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Recurrent Pregnancy Loss
Causes, Controversies, and Treatment
Series in Maternal-Fetal Medicine
About the Series

Published in association with the Journal of Maternal Fetal and Neonatal Medicine, the series in
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The Long-Term Impact of Medical Complications in Pregnancy: A Window into Maternal and
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Operative Obstetrics, Fourth Edition
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Neurology and Pregnancy: Clinical Management
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Recurrent Pregnancy Loss: Causes, Controversies, and Treatment, Third Edition
Howard J.A. Carp
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Recurrent Pregnancy Loss
Causes, Controversies, and Treatment
Third Edition
Edited by
Howard J.A. Carp, MB BS, FRCOG
Clinical Professor
Obstetrics and Gynecology
Sheba Medical Center, Tel Hashomer
and
Sackler School of Medicine
Tel Aviv University
Tel Aviv, Israel
Front cover: Disorganized embryo as seen on embryoscopy. Picture courtesy of Thomas Philipp, MD, Vienna, Austria.
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Contents
Preface.....................................................................................................................................................viii
Contributors............................................................................................................................................... ix
Part I Basic Principles

1. The Epidemiology of Recurrent Pregnancy Loss.......................................................................... 1
Ole B. Christiansen
2. The Signaling between Embryo and Mother as a Basis for the Development of Tolerance.... 13
Eytan R. Barnea
3. Recurrent Pregnancy Lossfrom Evidence-Based to Personalized Medicine........................... 22

Howard J.A. Carp
Part II Etiology
4. The Genetics of Spontaneous Abortions....................................................................................... 30
Joe Leigh Simpson
5. The Endometrial Factor in Recurrent Pregnancy Loss.............................................................. 43

Luiza Borges Manna and Ying Cheong
6. Fetal Structural Malformations and Recurrent Pregnancy Loss.............................................. 48

Howard J.A. Carp, Thomas Philipp, Micha Baum, and Michal Berkenstadt
7. The Endocrinology of Recurrent Pregnancy Loss....................................................................... 59

Nicola Pluchino, Serena Bellaminutti, Panagiotis Drakapoulos, Antonis Makrigiannakis,
and Andrea R. Genazzani
8. The Etiology of the Antiphospholipid Syndrome......................................................................... 70

Sara De Carolis, Giuseppina Monteleone, Cristina Garufi, Rotem Inbar, Miri Blank,
and Yehuda Shoenfeld
9. Defects in Coagulation Factors Leading to Recurrent Pregnancy Loss................................... 79

Aida Inbal and Howard J.A. Carp
10. The Immunobiology of Recurrent Miscarriage........................................................................... 89

Marighoula Varla-Leftherioti, Theodora Keramitsoglou, and Christina Tsekoura
11. Immune Testing in Recurrent Pregnancy Loss..........................................................................101

Jeffrey Braverman, Darren Ritsick, and Nadera Mansouri-Attia
12. Uterine Anomalies and Recurrent Pregnancy Loss...................................................................110

Daniel S. Seidman and Mordechai Goldenberg
v
vi Contents
13. The Male Factor in Recurrent Pregnancy Loss......................................................................... 126

Catherine F. Ingram, Nannan Thirumavalavan, Marc Goldstein, and Dolores J. Lamb
Part III The Developing Pregnancy

14. Ultrasound Follow-Up in Early Pregnancy................................................................................ 134
Akhila Vasudeva and Pratap Kumar
15. Threatened Miscarriage and Recurrent Pregnancy Loss.........................................................145

Howard J.A. Carp
16. The Role of Cerclage and Pessaries..............................................................................................153

Israel Hendler and Howard J.A. Carp
17. What Genetic Screening Is Appropriate in Recurrent Pregnancy Loss?............................... 164

Howard Cuckle
18. Obstetric Outcomes after Recurrent Pregnancy Loss...............................................................172

Rakefet Yoeli-Ullman, Howard J.A. Carp, and Shali Mazaki-Tovi
Part IV Management
19. Investigation Protocol for Recurrent Pregnancy Loss.............................................................. 184
Howard J.A. Carp
20. Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? Yes............................ 197
Ashok Kumar and Simar Kaur
21. Debate: Should Progestogens Be Used in Recurrent Pregnancy Loss? No............................. 202
Roy Mashiach
22. Human Chorionic Gonadotropin Supplementation in Recurrent Pregnancy Loss............... 206

Carlo Ticconi, Adalgisa Pietropolli, and C.V. Rao
23. Antiphospholipid Syndrome: Management of the Obstetric Patient........................................215

Ashley E. Benson and D. Ware Branch
24. Can Recurrent Pregnancy Loss Be Prevented by Antithrombotic Agents?........................... 223

Audrey A. Merriam and Michael J. Paidas
25. Empirical In Vitro Fertilization for Recurrent Pregnancy Loss: Is It a Valid Concept?.......231
Michal Kirshenbaum and Raoul Orvieto
26. Debate: Should PGT-A Still Be Performed in Recurrent Pregnancy Loss? Yes.................... 239
Carmen M. García-Pascual, Pilar López, Nasser Al-Asmar, Pere Mir, Lorena Rodrigo,
Carlos Simon, and Carmen Rubio
27. Debate: Should PGT-A Still Be Performed in Recurrent Pregnancy Loss? No..................... 243
Raoul Orvieto and Norbert Gleicher
Contents vii
28. Third Party Reproduction in Recurrent Pregnancy Loss........................................................ 249

Gautam Nand Allahbadia, Rubina Merchant, Akanksha Allahbadia Gupta, and A.H. Maham
Part V Immunotherapy
29. Leucocyte Immunotherapy for Recurrent Miscarriage............................................................ 257
Salim Daya
30. IVIg Treatment for Recurrent Pregnancy Loss........................................................................ 268

Carolyn B. Coulam
31. The Role of Filgrastim.................................................................................................................. 275

Fabio Scarpellini and Marco Sbracia
32. Opinion: Immunotherapy Has No Place in the Treatment of Recurrent

Pregnancy Loss.............................................................................................................................. 280
Micha Baum
Index....................................................................................................................................................... 287
Preface
Six years have passed since the second edition of this book, and
thirteen since the first edition. Major advances have necessitated a
new edition. Genetics, in particular, has evolved out of all recognition
with the introduction of higher and higher-resolution analyses which
are being employed to make more accurate diagnoses. These changes
are summarized in Chapters 4 and 6. However, the prevention of
genetic aberrations by PGT-A is a hotly debated issue; the two
sides are summarized in Chapters 26 and 27. The first two editions
contained numerous debates on controversial subjects in recurrent
pregnancy loss (RPL). Many of these contentious issues have now
reached a consensus and can be summarized in chapters rather than
debates. The guidelines of the various professional organizations
have narrowed their differences somewhat. However, none relates
to the resistant patient who continues to miscarry despite the various
treatment modalities recommended in the guidelines. The resistant
patient is addressed in Chapters 19 and 28. New chapters have been
added regarding structural anomalies, empirical in vitro fertilization,
and personalized medicine as opposed to evidence-based medicine and which immune assessment should
be used.
RPL remains a distressing problem. Patients understandably expect answers and solutions. The
physician often does not have these answers. Recommendations vary from inactivity and follow-up
to intensive investigation and treatment. Recommendations are confounded by the lack of a universal
definition of RPL and often failure to distinguish between good and poor prognosis patients. This edition,
like the previous editions of this book, tries to summarize the controversies and discuss the scientific
basis for various causes of RPL in depth and to clarify the various treatment modalities. It is hoped that
we have succeeded in this endeavor.
The book is planned for general gynecologists, and specialists working in the field. Each contributing
author is an authority on a specific area of recurrent pregnancy loss. All chapters have undergone major
revision to include the changes that have occurred since the second edition.
I would like to thank each author for the time and effort taken in preparing the manuscripts to make
the publication of this book possible. I would also like to thank those responsible in a more indirect way
for the publication of this book: my teachers over the years, and my collaborators. However, special
recognition goes to the greatest teachers and collaborators of all, the patients.
Howard J.A. Carp, MB BS, FRCOG
viii
Contributors
Nasser Al-Asmar D. Ware Branch

Igenomix Valencia University of Utah Health
Valencia, Spain Salt Lake City, Utah
Gautam Nand Allahbadia Jeffrey Braverman

Reproductive Endocrinology and IVF, MMC IVF Reproductive Immunology
and New York City, New York
Bourn Hall IVF
Jumeirah, Dubai, United Arab Emirates Sara De Carolis
and Department of Obstetrics, Gynaecology
Orchid Fertility and Andrology Services, DHCC and Pediatrics
and F. Policlinico Gemelli IRCCS
Dr. Amal Elias Fertility Center Università Cattolica del Sacro Cuore
and Rome, Italy
Millennium Medical Center MMC IVF
Dubai, United Arab Emirates Howard J.A. Carp
Department of Obstetrics and Gynecology
Eytan R. Barnea Sheba Medical Center
Society for the Investigation of Early Tel Hashomer, Israel
Pregnancy and
New York City, New York Sackler School of Medicine
Tel Aviv University
Micha Baum Tel Aviv, Israel
Sheba Medical Center
Tel Hashomer, Israel Ying Cheong
University of Southampton
Serena Bellaminutti and
Division of Obstetrics and Gynaecology Complete Fertility Southampton
University of Geneva Southampton, United Kingdom
Geneva, Switzerland
Ole B. Christiansen
Ashley E. Benson Department of Obstetrics and Gynaecology
University of Utah Health Center for Recurrent Pregnancy Loss of Western
Salt Lake City, Utah Denmark
Aalborg University Hospital
Michal Berkenstadt Aalborg, Denmark
Danek Gertner Institute of Human
Genetics Carolyn B. Coulam
Sheba Medical Center Clinical Immunology Laboratory
Tel Hashomer, Israel Rosalind Franklin University of Medicine and
Science
Miri Blank North Chicago, Illinois
Zabludowicz Center for Autoimmune Diseases
Sheba Medical Center Howard Cuckle
Sackler Faculty of Medicine Department of Obstetrics and Gynecology
Tel Aviv University Columbia University Medical Center
Tel Aviv, Israel New York City, New York
ix
x Contributors
Salim Daya Akanksha Allahbadia Gupta

McMaster University Indira IVF
Hamilton, Ontario, Canada New Delhi, India
and
Newlife Fertility Centre Israel Hendler
Mississauga, Ontario, Canada Obstetrics and Gynecology
Sackler Medical School
Panagiotis Drakapoulos Tel Aviv University
Center for Reproductive Medicine Tel Aviv, Israel
Universitair Ziekenhuis Brussel and
Brussels, Belgium Department of Obstetrics and Gynecology
Sheba Medical Center
Carmen M. García-Pascual Tel Hashomer, Israel
Igenomix Valencia
Valencia, Spain Aida Inbal
Thrombosis and Hemostasis Unit
Cristina Garufi Beilinson Hospital
Lupus Clinic, Rheumatology, Dipartimento di Rabin Medical Center
Medicina Interna e Specialità Mediche Petah Tikva, Israel
Sapienza Università di Roma and
Rome, Italy Sackler Faculty of Medicine
Tel Aviv University
Andrea R. Genazzani Tel Aviv, Israel
Division of Obstetrics and Gynaecology
University of Pisa Rotem Inbar
Pisa, Italy Zabludowicz Center for Autoimmune
Diseases and Department of Obstetrics and
Norbert Gleicher Gynecology
The Center for Human Reproduction Sheba Medical Center
and Tel Hashomer, Israel
Foundation for Reproductive Medicine
and Catherine F. Ingram
Stem Cell Biology and Molecular Embryology Baylor College of Medicine
Laboratory Houston, Texas
The Rockefeller University
Simar Kaur
New York City, New York
Department of Obstetrics and Gynaecology
and
Maulana Azad Medical College and Associated
Lok Nayak Hospital
Vienna University of Medicine
New Delhi, India
Vienna, Austria
Theodora Keramitsoglou
Mordechai Goldenberg
Department of Immunology and
Histocompatibility
Chaim Sheba Medical Center
Helena Venizelou Maternity Hospital
Tel Hashomer, Israel
Athens, Greece
and
Sackler School of Medicine Michal Kirshenbaum
Tel Aviv University Department of Obstetrics and Gynecology
Tel Aviv, Israel Chaim Sheba Medical Center
Marc Goldstein and
Department of Urology Sackler Faculty of Medicine
Weill Cornell School of Medicine Tel Aviv University
New York City, New York Tel Aviv, Israel
Contributors xi
Ashok Kumar Rubina Merchant

Department of Obstetrics and Gynaecology Rotunda—The Center for Human
Maulana Azad Medical College and Associated Reproduction
Lok Nayak Hospital Mumbai, India
New Delhi, India
Audrey A. Merriam
Pratap Kumar Division of Maternal-Fetal Medicine
Department of Obstetrics and Gynecology Department of Obstetrics, Gynecology and
Kasturba Medical College Reproductive Sciences
Manipal Academy of Higher Education Yale University
Manipal, Karnataka, India New Haven, Connecticut
Dolores J. Lamb Pere Mir

Department of Urology and Center for Igenomix Valencia
Reproductive Genomics Valencia, Spain
Weill Cornell School of Medicine
New York City, New York Giuseppina Monteleone
Department of Obstetrics, Gynaecology and
Pilar López Pediatrics
Igenomix Argentina F. Policlinico Gemelli IRCCS
Caba, Argentina Università Cattolica del Sacro Cuore
Rome, Italy
A.H. Maham
MMC IVF Raoul Orvieto
Dubai, United Arab Emirates Department of Obstetrics and Gynecology
Chaim Sheba Medical Center
Antonis Makrigiannakis
and
University of Crete
Family Planning and Fertility Regulation
Heraklion, Greece
Sackler Faculty of Medicine
Luiza Borges Manna Tel Aviv University
North Middlesex Hospital Tel Aviv, Israel
London, United Kingdom
and Michael J. Paidas
Obstetrics and Gynaecology Department of Obstetrics, Gynecology and
Wessex Deanery, United Kingdom Reproductive Sciences
Miller School of Medicine
Nadera Mansouri-Attia University of Miami
Braverman Reproductive Immunology Miami, Florida
New York City, New York
Thomas Philipp
Roy Mashiach Gynecology and Obstetrics
Department of Obstetrics and Gynecology Danube Hospital
Sheba Medical Center Vienna, Austria
Adalgisa Pietropolli
Shali Mazaki-Tovi Academic Department of Systems Medicine
Department of Obstetrics and Gynecology Tor Vergata University
Sheba Medical Center Rome, Italy
and Nicola Pluchino
Sackler School of Medicine Division of Obstetrics and Gynaecology
Tel Aviv University University of Geneva
Tel Aviv, Israel Geneva, Switzerland
xii Contributors
C.V. Rao Carlos Simon

Department of Cellular Biology and Igenomix Valencia
Pharmacology and
Herbert Wertheim College of Medicine Department of Obstetrics and Gynaecology
Florida International University Valencia University and INCLIVA
Miami, Florida Valencia, Spain
and
Darren Ritsick Department of Obstetrics and Gynaecology
Braverman Reproductive Immunology Stanford University
New York City, New York Stanford, California
Lorena Rodrigo Joe Leigh Simpson

Igenomix Valencia Department of Human and Medical Genetics
and Herbert Wertheim College of Medicine
Ronda Narciso Monturiol Florida International University
Valencia, Spain Miami, Florida
and
Carmen Rubio Reproductive Genetic Innovation (RGI)
Igenomix Valencia Northbrook, Illinois
Valencia, Spain
Nannan Thirumavalavan
Scott Department of Urology
Marco Sbracia
Baylor College of Medicine
Hungaria Center for Endocrinology and
Houston, Texas
Reproductive Medicine (CERM)
Rome, Italy
Carlo Ticconi
Academic Department of Surgical Sciences
Fabio Scarpellini
Tor Vergata University
Hungaria Center for Endocrinology and
Rome, Italy
Reproductive Medicine (CERM)
Rome, Italy Christina Tsekoura
Department of Immunology and
Daniel S. Seidman Histocompatibility
Department of Obstetrics and Gynecology Helena Venizelou Maternity Hospital
Chaim Sheba Medical Center Athens, Greece
and Marighoula Varla-Leftherioti
Sackler School of Medicine Department of Immunology and Histocompatibility
Tel Aviv University Helena Venizelou Maternity Hospital
Tel Aviv, Israel Athens, Greece
Yehuda Shoenfeld Akhila Vasudeva

Internal Medicine Department of Obstetrics and Gynecology
Zabludowicz Center for Autoimmune Diseases Kasturba Medical College
Sheba Medical Center Manipal Academy of Higher Education
Tel Hashomer, Israel Manipal, Karnataka, India
and
Research of Autoimmune Diseases Rakefet Yoeli-Ullman
Sackler Faculty of Medicine Department of Obstetrics and Gynecology
Tel Aviv University Sheba Medical Center
Tel Aviv, Israel Tel Hashomer, Israel
1
The Epidemiology of Recurrent Pregnancy Loss
Ole B. Christiansen
Substantial disagreement exists about spontaneous prognosis after recurrent pregnancy loss (RPL),
probably due to differences in monitoring intensity between studies. In future studies of prognosis in
RPL it is suggested that the live birth rate per time unit is introduced as the main outcome measure.
Introduction
The term miscarriage (or abortion) is used to describe a pregnancy that fails to progress, resulting
in death and expulsion of the embryo or fetus. The World Health Organization (WHO) definition [1]
stipulates that the fetus or embryo should weigh 500 g or less, a stage that corresponds to a gestational
age of 20 weeks. The European Society for Human Reproduction and Embryology (ESHRE) defines a
miscarriage as an intrauterine pregnancy demise prior to viability confirmed by ultrasound or histology,
whereas miscarriages, biochemical pregnancy losses, and pregnancies of unknown location (PULs)
are jointly termed pregnancy losses [2]. Recurrent miscarriage (RM) has traditionally been defined as
≥3 consecutive miscarriages, and recurrent pregnancy loss (RPL) as ≥3 pregnancy losses. However,
the American Society for Reproductive Medicine (ASRM) RPL defines RPL as ≥2 not necessarily
consecutive clinical miscarriages [3], and recently ESHRE’s RPL guideline group also defined RPL as
≥2 not necessarily consecutive pregnancy losses [4].
Including women with two previous pregnancy losses in studies of RPL is epidemiologically very
problematic. If the ASRM/ESHRE definition of >2 losses is used, the vast majority of patients will
have a good prognosis for live birth. The live birth rate after two consecutive pregnancy losses is
75%–80% in the next pregnancy [5,6] or within 3 years [7]. The ≥2 definition of RM/RPL assumes that
the prognosis for pregnancy losses is similar in women with the same number of previous consecutive
or nonconsecutive pregnancy losses, e.g., a woman with four pregnancy losses after a birth has the
same prognosis in the next pregnancy as a woman with three pregnancy losses followed by a live birth
followed by one miscarriage. Only one study [8] has addressed whether pregnancy losses prior to a live
birth have similar prognosis as those subsequent to a live birth. In a multivariate analysis of 127 patients
with unexplained secondary RPL, each pregnancy loss after the birth, and in particular the presence
of a second trimester miscarriage after the birth, increased the risk for subsequent pregnancy loss with
incidence rate ratio (IRR) = 1.14 (95% confidence interval [CI] 1.04–1.24, p = 0.002) and IRR = 2.15
(95% CI 1.57–2.94, p < 0.0001), respectively, whereas early and late pregnancy losses prior to the birth
did not exhibit any prognostic impact. According to this study [8], a patient with four pregnancy losses
after a birth will have a 50% chance of a live birth compared to a 90% chance in a patient with three
losses prior to but only one loss after the live birth. Knowledge about the prognosis is important for
designing valid trials.
1
2 Recurrent Pregnancy Loss
Epidemiologic Parameters Relevant for RPL

Occurrence
The prevalence of RM/RPL is the number of women in a population who, at a specific time point, meet
the definition, and the incidence is the number of new women per time unit suffering a new pregnancy
loss, and the prevalence or incidence is often expressed as the proportion of individuals at risk for the
disorder. The denominator could be all women in the population, women of fertile age, or women who
had attempted pregnancy at least two or three times. The estimate of the prevalence or incidence of RM/
RPL is uncertain since in most countries there is no nationwide registration of miscarriages or RM/RPL.
In addition, many early pregnancy losses are not treated in hospitals and are thus not registered. There
are a few older estimates of the prevalence of RM based on the definition of ≥3 consecutive miscarriages.
One of the most informative studies was the retrospective study by Alberman [9]. Nine out of 1097 female
doctors (0.8%) who had had three or four previous pregnancies reported ≥3 consecutive miscarriages.
However, since the study was summarized before 1990, many early miscarriages may not have been
registered due to the lack of highly sensitive hCG tests and ultrasound examinations at that time.
Other estimates of the prevalence of RM roughly concord with that of Alberman. In a group of 5901
women with ≥2 pregnancies screened for toxoplasma antibodies, 1.4% had experienced RM [10]. Data
from a questionnaire-based study [11] found in a sample of 493 women with ≥2 intrauterine pregnancies
that 0.6% had had ≥3 consecutive miscarriages and 1.8% had had ≥3, not necessarily consecutive, losses.
Overall, the prevalence of RM according to the old definitions seems to be between 0.6% and 1.4%.
A problem in adapting the new RPL definitions is that the number of women who meet the criteria
will expand substantially. In Alberman’s [9] study among 2062 women who had had two to four previous
pregnancies, 42 reported ≥2 not necessarily consecutive miscarriages (3.25%) which is significantly
higher than the 0.6%–1.4% prevalence of RM using the traditional definition. This suggests that adapting
the new RM/RPL definition will triple the prevalence of the diagnosis. The implications of this are
discussed later.
The observation that the RPL prevalence is >1% indicates that RPL is not a random event but a disorder
affecting women who have an increased risk of pregnancy loss. If RPL (according to the old definition)
were caused by a random accumulation of “sporadic” miscarriages mainly caused by fetal aneuploidy,
the prevalence of RPL would be 0.153 = 0.34% (based on a frequency of sporadic miscarriage of 15%
in the population [9]) rather than 1%. The 1% prevalence indicates that most RPL cases are caused by
nonrandom factors which increase the risk of pregnancy loss in each pregnancy.
Knowledge of changes in the incidence of RPL are important and can inform us about changes in
environmental or genetic risk factors of importance for pregnancy loss. Roepke et al. [12] in a nationwide
register-based study found that the incidence of women with three or more consecutive pregnancy losses
had increased significantly in Sweden from 2003 to 2012. If the denominator was all women in Sweden
aged 18–42 years in the period, the incidence increased from 0.042% to 0.069%, relative increase 74%
(p < 0.0001) and if the denominator was women with a least one pregnancy in the period, the incidence
increased from 0.55% to 0.82%, relative increase 58% (p < 0.0001). Changes in maternal age, body mass
index (BMI), and coding practice during the period could not explain the change.
Number of Previous Miscarriages

Prospective studies of RPL patients show remarkable consistency in finding an increasing risk of
pregnancy loss as the number of previous pregnancy losses increases. The chance of subsequent live birth
in untreated RPL patients with 3, 4, and ≥5 pregnancy losses has been reported to be 42%–86%, 41%–
72%, and 23%–51%, respectively [13–16] (Figure 1.1). The significant variability in the estimate of the
subsequent risk of pregnancy loss in RPL patients can probably be attributed to the time of ascertainment
of the pregnancies (Figure 1.2) since the average age of the patients and the duration of follow-up in the
various studies were not different. The information in Figure 1.2 is based on data directly supplied or
can unequivocally be deduced from the literature [14,16,17]. In studies where the patients were urged
to contact the department for inclusion in a treatment trial as soon as menstruation was overdue and the
The Epidemiology of Recurrent Pregnancy Loss 3
3 misc. 4 misc. 5+ misc.

100
80
Birth rate (%)
60
40
20
0
Ref. 13 Ref. 14 Ref. 15 Ref. 16
FIGURE 1.1 Subsequent birth rate according to the number of previous miscarriages in patients with RPL reported in
four studies (col. 1, ref. 15; col. 2, ref. 14; col. 3, ref. 13; col. 4, ref. 16).
pregnancy test was positive [16], almost all biochemical pregnancies were identified and the patients
would be registered as having a high pregnancy loss rate (47.1%) but a low nonpregnancy rate (14.7%)
during the observation period. In studies where the patients were told to call the department in gestational
week 6–7 and were included in treatment trials [17] or cohorts receiving standard care [14] only after
ultrasonographic demonstration of fetal heart action, most biochemical pregnancies would not be
ascertained and therefore significantly higher nonpregnancy rates (38.3%–55.6%) and significantly lower
pregnancy loss rates (11.1%–14.4%) would be registered compared with the former study (Figure 1.2).
The subsequent probability of live birth in RPL can best be estimated using data from the placebo arm
of placebo-controlled trials [16,17] (Figure 1.2) because in such trials the ascertainment of pregnancies
is generally better than in nonrandomized studies, as patients are included according to strict protocols
and are closely monitored in early pregnancy. Hence, more very early pregnancy losses are included in
placebo-controlled than in nonrandomized studies [18].
The negative prognostic effect of the number of previous pregnancy losses could be due to maternal
age being positively correlated to gravidity. However, in multivariate analyses of clinical parameters of
prognostic impact in RPL, the number of previous pregnancy losses has without exception remained the
strongest prognostic parameter even after adjustment for other risk factors [8,13,19,20].
Not pregnant Miscarriages Births
60
N = 45 N = 153 N = 34
50
*
**
40
Clinical
*
30
%
20
Preclinical
10 ** *
**
0
Ref. 17 Ref. 14 Ref. 16
FIGURE 1.2 Frequency of women registered as not being pregnant, miscarrying, or giving birth in three prospective
cohorts of untreated patients with RPL (col. 1, ref. 13; col. 2, ref. 14; col. 3, ref. 15; col. 4., ref. 16). Ref. 16 indicates the
proportion of both preclinical and clinical miscarriage; all miscarriages in ref. 17 (except one) and in ref. 14 were clinical.
*p = 0.001; **p < 0.0001, χ2 test.
Three miscarriages
Four miscarriages
Five miscarriages
Six or more miscarriages
100
Women with recurrent miscarriage and one or
more live births after first consultation (%)
80
60
40
20
0
0 5 10 15 20 25
Years elapsed after date of first consultation
FIGURE 1.3 Cumulative live birth rates per time unit in women with RPL according to the number of previous pregnancy
losses. (Reproduced with permission from Lund M et al. Obstet Gynecol. 2013;119. 37–43.)
Assessing the outcome of the first pregnancy after referral in order to assess prognosis is problematic.
A 100% follow-up is necessary and if very early biochemical pregnancies are included in the outcome
data (which they should), very close monitoring of the patients must be undertaken. In addition, the
outcome of the first pregnancy after referral is not clinically relevant since most patients have no problems
conceiving and will have further pregnancy attempts. For the patients, the only relevant outcome is a live
birth. We have proposed that the most relevant method of assessing prognosis is to calculate the chance
of a subsequent live birth per time unit after the date of first consultation. In countries with valid national
birth registers and the possibility of identifying all individuals in the registers through unique personal
identification numbers, an almost 100% follow-up of RPL women with regard to live births is possible.
In a study of 987 women with RPL [21], the chance for live birth after 5 years’ follow-up was 71.9%
after three, declining to 50.2% after ≥6 previous pregnancy losses (Figure 1.3). There was only a minor
additional improvement of the live birth chance after 5 years had elapsed.
Maternal Age
In a register-based study of 634,272 Danish women achieving pregnancy between 1978 and 1992 who
attended a hospital during pregnancy [22], the miscarriage rates in women with RPL were almost identical
in women of age 30–34 years and 35–39 years (38%–40%) but it increased to 70% in women of age 40–44
(Figure 1.4). It seems that the impact of age on the miscarriage rate is quite modest in RPL until age 40,
but beyond this age it is the strongest prognostic factor. In concordance with this, several multivariate
analyses [8,13,19] in RPL patients (almost all of whom were younger than 40), found that maternal age was
not a significant predictor of pregnancy loss after adjustment for other relevant independent variables. In
one study [8], the adjusted IRR for new pregnancy loss was 0.99 (95% CI 0.96–1.03) for each additional
year of age in patients younger than 40 years, indicating no impact at all.
Subgroups of RPL
Three different groups of women should be assessed separately: (a) the primary RPL group consists of
women with ≥3 consecutive pregnancy losses with no pregnancy progressing beyond 20 weeks’ gestation,
(b) the secondary RPL group consists of women who have had ≥3 pregnancy losses following a pregnancy,
80
70
60
50
Birth rate (%)
< 30 years
40 31–35 years
36–39 years
30
40–44 years
20
10
0
Ref. 22 Ref. 15
FIGURE 1.4 Subsequent birth rate according to maternal age in patients with RPL reported in two studies (col. 1, ref.
22; col. 2, ref. 15).
that progressed beyond 20 weeks’ gestation, which may have ended in live birth, stillbirth, or neonatal
death, and (c) the tertiary RPL group, which consists of women who have had several pregnancy losses
before a pregnancy that progressed beyond 20 weeks’ gestation followed by ≥3 pregnancy losses [18].
In some studies, secondary RPL is defined as RPL after a live birth [23] or a pregnancy that progressed
beyond gestational week 28; however, in this survey the 20-week cutoff will be used. Unfortunately,
many studies fail to distinguish patients with primary and secondary RPL. It is indeed possible that
secondary RPL is not a particular entity but just the clinical appearance of the RPL syndrome among
patients who, by chance, instead of delivering their child after three or four miscarriages deliver in the
first pregnancy and subsequently experience a series of miscarriages. However, there is support from
immunogenetic studies [24,25], NK cells [23,26], and immunotherapy [27,28] that secondary RPL is a
separate entity with characteristics different from primary RPL. If primary and secondary RPL have
different pathophysiological mechanisms, different prognoses would be expected. Summarizing the
placebo-treated patients included in the author’s placebo-controlled trials of immunotherapy [16,28],
the live birth rate in the first pregnancy was 17/35 = 48.6% in women with primary RPL compared with
11/34 = 32.4% in women with secondary RPL (not significant) when matched for the number of previous
miscarriages and age. Other studies have reported success rates [14,15] in the two subsets that are not
different, which is the commonly accepted view.
RPL patients with second trimester losses constitute a different subset. Drakeley et al. [29] found that
25% of their RPL patients had had at least one second trimester loss. Among 228 RPL patients admitted
to the RPL clinic in Copenhagen 2000–2004, 39 (17.1%) had experienced a mixture of first and second
trimester miscarriages but only three had suffered exclusively second trimester losses. Since almost all
patients with second trimester miscarriages had experienced first trimester miscarriages, early and late
RPL must have pathogenic factors that partially overlap. Several prospective studies indicate that a history
of second trimester pregnancy losses has a strong negative prognostic impact [8,30,31].
Familial Aggregation
Few studies have investigated the occurrence of RPL in families of RPL couples with normal chromosomes.
Results from published family studies are shown in Table 1.1. Johnson et al. [32], Alexander et al. [33],
and Ho et al. [34] compared the prevalence of RPL among relatives of women with RPL with the
corresponding prevalence in relatives of fertile controls. Christiansen et al. [35] obtained information
concerning relatives’ pregnancy outcomes from questionnaires completed by the relatives themselves,
and the stated pregnancy loses were confirmed from hospitals’ and practitioners’ records. The rate of
≥3 pregnancy losses in relatives was compared with an external control group [11]. Table 1.1 shows
that the risk of RPL in first-degree relatives of RPL patients is 2–7 times higher than in the background
TABLE 1.1
Proportion of Recurrent Pregnancy Loss (RPL) in Relatives of
Women with RPL
Reference and Kind RPL Rate in RPL Rate in
of Relatives Studied Relatives (%) Controls (%) P-value
Johnson et al. [32]
Blood relatives 12.2 7.3
Alexander et al. [33]
Mothers and sisters 7.0 0.0 0.02
Ho et al. [34]
First-degree relatives 1.4 0.2 0.0001
Christiansen et al. [35]
Sisters 10.6 1.8 0.00005
Brothers’ wives 6.3 1.8 NS
Abbreviation: NS, Not significant.
population. The relative frequency λ (= the frequency of RPL in relatives divided by the frequency in
the general population) is a measure of the degree of heritability of a disorder [36]. In the Danish study
[35], λ was 5.9 for sisters and 3.5 for brothers’ wives when comparisons are made with the population
prevalence [11], pointing toward a moderate degree of heritability of RPL.
Partner Specificity
It is commonly assumed that unexplained RPL is a partner-specific condition, and a criterion that all
pregnancies should be with the same partner has been included in the definition of primary and secondary
RPL by some authors [37]. However, no study has really addressed the question of partner specificity.
In a multivariate analysis [38], the authors’ group found that after adjustment for all relevant prognostic
factors, the chance of a subsequent live birth was not different in patients with secondary RPL who have
had all pregnancies with the same partner compared with those who have had two different partners,
casting doubt on the concept of partner specificity.
Clinical Associations
An association between RPL and perinatal complications has been reported in many studies. These
complications are fully described in Chapter 18 of this book. It is debatable whether the risk of intrauterine
growth restriction (IUGR) is associated with the previous consecutive miscarriages. However, Christiansen
et al. [39] found that the mean birth weight of women with RPL themselves was 3265 g compared with
3414 g in matched female controls (p < 0.025) and the mean birth weight of women with ≥5 miscarriages
at the time of admission was 2991 g (p < 0.001 compared with controls). The birth weights of the male
partners did not differ from the birth weight of matched male controls. These data strongly suggest that
the association between low birth weight and RPL is an inherent part of the RPL syndrome.
Lifestyle Factors
Lifestyle factors are rarely, if ever, major causes of RPL; however, studies have shown that many lifestyle
factors increase the risk of miscarriage. There is good evidence that obesity [40,41], high daily caffeine
intake [42–44], alcohol consumption [45], and use of nonsteroidal anti-inflammatory drugs [46,47]
increase the risk of miscarriage or RPL significantly. Social class and occupation also impact the rate
of miscarriage, with the greatest risk among women exposed to high physical or psychic stress during
work [48,49]. Several studies also indicate that a previous subfertility/infertility diagnosis or infertility
treatment may increase the risk of miscarriage [20,50].
Integration of Epidemiologic Knowledge in Research and Management of RPL

Occurrence
Knowing the incidence of RPL has several applications: it can be used for comparing risks of RPL
between different populations, and it can be used for comparing change in risk over time, which is
necessary for identifying risk factors. The alarming increase in the incidence of RPL in Sweden [12]
in the last 10 years should be sufficient to allocate funds in order to clarify which risk factors may have
increased in frequency. The focus could be on endocrine disruptors [51,52] or factors responsible for
the concomitant increase in the incidence of most autoimmune diseases. It has been suggested that less
exposure to helminthic infections during childhood in modern societies may have left the immune system
“uneducated” (resulting in a lack of regulatory T cells) thereby increasing the risk of autoimmune disease
and harmful immunity to the fetus and trophoblast.
Number of Miscarriages
The number of previous pregnancy losses is the most important prognostic factor in RPL and should
therefore be taken into account when planning therapeutic trials. The ideal trial should stratify for the
number of previous pregnancy losses, with randomization between control and experimental treatments
within each stratum. Stratifying the sample by the number of previous pregnancy losses may make it
easier to demonstrate the effect of the experimental intervention. It may then be easier to demonstrate
an effect in women with higher number of losses, as the spontaneous success rate is so much lower in
women with fewer losses [18,53].
Due to the new definitions of RPL as two or more losses, an increasing number of studies include women
with only two nonconsecutive pregnancy losses. Two pregnancy losses may in many cases be a chance
phenomenon. Sporadic miscarriages are due to chromosomal abnormalities in 43% of the cases [54]. Thus,
in theory, in 0.43 × 0.43 = 18.5% of women with two miscarriages the cause is due purely to embryonic
aneuploidy. Including women with only two early pregnancy losses will “dilute” the estimate of the risk factor
(in both case-control and cohort studies) or the treatment effect in controlled clinical trials. The proportion
of RPL patients in whom the disorder can be explained by an accumulation of “sporadic” pregnancy losses
declines with the number of previous losses [55]. Conversely, the proportion of euploid embryos increase
with the number of previous losses. This is supported by the fact that the frequency of many immunological
risk factors increases [24,56,57], the possible effect of immunotherapy increases [18,53], and the frequency
of aneuploid miscarriages declines [58] with the number of previous pregnancy losses.
Maternal Age
Because increased maternal age increases the subsequent pregnancy loss rate, therapeutic trials should
stratify for maternal age. However in RPL, age seems to impact on pregnancy outcome after age 40 [8,22]
(Figure 1.4) so it may be sufficient to undertake stratification according to age below and above 40 years.
Advanced maternal age is associated with several other disorders such as uterine fibroids and endocrine
and autoimmune abnormalities; therefore, maternal age should be accounted for in any trial.
Subgroups of RPL
If primary and secondary RPL and RPL with first and second trimester losses have different pathogenetic
backgrounds, the frequency of recognized risk factors for RPL and the efficacy of treatments may differ
between the groups. A series of studies have provided data suggesting that such differences exist (Table 1.2).
The factor V Leiden (FVL) genetic polymorphism is the most common cause of activated protein C
resistance (APCR), which is a risk factor for thrombosis and possibly associated with RPL [59]. Wramsby
et al. [60] found a significant association with primary but not secondary RPL, and Rai et al. [61] found
that APCR was significantly associated with the absence of a previous live birth. In a study of three
congenital thrombophilic factors (including FVL), 25.5% of women with primary RPL were positive
TABLE 1.2
Prevalence of Risk Factors or Effect of Treatments according to Subgroups of Patients
Prevalence/Effect in Prevalence/Effect in Late
Secondary vs. Primary RPL vs. Early Primary RPL
Parental chromosome abnormality Equal N/A
Antipaternal antibodies Higher Higher
Antiphospholipid antibodies Lower or equal Higher
Heriditary thrombophilia factors Lower Higher
NK cell activity Lower N/A
HLA-DRB1*03 Higher N/A
MBL deficiency N/A Higher
Allogeneic lymphocyte immunization Lower N/A
Treatment with i.v. immunoglobulin Higher N/A
Abbreviation: N/A, Cannot be estimated.
for at least one thrombophilic factor compared with 15.1% of women with secondary RPL [62]. Most
studies also claim a higher prevalence of thrombophilias in patients with second trimester miscarriages
compared to early losses [59,63].
In contrast, the prevalence of parental chromosome abnormalities is similar between primary and
secondary RPL. In a review [64] of 79 relevant studies, chromosome abnormalities were found in 3.7%
of secondary and 2.9% of primary RPL couples. Franssen et al. [65] also found that the prevalence of
parental chromosome abnormalities was similar in primary and secondary RPL. Consequently, parental
chromosome testing should be performed in both types of RPL.
A series of immunological parameters may be relevant in RPL and may have a different distribution
between the subgroups of RPL patients.
Antibodies
Alloantibodies directed against paternal/fetal human leukocyte antigens (HLAs) are produced with
increased gestational age [66,67]. Anti-HLA antibodies often persist for years and can therefore be found
more often in women with secondary compared with primary RPL [6]; however, they seem not to be
pathogenic [28,68].
Most autoantibodies can be found with increased prevalence in RPL and are associated with a poor
pregnancy prognosis [13]; however, few studies have differentiated between primary and secondary
RPL. In patients with primary RPL, the prevalence of positive anticardiolipin or antinuclear antibody
concentrations may be higher than in secondary RPL [13,69,70]. None of the differences were statistically
significant but future studies of autoantibodies in RPL should clearly distinguish between primary and
secondary RPL. There is, however, a consensus that antiphospholipid antibodies (aPL) display a stronger
association with late than early RPL [59,71].
NK Cells
NK cell numbers and cytotoxicity have been reported to predict a poor prognosis in RPL [72]. NK cell
activity has been reported to be increased in peripheral blood NK cells in primary but not secondary RPL
when compared with controls [25,26].
Class II HLA Alleles

Class II HLA alleles are associated with most autoimmune disorders. In the largest study of HLA-DRB1
alleles in patients with RPL [24], the immunological high-responder allele HLA-DRB1*03 was found
significantly more often in secondary RPL than in controls (32.4% and 21.0%, respectively; p < 0.006),
but not in primary RPL. In a later retrospective cohort study, Nielsen et al. [25] found that maternal
carriage of HLA class II alleles predisposing to immunity against male-specific HY antigens displayed a
strong prognostic impact in women with RPL after the birth of a boy [25]. Genetic polymorphism in the
MBL-2 gene are associated with low plasma levels of MBL and are more strongly associated with late
than first trimester RPL [73].
Immunotherapy also elicits different effects in primary and secondary RPL. The efficacy of paternal or
third-party leucocytes has been evaluated in a meta-analysis of placebo-controlled trials [74] showing that
immunotherapy did not improve the live birth rate in secondary RPL, whereas it significantly improved
the live birth rate in primary RPL [53]. In a meta-analysis [27], intravenous immune globulin (IVIg)
reduced the pregnancy loss rate significantly in women with secondary RPL (OR = 0.77 (95% CI 0.58–
1.02, p < 0.05) but not in patients with primary RPL. Unfortunately, these subgroup differences were not
taken into account in a recent Cochrane meta-analysis on immunotherapy in RPL [75], which concluded
that neither allogeneic lymphocyte immunization nor IVIg were efficient when all published studies were
analyzed as a single group.
Familial Aggregation
As discussed above, family studies (Table 1.1) support a multifactorial model for inheritance of RPL. The
development of many common diseases (e.g., arterial hypertension, diabetes mellitus, and schizophrenia)
is thought to be determined by a multifactorial model. One risk factor is not sufficient to cause disease
but when several intrinsic and extrinsic factors accumulate in an individual (or couple), the risk exceeds
a threshold level and disease develops. Both thrombophilic [76] and immunogenetic risk factors seem to
aggregate significantly more frequently than expected in RPL patients. Traditionally, the causes of RPL
have been assumed to be single causative factors, e.g., uterine malformations 10%, endocrine factors
10%, aPL 15%, etc. However, this model is probably inadequate, and the threshold of multiple factors
may be more appropriate [77]. In principle, RPL patients should be screened for all potential risk factors
and screening not stopped as soon as the first risk factor has been identified. The recognition that RPL
exhibits a high degree of heritability implies that susceptibility genes for RPL may be inherited by genetic
linkage analyses in families with several siblings experiencing RPL [7,78,79].
Partner Specificity
Early studies on HLA antigens in RPL assumed that increased HLA similarity between partners led to
inadequate maternal protective immune responses and fetal loss. However, after many studies on HLA
sharing in couples with RPL, the role of HLA sharing could not be confirmed [80,81]. If good quality
epidemiological studies showing little evidence of partner specificity in RPL had been performed [38]
prior to the HLA sharing studies, the theories of increased HLA sharing between RPL spouses may not
have developed.
Clinical Associations
A series of factors associated with RPL—aPL, hereditary thrombophilias, and MBL deficiency—have
also been associated with late miscarriage, low birthweight, and perinatal complications [57,59]. Since
RPL per se seems to be associated with perinatal complications and low birthweight, prospective studies
of the effect of the mentioned factors on perinatal complications should be adjusted for the confounding
effect of the number and type of previous miscarriages.
Lifestyle Factors
RPL is a complex disorder where lifestyle factors are expected to modify the effect of non-lifestyle
(intrinsic) factors previously discussed. The prevalence of the most important lifestyle factors among
patients and controls should be given in publications in order to document that the groups studied
for the occurrence of non-lifestyle risk factors or pregnancy outcome are comparable. Since it is
likely that smoking aggravates the effect of thrombophilic risk factors on the risk of pregnancy
loss, details of smoking habits should be reported in all studies of RPL and thrombophilia. It is
generally recognized that women with polycystic ovary syndrome (PCOS) exhibit an increased rate
of miscarriage and RPL. However, when adjustment for obesity is undertaken, the miscarriage rate in
PCOS does not seem to be dependent on polycystic ovarian pathology or PCOS-associated endocrine
abnormalities [41].
Conclusions
Epidemiologic studies can provide essential information for basic laboratory research, case-control
studies, or treatment trials. However, it seems that epidemiologic knowledge is rarely taken into account
in current clinical research and management of RPL.
The incidence of RPL has rarely been assessed but is a much more clinically important parameter than
the prevalence. It should be recognized that applying the new definitions of RPL suggested by ASRM
and ESHRE, the prevalence/incidence of RPL will probably triple, whereas the overall spontaneous
prognosis for live birth in the patients will increase to 75%–80%. However, the subset of patients with
the poor prognosis will not diminish, it will just be hidden in the mass of patients with a good prognosis.
Estimates of the future miscarriage risk in RPL vary significantly. Some studies have estimated the
prognosis too optimistically because preclinical pregnancy losses have been considered to be non-
pregnancy. Therefore, in future treatment trials the baby take-home rate per time unit may be a better
outcome measure than the pregnancy loss rate per pregnancy. The number of previous miscarriages is
not only the strongest prognostic factor but with an increased number of previous miscarriages fetal
aneuploidy seems to become less prevalent and maternal factors more prevalent. Therefore stratification
by the number of previous miscarriages is important in RPL studies.
In conclusion, at least three features should be included in future RPL research: recognition of the
multifactorial/polygenic background of RPL, recognition of the different pathogenetic features of
primary/secondary RPL, and recognition of the importance of the number of previous miscarriages.
Awareness of these features should eliminate the practice of combining data from too heterogeneous
RPL studies for meta-analysis.
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2
The Signaling between Embryo and Mother as
a Basis for the Development of Tolerance
Eytan R. Barnea
Introduction
In mammalian gestation, immunologic acceptance and tolerance are paramount for the successful
interaction between the embryo/graft and its maternal host. Initial immunologic awareness must take
place prior to implantation. The semipermeable zona pellucida forms rapidly post fertilization and
protects the embryo until it reaches the endometrium. The zona is surrounded by maternal immune cells,
and this unit transmits the message that fertilization has occurred. However, in in vitro fertilization (IVF)
and embryo transfer this mechanism is not operative. The main question is when and how the embryo-
maternal communication initiates and creates maternal recognition of pregnancy. Advances in assisted
reproduction suggest that the embryo is the dominant element in the reproductive process; its viability
and ability to signal are critical for embryo-maternal recognition. Furthermore, genetics does not appear
to play a predominant role; donor embryos or xeno-transfer embryos fare very well following transfer
with no discernable difference to semi-allogenic embryos.
Herein we focus on a peptide secreted from the earliest stages of gestation, preimplantation factor (PIF),
which fulfills three fundamental requirements. First, it is only secreted by viable embryos and is only
detected in the maternal circulation in viable pregnancies. Second, it has an essential role in pregnancy,
through autocrine effects on the embryo and as a promoter of implantation and trophoblast invasion.
Third, it regulates global maternal immune responses while preserving the antipathogen action. Evidence
is emerging that PIF can also be effective in the treatment of pregnancy pathologies and preclinical and
clinical nonpregnant immune disorders and transplantation.
Why Is There a Delay between Fertilization and Implantation?

The tube picks up the egg after fertilization when estradiol is at a peak. For implantation to take place,
progesterone must peak. Consequently, the embryo post fertilization needs to be delayed in order to
mature and develop until implantation can occur. Physiologically, maturation takes place in the tube.
However, as IVF has shown, if a four-cell stage embryo is replaced, maturation can take place in the
uterus for a few days until implantation occurs [1]. The delay in implantation suggests that embryo-derived
signaling must be present for the endometrium to become receptive. However, embryo signaling is not
specific for the uterus since implantation can take place in the tube, ovary, and abdominal cavity [2]. The
signal seems to be embryo-derived and the maternal system responds, and not vice-versa.
There are very few conditions that prevent conception and implantation. Even in severe diseases,
although the incidence of pregnancy is reduced significantly, the pregnancy can still develop [3–5], also
indicating that the maternal system is mostly receptive and does not guide the reproductive process.
In addition, there is no need for genetic compatibility, since cross-species fertilization can occur (e.g.,
horse and donkey, resulting in a mule). Embryo signaling overcomes the problem that only half the
genome is maternal and the other half is paternal, to the extreme example of cross-species pregnancy.
As earlier data have shown, the mother recognizes that conception has taken place upon the appearance
13
of a zygote, but not before. In organ transplantation, if the donor’s cells and organs are foreign, rejection
would immediately occur. In birds and mammals, fertilization is internal, and tolerance develops. The
sperm, although genetically and antigenically foreign, is not attacked by the maternal system. In birds,
later rejection is overcome as growth and development of the embryo takes place in an egg covered by a
shell [2]. In mammals, the zygote is surrounded by the zona pellucida, which prevents maternal immune
cells’ entrance. After hatching from the zona pellucida, protective mechanisms are operative to prevent
rejection. At hatching and implantation, the uterus should be sufficiently primed. Unless the embryo
signal is ineffective or the maternal organism is excessively hostile, the viable embryo will implant. The
uterus is not a privileged but a preferential site for the early embryo; the response would be completely
different in the presence of a foreign tissue. However, in cross-species embryo transfer, where the genetics
are completely different, specific embryo signaling allows the zygote to remain implanted and even
thrive until delivery. The hormones estrogen and progesterone are essential to mature the uterus for
implantation. However, they are insufficient to initiate pregnancy. The embryo must take a significant part
in this process though specific signaling. After implantation, there must be accommodation of the embryo
throughout pregnancy. In ectopic pregnancy, priming of the fallopian tube can also occur, as in the uterus.
Embryo-Specific Maternal Communication—PIF

The culture media in which embryos were grown in vitro have been shown to have immune regulatory
properties. Early pregnancy factor (EPF) (Chaperone 10, which facilitates protein folding) is probably
secreted by the ovum (bovine model) and can be detected in pregnant women’s sera 48 hours after
fertilization. In red deer (Cervus elaphus), EPF has been correlated with the number of viable fetuses [6].
Sera that contain EPF have significantly higher blastocyst development rates [7]. An additional embryo-
derived nonspecific compound is platelet-activating factor (PAF), a unique signaling phospholipid
required for fetal development [8]. PAF acts by binding to its receptor, PAF-R, which is a G-protein-
coupled receptor often found in immune cells. Embryo-derived signaling molecules can be detected in the
maternal circulation. The primary role of human chorionic gonadotropin (hCG) is to support the corpus
luteum, allowing maintenance of pregnancy. hCG also has a role in promoting placental angiogenesis
and syncytiotrophoblast formation. There are multiple forms of hCG which have diverse functions. The
hyperglycosylated form of hCG is involved in creating tolerance to the embryo by modulating endometrial
immunity. In dendritic cells, MHC class II, IL-10, and IDO expression is increased, which impairs T cell
proliferation. In patients undergoing IVF, circulating levels of various cytokines has been determined
following hCG injection. It was shown that hCG decreases the anti-inflammatory cytokines IL27 and
IL17; in contrast IL10 levels increased, as well circulating Treg cells [9]. hCG has also been shown to
increase IDO production particularly in dendritic cells, suggesting another mechanism for pregnancy
resistance to autoimmunity [10]. However, hCG is not embryo-specific; it is unique to humans. In rodents,
placental lactogen has the dominant role.
Our team has discovered an additional compound, preimplantation factor (PIF) [2,3,11,12]. PIF seems
to be embryo specific, secreted only by viable embryos, and can be detected in the maternal circulation.
This molecule provides the evidence that embryo-maternal communication starts at fertilization and lasts
throughout pregnancy until delivery [5,11–13]. PIF is found across many mammalian species: human, pig,
horse, cow, and mouse [5,13–17]. PIF levels have been detected in IVF culture and increase up to the blastocyst
stage [11,12,18]. Furthermore, PIF is detected in the extra-villous trophoblast where intimate contact is
established between embryo and the mother [14]. Hence, PIF first acts at a distance from the endometrium
and later through direct contact at the maternal-fetal interface. Therefore the correct signaling is transmitted
to the mother indicating the need for support until delivery. The data generated with IVF embryos provided
important information indicating that only a viable embryo will implant, while a non-viable embryo will not
[19]. If the pregnancy is healthy, PIF levels will increase until the second trimester and then decline [14]. In
contrast, in pathologic pregnancy—for example, miscarriage—the expression of PIF is low and prematurely
declines in preeclampsia and intrauterine growth restriction (IUGR) placentae [20]. Thus, embryo maternal
communication is altered in pathological pregnancy and may provide a danger signal to the mother that
pathology is present. In murine gestation the placenta and uNK cells release PIF-containing granules on day
The Signaling between Embryo and Mother as a Basis for the Development of Tolerance 15
14 in preparation for labor [17]. However, administration of lipopolysaccharide (LPS) led to re-expression
of PIF in the placenta. PIF administration is associated with a twofold reduction in fetal deaths compared
to controls. In premature labor, low expression is a reflection that embryo maternal communication is
disrupted, and there is a failure of effective fetal/maternal nutrient exchange. In most cases inflammation
is the cause, and innate immunity comes into operation and probably neither the mother nor fetus would
survive. By initiating a “rejection” presenting as labor, both mother and progeny have a chance of surviving
if the fetus is the right gestational age and not severely compromised.
PIF Autocrine, Autotrophic, and Protective Actions

Once formed, the zygote is surrounded by the zona pellucida. The embryo is then protected from the
maternal immune system. However, the mother becomes aware of the embryo a very short time after
fertilization due to signaling. Signaling has been shown with respect to platelet emargination [21]. Certain
proteins present in platelets are targeted by PIF (including platelet-derived growth factor beta [PDGFβ]).
In the presence of PIF, platelet and lymphocyte adhesion is increased [22–24]. From fertilization onward,
the secretion of PIF increases reflecting the health of the embryo, while embryos destined to miscarry
do not increase PIF levels in culture [12]. The main receptors to PIF are protein disulfide isomerase/
thioredoxin (PDI-T) and heat shock proteins (HSPs) [25]. In a recent study, the effect of an inhibitor of
PDI-T was assessed which increased oxidative stress of the embryos [26]. Bovine data has shown that
embryos cultured in large groups in the presence of this inhibitor led to growth arrest. When PIF was
added and cultures monitored for 8 days, the number of embryos reaching the blastocyst stage more than
doubled. PIF prevents the PDI-T protein transition from an oxidative to a redox form, thereby protecting
embryos from demise. As an adverse maternal environment can lead to recurrent pregnancy loss (RPL),
the protective effect of PIF was tested in the presence of embryo toxic serum derived from women with a
history of RPL. Early data indicated that if embryos were cultured in an optimized environment, progress
to the blastocyst stage was high (80%) [18]. If PIF was added to those cultures, even high concentrations
did not further improve embryo development. However, when PIF was added to the embryo culture media
that was exposed to RPL serum, PIF protected against embryo demise and also increased the number
of embryos that reached the blastocyst stage. Recognizing that oxygen radicals and toxins are of low
molecular weight (MW), the RPL serum was divided into two fractions, <3 kDa and >3 kDa [26]. The
low MW serum delayed embryo development until the blastocyst stage, whereas in the presence of the
>3 kDa serum fraction there was a significant increase in embryo demise. Embryo demise may have been
due to the presence of antibodies or other proteins in the RPL serum. Notably, in both circumstances
PIF effectively blocked the adverse effects of RPL serum. RPL serum has been tested for the presence of
anti-PIF antibodies, but no anti-PIF antibodies were found in RPL serum. Consequently, RPL is not due
to presence of circulating anti-PIF antibodies [26].
PIF has also been assessed for an autocrine effect. Anti-PIF antibody in the culture decreased
development to the blastocyst stage by 80% [12]. However, in singly cultured cow embryos, which rarely
develop to the blastocyst stage, the presence of PIF for 3 days followed by PIF-free media up to day 7
promoted progress to the blastocyst stage [18]. In analyzing embryo development to determine the exact
site of FITC-PIF uptake as the embryo advanced from the morula to the blastocyst stage, the major uptake
was observed in the leading edge of the blastocyst bulge, where the contact with the endometrium would
be expected (blastocyst extrusion) [25]. Hence, PIF has both an embryotropic and protective effect. This
effect is amplified following implantation when the zona is removed and the interaction between the
embryo and the mother is direct and therefore the most critical stage in reproduction takes place, leading
embryo implantation to succeed or to fail.
Receptive Uterine Environment Is Critical for Reproduction

In addition to the role of the embryo, a receptive uterine environment is also a clear requisite. There is
also an embryo-derived regulatory effect on the uterus [27–29]. Overall, PIF has a significant effect at
pre-, during, and post-implantation periods the requirement shifts from apposition to attachment and
invasion. Disturbance of any of these steps will lead to pregnancy loss: implantation failure, chemical
pregnancy, early or later miscarriage. Therefore, endometrial adaptation must be clearly coordinated.
PIF appears to have an important role in all these steps. The effect of PIF starts prior to implantation
since it increases endometrial integrin expression in human epithelial cells, a major receptivity marker
[28]. This pro-receptivity action is shown in human stromal cells that are activated by estrogen and
progesterone (human embryonic stem cell [hESC]). Detailed studies on gene, protein, and pathway
analysis indicate that PIF has a major effect on local immunity, adhesion, and apoptosis control, which
favor implantation. The increase in IRAKBP1 that interacts with TRL5 has antimicrobial activity. The
decrease in IL12RB2 reduces pro-inflammatory cytokine action. A local mild pro-inflammatory milieu
favors embryo embedding [27]. The effect on embryo adhesion genes was shown by increased Down
syndrome cell adhesion molecules such as SORBS2 and SORBS1 expression. Therefore, the embryo
through PIF creates a favorable endometrial environment. In first trimester decidua, the protective effects
are magnified. PIF leads to protection against an adverse maternal environment which would be highly
detrimental to the embryo during embryogenesis. The earliest embryonic structure is the notochord;
therefore special attention has been paid to determine whether PIF is involved in development of the
notochord. The data indicate that in both the implantation period and in the first trimester, PIF exerts
both neurotrophic and neuroprotective effects [31]. These neuroprotective effects may have implications
for postnatal life. The effect of PIF seemed to protect against childhood diseases including autism [29]
and adult neurodegenerative diseases [30], neonatal neurotrauma as observed in the premature infant,
and reversing advanced neuroinflammation leading to brain remyelination and reversal of paralysis
[5,30–35]. This suggests that PIF could help to reduce pregnancy pathologies by administration during
pregnancy [17].
Implantation Is Insufficient: Effective Trophoblast Invasion Is Also Required

In addition to improving endometrial receptivity, PIF also influences trophoblast invasion [14,20,29,35,36].
Invasion has to be regulated in order to prevent excessive invasion as seen in placenta accreta, or too
shallow an interaction which leads to coagulation disorders or abruption. [15]. The pro-invasive effect
of PIF was investigated on a transformed trophoblastic cell line using primary extravillous human
trophoblasts, which confirmed this effect on invasion. The effects of PIF involve metalloproteinases, tissue
inhibitor of metalloproteinase (TIMP), and integrins [14,20,29,36]. These ligands were also involved in
the effect of PIF on the endometrium [28]. Insights into the effect of PIF on trophoblast invasion were
provided by using specific inhibitors which defined the pathways involved: MAPK, IP3 K, and JAk-Stat
[14]. Furthermore, a detailed genomic analysis identified several associated genes that are affected in the
trophoblast [20]. PIF effectively upregulated Azurocidin-1, which has an important role in chemotactic
and antimicrobial activity. By promoting IL17F, a pro-inflammatory cytokine, trophoblast invasion is
supported, and the effect is also amplified by increased lincRNA MALAT-1 expression. By upregulating
T cell receptor alpha, similarly to that found in the endometrium, PIF regulates the immune response
[27]. PIF increases BCL2, resulting in anti-apoptosis, and affects associated pathways. These mechanisms
contrast with the effect of PIF on the endometrium where hESC cell apoptosis must take place in order to
accommodate the invading trophoblast. The increase in BCL2 involves p53-related pathways where p53
protein inhibition blocks the stimulatory effect of PIF on BCL2 and BAX, thereby establishing a direct
functional relationship. Thus, endogenous PIF that is present in the placenta appears to have a major local
regulatory and protective role that is disrupted in pathological placentae such as IUGR, where decreased
expression impairs fetal growth [20].
PIF Interaction and Synergy with Progesterone and HLA-G

Progesterone is essential for pregnancy development [37]. During implantation, the corpus luteum
increases progesterone secretion, which transforms the endometrium from the proliferative to the
secretory phase [38]. Data show that PIF as monotherapy primes the endometrium independent of
Another random document with
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bleus, verts, rouges, jaunes et violets. Je m’enhardissais jusqu’à
entrer dans le chœur, où je soulevais le couvercle de l’harmonium
dont jouait le frère Théodore. Je regardais les touches blanches et
noires, les registres sur lesquels étaient écrits des noms tels que
Bourdon, Clairon, Flûte, Clarinette, Hautbois. Je n’allais pourtant
point jusqu’à souffler du pied ni appuyer du doigt sur une touche : il
me semblait que l’église se serait écroulée pour m’ensevelir sous
ses ruines si j’avais eu l’audace de profaner un instrument dont
pouvait seul s’approcher un homme de la science du frère Théodore.
J’aimais les cérémonies religieuses, non par piété, mais par une
sorte de sentiment de la poésie que je ne pourrais définir. Ces
chants d’église me plaisaient, toujours graves, qu’ils fussent tristes
ou joyeux ; parfois j’aspirais vaguement à devenir un grand
organiste, dans le genre du frère Théodore.
V
— Madame, voulez-vous qu’Albert vienne s’amuser avec nous ?

C’était l’après-midi d’un jeudi d’octobre, le deuxième après la
rentrée des classes. Assis près de la cheminée où deux bûches se
consumaient lentement, Berlâne rêvait à vide, le menton appuyé sur
la paume des mains.
— Mais certainement, monsieur Georges ! dit Mme Dumas. Je ne
demande pas mieux : cela le distraira. Regardez-le donc ! Il est là à
s’abâtardir au coin du feu. Mais il est vraiment mal habillé pour sortir
avec vous. Je vais lui donner son autre blouse noire.
— Vous n’y pensez pas, madame ! répondit M. Georges. Pour
jouer on est toujours trop propre.
La porte de la salle à manger restée entr’ouverte, Mme Dumas
était assise à son comptoir, attendant les clients. M. Georges ne
venait rien acheter, mais elle était plus heureuse que s’il lui avait pris
d’un seul coup pour un louis de mercerie. Ce gamin de dix ans,
qu’elle appelait M. Georges, était le fils cadet des Labrosse,
bourgeois qui se fournissaient chez elle. Elle était leur humble
servante, comme de tous ceux qui voulaient bien lui donner leur
clientèle : même dans une petite ville on n’a entre les commerçants
que l’embarras du choix. Georges venait en éclaireur de la part de
Robert, son aîné. Je ne les aimais ni l’un ni l’autre. Comme Berlâne
et moi, ils étaient élèves de l’école des frères ; ils se distinguaient
parmi les moins appliqués à l’étude et les plus turbulents dans la
cour des récréations. Leurs deux camarades intimes, les fils Rouget,
étaient, au commencement du mois, partis pour le lycée. Et ils
restaient seuls, désemparés et s’ennuyant. Alors ils avaient pensé à
leur plus proche voisin. Ou, plutôt, c’était leur mère qui avait dû leur
dire, les voyant comme deux corps sans âme :
— Allez donc inviter ce pauvre Albert.
Mme Duras considéra que c’était un grand honneur pour elle, en
la personne de son fils. Elle déplorait qu’il restât toujours seul, et elle
souffrait à la pensée qu’il fût si déshérité que personne ne tînt à
rechercher sa compagnie.
Il se leva. Pourtant, lui non plus, il ne les aimait pas, les deux
Labrosse. Ils étaient de ceux qui, l’hiver précédent, lui avaient lancé
des boules de neige en criant : « Sur Berlâne ! » Mais, puisque sa
mère le lui ordonnait, sans protester il suivit « Monsieur Georges »
comme un petit domestique.
Les deux maisons se touchaient presque, mais qu’elles étaient
différentes ! Qu’il y avait loin de l’humble boutique à la demeure des
Labrosse avec ses deux étages ! Au rez-de-chaussée, les trois
fenêtres du salon donnaient sur la grand’rue. Que de fois,
lorsqu’elles étaient ouvertes, il avait, en passant, regardé les beaux
meubles vernis, les cadres dorés accrochés aux murs et la grande
table ronde, au milieu, chargée d’albums, dont il eût voulu caresser
le cuir épais ! Il ne lui venait même pas à l’idée qu’un jour il y pût
entrer. Et voici que Georges n’eut qu’à pousser deux portes, celle du
corridor, qu’il referma sur eux, puis une autre : et Berlâne se trouva
sur le seuil du salon, si interdit qu’il ne pensa pas tout de suite à
enlever sa casquette. Assise dans l’embrasure d’une fenêtre, Mme
Labrosse faisait du crochet avec Mlle Gertrude, sa fille, qui avait dix
ans. Étendu sur le canapé, les mains croisées sous la nuque, Robert
sifflotait comme un homme.
— Maman, dit Georges, voici Albert que je ramène. Il va jouer
avec nous.
— Tu es content, Albert ? lui demanda Mme Labrosse.
— Oh ! oui, madame ! répondit-il en devenant tout rouge.
Il était au supplice. Il regrettait son coin de cheminée ; quelle
bonne après-midi il eût passée là !
— Eh bien, mes enfants, allez ! dit-elle. Et n’oubliez pas de
rentrer pour vos quatre heures.
Il dut, à leur suite, traverser le salon en s’efforçant de ne point
glisser sur le parquet ciré. Il n’avait osé ni lever les yeux pour voir de
plus près les cadres dorés, ni avancer la main pour toucher le cuir
des albums.
— Qu’est-ce que nous allons faire à présent ? dit Robert,
lorsqu’ils furent dans la cour.
— Si nous allions dans le bois ? dit Georges. Nous emmènerions
le chien.
Roux comme un renard, presque aussi gros qu’un loup, Stop
était dans un tonneau, à l’entrée de la cour. Depuis qu’il les avait
vus, il aboyait en tirant sur sa chaîne. Berlâne avait, comme moi,
peur des chiens. Vous croyez qu’ils se promènent par les rues,
pacifiquement, en quête d’une borne ou d’un angle de mur ? Non. Ils
ont des intentions bien pires. Ils passent avec leurs mâchoires
ornées de crocs pour me mordre.
Il pensa dire :
— Il vaudrait peut-être mieux le laisser ici. Dans son tonneau il ne
doit pas être malheureux, pas plus que je ne l’étais tout à l’heure au
coin de la cheminée.
Mais il n’osa encore pas. Le chien avait ici beaucoup plus
d’importance que lui. Nul doute que, si Stop avait pu causer,
Georges ne fût pas venu inviter Berlâne.
Il faut voir cette après-midi d’un jeudi d’octobre. Tout le long du
chemin qui mène au bois, les feuilles jaunes, pour toujours
détachées de leurs branches, volent à l’aventure, égarées. Elles se
réunissent au pied de la haie. Les entendez-vous qui se concertent,
inquiètes ? Elles ne savent ce qui leur arrive.
Dans le ciel, des nuages d’un joli gris passent si vite qu’on a juste
le temps de les voir et de les saluer de loin.
Las de l’été, le soleil commence à prendre du repos, et le vent,
que l’on n’a guère entendu depuis l’hiver dernier, se remet à donner
de la voix, comme Stop, que voici revenir d’une course folle et qui
gambade autour de ses maîtres, autour de Berlâne, qui n’est pas
fier.
Berlâne essayait de sourire au chien pour se concilier ses
bonnes grâces, mais le moyen de voir sans trembler cette gueule
ouverte, langue pendante et dents pointues, de sentir cette chaude
haleine passer sur ses mains qu’il ne retirait pas de peur que Stop,
s’excitant au jeu, ne s’avisât de les lui happer ?
Mais Robert et Georges n’étaient-ils pas là pour le défendre ?
Non : ils ne faisaient pas plus attention à lui qu’à leur chien. Et
Berlâne se contentait de les écouter parler de l’école, des tours de
force que Lagache exécutait au trapèze et des bons tours qu’il jouait
au frère Stanislas. Lagache ? Un parisien dont les parents venaient
de s’installer ici. De tous nos camarades c’était certainement celui
dont Berlâne et moi nous avions le plus peur. Mais Berlâne se crut
obligé de sourire tout le temps que les deux Labrosse en parlèrent :
il souriait bien au chien !
Robert dut éprouver un violent besoin d’action. Il bondit en

poussant un cri qui ressemblait à un cri de guerre, Georges le suivit.
Berlâne, resté seul, hésita d’abord, puis se mit à courir lui aussi.
Mais il n’en avait guère l’habitude. Tout de suite essoufflé, il dut
s’arrêter : son cœur battait trop fort. Un instant il crut que sa tête
allait l’entraîner en avant.
— Eh bien, vrai, lui dit Robert quand il les eut rejoints, tu ne cours
pas vite !
Une fois de plus il essaya de sourire ; mais il ne réussit qu’à faire
une grimace et ne trouva rien à répondre.
Il s’assit près d’eux sur le socle d’une croix qui se dresse au
carrefour de quatre chemins.
— Si on en grillait une ? dit Robert qui tirait de sa poche un
paquet de cigarettes.
Georges en prit une.
— Une cigarette, Albert ?
— Merci bien, dit-il. Je ne pourrais pas fumer : ça me rendrait
malade. Et puis c’est défendu.
— Défendu, ricana Robert. Et par qui donc ?
— Mais maman m’a toujours dit qu’il ne faut pas fumer.
— Oh ! Ta mère… Eh bien, tu vois si ça me gêne, moi.
Heureux de poser à l’homme devant Berlâne, sur la pierre il fit
craquer une allumette : il lui semblait qu’il y eût entre eux non pas
deux ans, mais vingt, de différence.
— Et ta bonne amie, est-ce qu’il y a longtemps que tu ne l’as

embrassée ? lui demanda Robert entre deux bouffées.
Cette fois Berlâne ne sut quelle contenance prendre. Il rougit
beaucoup plus encore que dans le salon : il devint écarlate, comme
dans les grandes circonstances. Ses oreilles bourdonnèrent : que
venait-il d’entendre ! Sa « bonne amie » ! Mais cela aussi était
défendu, comme de fumer. Certes il éprouvait parfois de profonds
désirs de tendresse. Mais c’était en lui-même un jardin secret dont il
n’avait point la clef et qu’il n’apercevait qu’entouré d’un brouillard
bleu pâle. Et voici que quelqu’un brutalement déchirait le voile et
enfonçait la porte…
— Je n’en ai pas, répondit-il tremblant dans l’attente du nom que
n’allait pas manquer de prononcer Robert.
— Tu n’as pas de bonne amie ! Alors pourquoi est-ce que tu es
toujours fourré chez les Chovin, si ce n’est pour embrasser la
Marie ?
Berlâne respira : Robert ne savait rien.
— Laisse-le donc tranquille, dit Georges à son frère. Tu vois bien
que tu l’embêtes.
— Avec ça !… Il est bien content, au fond.
Ah ! oui, Berlâne était content ! Il trouvait l’après-midi
interminable. Est-ce que les Labrosse n’auraient pas pu se
promener sans lui ? Comme tous les fils de riches ils devaient avoir
à la maison beaucoup de jeux intéressants : tirs, soldats que l’on fait
défiler, ménageries avec arbres et animaux en bois peint. Il n’avait
peur des chiens que vivants. Sans doute eût-il encore préféré rester
seul, mais voir et toucher ces beaux objets eût été une
compensation à son ennui. Tandis qu’ici, que faisaient-ils ? C’était
cela qu’ils appelaient s’amuser ? Oui. Car ils étaient bien plus
heureux de fumer en cachette de leur mère que de tirer du fond d’un
placard ces jeux auxquels ils ne s’intéressaient plus : Robert, aux
environs de sa douzième année, Georges lui-même, moins âgé, ne
voulaient plus qu’on les prît pour des gamins. Leur enfance, ils la
considéraient l’un et l’autre comme terminée. Mais Berlâne, lui, était
toujours un enfant : peut-être le resterait-il toute sa vie ?
— Allons voir du côté de l’étang s’il y a des grenouilles, dit

Robert.
Ils entrèrent sous bois, et par des sentiers boueux aboutirent à
l’étang desséché que de récentes pluies avaient légèrement rempli.
Aussitôt qu’elles les eurent entendus, les grenouilles se hâtèrent
de sauter dans la vase : s’ils voulaient venir les y prendre, elles les
attendaient. Elles ignoraient que Berlâne eût peur d’elles, loin de leur
être un ennemi. Il eut un brusque haut-le-corps.
— Tu as donc peur des grenouilles ? lui dit Robert qu’au surplus
cela ne surprenait guère.
— Mais non ! répondit-il d’un tel accent que Robert pensa : Nous
allons bien rire, et dit à voix basse quelques mots à son frère.
Quelques grenouilles, plus confiantes, étaient restées parmi
l’herbe humide. Robert en saisit une et la tint par les pattes de
derrière. Berlâne la trouva horrible.
— Tu vois bien, dit Robert, que ce n’est pas méchant.
Il fit un signe à Georges qui maintint Berlâne par les bras, et il
rapprocha la grenouille de son visage.
— Non ! Non ! implorait Berlâne en se débattant.
Décidément ils avaient eu une bonne idée de l’amener avec eux :
jamais Stop ne se fût effrayé ainsi à la vue d’une grenouille. Et,
puisqu’ils le tenaient, ils ne le lâchèrent pas. Ce n’étaient pas de
méchants garçons, mais ils ne furent pas maîtres de cet instinct qui
souvent pousse les riches à faire des pauvres leurs souffre-douleurs.
Positivement Berlâne sentait le pauvre. Et ce n’était pas seulement
son attitude, mais son corps même, qui le désignaient comme la
victime nécessaire de toutes les plaisanteries et de toutes les
persécutions.
Robert ne se contenta point de rapprocher de son visage la
grenouille : il la lui promena sur la peau. Berlâne poussa un grand
cri, celui que j’aurais poussé dans les mêmes circonstances. Si
Georges avait cessé de le tenir, il serait tombé raide. Que c’était
amusant ! Robert, jetant la grenouille comme un instrument devenu
inutile, se tordait de rire. Il…
Mais qu’y avait-il donc ? Berlâne fermait les yeux ? Pâle
d’habitude, il était maintenant plus blanc qu’un mort. Ils le
regardèrent tous les deux en même temps.
— Il a tourné de l’œil ! dit Robert. Nous sommes frais ! Va vite
tremper ton mouchoir dans l’eau.
Ils lui mouillèrent les tempes.
Deux minutes après il ouvrit les yeux ; mais il ne se reconnut pas
tout de suite : il se rendit compte seulement qu’il sentait la vase. Puis
il se rappela tout. Stop, assis sur son séant, le regardait : depuis qu’il
avait vu de trop près l’horrible grenouille, Berlâne le trouva
sympathique.
— Tu peux te vanter de nous avoir flanqué une de ces
frousses !… dit Robert. C’était pour rire ! Il fallait nous dire que tu
n’es qu’une poule mouillée.
Mouillé, Berlâne l’était en effet. De l’eau avait coulé sur le col de
sa chemise et sur sa blouse : Mme Dumas avait bien fait de ne point
lui donner sa plus neuve. Il prit son mouchoir et s’essuya. Il n’avait
pas encore prononcé une seule parole : il en aurait été incapable. Il
frissonnait.
Le soleil, qui se dégagea de derrière les nuages, descendait vers
l’horizon occidental. Les arbres, qui n’avaient pas encore perdu
toutes leurs feuilles, étaient si serrés les uns contre les autres qu’on
devinait plutôt qu’on ne les voyait les obliques rayons de lumière.
C’était l’heure mélancolique qui dans les petites villes et au-
dessus des champs va sonner l’arrêt du travail et de la vie. Comme
le laboureur qui écoute l’angélus lointain, on se recueille dans le
silence ; partout les bougies s’allument comme pour une veillée
funéraire, et c’est ainsi que chaque soir semble ramener la fin du
monde.
Il sembla soudain à Berlâne qu’une grande tristesse se répandît
par les bois désertés, planât au-dessus de l’étang abandonné, et lui
entrât dans l’âme. Auprès des deux Labrosse moqueurs ou hostiles,
il lui sembla qu’il fût à cette heure à une infinie distance de sa
maison. Ses larmes jaillirent.
— Voilà que tu pleures à présent ? dit Robert. C’est le bouquet !
Allons-nous-en, Georges. Il nous suivra s’il veut.
Si Berlâne avait été un enfant grincheux comme on en voit
beaucoup, qui se privent de nourriture pour punir leurs parents de
les avoir grondés, il serait resté là, tout seul, quitte à avoir peur, pour
se venger des Labrosse en les obligeant à revenir sur leurs pas pour
le supplier de les suivre. Il n’en fit rien. Il leur emboîta le pas ; Stop
était resté derrière ses maîtres pour servir, eût-on dit, de trait d’union
entre eux trois. Stop n’était pas méchant ; Berlâne osa lui passer la
main sur la tête et ne fut pas mordu.
— Eh bien, mes enfants, vous êtes-vous bien amusés ? leur

demanda Mme Labrosse. Mais vous êtes en retard : quatre heures et
demie viennent de sonner.
— Nous ne sommes pas payés à l’heure, riposta Robert qui
traitait avec sa mère de puissance à puissance. En tout cas, nous ne
nous sommes pas ennuyés, n’est-ce pas, Albert ?
— Non, dit Berlâne.
— Qu’est-ce que vous avez fait ? lui demanda Mlle Gertrude qui
disposait sur la table de la vaste cuisine — Berlâne n’était pas un
invité de marque, — des assiettes et des verres.
Il baissa tout de suite les yeux. Il essaya de répondre, mais en
vain, trop ému pour ne pas bégayer : il lui était encore plus difficile
d’adresser la parole à Mlle Gertrude que de réciter une leçon. Avec
ses yeux presque verts qui luisaient dans son visage fin sous ses
cheveux blonds, Mlle Gertrude ressemblait à une jeune fée qui vole
en rasant les herbes de la prairie, et son écharpe bleue flotte
derrière elle au gré du vent du matin ou de la brise du soir.
— Tu es malade, Albert ? lui demanda Mme Labrosse.
— C’est le grand air. Il n’y est pas habitué, répondit Georges à sa
place.
— C’est vrai qu’il ne sort pas souvent, réfléchit Mlle Gertrude.
Plus que dans le salon Berlâne était au supplice. On lui servit des
confitures de coings, qu’il n’aimait pas et qu’il dut manger tout de
même. Assis entre Mme Labrosse et Mlle Gertrude, il maniait
gauchement sa petite cuiller.
— Nous te faisons donc peur ? lui dit Mme Labrosse. Il ne faut pas
être si timide !
Son embarras redoubla. Mlle Gertrude le regardait en souriant, et
Dieu sait si elle avait l’air terrible ! Un doigt de vin pur le réconforta
un peu, sans qu’il reprît confiance en lui-même. Quand le goûter fut
terminé, il eut pourtant le courage de dire :
— Je vous remercie beaucoup, madame. Maintenant je vais
rentrer.
Robert et Georges se gardèrent d’insister pour qu’il restât : ils
avaient assez de lui.
Sa mère lui demanda comme Mme Labrosse :
— T’es-tu bien amusé ?
Elle ajouta :
— Mais tu as ton col de chemise tout sale !
Il n’avait jamais bonne mine : elle ne remarqua point sa pâleur.
— C’est que nous avons joué au bord de l’eau, répondit-il. Je ne
me suis pas ennuyé.
— Tant mieux ! dit Mme Dumas. Je suis contente. Cela te distrait,
et c’est meilleur pour toi que de toujours rester seul. Je remercierai
Monsieur Georges et je lui demanderai qu’il pense à toi, de temps en
temps, quand ça ne les dérangera pas.
VI
Ce fut le recommencement des misères qu’il endura plusieurs

années de suite ; malgré sa timidité, il était trop fier pour s’en
plaindre, même pour en parler à sa mère. Il avait eu la chance que
jusqu’alors les Labrosse n’eussent pas fait trop attention à lui ; on
peut vivre presque porte à porte et être les uns pour les autres
comme des étrangers. Or il avait suffi que cette idée de l’emmener
avec eux leur fût venue pour que sa vie menaçât de changer et de
lui devenir intolérable. Il ne s’en alarma pas outre mesure. Les deux
Labrosse n’en avaient point parlé à leur mère, mais ils eurent vite
raconté à l’école la scène du bois dans ses menus détails, et
Berlâne fut considéré comme un capon tel qu’il était impossible que
la terre en portât un semblable. Il eut beau se faire violence et
affecter de prendre part à tous les jeux les plus périlleux : à lui
étaient réservés les coups les plus secs des balles le plus durement
rembourrées de chiffons et de son, les positions les plus fatigantes
au jeu de saute-mouton, et l’hiver il redevint plus d’une fois une cible
vivante pour les amateurs de boules de neige. Il retenait des larmes
de dépit quand il entendait Robert crier, en le désignant aux plus
enragés :
— Sur Berlâne ! Sur Berlâne !
Il n’était plus l’Albert du jeudi d’octobre : il était redevenu Berlâne,
le pauvre gamin ridicule vers qui convergeaient moqueries, rires et
coups.
Les Labrosse se gardèrent bien de revenir l’inviter le jeudi : Mme
Dumas avait eu beau les remercier et leur faire comprendre que,
s’ils voulaient recommencer, elle en serait heureuse et fière. Il ne le
regretta point : il avait trop souffert dans le salon et au bois. Et
pourtant il lui semblait qu’à la longue, et surtout l’hiver, il aurait fini
par s’habituer à l’atmosphère de luxe de leur maison, et qu’à force
de regarder Mlle Gertrude il aurait cessé d’avoir peur d’elle : peut-
être même aurait-il pu avoir avec elle de longues conversations. Au
lieu de cela il ne l’apercevait guère que le dimanche, et jamais il
n’aurait osé lui adresser la parole. Mme Labrosse, pas fière, causait
un peu avec Mme Dumas qui lui répondait humblement. Mais il n’était
pas question de lui. Elle ne disait pas :
— Il faudra nous envoyer Albert.
Sans doute avait-il laissé à Mme Labrosse une mauvaise
impression.
Son refuge resta la boutique des Chovin. Les premières fois qu’il
y retourna, il ne put regarder Marie sans rougir : était-il possible
qu’elle fût sa bonne amie ! Par des bouts de conversations qu’il
saisissait à l’école, il devinait bien qu’une bonne amie c’est une
gamine, une jeune fille qu’on embrasse en cachette. Avec Marie,
jamais cette idée ne lui serait venue. Avec Mlle Gertrude encore bien
moins, mais pour des motifs tout différents. Elle était si jolie !
Mais en lui s’opérait une transformation que j’étais loin de
soupçonner.
VII
Peut-être sa mère y fut-elle pour quelque chose ? Je n’en sais

rien.
Mme Dumas ne faisait point partie du groupe des femmes
pieuses que l’on rencontrait plus souvent à l’église ou à la chapelle
des sœurs que chez elles. Les nécessités de la vie, surtout depuis la
mort de son mari, l’obligeaient à quitter le moins possible sa
boutique. Mais elle ne manquait pas de fermer sa porte, le
dimanche, pour assister à la messe et aux vêpres. En semaine elle
s’associait par la pensée à celles qui, plus libres, avaient le bonheur
de pouvoir réciter en commun le Rosaire devant la statue de Notre-
Dame de Lourdes et faire chaque vendredi l’exercice du chemin de
la croix.
De beaucoup de manières notre maison était en quelque sorte
une succursale de l’église.
Mon père étant sacristain, il ne se passait pour ainsi dire pas de
jour que soit le curé-doyen, soit le vicaire ne vinssent frapper à la
porte pour annoncer un baptême, un enterrement, un mariage ;
souvent ils s’arrêtaient, prenaient une chaise et restaient longtemps
à causer avec ma mère. C’étaient aussi des gens qui venaient, à
toute heure du jour, se renseigner sur les offices, quand par exemple
ils avaient entendu, la veille au soir, les cloches sonner un glas :
c’était un enterrement pour le lendemain matin, et il y avait de ce fait
une messe supprimée, soit celle du vicaire, soit celle du doyen.
Ma mère faisait partie du groupe des femmes pieuses à qui il ne
suffisait point d’assister le dimanche à la messe. Et ce n’étaient ni
les crucifix, ni les bénitiers, ni les images de sainteté qui manquaient
chez nous. Sur le manteau de notre cheminée il y avait deux
statuettes de la Vierge et de saint Joseph, et sur le coin d’une petite
table de nombreux livres de piété. Ma mère savait nettement
différencier le bien du mal. Elle n’ignorait point que ce fût un péché
de jurer par « sacristie ! » au lieu de « sapristi ! » Chaque année, des
voisines venaient se renseigner auprès d’elle sur la façon
d’accommoder les plats le Vendredi saint ; celles qui emploient le
beurre encourent la colère de Dieu. Elle savait qu’il est nécessaire
de réciter sa prière le matin en se levant et le soir avant de se
coucher ; qu’il ne faut pas se mettre à table sans faire le signe de la
croix, parce que c’est à Dieu, et à lui seul, que nous devons de nous
asseoir devant la nourriture : que chacun de nous est
continuellement guetté par le démon contre les attaques duquel le
protège son ange gardien. Elle parlait du ciel, du purgatoire et de
l’enfer comme de pays dont elle eût connu les moindres accidents,
et plus d’une fois je frissonnai à l’évocation terrible de Lucifer
siégeant, le trident à la main, dans des régions souterraines emplies
des lamentations et des cris de haine des damnés. Elle ne cessait
de me diriger dans la voie du bien. C’était mal de courir avec les
petites filles, mal d’assister aux bals publics et de regarder danser,
mal d’entrer dans l’église sans prendre de l’eau bénite, mal de dire
« le curé » au lieu de « Monsieur le curé », mal de prononcer de gros
mots comme faisaient les élèves de l’école communale, mal de trop
boire et de fumer, et après cela elle me reprochait de n’être pas
comme les autres !
Depuis un an j’étais enfant de chœur, et depuis six mois
j’apprenais le latin. Dans une petite ville personne n’ignore ce que
cela signifie : le latin est la langue de l’Église. Le moment venu je
partirais donc pour le séminaire. On ne m’avait pas demandé si cela
me convenait.
Or, quelques soirs après la Toussaint de cette même année, ma
mère me dit, comme nous nous mettions à manger la soupe :
— Mme Dumas est venue me voir tout à l’heure. Elle voudrait
qu’Albert soit enfant de chœur. Il paraît que c’est lui qui en a parlé le
premier. Elle dit qu’il fera tout son possible pour apprendre.
J’en fus étonné. Il n’y avait pas encore tout à fait un an qu’il était
sorti de l’école communale. Il était arrivé chez les frères avec la
certitude de sa supériorité sur nous qui étions officiellement des
enfants pieux puisque chaque matin nous récitions en commun la
prière. Il avait donc changé sans que je m’en fusse aperçu ? Cela
m’eût d’ailleurs été impossible, puisque je ne le fréquentais pas, tout
en m’abstenant de me moquer de lui : mes rires ne se seraient-ils
pas retournés contre moi ? Mais je ne pouvais admettre qu’il se
rapprochât de moi.
Je dis avec orgueil :
— Lui, apprendre les réponses de la messe ? Il est bien trop bête
pour ça !
Ma mère ne manqua point de me réprimander.
— Ce n’est qu’au bon Dieu, dit-elle, qu’on doit l’intelligence que
l’on a. Et il ne nous tiendra compte plus tard que de notre bonne
volonté et des efforts que nous aurons faits pour le bien servir.
Puisque Albert demande à être enfant de chœur sans que personne
lui en ait parlé, c’est qu’il a été touché par la grâce. Car ce n’est bien
sûr pas l’instituteur qui a pu lui mettre cette idée dans la tête. Avec
du travail on arrive à tout, et il apprendra à servir la messe aussi
bien que toi, et peut-être mieux, car tu ne te tiens guère bien.
Demain j’en dirai un mot à M. le curé.
En ce qui me concernait, elle avait raison. Moi qui n’ai jamais su
ce que c’était que la piété, je baignais de toutes parts dans une
atmosphère religieuse. Sans doute n’étais-je pas, à neuf ans ni à
quatorze, un précoce incrédule, et je n’aurais contesté l’existence de
Dieu, ni de ses saints, ni des anges, ni même du démon : mais il
m’était impossible d’avoir de ces élans affectueux qui vous font
tomber à genoux, les mains jointes devant le visage soudain inondé
de larmes. Sans trembler d’effroi je servais la messe, et, sans être
persuadé de la sublimité d’une vocation que je ne sentais pas,
j’apprenais le latin. Je n’en eus que plus de mépris pour Berlâne. Il
n’était pas intelligent : il ne lui manquait vraiment plus que de devenir
pieux.
Mais ce fut bien pis quand, quelques jours après, ma mère dit :
— Il paraît qu’Albert voudrait aussi apprendre le latin.
Elle rayonnait : c’était une vocation de plus — en comptant la
mienne, — dans le canton. C’était pour elle le dernier coup de la
grâce. Pour moi ce fut le coup de grâce. Ainsi, maintenant, j’allais
l’avoir toujours auprès de moi ? Nous étions une cinquantaine
d’élèves à l’école des frères. Il deviendrait peut-être bientôt, — dès
la première vacance, — un des six enfants de chœur. Et tout de
suite — car je ne doutais pas que « Monsieur le curé » n’acceptât, —
il allait être, avec moi, le seul qui apprît le latin ? Il allait s’attacher à
mes pas, comme mon ombre. D’un pied léger il s’engageait dans
cette voie où l’on m’avait poussé par les épaules. Quel imbécile,
décidément !
VIII
Nous prenions nos leçons dans la chambre qu’occupait au

presbytère l’abbé Bichelonne, le vicaire. Il avait l’accent auvergnat.
Là-bas surabondance, ici disette de vocations : le diocèse de
Clermont-Ferrand prêtait chaque année à celui de Nevers quelques
jeunes prêtres.
Berlâne faisait un petit détour pour me cueillir en passant : je suis
sûr qu’il n’aurait pas reculé devant une lieue. La première fois que
nous prîmes contact — il y avait juste un an qu’il était arrivé chez les
frères, — il me dit respectueusement :
— Je suis heureux de ce que maintenant nous nous allons être
ensemble.
Qu’est-ce qui me retint de lui répondre :
— Eh bien, moi, je ne le suis pas !
Fut-ce manque de cruauté ? Ou que, malgré moi, cette marque
de déférence me toucha ? Je n’en sais rien. Mais je gardai le
silence. Ma supériorité sur lui m’en donnait le droit. Il attendit,
comme l’année dernière dans la cour de l’école. Quoi ? Une bonne
parole, sans doute. J’évitai de le regarder. Sinon je n’aurais peut-être
pas pu m’empêcher de lui répondre :
— Moi aussi.
Mais je ne voulais à aucun prix qu’il se raccrochât à moi comme
un chien galeux que tout le monde repousse, ni qu’il s’imaginât, lui
dont personne n’acceptait la compagnie, que je pouvais être
heureux de le voir auprès de moi : j’avais envie de le renvoyer aux
Chovin et à leur boutique. Hélas ! La douceur et la misère m’ont
toujours ému. Et tout ce que je pouvais faire, c’était de ne pas
répondre.
Le jeudi, jour de la leçon de latin, je partais le plus souvent un
quart d’heure plus tôt, quitte à muser sous les sapins, autour de ce
petit arbre de la Liberté avec qui j’avais fait connaissance de plus
près, depuis l’époque, pas si éloignée pourtant, où j’allais à la salle
d’asile. Mes livres et mon cahier sous le bras, pour m’esquiver je
profitais d’un moment d’inattention de ma mère. Elle tenait à ce que
j’attendisse Berlâne. Elle avait cessé de voir en lui « l’original » qui
me ressemblait pour ne plus prendre garde qu’à ses qualités qui
n’étaient pas les miennes : l’obéissance et la piété.
Quand nous montions ensemble vers le presbytère, j’affectais de
ne pas entendre les questions qu’il me posait de sa voix la plus
douce. Je sifflotais comme un homme. Avec lui, seul à seul, j’avais
beau jeu à prendre des airs de matamore ! Pourtant, je l’ai bien vu
depuis et je m’en doutais alors, ce n’était comme moi qu’un pauvre
gamin qui eût été si heureux de rencontrer une vraie sympathie !
Mais je ne voulais pas de la sienne, pas plus que je n’aurais voulu
faire plaisir à ma mère en devenant tout à fait obéissant. S’il ne me
l’avait pas offerte, peut-être l’aurais-je recherché, qui sait ? Mais
non : nous nous ressemblions sur trop de points pour pouvoir nous
entendre. Certes, il n’aurait tenu qu’à moi que nous fussions amis :
seulement mon orgueil montait bonne garde.
J’avais plaisir à l’entendre bégayer et bafouiller, confondant les

cas des déclinaisons et les temps des conjugaisons. Ses premiers
thèmes fourmillèrent de barbarismes, ses premières versions de
contresens. A la fin des fins, l’abbé Bichelonne s’emportait et
Berlâne, tirant son mouchoir, s’essuyait les yeux. Je haussais les
épaules. S’il s’était évanoui, comme le jour de la grenouille, je crois
que je l’aurais giflé : peut-être cela l’eût-il fait revenir plus
promptement à lui. Moi aussi, plus d’une fois, j’avais pleuré dans
cette même chambre. Maintenant encore, maintenant surtout que
j’abordais le De viris et que je me dépêtrais tant mal que bien au
travers de textes tout exprès, me semblait-il, semés d’embûches, les
larmes souvent me venaient aux yeux : l’abbé Bichelonne était
prompt à se mettre en colère. Il ne comprenait point que l’on ne
comprît pas. Mais devant Berlâne je me tenais à quatre : pour rien
au monde je n’aurais voulu qu’il me vît pleurer.
Plus avancé que lui, j’aurais pu l’aider à faire ses devoirs et lui
éviter ainsi quelques observations. Je m’en gardais bien. Mais je
devins moi-même son professeur, ayant été chargé de lui apprendre
à servir la messe.
Il fallait qu’il sût par cœur tous les répons à l’officiant, depuis Ad
Deum qui laetificat juventutem meam, jusqu’au dernier Deo gratias.
De ses journées il ne perdait pas une minute.
Si ma mère avait de l’estime pour lui, Mme Dumas en avait pour
moi. Elle trouvait en moi telle qualité qui manquait à son pauvre
Albert. Elle me faisait des confidences comme à quelqu’un qui déjà
comprend tout.
— Le soir, me disait-elle, il faut que je le déshabille de force ;
autrement il ne se coucherait pas. Ainsi, croyez-vous !…
Parfois elle disait à ma mère :
— Ah ! Madame, si mon Albert ressemblait à votre fils !…
Mais elle, dédaigneuse :
— Ne vous plaignez donc pas, Madame Dumas. Chacun a les
facultés que le Bon Dieu lui a données. Et j’aimerais bien mieux le
voir obéissant et pieux comme Albert.
C’était dur pour lui. Il lui fallait penser non seulement à ses
leçons et à ses devoirs de latin et de l’école, mais aussi à ses
répons au milieu desquels il s’embrouillait. Dans le Confiteor, d’une
longueur invraisemblable pour lui, il perdait pied, faisant passer les
saints Pierre et Paul après le bienheureux Michel archange. Moi,
vieux routier, qui depuis un an avais servi plus de quatre cents

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Recurrent Pregnancy Loss: Causes,

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About the Series

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Part I Basic Principles

3. Recurrent Pregnancy Loss­from Evidence-Based to Personalized Medicine........................... 22

5. The Endometrial Factor in Recurrent Pregnancy Loss.............................................................. 43

6. Fetal Structural Malformations and Recurrent Pregnancy Loss.............................................. 48

7. The Endocrinology of Recurrent Pregnancy Loss....................................................................... 59

8. The Etiology of the Antiphospholipid Syndrome......................................................................... 70

9. Defects in Coagulation Factors Leading to Recurrent Pregnancy Loss................................... 79

10. The Immunobiology of Recurrent Miscarriage........................................................................... 89

11. Immune Testing in Recurrent Pregnancy Loss..........................................................................101

12. Uterine Anomalies and Recurrent Pregnancy Loss...................................................................110

13. The Male Factor in Recurrent Pregnancy Loss......................................................................... 126

Part III The Developing Pregnancy

15. Threatened Miscarriage and Recurrent Pregnancy Loss.........................................................145

16. The Role of Cerclage and Pessaries..............................................................................................153

17. What Genetic Screening Is Appropriate in Recurrent Pregnancy Loss?............................... 164

18. Obstetric Outcomes after Recurrent Pregnancy Loss...............................................................172

22. Human Chorionic Gonadotropin Supplementation in Recurrent Pregnancy Loss............... 206

23. Antiphospholipid Syndrome: Management of the Obstetric Patient........................................215

24. Can Recurrent Pregnancy Loss Be Prevented by Antithrombotic Agents?........................... 223

28. Third Party Reproduction in Recurrent Pregnancy Loss........................................................ 249

30. IVIg Treatment for Recurrent Pregnancy Loss........................................................................ 268

31. The Role of Filgrastim.................................................................................................................. 275

32. Opinion: Immunotherapy Has No Place in the Treatment of Recurrent

Howard J.A. Carp, MB BS, FRCOG

Nasser Al-Asmar D. Ware Branch

Gautam Nand Allahbadia Jeffrey Braverman

Salim Daya Akanksha Allahbadia Gupta

Ashok Kumar Rubina Merchant

Dolores J. Lamb Pere Mir

C.V. Rao Carlos Simon

Lorena Rodrigo Joe Leigh Simpson

Yehuda Shoenfeld Akhila Vasudeva

Epidemiologic Parameters Relevant for RPL

Number of Previous Miscarriages

3 misc. 4 misc. 5+ misc.

Not pregnant Miscarriages Births

Integration of Epidemiologic Knowledge in Research and Management of RPL

Class II HLA Alleles

Why Is There a Delay between Fertilization and Implantation?

Embryo-Specific Maternal Communication—PIF

PIF Autocrine, Autotrophic, and Protective Actions

Receptive Uterine Environment Is Critical for Reproduction

Implantation Is Insufficient: Effective Trophoblast Invasion Is Also Required

PIF Interaction and Synergy with Progesterone and HLA-G

— Madame, voulez-vous qu’Albert vienne s’amuser avec nous ?

Robert dut éprouver un violent besoin d’action. Il bondit en

3. Recurrent Pregnancy Lossfrom Evidence-Based to Personalized Medicine........................... 22