 21.
Am J Obstet Gynecol 2006. https://doi.org/10.1016/j.
ajog.2005.11.042.
[108] Kagan KO, Cicero S, Staboulidou I, Wright D, Nico-
laides KH. Fetal nasal bone in screening for trisomies
21, 18 and 13 and Turner syndrome at 11–13 weeks of
gestation. Ultrasound Obstet Gynecol 2009. https://
doi.org/10.1002/uog.6318.
[109] Lachmann R, Chaoui R, Moratalla J, Picciarelli G,
Nicolaides KH. Posterior brain in fetuses with open
spina bifida at 11 to 13 weeks. Prenat Diagn 2011.
https://doi.org/10.1002/pd.2632.
Another random document with
no related content on Scribd:
she assumed the charge of the paper, she printed it with her own
motto as the heading, Vox Populi Vox Dei.
William Goddard drifted to Philadelphia, where he published the
Pennsylvania Chronicle for a short season, and in 1773 he removed
to Baltimore and established himself in the newspaper business
anew, with only, he relates, “the small capital of a single solitary
guinea.” He found another energetic business woman, the widow
Mrs. Nicholas Hasselbaugh, carrying on the printing-business
bequeathed to her by her husband; and he bought her stock in trade
and established The Maryland Journal and Baltimore Advertiser. It
was the third newspaper published in Maryland, was issued weekly
at ten shillings per annum, and was a well-printed sheet. But William
Goddard had another bee in his bonnet. A plan was formed just
before the Revolutionary War to abolish the general public post-
office and to establish in its place a complete private system of post-
riders from Georgia to New Hampshire. This system was to be
supported by private subscription; a large sum was already
subscribed, and the scheme well under way, when the war ended all
the plans. Goddard had this much to heart, and had travelled
extensively through the colonies exploiting it. While he was away on
these trips he left the newspaper and printing-house solely under the
charge of his sister Mary Katharine Goddard, the worthy daughter of
her energetic mother. From 1775 to 1784, through the trying times of
the Revolution, and in a most active scene of military and political
troubles, this really brilliant woman continued to print successfully
and continuously her newspaper. The Journal and every other work
issued from her printing-presses were printed and published in her
name, and it is believed chiefly on her own account. She was a
woman of much intelligence and was also practical, being an expert
compositor of types, and fully conversant with every detail of the
mechanical work of a printing-office. During this busy time she was
also postmistress of Baltimore, and kept a bookshop. Her brother
William, through his futile services in this postal scheme, had been
led to believe he would receive under Benjamin Franklin and the new
government of the United States, the appointment of Secretary and
Comptroller of the Post Office; but Franklin gave it to his own son-in-
law, Richard Bache. Goddard, sorely disappointed but pressed in
money matters, felt forced to accept the position of Surveyor of Post
Roads. When Franklin went to France in 1776, and Bache became
Postmaster-General, and Goddard again was not appointed
Comptroller, his chagrin caused him to resign his office, and naturally
to change his political principles.
He retired to Baltimore, and soon there appeared in the Journal an
ironical piece (written by a member of Congress) signed Tom Tell
Truth. From this arose a vast political storm. The Whig Club of
Baltimore, a powerful body, came to Miss Goddard and demanded
the name of the author; she referred them to her brother. On his
refusal to give the author’s name, he was seized, carried to the
clubhouse, bullied, and finally warned out of town and county. He at
once went to the Assembly at Annapolis and demanded protection,
which was given him. He ventilated his wrongs in a pamphlet, and
was again mobbed and insulted. In 1779, Anna Goddard printed
anonymously in her paper Queries Political and Military, written
really by General Charles Lee, the enemy and at one time
presumptive rival of Washington. This paper also raised a
tremendous storm through which the Goddards passed triumphantly.
Lee remained always a close friend of William Goddard, and
bequeathed to him his valuable and interesting papers, with the
intent of posthumous publication; but, unfortunately, they were sent
to England to be printed in handsome style, and were instead
imperfectly and incompletely issued, and William Goddard received
no benefit or profit from their sale. But Lee left him also, by will, a
large and valuable estate in Berkeley County, Virginia, so he retired
from public life and ended his days on a Rhode Island farm. Anna
Katharine Goddard lived to great old age. The story of this
acquaintance with General Lee, and of Miss Goddard’s connection
therewith, forms one of the interesting minor episodes of the War.
Just previous to the Revolution, it was nothing very novel or
unusual to Baltimoreans to see a woman edit a newspaper. The
Maryland Gazette suspended on account of the Stamp Act in 1765,
and the printer issued a paper called The Apparition of the Maryland
Gazette which is not Dead but Sleepeth; and instead of a Stamp it
bore a death’s head with the motto, “The Times are Dismal, Doleful,
Dolorous, Dollarless.” Almost immediately after it resumed
publication, the publisher died, and from 1767 to 1775 it was carried
on by his widow, Anne Katharine Green, sometimes assisted by her
son, but for five years alone. The firm name was Anne Katharine
Green & Son: and she also did the printing for the Colony. She was
about thirty-six years old when she assumed the business, and was
then the mother of six sons and eight daughters. Her husband was
the fourth generation from Samuel Green, the first printer in New
England, from whom descended about thirty ante-Revolutionary
printers. Until the Revolution there was always a Printer Green in
Boston. Mr. Green’s partner, William Rind, removed to Williamsburg
and printed there the Virginia Gazette. At his death, widow
Clementina Rind, not to be outdone by Widow Green and Mother
and Sister Goddard, proved that what woman has done woman can
do, by carrying on the business and printing the Gazette till her own
death in 1775.
It is indeed a curious circumstance that, on the eve of the
Revolution, so many southern newspapers should be conducted by
women. Long ere that, from 1738 to 1740, Elizabeth Timothy, a
Charleston woman, widow of Louis Timothy, the first librarian of the
Philadelphia Library company, and publisher of the South Carolina
Gazette, carried on that paper after her husband’s death; and her
son, Peter Timothy, succeeded her. In 1780 his paper was
suspended, through his capture by the British. He was exchanged,
and was lost at sea with two daughters and a grandchild, while on
his way to Antigua to obtain funds. He had a varied and interesting
life, was a friend of Parson Whitefield, and was tried with him on a
charge of libel against the South Carolina ministers. In 1782 his
widow, Anne Timothy, revived the Gazette, as had her mother-in-law
before her, and published it successfully twice a week for ten years
till her death in 1792. She had a large printing-house, corner of
Broad and King Streets, Charleston, and was printer to the State;
truly a remarkable woman.
Peter Timothy’s sister Mary married Charles Crouch, who also was
drowned when on a vessel bound to New York. He was a sound
Whig and set up a paper in opposition to the Stamp Act, called The
South Carolina Gazette and Country Journal. This was one of the
four papers which were all entitled Gazettes in order to secure
certain advertisements that were all directed by law “to be inserted in
the South Carolina Gazette.” Mary Timothy Crouch continued the
paper for a short time after her husband’s death; and in 1780 shortly
before the surrender of the city to the British, went with her printing-
press and types to Salem, where for a few months she printed The
Salem Gazette and General Advertiser. I have dwelt at some length
on the activity and enterprise of these Southern women, because it
is another popular but unstable notion that the women of the North
were far more energetic and capable than their Southern sisters;
which is certainly not the case in this line of business affairs.
Benjamin and James Franklin were not the only members of the
Franklin family who were capable newspaper-folk. James Franklin
died in Newport in 1735, and his widow Anne successfully carried on
the business for many years. She had efficient aid in her two
daughters, who were quick and capable practical workers at the
compositor’s case, having been taught by their father, whom they
assisted in his lifetime. Isaiah Thomas says of them:—
A gentleman who was acquainted with Anne Franklin and
her family, informed me that he had often seen her daughters
at work in the printing house, and that they were sensible and
amiable women.
We can well believe that, since they had Franklin and Anne
Franklin blood in them. This competent and industrious trio of
women not only published the Newport Mercury, but were printers
for the colony, supplying blanks for public offices, publishing
pamphlets, etc. In 1745 they printed for the Government an edition of
the laws of the colony of 340 pages, folio. Still further, they carried on
a business of “printing linens, calicoes, silks, &c., in figures, very
lively and durable colors, and without the offensive smell which
commonly attends linen-printing.” Surely there was no lack of
business ability on the distaff side of the Franklin house.
Boston women gave much assistance to their printer-husbands.
Ezekiel Russel, the editor of that purely political publication, The
Censor, was in addition a printer of chap-books and ballads which
were sold from his stand near the Liberty Tree on Boston Common.
His wife not only helped him in printing these, but she and another
young woman of his household, having ready pens and a biddable
muse, wrote with celerity popular and seasonable ballads on passing
events, especially of tragic or funereal cast; and when these ballads
were printed with a nice border of woodcuts of coffins and death’s
heads, they often had a long and profitable run of popularity. After
his death, Widow Russel still continued ballad making and monging.
It was given to a woman, Widow Margaret Draper, to publish the
only newspaper which was issued in Boston during the siege, the
Massachusetts Gazette and Boston News Letter. And a miserable
little sheet it was, vari-colored, vari-typed, vari-sized; of such poor
print that it is scarcely readable. When the British left Boston,
Margaret Draper left also, and resided in England, where she
received a pension from the British government.
The first newspaper in Pennsylvania was entitled The American
Weekly Mercury. It was “imprinted by Andrew Bradford” in 1719. He
was a son of the first newspaper printer in New York, William
Bradford, Franklin’s “cunning old fox,” who lived to be ninety-two
years old, and whose quaint tombstone may be seen in Trinity
Churchyard. At Andrew’s death in 1742, the paper appeared in
mourning, and it was announced that it would be published by “the
widow Bradford.” She took in a partner, but speedily dropped him,
and carried it on in her own name till 1746. During the time that
Cornelia Bradford printed this paper it was remarkable for its good
type and neatness.
The Connecticut Courant and The Centinel were both of them
published for some years by the widows of former proprietors.
The story of John Peter Zenger, the publisher of The New York
Weekly Journal, is one of the most interesting episodes in our
progress to free speech and liberty, but cannot be dwelt on here. The
feminine portion of his family was of assistance to him. His daughter
was mistress of a famous New York tavern that saw many
remarkable visitors, and heard much of the remarkable talk of
Zenger’s friends. After his death in 1746, his newspaper was carried
on by his widow for two years. Her imprint was, “New York; Printed
by the Widow Cathrine Zenger at the Printing-Office in Stone Street;
Where Advertisements are taken in, and all Persons may be
supplied with this Paper.”
The whole number of newspapers printed before the Revolution
was not very large; and when we see how readily and successfully
this considerable number of women assumed the cares of
publishing, we know that the “newspaper woman” of that day was no
rare or presumptuous creature, any more than is the “newspaper-
woman” of our own day, albeit she was of very different ilk; but the
spirit of independent self-reliance, when it became necessary to
exhibit self-reliance, was as prompt and as stable in the feminine
breast a century and a half ago as now. Then, as to-day, there were
doubtless scores of good wives and daughters who materially
assisted their husbands in their printing-shops, and whose work will
never be known.
There is no doubt that our great-grandmothers possessed
wonderful ability to manage their own affairs, when it became
necessary to do so, even in extended commercial operations. It is
easy to trace in the New England coast towns one influence which
tended to interest them, and make them capable of business
transactions. They constantly heard on all sides the discussion of
foreign trade, and were even encouraged to enter into the discussion
and the traffic. They heard the Windward Islands, the Isle of France,
and Amsterdam, and Canton, and the coast of Africa described by
old travelled mariners, by active young shipmasters, in a way that
put them far more in touch with these far-away foreign shores, gave
them more knowledge of details of life in those lands, than women of
to-day have. And women were encouraged, even urged, to take an
active share in foreign trade, in commercial speculation, by sending
out a “venture” whenever a vessel put out to sea, and whenever the
small accumulation of money earned by braiding straw, knitting
stockings, selling eggs or butter, or by spinning and weaving, was
large enough to be worth thus investing; and it needed not to be a
very large sum to be deemed proper for investment. When a ship
sailed out to China with cargo of ginseng, the ship’s owner did not
own all the solid specie in the hold—the specie that was to be
invested in the rich and luxurious products of far Cathay.
Complicated must have been the accounts of these transactions, for
many were the parties in the speculation. There were no giant
monopolies in those days. The kindly ship-owner permitted even his
humblest neighbor to share his profits. And the profits often were
large. The stories of some of the voyages, the adventures of the
business contracts, read like a fairy tale of commerce. In old letters
may be found reference to many of the ventures sent by women.
One young woman wrote in 1759:—
Inclos’d is a pair of Earrings. Pleas ask Captin Oliver to
carry them a Ventur fer me if he Thinks they will fetch
anything to the Vally of them; tell him he may bring the effects
in anything he thinks will answer best.
One of the “effects” brought to this young woman, and to hundreds
of others, was a certain acquaintance with business transactions, a
familiarity with the methods of trade. When the father or husband
died, the woman could, if necessary, carry on his business to a
successful winding-up, or continue it in the future. Of the latter
enterprise many illustrations might be given. In the autumn of 1744 a
large number of prominent business men in Newport went into a
storehouse on a wharf to examine the outfit of a large privateer. A
terrible explosion of gunpowder took place, which killed nine of them.
One of the wounded was Sueton Grant, a Scotchman, who had
come to America in 1725. His wife, on hearing of the accident, ran at
once to the dock, took in at a glance the shocking scene and its
demands for assistance, and cutting into strips her linen apron with
the housewife’s scissors she wore at her side, calmly bound up the
wounds of her dying husband. Mr. Grant was at this time engaged in
active business; he had agencies in Europe, and many privateers
afloat. Mrs. Grant took upon her shoulders these great
responsibilities, and successfully carried them on for many years,
while she educated her children, and cared for her home.
A good example of her force of character was once shown in a
court of law. She had an important litigation on hand and large
interests at stake, when she discovered the duplicity of her counsel,
and her consequent danger. She went at once to the court-room
where the case was being tried; when her lawyer promptly but vainly
urged her to retire. The judge, disturbed by the interruption, asked
for an explanation, and Mrs. Grant at once unfolded the knavery of
her counsel and asked permission to argue her own case. Her
dignity, force, and lucidity so moved the judge that he permitted her
to address the jury, which she did in so convincing a manner as to
cause them to promptly render a verdict favorable to her. She
passed through some trying scenes at the time of the Revolution
with wonderful decision and ability, and received from every one the
respect and deference due to a thorough business man, though she
was a woman.
In New York the feminine Dutch blood showed equal capacity in
business matters; and it is said that the management of considerable
estates and affairs often was assumed by widows in New
Amsterdam. Two noted examples are Widow De Vries and Widow
Provoost. The former was married in 1659, to Rudolphus De Vries,
and after his death she carried on his Dutch trade—not only buying
and selling foreign goods, but going repeatedly to Holland in the
position of supercargo on her own ships. She married Frederick
Phillipse, and it was through her keenness and thrift and her
profitable business, as well as through his own success, that
Phillipse became the richest man in the colony and acquired the
largest West Indian trade.
Widow Maria Provoost was equally successful at the beginning of
the eighteenth century, and had a vast Dutch business
correspondence. Scarce a ship from Spain, the Mediterranean, or
the West Indies, but brought her large consignments of goods. She
too married a second time, and as Madam James Alexander filled a
most dignified position in New York, being the only person besides
the Governor to own a two-horse coach. Her house was the finest in
town, and such descriptions of its various apartments as “the great
drawing-room, the lesser drawing-room, the blue and gold leather
room, the green and gold leather room, the chintz room, the great
tapestry room, the little front parlour, the back parlour,” show its size
and pretensions.
Madam Martha Smith, widow of Colonel William Smith of St.
George’s Manor, Long Island, was a woman of affairs in another
field. In an interesting memorandum left by her we read:—
Jan ye 16, 1707. My company killed a yearling whale made
27 barrels. Feb ye 4, Indian Harry with his boat struck a whale
and called for my boat to help him. I had but a third which was
4 barrels. Feb 22, my two boats & my sons and Floyds boats
killed a yearling whale of which I had half—made 36 barrels,
my share 18 barrels. Feb 24 my company killed a school
whale which made 35 barrels. March 13, my company killed a
small yearling made 30 barrels. March 17, my company killed
two yearlings in one day; one made 27, the other 14 barrels.
We find her paying to Lord Cornbury fifteen pounds, a duty on “ye
20th part of her eyle.” And she apparently succeeded in her
enterprises.
In early Philadelphia directories may be found the name of
“Margaret Duncan, Merchant, No. 1 S. Water St.” This capable
woman had been shipwrecked on her way to the new world. In the
direst hour of that extremity, when forced to draw lots for the scant
supply of food, she vowed to build a church in her new home if her
life should be spared. The “Vow Church” in Philadelphia, on
Thirteenth Street near Market Street, for many years proved her
fulfilment of this vow, and also bore tribute to the prosperity of this
pious Scotch Presbyterian in her adopted home.
Southern women were not outstripped by the business women of
the north. No more practical woman ever lived in America than Eliza
Lucas Pinckney. When a young girl she resided on a plantation at
Wappoo, South Carolina, owned by her father, George Lucas. He
was Governor of Antigua, and observing her fondness for and
knowledge of botany, and her intelligent power of application of her
knowledge, he sent to her many tropical seeds and plants for her
amusement and experiment in her garden. Among the seeds were
some of indigo, which she became convinced could be profitably
grown in South Carolina. She at once determined to experiment, and
planted indigo seed in March, 1741. The young plants started finely,
but were cut down by an unusual frost. She planted seed a second
time, in April, and these young indigo-plants were destroyed by
worms. Notwithstanding these discouragements, she tried a third
time, and with success. Her father was delighted with her enterprise
and persistence, and when he learned that the indigo had seeded
and ripened, sent an Englishman named Cromwell—an experienced
indigo-worker—from Montserrat to teach his daughter Eliza the
whole process of extracting the dye from the weed. Vats were built
on Wappoo Creek, in which was made the first indigo formed in
Carolina. It was of indifferent quality, for Cromwell feared the
successful establishment of the industry in America would injure the
indigo trade in his own colony, so he made a mystery of the process,
and put too much lime in the vats, doubtless thinking he could
impose upon a woman. But Miss Lucas watched him carefully, and in
spite of his duplicity, and doubtless with considerable womanly
power of guessing, finally obtained a successful knowledge and
application of the complex and annoying methods of extracting
indigo,—methods which required the untiring attention of sleepless
nights, and more “judgment” than intricate culinary triumphs. After
the indigo was thoroughly formed by steeping, beating, and washing,
and taken from the vats, the trials of the maker were not over. It must
be exposed to the sun, but if exposed too much it would be burnt, if
too little it would rot. Myriads of flies collected around it and if
unmolested would quickly ruin it. If packed too soon it would sweat
and disintegrate. So, from the first moment the tender plant
appeared above ground, when the vast clouds of destroying
grasshoppers had to be annihilated by flocks of hungry chickens, or
carefully dislodged by watchful human care, indigo culture and
manufacture was a distressing worry, and was made still more
unalluring to a feminine experimenter by the fact that during the
weary weeks it laid in the “steepers” and “beaters” it gave forth a
most villainously offensive smell.
Soon after Eliza Lucas’ hard-earned success she married Charles
Pinckney, and it is pleasant to know that her father gave her, as part
of her wedding gift, all the indigo on the plantation. She saved the
whole crop for seed,—and it takes about a bushel of indigo seed to
plant four acres,—and she planted the Pinckney plantation at
Ashepoo, and gave to her friends and neighbors small quantities of
seed for individual experiment; all of which proved successful. The
culture of indigo at once became universal, the newspapers were full
of instructions upon the subject, and the dye was exported to
England by 1747, in such quantity that merchants trading in Carolina
petitioned Parliament for a bounty on Carolina indigo. An act of
Parliament was passed allowing a bounty of sixpence a pound on
indigo raised in the British-American plantations and imported
directly to Great Britain. Spurred on by this wise act, the planters
applied with redoubled vigor to the production of the article, and
soon received vast profits as the rewards of their labor and care. It is
said that just previous to the Revolution more children were sent
from South Carolina to England to receive educations, than from all
the other colonies,—and this through the profits of indigo and rice.
Many indigo planters doubled their capital every three or four years,
and at last not only England was supplied with indigo from South
Carolina, but the Americans undersold the French in many European
markets. It exceeded all other southern industries in importance, and
became a general medium of exchange. When General Marion’s
young nephew was sent to school at Philadelphia, he started off with
a wagon-load of indigo to pay his expenses. The annual dues of the
Winyah Indigo Society of Georgetown were paid in the dye, and the
society had grown so wealthy in 1753, that it established a large
charity school and valuable library.
Ramsay, the historian of South Carolina, wrote in 1808, that the
indigo trade proved more beneficial to Carolina than the mines of
Mexico or Peru to old or new Spain. By the year of his writing,
however, indigo (without waiting for extermination through its modern
though less reliable rivals, the aniline dyes) had been driven out of
Southern plantations by its more useful and profitable field neighbor,
King Cotton, that had been set on a throne by the invention of a
Yankee schoolmaster. The time of greatest production and export of
indigo was just previous to the Revolution, and at one time it was
worth four or five dollars a pound. And to-day only the scanty records
of the indigo trade, a few rotting cypress boards of the steeping-vats,
and the blue-green leaves of the wild wayside indigo, remain of all
this prosperity to show the great industry founded by this remarkable
and intelligent woman.
The rearing of indigo was not this young girl’s only industry. I will
quote from various letters written by her in 1741 and 1742 before her
marriage, to show her many duties, her intelligence, her versatility:—
Wrote my father on the pains I had taken to bring the
Indigo, Ginger, Cotton, Lucern, and Casada to perfection and
had greater hopes from the Indigo, if I could have the seed
earlier, than any of ye rest of ye things I had tried.
I have the burthen of 3 Plantations to transact which
requires much writing and more business and fatigue of other
sorts than you can imagine. But lest you should imagine it too
burthensome to a girl in my early time of life, give me leave to
assure you I think myself happy that I can be useful to so
good a father.
Wont you laugh at me if I tell you I am so busy in providing
for Posterity I hardly allow myself time to eat or sleep, and
can but just snatch a moment to write to you and a friend or
two more. I am making a large plantation of oaks which I look
upon as my own property whether my father gives me the
land or not, and therefore I design many yeer hence when
oaks are more valuable than they are now, which you know
they will be when we come to build fleets. I intend I say two
thirds of the produce of my oaks for a charity (Ill tell you my
scheme another time) and the other third for those that shall
have the trouble to put my design in execution.
I have a sister to instruct, and a parcel of little negroes
whom I have undertaken to teach to read.
The Cotton, Guinea Corn, and Ginger planted was cutt off
by a frost. I wrote you in a former letter we had a good crop of
Indigo upon the ground. I make no doubt this will prove a
valuable commodity in time. Sent Gov. Thomas daughter a
tea chest of my own doing.
 I am engaged with the Rudiments of Law to which I am but
a stranger. If you will not laugh too immoderately at me I’ll
trust you with a Secrett. I have made two Wills already. I know
I have done no harm for I conn’d my Lesson perfect. A widow
hereabouts with a pretty little fortune teazed me intolerably to
draw a marriage settlement, but it was out of my depth and I
absolutely refused it—so she got an able hand to do it—
indeed she could afford it—but I could not get off being one of
the Trustees to her settlement, and an old Gentⁿ the other. I
shall begin to think myself an old woman before I am a young
one, having such mighty affairs on my hands.
I think this record of important work could scarce be equalled by
any young girl in a comparative station of life nowadays. And when
we consider the trying circumstances, the difficult conditions, in
which these varied enterprises were carried on, we can well be
amazed at the story.
Indigo was not the only important staple which attracted Mrs.
Pinckney’s attention, and the manufacture of which she made a
success. In 1755 she carried with her to England enough rich silk
fabric, which she had raised and spun and woven herself in the
vicinity of Charleston, to make three fine silk gowns, one of which
was presented to the Princess Dowager of Wales, and another to
Lord Chesterfield. This silk was said to be equal in beauty to any silk
ever imported.
This was not the first American silk that had graced the person of
English royalty. In 1734 the first windings of Georgia silk had been
taken from the filature to England, and the queen wore a dress made
thereof at the king’s next birthday. Still earlier in the field Virginia had
sent its silken tribute to royalty. In the college library at Williamsburg,
Va., may be seen a letter signed “Charles R.”—his most Gracious
Majesty Charles the Second. It was written by his Majesty’s private
secretary, and addressed to Governor Berkeley for the king’s loyal
subjects in Virginia. It reads thus:—
Trusty and Well beloved, We Greet you Well. Wee have
received wᵗʰ much content ye dutifull respects of Our Colony
 in ye present lately made us by you & ye councill there, of ye
first product of ye new Manufacture of Silke, which as a
marke of Our Princely acceptation of yoʳ duteys & for yoʳ
particular encouragement, etc. Wee have been commanded
to be wrought up for ye use of Our Owne Person.
And earliest of all is the tradition, dear to the hearts of Virginians,
that Charles I. was crowned in 1625 in a robe woven of Virginia silk.
The Queen of George III. was the last English royalty to be similarly
honored, for the next attack of the silk fever produced a suit for an
American ruler, George Washington.
The culture of silk in America was an industry calculated to attract
the attention of women, and indeed was suited to them, but men
were not exempt from the fever; and the history of the manifold and
undaunted efforts of governor’s councils, parliaments, noblemen,
philosophers, and kings to force silk culture in America forms one of
the most curious examples extant of persistent and futile efforts to
run counter to positive economic conditions, for certainly physical
conditions are fairly favorable.
South Carolina women devoted themselves with much success to
agricultural experiments. Henry Laurens brought from Italy and
naturalized the olive-tree, and his daughter, Martha Laurens
Ramsay, experimented with the preservation of the fruit until her
productions equalled the imported olives. Catharine Laurens
Ramsay manufactured opium of the first quality. In 1755 Henry
Laurens’ garden in Ansonborough was enriched with every curious
vegetable product from remote quarters of the world that his
extensive mercantile connections enabled him to procure, and the
soil and climate of South Carolina to cherish. He introduced besides
olives, capers, limes, ginger, guinea grass, Alpine strawberries
(bearing nine months in the year), and many choice varieties of
fruits. This garden was superintended by his wife, Mrs. Elinor
Laurens.
Mrs. Martha Logan was a famous botanist and florist. She was
born in 1702, and was the daughter of Robert Daniel, one of the last
proprietary governors of South Carolina. When fourteen years old,
she married George Logan, and all her life treasured a beautiful and
remarkable garden. When seventy years old, she compiled from her
knowledge and experience a regular treatise on gardening, which
was published after her death, with the title The Garden’s Kalendar.
It was for many years the standard work on gardening in that locality.
Mrs. Hopton and Mrs. Lamboll were early and assiduous flower-
raisers and experimenters in the eighteenth century, and Miss Maria
Drayton, of Drayton Hall, a skilled botanist.
The most distinguished female botanist of colonial days was Jane
Colden, the daughter of Governor Cadwallader Colden, of New York.
Her love of the science was inherited from her father, the friend and
correspondent of Linnæus, Collinson, and other botanists. She
learned a method of taking leaf-impressions in printers’ ink, and sent
careful impressions of American plants and leaves to the European
collectors. John Ellis wrote of her to Linnæus in April, 1758:—
This young lady merits your esteem, and does honor to
your system. She has drawn and described four hundred
plants in your method. Her father has a plant called after her
Coldenia. Suppose you should call this new genus Coldenella
or any other name which might distinguish her.
Peter Collinson said also that she was the first lady to study the
Linnæan system, and deserved to be celebrated. Another tribute to
her may be found in a letter of Walter Rutherford’s:—
From the middle of the Woods this Family corresponds with
all the learned Societies in Europe. His daughter Jenny is a
Florist and Botanist. She has discovered a great number of
Plants never before described and has given their Properties
and Virtues, many of which are found useful in Medicine and
she draws and colours them with great Beauty. Dr. Whyte of
Edinburgh is in the number of her correspondents.
N. B. She makes the best cheese I ever ate in America.
The homely virtue of being a good cheese-maker was truly a
saving clause to palliate and excuse so much feminine scientific
knowledge.
 CHAPTER III.
“DOUBLE-TONGUED AND NAUGHTY WOMEN.”

I am much impressed in reading the court records of those early

days, to note the vast care taken in all the colonies to prevent lying,
slandering, gossiping, backbiting, and idle babbling, or, as they
termed it, “brabling;” to punish “common sowers and movers”—of
dissensions, I suppose.
The loving neighborliness which proved as strong and as
indispensable a foundation for a successful colony as did godliness,
made the settlers resent deeply any violations, though petty, of the
laws of social kindness. They felt that what they termed “opprobrious
schandalls tending to defamaçon and disparagment” could not be
endured.
One old author declares that “blabbing, babbling, tale-telling, and
discovering the faults and frailities of others is a most Common and
evill practice.” He asserts that a woman should be a “main store
house of secresie, a Maggazine of taciturnitie, the closet of
connivence, the mumbudget of silence, the cloake bagge of rouncell,
the capcase, fardel, or pack of friendly toleration;” which, as a whole,
seems to be a good deal to ask. Men were, as appears by the
records, more frequently brought up for these offences of the tongue,
but women were not spared either in indictment or punishment. In
Windsor, Conn., one woman was whipped for “wounding” a neighbor,
not in the flesh, but in the sensibilities.
In 1652 Joane Barnes, of Plymouth, Mass., was indicted for
“slandering,” and sentenced “to sitt in the stockes during the Courts
pleasure, and a paper whereon her facte written in Capitall letters to
be made faste vnto her hatt or neare vnto her all the tyme of her
sitting there.” In 1654 another Joane in Northampton County, Va.,
suffered a peculiarly degrading punishment for slander. She was
“drawen ouer the Kings Creeke at the starne of a boate or Canoux,
also the next Saboth day in the tyme of diuine seruis” was obliged to
present herself before the minister and congregation, and
acknowledge her fault, and ask forgiveness. This was an old Scotch
custom. The same year one Charlton called the parson, Mr. Cotton,
a “black cotted rascal,” and was punished therefor in the same way.
Richard Buckland, for writing a slanderous song about Ann Smith,
was similarly pilloried, bearing a paper on his hat inscribed Inimicus
Libellus, and since possibly all the church attendants did not know
Latin, to publicly beg Ann’s forgiveness in English for his libellous
poesy. The punishment of offenders by exposing them, wrapped in
sheets, or attired in foul clothing, on the stool of repentance in the
meeting-house in time of divine service, has always seemed to me
specially bitter, unseemly, and unbearable.
It should be noted that these suits for slander were between
persons in every station of life. When Anneke Jans Bogardus (wife of
Dominie Bogardus, the second established clergyman in New
Netherlands), would not remain in the house with one Grietje van
Salee, a woman of doubtful reputation, the latter told throughout the
neighborhood that Anneke had lifted her petticoats when crossing
the street, and exposed her ankles in unseemly fashion; and she
also said that the Dominie had sworn a false oath. Action for slander
was promptly begun, and witnesses produced to show that Anneke
had flourished her petticoats no more than was seemly and tidy to
escape the mud. Judgment was pronounced against Grietje and her
husband. She had to make public declaration in the Fort that she
had lied, and to pay three guilders. The husband had to pay a fine,
and swear to the good character of the Dominie and good carriage of
the Dominie’s wife, and he was not permitted to carry weapons in
town,—a galling punishment.
Dominie Bogardus was in turn sued several times for slander,—
once by Thomas Hall, the tobacco planter, simply for saying that
Thomas’ tobacco was bad; and again, wonderful to relate, by one of
his deacons—Deacon Van Cortlandt.
Special punishment was provided for women. Old Dr. Johnson
said gruffly to a lady friend: “Madam, there are different ways of
restraining evil; stocks for men, a ducking-stool for women, pounds
for beasts.” The old English instrument of punishment,—as old as
the Doomsday survey,—the cucking-stool or ducking-stool, was in
vogue here, was insultingly termed a “publique convenience,” and
was used in the Southern and Central colonies for the correction of
common scolds. We read in Blackstone’s Commentaries, “A
common scold may be indicted and if convicted shall be sentenced
to be placed in a certain engine of correction called the trebucket,
castigatory, or cucking-stool.” Still another name for this “engine”
was a “gum-stool.” The brank, or scold’s bridle,—a cruel and
degrading means of punishment employed in England for “curst
queans” as lately as 1824,—was unknown in America. A brank may
be seen at the Guildhall in Worcester, England. One at Walton-on-
Thames bears the date 1633. On the Isle of Man, when the brank
was removed, the wearer had to say thrice, in public, “Tongue, thou
hast lied.” I do not find that women ever had to “run the gauntelope”
as did male offenders in 1685 in Boston, and, though under another
name, in several of the provinces.
Women in Maine were punished by being gagged; in Plymouth,
Mass., and in Easthampton, L. I., they had cleft sticks placed on their
tongues in public; in the latter place because the dame said her
husband “had brought her to a place where there was neither gospel
nor magistracy.” In Salem “one Oliver—his wife” had a cleft stick
placed on her tongue for half an hour in public “for reproaching the
elders.” It was a high offence to speak “discornfully” of the elders and
magistrates.
The first volume of the American Historical Record gives a letter
said to have been written to Governor Endicott, of Massachusetts, in
1634 by one Thomas Hartley from Hungar’s Parish, Virginia. It gives
a graphic description of a ducking-stool, and an account of a ducking
in Virginia. I quote from it:—
The day afore yesterday at two of ye clock in ye afternoon I
saw this punishment given to one Betsey wife of John Tucker,
who by ye violence of her tongue had made his house and ye
neighborhood uncomfortable. She was taken to ye pond
where I am sojourning by ye officer who was joyned by ye
 magistrate and ye Minister Mr. Cotton, who had frequently
admonished her and a large number of People. They had a
machine for ye purpose yᵗ belongs to ye Parish, and which I
was so told had been so used three times this Summer. It is a
platform with 4 small rollers or wheels and two upright posts
between which works a Lever by a Rope fastened to its
shorter or heavier end. At the end of ye longer arm is fixed a
stool upon which sᵈ Betsey was fastened by cords, her gown
tied fast around her feete. The Machine was then moved up
to ye edge of ye pond, ye Rope was slackened by ye officer
and ye woman was allowed to go down under ye water for ye
space of half a minute. Betsey had a stout stomach, and
would not yield until she had allowed herself to be ducked 5
severall times. At length she cried piteously Let me go Let me
go, by Gods help I’ll sin no more. Then they drew back ye
machine, untied ye Ropes and let her walk home in her
wetted clothes a hopefully penitent woman.
I have seen an old chap-book print of a ducking-stool with a “light
huswife of the banck-side” in it. It was rigged much like an old-
fashioned well-sweep, the woman and chair occupying the relative
place of the bucket. The base of the upright support was on a low-
wheeled platform.
Bishop Meade, in his Old Churches, Ministers, and Families of
Virginia, tells of one “scolding quean” who was ordered to be ducked
three times from a vessel lying in James River. Places for ducking
were prepared near the Court Houses. The marshal’s fee for ducking
was only two pounds of tobacco. The ducking-stools were not kept in
church porches, as in England. In 1634 two women were sentenced
to be either drawn from King’s Creek “from one Cowpen to another
at the starn of a boat or kanew,” or to present themselves before the
congregation, and ask forgiveness of each other and God. In 1633 it
was ordered that a ducking-stool be built in every county in
Maryland. At a court-baron at St. Clements, the county was
prosecuted for not having one of these “public conveniences.” In
February, 1775, a ducking-stool was ordered to be placed at the
confluence of the Ohio and Monongahela Rivers, and was doubtless
used. As late as 1819 Georgia women were ducked in the Oconee
River for scolding. And in 1824, at the court of Quarter Sessions, a
Philadelphia woman was sentenced to be ducked, but the
punishment was not inflicted, as it was deemed obsolete and
contrary to the spirit of the times. In 1803 the ducking-stool was still
used in Liverpool, England, and in 1809 in Leominster, England.
One of the last indictments for ducking in our own country was that
of Mrs. Anne Royall in Washington, almost in our own day. She was
a hated lobbyist, whom Mr. Forney called an itinerant virago, and
who became so abusive to congressmen that she was indicted as a
common scold before Judge William Cranch, and was sentenced by
him to be ducked in the Potomac. She was, however, released with a
fine.
Women curst with a shrewish tongue were often punished in
Puritan colonies. In 1647 it was ordered that “common scoulds” be
punished in Rhode Island by ducking, but I find no records of the
punishment being given. In 1649 several women were prosecuted in
Salem, Mass., for scolding; and on May 15, 1672, the General Court
of Massachusetts ordered that scolds and railers should be gagged
or “set in a ducking-stool and dipped over head and ears three
times,” but I do not believe that this law was ever executed in
Massachusetts. Nor was it in Maine, though in 1664 a dozen towns
were fined forty shillings each for having no “coucking-stool.” Equally
severe punishments were inflicted for other crimes. Katharine Ainis,
of Plymouth, was publicly whipped on training day, and ordered to
wear a large B cut in red cloth “sewed to her vper garment.” In 1637
Dorothy Talbye, a Salem dame, for beating her husband was
ordered to be bound and chained to a post. At a later date she was
whipped, and then was hanged for killing her child, who bore the
strange name of Difficulty. No one but a Puritan magistrate could
doubt, from Winthrop’s account of her, that she was insane. Another
“audatious” Plymouth shrew, for various “vncivill carriages” to her
husband, was sentenced to the pillory; and if half that was told of her
was true, she richly deserved her sentence; but, as she displayed
“greate pensiveness and sorrow” before the simple Pilgrim
magistrates, she escaped temporarily, to be punished at a later date