RIFbook

Recurrent Implantation Failure
Recurrent Implantation Failure
Edited by
Efstratios M. Kolibianakis, MD, MSc, PhD
Associate Professor, Obstetrics and Gynecology and Assisted Reproduction
Unit for Human Reproduction
Medical School
Aristotle University of Thessaloniki
Thessaloniki, Greece
Christos A. Venetis, MD, MSc(Res),

MSc(HCM), PhD, FRANZCOG
Consultant Gynaecologist and Fertility Specialist and Senior Lecturer
Centre for Big Data Research in Health and School
of Women’s and Children’s Health
University of New South Wales (UNSW) Faculty of Medicine
Sydney, Australia
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Library of Congress Cataloging-in-Publication Data
Names: Kolibianakis, Efstratios M., editor. | Venetis, Christos A., editor.

Title: Recurrent implantation failure/edited by Efstratios M. Kolibianakis and Christos A. Venetis.
Other titles: Recurrent implantation failure (Kolibianakis)
Description: Boca Raton : CRC Press, [2019] | Includes bibliographical references and index.
Identifiers: LCCN 2019005130| ISBN 9781138055780 (hardback : alk. paper) | ISBN 9781138055766
(pbk. : alk. paper)
Subjects: | MESH: Embryo Transfer—adverse effects | Embryo Loss | Embryo Implantation—
physiology | Infertility, Female—etiology | Infertility, Female—therapy | Treatment Failure
Classification: LCC RG135 | NLM WQ 208 | DDC 618.1/780599—dc23
LC record available at https://lccn.loc.gov/2019005130
Visit the Taylor & Francis Web site at

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Contents
Contributors vii
1 What is recurrent implantation failure? 1

Basil C. Tarlatzis, Julia K. Bosdou, and Efstratios M. Kolibianakis
2 The psychological consequences of recurrent implantation failure 9
Alice D. Domar and Jeremy J. Cottle
3 The role of lifestyle factors in recurrent implantation failure 14
Vicki Nisenblat and Robert J. Norman
4 Endocrine causes of recurrent implantation failure 27
Gesthimani Mintziori and Dimitrios G. Goulis
5 Congenital uterine anomalies and recurrent implantation failure 35
Grigoris F. Grimbizis, Sara Y. Brucker, and Rudi Campo
6 Immunological causes of recurrent implantation failure: Comprehensive insights into
mechanisms and therapeutic approaches 42
Ari Kim, Wael Saab, Nayoung Sung, and Joanne Kwak-Kim
7 Acquired uterine conditions, reproductive surgery, and recurrent implantation failure 55
Dimitra Aivazi, Eleni Tsakalidou, and Grigoris F. Grimbizis
8 Thrombophilia: Diagnosis and management in women with recurrent implantation failure 72
Kay Neumann and Georg Griesinger
9 Andrological causes of recurrent implantation failure 79
Chrisanthi Marakaki, Georgios A. Kanakis, and Dimitrios G. Goulis
10 Diagnostic evaluation of the couple with recurrent implantation failure 93
Engin Turkgeldi, Sule Yildiz, and Baris Ata
11 Optimizing embryo culture for recurrent implantation failure 107
Katerina Chatzimeletiou
12 Optimizing endometrial receptivity for patients with recurrent implantation failure:
The role of progesterone on the day of triggering final oocyte maturation 113
Christos A. Venetis, Julia K. Bosdou, and Efstratios M. Kolibianakis
13 The role of late follicular phase luteinizing hormone and estradiol on embryo implantation:
Implications for recurrent implantation failure 127
Julia K. Bosdou, Christos A. Venetis, and Efstratios M. Kolibianakis
14 Manipulating the endometrium: Can recurrent implantation failure be managed
by inducing endometrial inflammation or by the use of novel research therapies? 135
Antonis Makrigiannakis and Thomas Vrekoussis
15 The embryo in recurrent implantation failure: Genetic selection and strategies
for improving its implantation potential 141
Katerina Chatzimeletiou
16 Is oocyte donation efficient in patients with recurrent implantation failure? 150
Gustavo Nardini Cecchino and Juan Antonio García-Velasco
17 Sperm donation and recurrent implantation failure 158
Christos P. Tsametis and Dimitrios G. Goulis
18 Optimizing embryo transfer technique for recurrent implantation failure management 166
Frank J. Broekmans
v
vi Contents
19 When should patients abandon treatment? 180

Konstantinos Dafopoulos
20 Proposed management of patients with recurrent implantation failure and
directions for future research 186
Christos A. Venetis and Efstratios M. Kolibianakis
Index 195
Contributors
Dimitra Aivazi Jeremy J. Cottle

Gynecologist Domar Center for Mind/Body Health
Thessaloniki, Greece at Boston IVF
and
Baris Ata Beth Israel Deaconess Medical Center
Department of Obstetrics and Gynecology Harvard Medical School
Koç University Hospital Boston, Massachusetts
Istanbul, Turkey
Konstantinos Dafopoulos
Julia K. Bosdou Assisted Reproduction Unit
Unit for Human Reproduction University of Thessaly
First Department of Obstetrics and Gynecology Larissa, Greece
Medical School
Aristotle University of Thessaloniki Alice D. Domar
Thessaloniki, Greece Domar Center for Mind/Body Health
at Boston IVF
Frank J. Broekmans and
Department of Reproductive Medicine Beth Israel Deaconess Medical Center
University Medical Center Utrecht Harvard Medical School
Utrecht, The Netherlands Boston, Massachusetts
Sara Y. Brucker
Dimitrios G. Goulis
Medical Director
Unit of Reproductive Endocrinology and Unit
Research Center for Women’s Health
of Human Reproduction
Universitätsklinikum Tübingen
First Department of Obstetrics and
Tübingen, Germany
Gynecology
Medical School
Rudi Campo
Clinical Director
Leuven Institute for Fertility and Embryology
Leuven, Belgium
Georg Griesinger
Gustavo Nardini Cecchino Department of Gynaecological Endocrinology
Department of Reproductive Endocrinology and Reproductive Medicine
and Infertility University Hospital of Schleswig-Holstein
IVIRMA—Madrid Lübeck, Germany
Madrid, Spain
Grigoris F. Grimbizis
Katerina Chatzimeletiou President of the European Society for
First Department of Obstetrics and Gynaecology Gynaecological Endoscopy
Aristotle University Medical School First Department of Obstetrics and
and Gynaecology
Papageorgiou General Hospital Aristotle University of Thessaloniki
Thessaloniki, Greece Thessaloniki, Greece
vii
viii Contributors
Georgios A. Kanakis Kay Neumann

Department of Endocrinology Department of Obstetrics and Gynecology
Diabetes and Metabolism Schleswig-Holstein
Athens Naval and Veteran Affairs Hospital University Lübeck, Germany
Athens, Greece
Vicki Nisenblat
Ari Kim Discipline of Obstetrics and Gynaecology
Reproductive Medicine and Immunology School of Medicine
Department of Obstetrics and Gynecology Robinson Research Institute
Department of Microbiology and Adelaide, Australia
Immunology
Chicago Medical School Robert J. Norman
Rosalind Franklin University of Medicine and Discipline of Obstetrics and Gynaecology
Science School of Medicine
Vernon Hills, Illinois Robinson Research Institute
Adelaide, Australia
Efstratios M. Kolibianakis
Sule Yildiz
Unit for Human Reproduction
Department of Obstetrics and Gynecology
First Department of Obstetrics and Gynecology
Koc University Hospital
Medical School
Istanbul, Turkish Republic
Thessaloniki, Greece Wael Saab
Center for Reproductive and Genetic Health
Joanne Kwak-Kim
London, United Kingdom
Reproductive Medicine and Immunology
Department of Obstetrics and Gynecology Nayoung Sung
Department of Microbiology and Reproductive Medicine and Immunology
Immunology Department of Obstetrics and Gynecology
Chicago Medical School Department of Microbiology and Immunology
Rosalind Franklin University of Medicine and Chicago Medical School
Science Rosalind Franklin University of Medicine and
Vernon Hills, Illinois Science
Vernon Hills, Illinois
Antonis Makrigiannakis
Department of Obstetrics and Gynecology Eleni Tsakalidou
Medical School Department of Gynaecological Endocrinology
University of Crete and Reproductive Medicine
Heraklion, Greece University Hospital of Schleswig-Holstein
Lübeck, Germany
Chrisanthi Marakaki
Department of Endocrinology Christos P. Tsametis
Diabetes and Metabolism Unit of Reproductive Endocrinology
Athens Naval and Veteran Affairs Hospital First Department of Obstetrics and Gynecology
Athens, Greece Aristotle University of Thessaloniki
Gesthimani Mintziori
Unit of Reproductive Endocrinology and Unit of Basil C. Tarlatzis
Human Reproduction Unit for Human Reproduction
First Department of Obstetrics and Gynecology First Department of Obstetrics and Gynecology
Medical School Medical School
Aristotle University of Thessaloniki Aristotle University of Thessaloniki
Thessaloniki, Greece Thessaloniki, Greece
Contributors ix
Engin Turkgeldi Christos A. Venetis

Department of Obstetrics and Gynecology Centre for Big Data Research in Health
Koç University Hospital School of Women’s and Children’s Health
and University of New South Wales (UNSW)
Department of Obstetrics and Gynecology Faculty of Medicine
Koç University School of Medicine Sydney, Australia
Istanbul, Turkish Republic
Thomas Vrekoussis
Juan Antonio García-Velasco Department of Obstetrics and Gynecology
Department of Reproductive Endocrinology Medical School
and Infertility University of Crete
IVIRMA—Madrid Heraklion, Greece
and
Department of Gynecology
Faculty of Medicine of the Rey Juan Carlos
University
Madrid, Spain
What is recurrent implantation
failure?
1
BASIL C. TARLATZIS, JULIA K. BOSDOU,
and EFSTRATIOS M. KOLIBIANAKIS
INTRODUCTION
Successful implantation
Implantation is a complex phenomenon, still not thoroughly understood, involving the embryo
and the endometrium.1 Implantation has been considered as a gradual process requiring embryo
adhesion to the luminal surface of the endometrium, followed by invasion of the trophectoderm
cells from the embryo, through the luminal epithelium, into the deeper layer of the endometrium.2,3
These complex molecular interactions between the uterus and the mature blastocyst are neces-
sary to establish the uteroplacental circulation and finally lead to implantation, approximately six
or seven days after fertilization.1,4 By the 10th day after fertilization, the blastocyst is completely
embedded in the stromal tissue of the uterus, leading to cytotrophoblast invasion into the entire
endometrium and the inner third of the myometrium, as well as the uterine vasculature.5
In clinical practice, implantation can be confirmed by a rising human chorionic gonadotro-
phin (hCG) level, which occurs eight to ten days after ovulation or 14 days after oocyte retrieval
in assisted reproductive technology (ART) cycles.6 It is considered to be successful when there is
ultrasonographic evidence of an intrauterine gestational sac after five weeks of gestation, or usually
three weeks after oocyte retrieval in ART cycles.2
Unfortunately, pregnancy achievement in the human is a relatively inefficient process, with a
probability of conception approximately 25%–30% per cycle.7 Thus, it is more likely for a human
embryo not to implant than to lead to establishment of pregnancy.1
Implantation failure
From a clinical point of view, implantation failure may occur very early during the adhesion stage,
with no detectable hCG production and, thus, no objective evidence of pregnancy. It may also
occur at a later stage, after initiation of hCG production from the implanting embryo, without,
however, formation of an intrauterine gestational sac visible on ultrasonography, which is clinically
recognized as biochemical pregnancy.2,3
Implantation failure may be due to embryo and/or maternal factors. Problems due to the embryo
can be associated either with poor oocyte quality or with paternal factors.8 On the other hand,
implantation problems associated with the female may be due to uterine, tubal, or immunological
factors; endometriosis; or thrombophilia.9,10 Surprisingly, the high implantation rate of donated
oocytes in women of advanced reproductive age suggests that oocyte quality rather than endome-
trial receptivity largely determines the success of implantation.1,11
In non-oocyte donation cycles and despite the technological advances in in vitro fertilization
(IVF), 3 only about one-quarter of IVF cycles will result in live births, leading many couples
to experience multiple IVF failures. After each failed IVF attempt, pregnancy rates decrease
significantly in subsequent cycles, with the most remarkable decrease after the third cycle.12
Failure to achieve pregnancy after multiple IVF attempts is recognized as recurrent implantation
failure (RIF). 2
1
2 What is recurrent implantation failure?
FACTORS CONTRIBUTING TO RIF

The embryo
The embryo has been implicated in RIF in the presence of poor oocyte or spermatozoa quality. Poor
oocyte quality has been associated with advanced maternal age, known to lead to a low number of
oocytes that are usually aneuploid.13 On the other hand, poor spermatozoa quality has been associ-
ated with sperm DNA damage, also leading to the formation of poor-quality embryos.14
Uterine pathologies and endometrial receptivity

A number of congenital uterine abnormalities, including Müllerian-duct malformations as well as
submucous fibroids, endometrial polyps, intrauterine adhesions, or adenomyosis, may seriously
affect endometrial receptivity and contribute to RIF.15,16 A functioning and receptive endome-
trium is of crucial importance for successful embryo implantation. Endometrial receptivity refers
to the synchronized interaction among ovarian hormones, growth factors, adhesion molecules,
cytokines, and lipids, allowing embryo adhesion, invasion, placentation, and therefore pregnancy
initiation to occur.9,17 Impaired endometrial receptivity is responsible for approximately two-thirds
of implantation failures.18
Whether or not a thin endometrium is associated with impaired endometrial receptivity is still a
matter of debate.19 The minimal adequate endometrial thickness for successful implantation varies
between studies, ranging from 6–8 mm.20,21 Significantly higher embryo implantation and preg-
nancy rates have been demonstrated in patients with an endometrial thickness of >9 mm com-
pared with those with a thickness <9 mm.22–24 On the other hand, two large prospective studies
failed to confirm such an association.25,26
Hydrosalpinges
Hydrosalpinges have also been recognized as a contributing factor to RIF due to their harmful
impact on implantation. Hydrosalpinges may negatively influence implantation either by a direct
toxic effect on the preimplantation embryo or mechanically by flushing the embryo out of the
uterus. Moreover, it has been shown that endometrial receptivity is impaired in the presence of
hydrosalpinges due to the abnormal expression of cytokines, which are necessary for successful
implantation.27,28
Immunological factors/thrombophilia
Endometrial decidualization is a process in which the differentiation of endometrial stromal cells
occurs. It is of vital importance for pregnancy establishment and maintenance, enabling maternal
immune tolerance, fetus survival, and placentation process regulation.29 In this respect, the impact
of various immunological factors on implantation has attracted a lot of attention as a cause of RIF,
although no consensus exists on whether or not immunological investigations and treatments may
be beneficial.2,30
The role of thrombophilia in implantation failure has been also the focus of research.31–33 The
association of inherited thrombophilia, antiphospholipid or other autoantibodies, with RIF has
been investigated with, however, controversial results.34,35 Hence, the value of thrombophilia
screening in patients with RIF still remains a matter of debate.
VARIABLES USED IN THE DEFINITION OF RIF

Quality and number of embryos transferred
Generally, the probability of an embryo to implant is 30%, and thus, in order to increase the prob-
ability of pregnancy, it is imperative that the best-quality available embryo(s) are selected for
Definition of RIF 3
transfer. A good-quality embryo has been defined as an embryo with the number of cells corre-
sponding to the day of its development, blastomeres of equal size with regular distribution, and less
than 10% fragmentation.36 In addition, day-5 embryos (blastocysts) have been graded according to
the expansion and quality of the inner cell mass and the trophectoderm.36
Due to the higher implantation potential of a blastocyst compared with that of a cleavage-stage
embryo, the developmental stage of the embryo transferred has also been taken into account in the
definition of RIF.2,37 Single-blastocyst transfer has been suggested to result in a similar probability
of live birth compared to the transfer of two cleavage-stage embryos.38–41
Several definitions of RIF have been proposed including various numbers of embryos
transferred per cycle or a cumulative number of embryos transferred overall. Controversy exists
regarding the inclusion or not of frozen embryos in the number of total embryos used to define
RIF. It has been suggested that frozen embryos should not be included in the definition of RIF
due to their lower implantation rate compared with fresh embryos.42 However, the opposite has
also been suggested since transfer of frozen embryos contributes to the cumulative pregnancy
rate achieved.43–45
Number of unsuccessful IVF/ICSI cycles

Many investigators have preferred to base the definition of RIF on the number of unsuccessful IVF/
intracytoplasmic sperm injection (ICSI) cycles alone using different numbers, ranging from one to
multiple cycles, usually in combination with other variables.
Maternal age
Maternal age has been incorporated into the definition of RIF, given the fact that maternal age is
closely related to embryo quality.46 It has been demonstrated that implantation rates remain con-
stant until the age of 35, followed by a linear decrease of 2.77% per year.47 Moreover, it has been
shown that aneuploidy significantly increases with maternal age, and thus, implantation failure
in women of advanced maternal age may be largely attributed to this problem.48 Although the age
limit of 40 years has been proposed as an upper age limit for defining RIF in combination with
other factors,2 it still has not been widely adopted.
DEFINITION OF RIF
As already discussed, many variables have been used for the definition of RIF, including number
of embryos transferred, number of IVF attempts, embryo quality, or maternal age.7 Using these
variables in different combinations, several definitions of RIF have been proposed without, unfor-
tunately, a universally accepted definition being present. Consequently, the exact prevalence of
patients with RIF is difficult to be determined.
According to an early definition, dated more than 20 years ago, RIF is defined as a cumulative trans-
fer of eight cleavage-stage embryos or four blastocysts, not leading to a positive pregnancy test 14 days
after oocyte pick-up (OPU).49 On the other hand, the ESHRE Preimplantation Genetic Diagnosis (PGD)
Consortium has defined RIF as a failure to achieve clinical pregnancy after ≥3 unsuccessful transfers
of high-quality embryos or after the cumulative transfer of ≥10 embryos in multiple transfers.50
In a more recent approach, RIF has been defined as the failure of pregnancy achievement after
the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles in
women under the age of 40 years.2
A systematic review performed in 2014 attempted to standardize the definition criteria for RIF
by evaluating all published definitions, aiming to assist in the design and to enhance the quality
of future studies focusing on RIF.37 A total of 119 studies, with a significant heterogeneity in the
definition of RIF, were included in this systematic review. All definitions of RIF in these studies
were arbitrary. The studies were categorized in multiple definition groups, based on the number
of unsuccessful cycles or the number of unsuccessful cycles in combination with the number of
embryos transferred (Table 1.1).
The number of failed cycles, used as a single variable to define RIF, ranged from two to seven.
The most common single-variable definition of RIF in the above systematic review was that using
“three or more unsuccessful or failed cycles.”
Regarding the combination of the number of embryos transferred with the number of
unsuccessful cycles, this ranged considerably from one to more than three embryos transferred
or transfer of four to 10 or more cleavage-stage embryos, cumulatively. The most commonly
Table 1.1 Studies using a definition of RIF based on the number of unsuccessful cycles alone or
on the combination of unsuccessful cycles with the number of embryos transferred/cumulative
transfer of embryos.
Number of unsuccessful cycles alone

≥2 failed treatment cycles ≥3 failed treatment cycles
Gianaroli et al. (1997) De Placido et al. (1991)
Kahraman et al. (2000) Rubio et al. (2001)
Stephenson and Fluker (2000) Levran et al. (2002)
Matsubayashi et al. (2001)a Ledee-Bataille et al. (2004a)a
Spandorfer et al. (2002) Ledee-Bataille et al. (2004b)
Martinuzzo et al. (2005)a Loutradis et al. (2004)a
Petersen et al. (2005) Pantos et al. (2004)
Fukui et al. (2006)a Rufas-Sapir et al. (2004)
Vaquero et al. (2006) Ledee-Bataille et al. (2005)a
Foulk et al. (2007)a Ghobara et al. (2006)
Hiraoka et al. (2008) Platteau et al. (2006)
Kalu et al. (2008)a Qublan et al. (2006)
Valojerdi et al. (2008) Mantzouratou et al. (2007)
Urman et al. (2009) Matsubayashi et al. (2007)
Eyheremendy et al. (2010) Van den Heuvel et al. (2007)
Tsoumpou et al. (2010)a Weissman et al. (2007)
Schoolcraft et al. (2010) Kling et al. (2008a)
Berker et al. (2011) Kling et al. (2008b)
Chou et al. (2011)a Pagidas et al. (2008)
Huang et al. (2011) Prakash et al. (2008)a
Lodigiani et al. (2011) Yakin et al. (2008)
Oliveira et al. (2011)a Brosens et al. (2009)a
Takahashi et al. (2011) Debrock et al. (2009)
Ivanov et al. (2012) Jee et al. (2009)
Sermondade et al. (2012) Achache et al. (2010a)a
Achache et al. (2010b)
Germeyer et al. (2010)a
Choi et al. (2011)
Kim et al. (2011)a
Rajaei et al. (2011)
Tiboni et al. (2011)
Sudoma et al. (2011)a
Sacks et al. (2012)
Scarpellini and Sbracia (2012)a
(Continued)
Conclusions 5
Table 1.1 (Continued) Studies using a definition of RIF based on the number of unsuccessful
cycles alone or on the combination of unsuccessful cycles with the number of embryos transferred/
cumulative transfer of embryos.
Combination of the number of unsuccessful cycles and the number of embryos transferred
≥2 unsuccessful cycles & ≥3–7 unsuccessful cycles & ≥3–5 unsuccessful cycles &
transfer of ≥1–2 embryos transfer of ≥1–2 embryos transfer of ≥3 embryos
Kwak-Kim et al. (2003) Stein et al. (1995)a Carp et al. (1994)
Kahraman et al. (2004)a Creus et al. (1998) Huang et al. (1999)a
Koscinski et al. (2006)a Primi et al. (2004)a
Arefi et al. (2008)a Elram et al. (2005)a
Aletebi (2010)a Matteo et al. (2007)a
Johnston-MacAnanny et al. Varla-Leftherioti et al. (2007)
(2010)a Thum et al. (2008)a
Yang et al. (2010) Brinsden et al. (2009)
Simur et al. (2009)a
Chernyshov et al. (2010)a
Maritnez-Zamora et al. (2011)a
Combination of the number of unsuccessful cycles and the cumulative transfer of embryos
≥2 unsuccessful cycles & ≥2–3 unsuccessful cycles & Several unsuccessful cycles
cumulative transfer of cumulative transfer of >9–10 and/or cumulative transfer of
>4–6 embryos embryos >10 embryos
Sharif and Ghunaim (2010)a Pehlivan et al. (2003)a Raziel et al. (2002)
Fukui et al. (2008) Taranissi et al. (2005) Voullaire et al. (2002)
Quenby et al. (2007)a Ledee et al. (2008)a
Blockeel et al. (2008)a Tuckerman et al. (2010)a
Koler et al. (2009)a Dos Santos et al. (2012)a
Karimzadeh et al. (2009)a
Fragouli et al. (2010)a
Source: Compiled from data in Polanski LT et al. Reprod Biomed Online. 2014;28: 409–23.
a Good/high embryo quality.
used combination of the number of embryos transferred with the number of unsuccessful cycles
was “two or more embryos transferred during three or more unsuccessful or failed cycles”
(Table 1.1).
Employing the number of embryos transferred cumulatively in combination with the number of
unsuccessful cycles, the most common definition was “cumulative transfer of 10 or more embryos
during three or more unsuccessful or failed cycles” (Table 1.1).
CONCLUSIONS
Successful implantation is considered to stem from an efficient combination of various factors.
Repeated failure of any of these factors or of their combination might decrease the chance of
implantation and eventually lead to recurrent implantation failure.
Unfortunately, an enormous variability in the definition of RIF exists, necessitating the adoption
of a widely accepted definition. This would assist in enhancing our understanding of implanta-
tion and in developing effective treatments that would decrease significantly the psychological,
emotional, and financial burden of couples with RIF experience. Until then, the evaluation of any
proposed treatment for RIF should always take into account the definition used.
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offspring. Biol Reprod. 2008;78(4):761–72.
15. Valdes CT, Schutt A, Simon C. Implantation failure of endometrial origin: It is not pathology,
but our failure to synchronize the developing embryo with a receptive endometrium. Fertil
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16. Taylor E, Gomel V. The uterus and fertility. Fertil Steril. 2008;89(1):1–16.
17. Miravet-Valenciano JA, Rincon-Bertolin A, Vilella F, Simon C. Understanding and improv-
ing endometrial receptivity. Curr Opin Obstet Gynecol. 2015;27(3):187–92.
18. Simon C, Moreno C, Remohi J, Pellicer A. Cytokines and embryo implantation. J Reprod
Immunol. 1998;39(1–2):117–31.
19. Revel A. Defective endometrial receptivity. Fertil Steril. 2012;97(5):1028–32.
20. Simon A, Laufer N. Repeated implantation failure: Clinical approach. Fertil Steril.
2012;97(5):1039–43.
21. Chen SL, Wu FR, Luo C et al. Combined analysis of endometrial thickness and pattern in
predicting outcome of in vitro fertilization and embryo transfer: A retrospective cohort study.
Reprod Biol Endocrinol. 2010;8:30.
22. Noyes N, Liu HC, Sultan K, Schattman G, Rosenwaks Z. Endometrial thickness appears
to be a significant factor in embryo implantation in in-vitro fertilization. Hum Reprod.
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23. Kovacs P, Matyas S, Boda K, Kaali SG. The effect of endometrial thickness on IVF/ICSI out-
come. Hum Reprod. 2003;18(11):2337–41.
24. Richter KS, Bugge KR, Bromer JG, Levy MJ. Relationship between endometrial thickness
and embryo implantation, based on 1,294 cycles of in vitro fertilization with transfer of two
blastocyst-stage embryos. Fertil Steril. 2007;87(1):53–9.
25. Khalifa E, Brzyski RG, Oehninger S, Acosta AA, Muasher SJ. Sonographic appearance of
the endometrium: the predictive value for the outcome of in-vitro fertilization in stimulated
cycles. Hum Reprod. 1992;7(5):677–80.
26. Oliveira JB, Baruffi RL, Mauri AL, Petersen CG, Campos MS, Franco JG, Jr. Endometrial
ultrasonography as a predictor of pregnancy in an in-vitro fertilization programme. Hum
Reprod. 1993;8(8):1312–5.
27. Seli E, Kayisli UA, Cakmak H et al. Removal of hydrosalpinges increases endometrial leukae-
mia inhibitory factor (LIF) expression at the time of the implantation window. Hum Reprod.
2005;20(11):3012–7.
28. Bildirici I, Bukulmez O, Ensari A, Yarali H, Gurgan T. A prospective evaluation of the effect
of salpingectomy on endometrial receptivity in cases of women with communicating hydro-
salpinges. Hum Reprod. 2001;16(11):2422–6.
29. Ledee N, Petitbarat M, Chevrier L et al. The uterine immune profile may help women with
repeated unexplained embryo implantation failure after in vitro fertilization. Am J Reprod
Immunol. 2016;75(3):388–401.
30. Pathare ADS, Zaveri K, Hinduja I. Downregulation of genes related to immune and inflam-
matory response in IVF implantation failure cases under controlled ovarian stimulation. Am
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31. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated
implantation failure following IVF-ET. Hum Reprod. 2006;21(12):3036–43.
32. Lattova V, Dostal J, Peskova M, Sobek A, Jr., Prochazka M. [Recurrent implantation fail-
ure and thrombophilia]. Ceska gynekologie/Ceska lekarska spolecnost J Ev Purkyne. 2015;
80(1):5–10.
33. Bellver J, Soares SR, Alvarez C et al. The role of thrombophilia and thyroid autoimmunity
in unexplained infertility, implantation failure and recurrent spontaneous abortion. Hum
Reprod. 2008;23(2):278–84.
34. Qublan HS, Eid SS, Ababneh HA et al. Acquired and inherited thrombophilia: Implication in
recurrent IVF and embryo transfer failure. Hum Reprod. 2006;21(10):2694–8.
35. Coulam CB, Jeyendran RS, Fishel LA, Roussev R. Multiple thrombophilic gene mutations are
risk factors for implantation failure. Reprod Biomed Online. 2006;12(3):322–7.
36. Cutting R, Morroll D, Roberts SA et al. Elective single embryo transfer: Guidelines for prac-
tice British Fertility Society and Association of Clinical Embryologists. Hum Fertil (Camb).
2008;11(3):131–46.
37. Polanski LT, Baumgarten MN, Quenby S, Brosens J, Campbell BK, Raine-Fenning NJ. What
exactly do we mean by “recurrent implantation failure”? A systematic review and opinion.
Reprod Biomed Online. 2014;28(4):409–23.
38. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage
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39. Csokmay JM, Hill MJ, Chason RJ et al. Experience with a patient-friendly, mandatory, single-
blastocyst transfer policy: The power of one. Fertil Steril. 2011;96(3):580–4.
40. Gardner DK, Surrey E, Minjarez D, Leitz A, Stevens J, Schoolcraft WB. Single blastocyst
transfer: A prospective randomized trial. Fertil Steril. 2004;81(3):551–5.
41. Zander-Fox DL, Tremellen K, Lane M. Single blastocyst embryo transfer maintains compa-
rable pregnancy rates to double cleavage-stage embryo transfer but results in healthier preg-
nancy outcomes. Aust N Z J Obstet Gynaecol. 2011;51(5):406–10.
42. Tan BK, Vandekerckhove P, Kennedy R, Keay SD. Investigation and current management of
recurrent IVF treatment failure in the UK. BJOG. 2005;112(6):773–80.
43. Horne G, Critchlow JD, Newman MC, Edozien L, Matson PL, Lieberman BA. A prospective
evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum Reprod.
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44. Lieberman BA, Troup SA, Matson PL. Cryopreservation of embryos and pregnancy rates after
IVF. Lancet. 1992;340(8811):116.
45. de Mouzon J, Goossens V, Bhattacharya S et al. Assisted reproductive technology in Europe,
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46. Devroey P, Godoy H, Smitz J et al. Female age predicts embryonic implantation after ICSI:
A case-controlled study. Hum Reprod. 1996;11(6):1324–7.
47. Spandorfer SD, Chung PH, Kligman I, Liu HC, Davis OK, Rosenwaks Z. An analysis of the
effect of age on implantation rates. J Assist Reprod Genet. 2000;17(6):303–6.
48. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmen-
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1995;64(2):382–91.
49. Coulam CB. Implantation failure and immunotherapy. Hum Reprod. 1995;10(6):1338–40.
50. Thornhill AR, deDie-Smulders CE, Geraedts JP et al. ESHRE PGD Consortium “Best practice
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The psychological
consequences of recurrent
2
implantation failure
ALICE D. DOMAR and JEREMY J. COTTLE
INTRODUCTION
Although the goal of every initiated in vitro fertilization (IVF) cycle is a viable singleton pregnancy,
in many cases that won’t be the outcome. With the exception of the most ideal circumstances, most
patients who garner the physical, financial, and psychological resources to initiate treatment will
meet with disappointment. For those who have the stamina to try again, some will have the same
experience. And for the patients who undergo cycle after cycle, which happens often when insur-
ance covers multiple attempts, there are still going to be individuals and couples who never achieve
genetic parenthood. How does one cope when one has such an experience? What are the short- and
long-term psychological consequences of multiple IVF failures?
A recent research study should serve as a wake-up call about the long-term psychological
impact of IVF treatment, most especially in women who never achieved a pregnancy.1 In this
study of 470 Swedish women who had undergone at least one IVF cycle 20–23 years before being
assessed, all women, when compared to a reference group, were at increased risk for depression,
obsessions-compulsions, and somatization. However, the women who had not conceived had
significantly higher levels of depression and phobic anxiety. Given that their IVF failure(s) had
happened more than two decades previously, it is alarming that they were still experiencing
such profound psychological distress. In a similar study, but with follow-up four to five years
after IVF treatment, individuals who had experienced IVF failure were compared to those who
had succeeded, versus a control group that had not undergone IVF treatment. 2 Women who had
experienced IVF failure had higher levels of both depression and anxiety, as well as a lower sense
of coherence than women in the successful group. They also reported more depression and less
coherence than the control group.
We know that women who experience IVF failure report high levels of emotional distress. The
purpose of this chapter is to describe what we know about the impact on women who experience
recurrent implantation failure.
THE PSYCHOLOGICAL IMPACT OF RECURRENT FAILURE

Unfortunately, recurrent implantation failure (RIF) has not caught the attention of mental health
researchers in the reproductive medicine field. A PubMed search including the words “recurrent
implantation failure” with “depression,” “anxiety,” or “distress” turned up zero references. Another
search on “post IVF syndrome” also did not turn up any studies on psychological distress as a fac-
tor. A recent study might explain the paucity in the field. In a recent prospective cohort study of 174
women undergoing IVF, participants were assessed for major depressive disorder (MDD) repeat-
edly over an 18-month study period, and 39% of women met the criteria for MDD on at least one
time point.3 Although the mean number of failed treatment cycles was 2.5, there was no association
between the number of failed cycles and the development of MDD. The only variable that predicted
the development of depressive symptoms was a past history of MDD. Thus, it was hypothesized
9
10 The psychological consequences of recurrent implantation failure
that it is the infertility experience itself, especially in women with a prior history of depression, that
leads to depressive symptoms, not the experience of multiple failed cycles.
However, there was one study that did assess the psychological status of women who had expe-
rienced RIF with women who had experienced recurrent pregnancy loss (RPL), comparing both
groups to fertile controls.4 As expected, both the RIF and RPL participants reported significantly
higher levels of psychological distress than the control group. The only significant difference
between the RPL and RIF patients was that the perceived stress score of the RPL group was higher
than that of the controls, and that of the RIF patients was not.
In a study which examined the impact of IVF failure on emotional status, 64 couples who had
experienced at least one failed cycle were compared to 56 couples who had not yet had an unsuc-
cessful cycle.5 The two groups did not differ with respect to anxiety and depression scores.
IVF FAILURE = EMOTIONAL DISTRESS?

Although there have been millions of women and men who have undergone unsuccessful IVF
treatment, and there are 1333 citations in PubMed on “IVF failure,” there are no citations on mea-
sures of depression or anxiety and only three on stress. However, the heightened levels of depres-
sion and anxiety of individuals undergoing IVF is well known. In one of the larger studies on
the prevalence of distress in individuals undergoing infertility treatment, 56.5% of women and
32.1% of men reported clinical levels of depressive symptoms and 75.9% of women and 60.6% of
men reported clinical anxiety symptoms.6 The majority of other recent research on the psychologi-
cal profiles of infertility patients shows similar findings—infertility patients routinely experience
symptoms of anxiety and depression.
It is understandable that IVF failure commonly leads to intensive feelings of sadness and loss
of hope. In a survey of 66 women who had recently experienced at least one IVF failure, the most
frequently reported grief responses were bargaining, acceptance, depression, anger, denial, and iso-
lation.7 In a study on 372 Chinese women who had at least one unsuccessful IVF cycle, participants
scored significantly higher on assessments of depression after the failed cycle compared to levels
prior to treatment.8 What is alarming about this study is that 13% of the participants reported ideas
about self-harm. In fact, self-harm and suicidal ideation are not uncommon in infertile women.
In a recent study on the prevalence of suicidal risk, a total of 9.4% of women receiving infertility
treatment reported suicidal ideation and/or attempts.9 In a small study of 21 couples who had expe-
rienced unsuccessful IVF treatment who were compared to 20 successful couples, the unsuccessful
couples reported a lower quality of life, with the women reporting the lowest scores.10
There have been a number of studies that assessed the emotional status of women who stopped
treatment, although the reason for the failure was not cited. In one of the earliest studies on 40
couples with an average length of follow-up of 1.5 years, although 60% of the women reported
symptoms of depression, the severity of the symptoms did decrease over time.11 However, 13% of
the women had thought about suicide because of their inability to have children.
In another follow-up study of IVF treatment failure, 76 women were assessed four to nine years
after treatment.12 In comparison to social norms, the 52 women who never conceived or adopted
reported significantly higher levels of stress but also higher self-esteem. When the successful IVF
patients were compared to the unsuccessful ones however, the unsuccessful ones reported them-
selves as significantly more stressed, more depressed, and with lower levels of life satisfaction and
self-esteem.
There are a number of factors that can have an impact on emotional recovery after unsuccessful
treatment. In a study of 187 women who were assessed prior to treatment, the women who reported
the most negative psychological symptoms after failed treatment had the highest neuroticism
scores pretreatment.13 Other factors associated with more distress were feelings of helplessness and
marital dissatisfaction, while social support and acceptance of the situation were associated with
less distress. In another study of 184 women who had failed IVF treatment, immediately after the
last failed cycle, marital quality was positively related to depression while resilience was inversely
Psychological interventions to reduce distress 11
correlated.14 During the follow-up period, however, marital quality became negatively correlated
with depressive symptoms. The authors concluded that marital quality may allow patients to expe-
rience the psychological consequences of failure while resilience can tamper distress. Longer term,
however, marital quality can enhance the psychological recovery.
Making the decision to end treatment is a complicated process for many individuals and couples.
For those without insurance, the main reason for treatment termination is financial, but for those
with insurance or significant resources, it can be a challenging decision. In a study of 25 women
who decided to end treatment, many reported that they had had unrealistic expectations of the pos-
sibilities of success.15 The main benefit of deciding to stop treatment was the end of the emotional
pain of IVF, but many of the women found it challenging to confront issues that they had previ-
ously not been able to contemplate. Interestingly, the women who adopted perceived less pressure
from others about their decision than those who remained childless. In another study of women
who experienced treatment failure and were followed for three to five years, those who didn’t con-
ceive but did focus on new life goals had lower levels of depression and anxiety than those who
continued to try to conceive.16
A review, published in 2002, proposed a theory to explain why women may not express signifi-
cantly more distress after multiple failed cycles when compared to one.17 It is possible that when an
individual is exposed to multiple failures, it forces them to truly confront their infertility and move
into acceptance.
PSYCHOLOGICAL INTERVENTIONS TO REDUCE DISTRESS

There are a number of meta-analyses that examined the impact of various psychological interven-
tions on distress levels of infertile women, but none specifically for those who have experienced
RIF. In addition, the results of the different analyses don’t necessarily agree with each other, mostly
because of the studies selected for inclusion.
In the first meta-analysis on this topic, 25 studies were included.18 The conclusions were that there
were no differences in efficacy between men and women, psychological interventions were not asso-
ciated with improvements in pregnancy rates but were for psychological symptoms, s kills-based
interventions were the most effective, and group interventions were better than individual.
Another review published six years later included only 21 studies and had different conclusions.19
These conclusions were that psychological interventions were not correlated to less distress and that
non-ART (assisted reproductive technology) patients who received an intervention did experience
higher pregnancy rates. In addition, the interventions of six or more sessions were more effective
than shorter ones.
A 2016 review included 20 randomized trials and concluded that there were significant meth-
odological issues with the studies that had significant findings for psychological improvement
or increased pregnancy rates.20 The authors strongly encouraged more research on interventions
aimed during the waiting period between transfer and pregnancy test. A Cochrane review, pub-
lished in 2016, also reported on methodological limitations in the research in this area.21
However, the largest review to date had more definitive conclusions.22 This review included 39
studies on 2746 men and women. Eligible studies assessed the impact of various psychological
interventions on pregnancy rates and/or psychological symptoms including anxiety, depression,
stress, and marital function. The conclusions were that there were significant impacts of psycho-
logical interventions on both psychological symptoms and pregnancy rates. The effect size for
pregnancy was large (p < 0.001). The effect sizes were larger for women than men and the high-
est pregnancy rates were associated with the greatest decreases in anxiety. The authors reported
that there were no obvious differences between cognitive behavioral therapy (CBT), mind/body
interventions, and other intervention types but, in their conclusions, noted that the most effective
intervention included instruction in CBT.
Finally, a recent small review had similar findings, including the association of psychological
interventions with less distress and higher pregnancy rates.23
12 The psychological consequences of recurrent implantation failure
Unfortunately, the research on psychological interventions with infertility patients has focused
on the treatment and waiting phases, rather than on women who had stopped treatment due to lack
of success. It is entirely plausible that any of these interventions, perhaps most likely CBT, could
lessen the short- and long-term suffering of women who experienced treatment failure. Given that
past research has indicated that women who move ahead after infertility by focusing on different
life goals have more positive psychological outcomes, future research should focus on interventions
that encourage such reframing.
ALTERNATIVE FORMS OF INTERVENTIONS

It can be challenging to recruit infertile women to attend on-site interventions, either for research
purposes or in a clinical setting. Although insured infertile women who drop out of treatment
report that they desire stress-reduction interventions, the vast majority of women, either during
treatment or after treatment failure, do not participate in such interventions.24 Thus, the possibility
of more advanced technological interventions has been proposed. In a pilot study on the efficacy of
an online mind/body intervention, participants who were randomized to the intervention group
reported significantly less psychological distress and higher pregnancy rates than women who were
randomized to the control group.25 A new app, FertiCalm, which is designed to apply cognitive
behavioral and relaxation techniques to women experiencing infertility, has one section devoted
exclusively to coping after treatment failure. However, there are no data yet on the efficacy of any
apps on improving the psychological status of women who use them.
THE PSYCHOLOGICAL IMPLICATIONS OF RECURRENT IMPLANTATION FAILURE

Although the vast majority of research studies on the psychological consequences of IVF failure
do not differentiate between recurrent implantation failure versus other causes, it is most plausible
that the psychological impact of unsuccessful treatment is fairly universal. Unsuccessful treatment
is associated with significant distress, and in some women, this distress can be manifested with
suicidal thoughts, with most women expressing symptoms of anxiety, low self-esteem, and depres-
sion.26 However, most women do adapt over time. Unfortunately, a subset continues to report
symptoms for decades.
It is clear that research in this field is inadequate; although multiple studies confirm the long-
lasting psychological consequences of treatment failure, there are no intervention studies that aim
at decreasing this distress long after treatment is terminated. Since many women appear to be loath
to physically attend any kind of intervention designed to decrease distress, more innovative forms
of therapy must be assessed. Online interventions and apps need to be subjected to randomized
controlled trials in an effort to determine the most effective way to improve the psychological status
of women who fail high-tech treatment, both for short- and long-term efficacy. Women should not
have to suffer for decades after failing to conceive.
REFERENCES
1. Vikstrom J, Josefsson A, Bladh M, Sydsio G. Mental health in women 20–23 years after IVF
treatment: A Swedish cross-sectional study. BMJ Open. 2015;5(10):e009426.
2. Johansson M, Adolfsson A, Berg M et al. Gender perspective on quality of life, compari-
sons between groups 4–5.5 years after unsuccessful or successful IVF treatment. Acta Obstet
Gynecol Scand. 2010;89:683–91.
3. Holley SR, Pasch LA, Bleil ME et al. Prevalence and predictors of major depressive disorder
for fertility treatment patients and their partners. Fertil Steril. 2015;103:1332–9.
4. Coughlan C, Walters S, Ledger W, Li TC. A comparison of psychological stress among women
with and without reproductive failure. Int J Gynaecol Obstet. 2014;124:143–7.
5. Karaca N, Karabulut A, Ozkan S et al. Effect of IVF failure on quality of life and emotional
status in infertile couples. Eur J Gynecol Reprod Biol. 2016;206:158–63.
References 13
6. Pasch LA, Holley SR, Bleil ME et al. Addressing the needs of fertility treatment patients and
their partners: Are they informed of and do they receive mental health services? Fertil Steril.
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7. Lee SH, Wang SC, Kuo CP et al. Grief responses and coping strategies among infertile women
after failed in vitro fertilization treatment. Scand J Caring Sci. 2010;3:507–13.
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undergoing treatment with assisted reproductive technology and the impact of treatment fail-
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9. Shani C, Yelena S, Reut BK et al. Suicidal risk among infertile women undergoing in-vitro
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and GIFT treatment. Patient Educ Couns. 1997;31:7–18.
11. Baram D, Tourletot E, Muechler J, Huang KE. Psychological adjustment following unsuccess-
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12. Bryson CA, Sykes DH, Traub AI. In vitro fertilization: A long-term follow-up after treatment
failure. Hum Fertil. 2000;3:214–20.
13. Verhaak CM, Smeenk JM, Evers AW et al. Predicting emotional response to unsuccessful
fertility treatment: A prospective study. J Behav Med. 2005;28:181–90.
14. Chochovski J, Moss SA, Charman DP. Recovery after unsuccessful in vitro fertilization:
The complex role of resilience and marital relationships. J Psychosom Obstet Gynaecol.
2013;34:122–8.
15. Peddie VL, van Teijingen E, Bhattacharya S. A qualitative study of women’s decision-making
at the end of IVF treatment. Hum Reprod. 2005;20:1944–51.
16. Verhaak CM, Smeenk JM, Nahuis MJ et al. Long-term psychological adjustment to IVF/ICSI
treatment in women. Hum Reprod. 2007;22:305–8.
17. Bryson CA, Traub AI. Post IVF syndrome? Psychological implications of failed IVF. The Obs
Gyn. 2002;4:201–4.
18. Boivin J. A review of psychosocial interventions in infertility. Soc Sco Med. 2003;57:2325–41.
19. Hammerli K, Znoj H, Barth J. The efficacy of psychological interventions for infertile
patients: A meta-analysis examining mental health and pregnancy rate. Hum Reprod Update.
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20. Ying L, Wu LH, Loke AY. The effects of psychosocial interventions on the mental health,
pregnancy rates and marital function of infertile couples undergoing in vitro fertilization: A
systematic review. J Assist Reprod Genet. 2016;33(6):689–701.
21. Verkujilen J, Verhaak C, Nelen WL et al. Psychological interventions and education interven-
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22. Frederiksen Y, Farver-Vestergaard I, Skovgard NG et al. Efficacy of psychosocial interventions
for psychological and pregnancy outcomes in infertile women and men: A systematic review
and meta-analysis. BMJ Open. 2015;5(1):e006592.
23. Chow KM, Cheung MC, Cheung IK. Psychosocial interventions for infertile couples: A criti-
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24. Domar AD, Smith K, Conboy L et al. A prospective investigation into the reasons why
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atic review of 25 years of research. Hum Reprod Update. 2007;13:27–36.
The role of lifestyle factors in
recurrent implantation failure
3
VICKI NISENBLAT and ROBERT J. NORMAN
INTRODUCTION
Implantation is a remarkably complex, highly organized process that relies on precise coordination
between the embryo and the host endometrium. Although over the last decade our understanding
of the implantation process has improved dramatically, many unanswered questions remain, and
we still have little knowledge about the molecular mechanisms involved in endometrial receptivity
and in regulation of embryo-endometrium crosstalk.1
Recurrent implantation failure (RIF) is referred to a clinical condition when implantation fails
repeatedly despite several transfer cycles of good-quality embryos.2 Multiple etiological factors of
RIF have been described, of which suboptimal embryonic capacity and reduced endometrial recep-
tivity are thought to be the key determinants. In a substantial proportion of couples, however, the
exact cause of RIF is not identified, as we do not have universally accepted markers that accurately
assess embryo competence and receptive status of the endometrium.3 Limited understanding of the
cellular events underlying implantation failure results in lack of effective therapeutic and preven-
tive strategies, which is frustrating for both patients and health-care providers.
There is an increasing concern that modern lifestyle and changes within the environment influ-
ence reproductive function of the couple and can have a detrimental effect on pregnancy out-
comes.4–7 Although the couples undergoing assisted reproductive technology (ART) treatments
are thought to be more conscious of their health and are less likely to engage in negative lifestyle
behaviors, a survey of more than 12,000 ART patients in United States revealed that during fertility
treatment, up to 23% of patients drank alcohol, up to 7% smoked, 68% consumed caffeine, and less
than 1% used recreational drugs.8
Both oocytes and sperm are susceptible to the environmental influences, which can subse-
quently affect embryo development.9,10 Nutrition and other factors originating from the environ-
ment can adversely influence maternal health, the intrauterine milieu, and, possibly, maternal
response to the implanting embryo.11 It has been recently proposed that women with RIF have a
different metabolic status compared with women who have highly receptive endometria.12 RIF has
been associated with higher levels of serum glucose and altered levels of the metabolites related
to energy, amino acid, and lipid metabolism, suggesting a possibility of metabolic dysfunction.12
At the endometrial level, impaired glucose uptake has been implicated in abnormal decidualiza-
tion, poor implantation, and pregnancy loss,13 whereas normal glucose metabolism was associated
with successful decidualization.14 Certain lifestyle factors such as smoking, alcohol, and obesity
are known to induce chronic oxidative stress, which has been extensively linked with reproductive
dysfunction on oocyte, tubal, and endometrial levels.15 Elevated levels of adipic acid, a by-product
metabolite of reactive oxygen species (ROS) has been detected in the circulation of women with
RIF, indicating that women with RIF have an increased oxidative stress state, and the condition can
be possibly linked with environmentally induced oxidative damage.12
Taken together, it is not unreasonable to assume that lifestyle, environmental, and occupational
factors contribute to RIF. Currently, however, there is no clear evidence on association between
lifestyle or environmental factors and RIF, with the ART outcomes and recurrent pregnancy loss
14
Obesity 15
being a primary focus in most studies. Although repeat in vitro fertilization (IVF) failure, recurrent
miscarriage, and RIF represent distinct clinical entities, their underlying causes overlap, and sup-
posedly these conditions have a similar link to the environmental exposures.
This chapter provides an up-to-date overview of the lifestyle, occupational, and environmental
factors known to adversely affect reproductive function and discusses their possible contribution to
RIF. The evaluated factors include obesity, caffeine intake, smoking, alcohol consumption, psycho-
logical stress, periodontal disease, and endocrine-disruptive chemicals as environmental pollutants.
OBESITY
Obesity is widely recognized as a global epidemic affecting around third of women and men of
reproductive age.16 The clinical impact of obesity on reproduction, pregnancy outcomes and health
of the mother and offspring have been well characterized.
Obesity increases the rate of anovulation, has a pronounced negative effect on endocrine and fer-
tility parameters in women with polycystic ovarian syndrome (PCOS), and decreases the probabil-
ity of spontaneous conception in the absence of ovulatory dysfunction.17,18 In women undergoing
ART treatments, obesity has been associated with decreased ovarian response, higher rate of cycle
cancellations, lower oocyte yield, larger number of immature oocytes, impaired fertilization rate,
and poor embryo development.19 The analysis of 239,126 fresh IVF cycles revealed that increased
body mass index (BMI) was associated with worsening of implantation, pregnancy, and live birth
rates.20 Probability of live birth is inversely related to severity of obesity, with a 50% decrease in the
odds for live birth in women with BMI >40 kg/m2.21
Compelling evidence links obesity with a risk of miscarriage.20,22–25 A recent umbrella review of
systematic reviews and meta-analyses on the association between obesity and any type of obstet-
ric or gynecological morbidity demonstrated highly suggestive evidence for a negative association
between BMI >25 kg/m2 and increased risk of miscarriage among IVF patients.22 A systematic
review of 30 studies encompassing 115,158 participants concluded that paternal obesity negatively
affects fertility and is associated with reduced live birth and increased pregnancy loss in ART
cycles.26 Obese individuals don’t seem to have an increased rate of aneuploid embryos27 and have a
high frequency of euploid miscarriage,23,25 implying that the negative effect of obesity on reproduc-
tive outcomes relies on factors other than embryo aneuploidy.
Obesity-related hyperinsulinemia stimulates production of ovarian androgens that aromatize
to estrogen in adipose tissue and lead to negative feedback on the hypothalamic-pituitary-ovarian
axis. Ovarian steroidogenesis is also affected by leptin, a cell-signaling protein produced in adipose
tissue, acting on the level of the hypothalamus.19
Raised inflammatory markers, lipids, and insulin have been observed in the follicular fluid of
obese women.28 In animal models, obesity has been linked with oocyte mitochondrial abnormali-
ties reflective of metabolic stress, derangements in the meiotic spindle, abnormal transcriptional
activity in cumulus-oocyte complexes, and increased apoptosis of granulosa cells.19 In addition,
the negative influence of obesity on oocyte competence and on the preimplantation embryo is
mediated by leptin-induced apoptosis and lipotoxicity. Lipotoxicity is manifested by accumula-
tion of excess fatty acids outside adipocytes with subsequent toxic damage to the surrounding cells
through increase in oxidative stress, activation of the apoptosis cascade, and induction of inflam-
matory pathways.19
In males, increased BMI has been associated with a decrease in androgens, an increase in estra-
diol due to aromatization of testosterone in adipose tissue, and an inverse correlation with semen
parameters.26,29,30 Impaired steroidogenesis, oxidative stress, insulin resistance, apoptosis, and
inflammation are thought to mediate the adverse effects on sperm parameters and DNA integrity.31
The data from animal and human in vitro experiments demonstrate that obesity is implicated in
endometrial decidualization defects and possibly affects endometrial receptivity.32 The exact mech-
anism is unclear, but it has been proposed that leptin may be involved in endometrial derangements
by modulating cellular signaling involved in proliferation and apoptosis.33 Furthermore, obesity
16 The role of lifestyle factors in recurrent implantation failure
induces low-grade systemic inflammation, which in turn can negatively influence implantation
and embryo development. The studies on oocyte donation with normal-weight donors that isolate
the effect of embryo on implantation and hence reflect the effect of obesity on endometrial milieu
are equivocal. A systematic review of five studies encompassing 4758 women reported no difference
in implantation rate between obese recipients and controls with normal BMI.34 In contrast, a more
recent cohort analysis of 9587 oocyte donation cycles demonstrated significantly reduced implan-
tation, clinical pregnancy, and live birth rate in obese recipients.24
Despite obvious deleterious effects of obesity on a wide range of reproductive outcomes indepen-
dent of methods of conception, there has been very little exploration of the role of obesity in RIF.
Theorizing that gametes and embryos from obese individuals are subject to alterations, coupled
with clear evidence on increased rate of euploid miscarriages, it can be supposed that obesity may
contribute to RIF, although there are no data to support a direct association.
Weight-reduction strategies include lifestyle changes through diet and exercise as a first-line
option and additional interventions such as medical therapies and bariatric surgery. To date, most
studies that have evaluated weight-loss interventions in a context of fertility were significantly
underpowered with high dropout rate and generally achieved modest weight loss.35 Weight reduc-
tion has been positively associated with improved endocrine parameters, increased rate of ovula-
tion, and higher likelihood of spontaneous pregnancy in anovulatory women, although the results
with regard to the effect of interventions on live birth or miscarriage rate are largely disappoint-
ing.36,37 It also remains unclear what degree of weight loss improves reproductive performance and
what is the optimal timing and type of intervention.36 The interventional meta-analysis concluded
that the existing studies on preconception weight-loss interventions failed to demonstrate improve-
ment in the outcomes of fertility treatments.36 More work is clearly required for firm conclusions
to be drawn as to whether fertility-related outcomes are amenable to risk-reduction interventions.
Guided by the evidence on multiple health benefits of normal weight, and considering repro-
ductive challenges and obstetrics complications of obesity, multiple societies advocate that obese
individuals should be engaged in weight-loss programs before attempting conception.38–40
CAFFEINE
Caffeine is present in multiple food products and beverages and is naturally occurring in many
plants. The main sources of caffeine include coffee, tea, chocolate, and soft or “energy” drinks.
Caffeine is consumed by up to 90% of reproductive-aged women with 7%–18% consuming above
300 mg/day.41 Caffeine has been associated with a negative effect on fertility and pregnancy,
although the data are inconsistent, and the findings of most studies are hampered by serious
methodological flaws.5–7,41 A meta-analysis of 60 publications demonstrated a modest association
between caffeine intake (within the range of intake currently recommended in most countries) and
adverse reproductive outcomes.42 Likewise, there was no clear negative association between caf-
feine and sperm parameters or sperm DNA damage.43 The umbrella review of 201 meta-analyses
of observational studies and 17 meta-analyses of interventions concluded that coffee intake within
usual levels is generally safe, except for the relationship of high consumption during pregnancy
with pregnancy loss and low birthweight.44 The negative effect of increased caffeine consumption
on pregnancy loss was suggested in a more recent systematic review of 35 studies on dose-response
association between caffeine and reproductive events, with significant risk observed for a caffeine
dose of 300 mg/day and above.45
The relationship between caffeine and ART outcomes is unconvincing. In high coffee and tea
consumers, caffeine was detected in follicular fluid and was associated with reduced number of
oocytes and good-quality embryos but did not affect live birth rate.46 One study in 221 IVF couples
showed that caffeine consumption in women led to reduced live births and an increased chance
of miscarriage in a dose-dependent manner, although a negative effect on IVF endpoints were not
observed with male caffeine intake.47 These findings were not confirmed by others, and there are no
publications on caffeine consumption in couples with RIF.44,45
Alcohol 17
The proposed biological mechanisms by which caffeine could influence reproductive events
involve alterations in secretion and metabolism of reproductive hormones and disruption of ovar-
ian function.45 Increased levels of circulating catecholamines, homocysteine, cholesterol, and cel-
lular c-AMP have also been reported, although the magnitude of these changes with the typically
consumed caffeine doses and their direct effect on the embryo or endometrium are unclear.45
The evidence linking preconception caffeine restriction with reproductive outcomes is inade-
quate, and there are no evidence-based recommendations for caffeine intake.48 Most guidelines
recommend that women who are pregnant or trying to become pregnant limit caffeine consump-
tion to less than 200–300 mg per day, and there is no evidence to suggest caffeine avoidance.38–41
SMOKING
The epidemiological data indicate that about 2%–6% of women undergoing ART smoke, and a large
proportion of the nonsmokers are exposed to second-hand smoke.8,49 Smoking has been repeat-
edly associated with decreased fertility and reduced success rate of ART cycles.5–7,49 Several meta-
analyses reported that clinical pregnancy and live birth rate were reduced by approximately half
in smokers compared with nonsmokers, with about a three-fold increase in miscarriage risk.50,51
A growing body of literature suggests a detrimental effect of second-hand tobacco smoke expo-
sure on fertility and IVF outcomes, which is similar to that observed in smokers.52 Cotinine, a
metabolite of nicotine, was detected in follicular fluid of nonsmoking women who reported
second-hand exposure to smoke and was associated with 52% higher risk of implantation failure
and a 24% reduction in live births.53 Likewise, a reduction greater than 50% in live birth rate was
demonstrated among IVF couples in which only male partners smoked, and this effect was only
mildly improved with intracytosplasmic sperm injection (ICSI).54 The influence of smoking on
uterine receptivity was demonstrated in oocyte donation cycles, where the recipients who smoked
had a lower chance to conceive proportionally to the number of cigarettes smoked.55 Both active
and passive smoking were reported in association with reduced implantation and increased risk of
implantation failure.52 Despite compelling evidence that smoking has a negative effect on fertility
and implantation, the extent to which smoking contributes to RIF is less clear.
Smoking is thought to exert its negative effect through the heavy metals, polycyclic hydrocarbons,
nitrosamines, and aromatic amines. Impaired folliculogenesis, altered steroidogenesis, oocyte tox-
icity, and increased oxidative stress are among the proposed mechanisms of the deleterious effects
of smoking on IVF outcomes.4,6 Cigarette smoking has been associated with factors that can poten-
tially affect embryo development and implantation, including impaired oocyte maturation, sperm
DNA damage, delayed embryo cleavage events, increased risk of embryonic aneuploidy, and altered
embryo gene expression.4,6,10 The data from animal models and endometrial cell lines indicate that
dose-dependent exposure to cigarette smoke directly affects the endometrium via altered angio-
genesis, antiestrogenic effects, dysregulated endometrial gene expression, increased apoptosis, and
aberrations of various growth factors.56
Smoking cessation in both partners is considered an important strategy to improve fertility and
is a universal recommendation for the couples presenting for preconception or fertility counseling.
Current evidence indicates that reproductive risks associated with smoking are reversible within
up to one year of smoking cessation, although the uptake of smoking reduction strategies is dis-
appointedly low.57,58 The data on smoking reduction strategies in couples who attempt concep-
tion through ART are sparse, and the effect of these interventions on reproductive outcomes and
broader lifestyle changes remains to be established.
ALCOHOL
Alcohol is one of the most frequently abused substances in the Western world. Epidemiological
reports demonstrate that around 25% of couples planning a pregnancy consume alcohol at poten-
tially unsafe levels and a large proportion of the population reports mild to moderate intake.5
Numerous studies have documented the negative effect of male and female alcohol consumption
on reduced fertility, increased risk of spontaneous miscarriage, and adverse neonatal outcomes.5–7
The safe level of consumption associated with reproductive risk is unclear, and the most suscep-
tible window of exposure has not been identified. Some investigators have reported that alcohol
consumption levels as low as one drink per week reduced the chance for conception, but this has
not been confirmed by others.5,59 A large, prospective, observational study in 27,580 pregnancies
reported no association between low to moderate alcohol intake pre-pregnancy (<12 g/d) and early
or late pregnancy loss, irrespective of maternal age, BMI, and smoking status.60 A systematic review
of 15 observational studies in a total of 16,395 participants demonstrated that semen volume and
normal morphology were affected by occasional alcohol consumption in fertile or infertile men, but
daily drinking had a definite adverse effect versus both occasional and never alcohol consumers.61
Among the couples participating in IVF programs, up to 45% of women and 67% of men reported
alcohol consumption during their treatment, of which 4% women and 9% men drank daily.62 The
direct effect of alcohol on IVF is less clear, and there is conflicting evidence on the impact of alcohol
intake on treatment outcome.5,59 Alcohol drinkers had lower numbers of oocytes retrieved, lower
fertilization rates, abnormal blastocyst development, and lower implantation rates.62 Consumption
of four or more drinks per week was associated with 16% lower chance for live birth following IVF,
with a more prominent effect (21% reduction in live birth) if both partners drank.62 Conversely,
several studies failed to confirm a negative association between alcohol and live birth rate in IVF
cohorts.59,63
The exact mechanism is unclear, although chronic alcohol consumption is thought to affect
feedback regulation of the hypothalamic-pituitary-gonadal axis by increasing circulating estrogen
levels, with subsequent consequences for folliculogenesis and oocyte maturation. Alcohol has been
shown to affect sperm parameters in some, but not other, studies59,61 and has been associated with
increased sperm DNA damage and deranged male reproductive hormones.64
In animal models, excess alcohol exposure resulted in amenorrhea, uterine atrophy, decreased
ovarian weights, and suppression of luteinizing hormone (LH) levels.6 Ethanol-exposed male ani-
mals had reduced testicular volume, lower testosterone, gamete abnormalities, and decreased litter
size.6,7 Oxidative damage has been observed in both the testes and epididymides of the exposed ani-
mals.65 The adverse effects on implantation were manifested by production of aneuploid embryos,
delayed attachment of blastocysts, absence of the decidual reaction, and resynchronization of the
implantation process.6,7
Based on the evidence derived from animal studies, there is biological plausibility to support an
adverse effect of alcohol on implantation. However, little is known on the effect of alcohol intake on
implantation defects in humans, and the association of male or female alcohol consumption with
RIF is unclear.
Excessive alcohol consumption is linked with higher rates of mental-health disorders and poly-
substance abuse, while consumption during pregnancy may lead to serious health problems in the
unborn child. Most guidelines recommend a cautious approach to alcohol consumption in couples
planning pregnancy, which includes avoiding binge drinking, limiting number of weekly standard
drinks, or complete abstinence for both partners.38–40 The therapeutic strategies to reduce alcohol
intake include counseling, psychosocial intervention, and pharmacological therapy, although there
are limited data on the effectiveness of preconception interventions and their impact on reproduc-
tive outcomes.66,67
PSYCHOLOGICAL STRESS
Infertility is a stressful experience. Fertility treatments are commonly associated with significant
stress for the couple, which can be further exacerbated by underlying anxiety, or a range of societal,
energetic, and psychological stressors. Overall, one-fifth of the couples undergoing ART have clini-
cally significant psychological distress or anxiety.68 Failed treatments result in higher psychological
burden, which can lead to treatment discontinuation or higher levels of depression and anxiety in
subsequent pregnancy. Women experiencing RIF had increased stress levels compared to fertile
Periodontal disease 19
controls, similarly to those in women with recurrent pregnancy loss, but the investigators were not
able to demonstrate whether increased stress was a cause or consequence of reproductive failure.69
Increased stress levels were associated with deranged levels of reproductive hormones and
anovulation,70 while modified behavior led to restored ovulation.71 Long working hours and mov-
ing of heavy loads were also linked with increased time to conceive, with a more pronounced nega-
tive effect in overweight or obese women.72 The literature on the effect of stress on pregnancy loss is
conflicting, and there is no conclusive evidence regarding the effect of stress on IVF outcomes.69,73
One systematic review of 43 studies found a correlation between psychological stress and negative
IVF outcomes, although the authors reported substantial inter-study heterogeneity with respect to
the outcome measures and the tools of stress measurement.7 A more recent meta-analysis concluded
that pretreatment emotional distress in women undergoing ART did not compromise treatment
outcomes.74 Similarly, The Cochrane Library systematic review evaluated 39 studies comprising
4925 participants and reported uncertainty regarding the benefits of psychological or educational
interventions on mental health or reproductive outcomes in subfebrile couples, mainly due to the
overall low-quality evidence.75
Stress may affect reproductive function through a corticotrophin releasing hormone (CRH)-
induced effect on the hypothalamic-pituitary-gonadal axis, and this can be further modified by
other stress-related mediators (namely, glucocorticoids, opioid peptides, and catecholamines).4,73 It
is thought that deleterious effect of stress is mediated by cortisol and prolactin, although the exact
neuroendocrine mechanism is unclear.76 In women subjected to stress, urine cortisol levels nega-
tively correlate with the levels of luteal progesterone, which could subsequently interfere with endo-
metrial decidualization.70 Stress mediators may also alter cytokine expression in the endometrium
and increase activity of uterine natural killer (NK) cells, which could contribute to impaired endo-
metrial receptivity.73 Detection of adrenergic receptors in mice preimplantation embryos suggests
that stress-related release of maternal catecholamine could affect early embryonic development
and influence embryo implantation.77 Despite a plausible pathophysiological mechanism, little is
known about the effect of stress and stress-relieving therapy on RIF. There are little epidemiologi-
cal data, and no human studies have yet been performed to assess the effect of stress hormones on
embryo developmental competence and implantation.
Considering broad social impact of emotional distress and its known association with discon-
tinuation of fertility treatments, support and appropriate counseling should be available for every
couple undergoing fertility treatments, irrespective of their treatment outcomes. Until new evi-
dence emerges, there are no recommended psychological interventions specifically targeted to the
couples with RIF.
PERIODONTAL DISEASE
The relationship between periodontal disease and its systemic sequalae has been an area of research
interest over the past two decades.78 Periodontal disease affects around 10% of the population, with
a higher prevalence in smokers and low-income groups. It has been suggested that periodontal dis-
ease may have a negative impact on fecundity and time to conception, and it is linked with increased
risk of preterm birth, low birth weight, and miscarriage by most but not all of the studies.79,80
The hypothesized connection between periodontal disease and adverse reproductive outcomes
posits that oral bacteria cause systemic bacteremia that initiates an inflammatory cascade with
local and systemic production of cytokines, which can lead to intrauterine inflammation, reduced
embryo implantation or placental infection.79 Local and systemic oxidative stress have been impli-
cated in the pathogenesis of periodontitis and may compromise intrauterine milieu.81
Interventional randomized controlled trials and systematic reviews of these studies have reached
mixed conclusions with regard to effect of periodontal treatment on reproductive outcomes, mainly
due to multiple confounders and the multifactorial nature of the condition.82 It remains unclear
what subgroups of women are the most responsive to treatment, which treatment works the best,
and when is the most optimal time for intervention to be effective. Even though periodontal disease
has not been specifically evaluated in couples with RIF, in view of long-term health benefits, oppor-
tunistic counseling regarding oral health should be offered to the patients in every care setting.
ENDOCRINE DISRUPTIVE CHEMICALS

There is growing awareness that multiple chemicals within the environment interfere with hor-
mone action and may affect reproductive function and fertility. Environmental endocrine disrupt-
ing chemicals (EDCs) have been detected in hair or biological fluids of a large proportion of the
population worldwide.83
EDCs may exert their effect on reproduction through binding to hormone receptors, by altering
cytochrome P450 activity, by modulating estrogen signaling, or by interfering with thyroid func-
tion.83,84 EDCs have been also shown to affect mitochondrial function, promote oxidative stress, and
induce apoptosis. It has been suggested that some of these compounds may have transgenerational
influences through epigenetic modifications by inducing DNA methylation, histone modification and
dysregulation of noncoding RNAs. More than 800 potentially hazardous chemicals have been iden-
tified, of which bisphenol A (BPA), the phthalates, certain pesticides, the polychlorinated biphenyls
(PCBs), and heavy metals are the most extensively evaluated in the context of reproductive toxicity.4
BPA and phthalates

BPA and phthalates are abundant synthetic compounds widely present in various household prod-
ucts, cosmetics, plastics, packaging, and pharmaceuticals. Exposure occurs through foods from
plastic containers or via transdermal contact.
BPA exposure has been linked with a higher prevalence of PCOS, endometriosis, recurrent mis-
carriage, and infertility.85,86 Increased concentrations of BPAs in blood or urine in women under-
going IVF were associated with a lower number of antral follicles and mature oocytes, decreased
fertilization, and deranged implantation.83 Likewise, increased peripheral levels of phthalates have
been associated with implantation failure and adverse IVF outcomes.87 Epidemiological data on the
effect of BPA and phthalates on sperm parameters and DNA damage are equivocal, although most
studies reported dose-dependent negative associations.83 In animal models, BPA and phthalates
demonstrated adverse effects on steroidogenesis, oocyte, sperm, embryo, and endometrium, but
mechanisms underlying their reproductive toxicity in humans are unclear. It has been proposed
that BPA affects implantation via downregulation of endometrial HOXA10, abnormal expression of
endometrial tight-junction proteins, and by increasing the rate of embryo aneuploidy.88,89
Pesticides
Atrazine is the most common pesticide suspected to have an EDC effect. It is widely used in com-
mercial agriculture and remains in soil and water for a long time. The use of another once com-
monly used pesticide, dichloro-diphenyl-trichloroethane (DDT), has been banned for years in
many countries, but it persists within the environment for decades due to resistance to degrada-
tion. Unfiltered drinking water is the main source of contact, and there is a possibility for occupa-
tional exposure in individuals who work with pesticides or in pesticide-treated areas.
Exposure to pesticides has been linked with pubertal abnormalities, menstrual irregularities,
decreased fecundity, miscarriage, and risk of endometriosis in human and animal studies.4,83
Occupational exposure to certain pesticides, including DDT, is inversely associated with sperm
parameters and male reproductive hormones.83 The authors of a systematic review of the avail-
able literature concluded that DDT did not seem to affect oocyte quality, fertilization, embryo
development, or pregnancy rate in IVF patients.90 More recent data showed a significantly reduced
implantation rate in women with high follicular fluid of DDT and several other pesticides (lindane,
diazinon, and chlorpyrifos).91 The authors also found a significant inverse correlation between fol-
licular fluid concentrations of the examined pesticides (DDT, lindane, diazinon, malathion, chlor-
pyrifos, bioallethrin, pretilachor, and b-cyfluthrin) on endometrial thickness. However, the effect
of these chemicals on clinical pregnancy and live birth rate has not been evaluated.
Conclusions 21
PCBs
PCBs are organic compounds that have been widely used in electrical equipment and different
industries, are a recognized carcinogen in humans, and are currently banned in most countries.
PCBs, however, are continuously detected within the soil, water, buildings, and animal products
because of the long half-life and the ability to accumulate in adipose tissue. Therefore, both non-
occupational exposures via contaminated food and occupational exposure during the contact with
old electric equipment can still occur.92
Human and animal studies demonstrated that PCBs reduce circulating levels of thyroid hor-
mone and exhibit estrogenic and antiandrogenic properties, with a more prominent negative effect
in obese individuals due to fat tissue affinity.4,83 PCBs are inversely associated with sperm param-
eters and sperm DNA damage at any level of exposure.92
Several recent studies reported an association between PCBs and decreased implantation after
ART treatments. High concentrations of PCBs in follicular fluid from 94 women were related to
thinner endometrium, fewer retrieved oocytes, lower fertilization rate with ICSI, and decreased
implantation rate, but they did not significantly influence clinical pregnancy rate.91 The latter could
be explained by the limited sample size, which could lead to a type II error. Additional prospec-
tive valuation measured 43 PCBs in follicular fluid from 32 IVF patients and identified significant
inverse associations between levels of PCBs and all IVF endpoints, such as ovarian response, num-
ber of oocytes retrieved, endometrial thickness, oocyte fertilization, embryo quality, implanta-
tion, and live birth, after adjusting for BMI, smoking, race, and age.93 Dose-dependent decrease in
implantation rate with increased serum levels of several PCBs was also observed in 827 ART cycles
in 765 women.94
Heavy metals
Environmental heavy metals, including cadmium (Cd), mercury (Hg), lead (Pb), arsenic (As),
and copper (Cu), are derived from multiple sources, of which industrial pollution and smoking
are the most common. Heavy metals can accumulate in water or animal products and persist for
years. Current evidence supports a negative association between cadmium, lead, arsenic, and cop-
per with reduced fecundability, spontaneous miscarriage, and male infertility,95,96 but this adverse
relation was not confirmed for exposure to reasonably accepted environmental levels of these met-
als.97 Exposure to mercury in population at risk (high fish consumers and dental practitioners) has
been linked with reduced fecundability in a dose-dependent fashion, but typical environmental
exposure did not seem to alter ovulation, and it did not affect ovarian response, fertilization rate,
embryo development, or pregnancy rates in women undergoing ART.4,95 This is in agreement with
a study in rats that showed an altered estrous cycle with short-term exposure to mercury, but no
significant effects on implantation or pregnancy.98
The biological mechanism by which environmental heavy metals influence implantation and the
exact dose-response association are unclear. Heavy metals within cigarette smoke have been found
within the endometrium of women who smoke, and the findings are proportional to the magni-
tude of exposure.99 It is possible that heavy metals from the other environmental sources aggregate
within the endometrium and induce immune response or oxidative stress in a dose-dependent
manner, which is likely to disturb the endometrial environment and lead to impaired implantation
or abnormal maintenance of pregnancy. Currently, however, there is a relative paucity of informa-
tion concerning the effect of EDCs on implantation, and studies on exposure to EDCs in couples
with RIF are lacking.
CONCLUSIONS
In summary, detrimental effects on fertility and IVF success rates have been demonstrated for
obesity, smoking, and heavy alcohol consumption. It remains unclear whether and to what extent
these lifestyle factors affect implantation in couples with RIF. It can be supposed that EDCs such as
BPA, phthalates, PCB, and heavy metals adversely affect implantation and may contribute to RIF,
although the dose-effect association and the critical timing of exposure are unknown. There is no
convincing evidence to support negative effects of pesticides and heavy metals at low to moderate,
accepted environmental levels on implantation, and a possible negative impact of heavy occupa-
tional or unique geographical exposures remains to be determined. The data derived from animal
models and studies on pregnancy loss provide indirect evidence for the contribution of stress and
periodontal disease to RIF. There seems to be little association, if any, between low to moderate caf-
feine and alcohol consumption and reproductive events, including implantation failure; however
the effects of excessive caffeine intake are still unclear.
More experimental and epidemiological studies are required to estimate the magnitude of the
effect of these factors in RIF that would help to refine the counseling of the affected couples. It
would be also interesting to identify individual susceptibility factors that modify the dose-response
relationship between different environmental factors and RIF. Furthermore, it needs to be deter-
mined whether or not there exists a direct link between environmental exposures, metabolic dys-
regulation, and genetic factors that affect implantation in RIF women.
Preconception interventions for the majority of adverse lifestyle behaviors have not been assessed
in the couples seeking fertility treatments, and there are no reports on specific interventions in the
context of RIF. There is a need to evaluate the efficacy, intensity, and timing of lifestyle interven-
tions that could potentially improve a likelihood of reproductive success in different subpopula-
tions of infertile couples and in those presenting with RIF.
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Endocrine causes of recurrent
4
GESTHIMANI MINTZIORI and DIMITRIOS G. GOULIS
INTRODUCTION
Implantation is a critical step in the reproduction process and requires synchronized development
of both the embryo and the endometrium. A series of well-coordinated events are taking place
simultaneously, such that failure of one or more of these events or of their coordination results in
no further development of the embryo taking place. A spectrum of causes can result in recurrent
implantation failure (RIF), including anatomic, endometrial, endocrine, immunologic, and hema-
tologic factors.1 Similarly, a number of hormones have been implicated in the pathogenesis of RIF,
including human chorionic gonadotropin (hCG), progesterone (PR), kisspeptin, activin A, activin
B, follistatin, and periostin.2
Nuclear-located receptors for progesterone (PR-A and PR-B) and estrogen (ER-α and ER-β) as
well as non-nuclear PRs, involved in rapid progesterone responses, are expressed in the human
endometrium. Their expression varies throughout the menstrual cycle.3 Other hormonal receptors
located in the human endometrium include thyroid stimulating hormone (TSH), thyroid hormone,
and insulin receptors.4,5 Though their exact role in the human endometrium is not fully under-
stood, they underline the complex interactions between reproductive and endocrine systems.
Certain endocrine disorders have been suggested to be involved in the pathogenesis of RIF,
such as thyroid disease, vitamin D deficiency, diabetes mellitus (DM), polycystic ovary syndrome
(PCOS), and obesity. The current chapter aims to review the evidence of the association of endo-
crine disorders with RIF.
THYROID DISEASE
Thyroid diseases are common among women of reproductive age. Hypothyroidism or the presence
of thyroid autoimmunity (TAI) have been linked to adverse reproductive outcomes, either after
natural conception or in vitro fertilization (IVF). The close link between thyroid and reproduction
is underlined by the wide expression of thyroid hormone and TSH receptors (TΗRs and TSHRs,
respectively) in reproductive tissues. THRs and TSHRs are distributed in human endometrium,
granulosa, and ovarian stromal cells, and their distribution differs according to the different devel-
opmental stages of the follicle.4 It has even been suggested that the human ovarian follicle is an
independent thyroid hormone producing unit6 and that thyroid hormones may influence implan-
tation and early development of the blastocyst, through endometrial tissue proliferation and matu-
ration.4 It has been shown that thyroid hormones increase bovine blastocyst formation and embryo
quality,7 as well as having an impact on angiogenesis and immune function.4 Furthermore, TSH
increases the expression of key factors for embryo implantation, namely, the leukemia inhibitory
factor (LIF) and its receptor (LIFR) in endometrial stromal cells.8
In vitro studies of blastocyst cultures in the presence of thyroid hormone have demonstrated that
thyroid hormone influences both the blastocyst and the endometrium at the time of implantation.9
In vivo studies of methimazole-induced (MMI) hypothyroid, pregnant mice have demonstrated
that hypothyroidism may influence implantation and later adversely affect placental activity.9
In the setting of IVF/intracytoplasmic sperm injection (ICSI), ovarian stimulation (OS) leads to
increased estradiol (E2) concentrations. High E2 concentrations result to an increase in thyroid-binding
27
28 Endocrine causes of recurrent implantation failure
globulin (TBG) and this, in turn, leads to a decrease in free TH concentrations. As a result, TSH pro-
duction is enhanced.10,11 A systematic review on thyroid function before and after OS has demon-
strated that TSH concentrations may rise during or within one month after OS, above the threshold
of 2.5 mU/L suggested as the norm for the first trimester of pregnancy.12 When women undergoing
OS were studied closely, at five different time points during OS, TSH concentrations did not change
throughout OS (p = 0.066). However, this lack of significance may be due to the small sample size
(n = 42) of the aforementioned study.13 In the same study, however, TSH was increased in women who
became pregnant in comparison to their baseline concentrations (3.41 ± 1.20 vs. 2.01 ± 0.95 mIU/mL,
p = 0.001).13 This observation, if confirmed, is of major clinical significance as it would affect the man-
agement of women undergoing OS, which should include universal screening.
In women with subclinical hypothyroidism, levothyroxine (LT4) supplementation resulted in a
higher delivery rate (pooled relative risk [RR] 2.76, 95% confidence interval [CI] 1.20–6.44; p = 0.018;
I2 = 70%) and a lower miscarriage rate (pooled RR 0.45, 95% CI 0.24–0.82; p = 0.010; I2 = 26%).
Even in the case of euthyroidism, it has been suggested that thyroid hormone replacement for TSH
concentrations above 2.5 µIU/mL (high normal concentration) is a predictive factor for pregnancy
rate following intrauterine insemination (IUI).14 However, evidence of the association of clinical
pregnancy or live birth rate and high normal baseline TSH concentrations is conflicting.14,15
Women with TAI carry a higher risk regarding the reproductive outcome after IVF/ICSI, as dem-
onstrated by a meta-analysis of nine studies involving 4396 women.16–19 According to this meta-
analysis, the live birth rate (LBR) of women with TAI was lower in comparison to that of those
without TAI (odds ratio [OR] 0.73, 95% CI 0.54–0.99; p = 0.04; I2 = 41%). In another meta-analysis
of four prospective studies involving 1098 women undergoing IVF, the presence of TAI was associ-
ated with an increased risk for spontaneous miscarriage.20 In a study of 72 women with RIF, it was
shown that women with TAI had a decreased percentage of CD3+CD8+ T cytotoxicity (Tc) cells and
an increased T helper (Th)/Tc ratio compared with women without TAI.21 This is clinically impor-
tant as a lower percentage of CD3+CD8+ Tc and higher Th/Tc ratio has been associated with adverse
pregnancy outcome, including recurrent spontaneous abortion.22 Furthermore, decreased Tc cells in
women with TAI may result to an increase in the Th1/Th2, thus leading to implantation failure.21,23,24
VITAMIN D DEFICIENCY
There is increasing evidence demonstrating the role of vitamin D in female fertility and preg-
nancy.25 Vitamin D may also play a role in implantation, as there is evidence on its role as an
immune modulator,26 and vitamin D metabolizing enzymes are present in human endometrium.25
Vitamin D receptor (VDR) and 1α-hydroxylase are also expressed in reproductive tissues such as
the human placenta, endometrium, and ovaries.27 CYP2R1, the enzyme that converts inert vitamin
D into 25(OH)D, is expressed in endometrial cells, with an increasing expression during the secre-
tory phase over the the proliferative phase.28 An animal study has shown the expression of VDR,
CYP24A1—the enzyme that degrades 1,25(OH)2D and 25(OH)D—CYP2R1, and CYP27B1—which
also converts inert vitamin D into 25(OH)D—at the mRNA level in the endometrium of pigs. Also,
interestingly, calcitriol is synthesized in human endometrial decidua.29 It has been suggested that
calcitriol influences specific genes’ expression (i.e., HOXA10 and the calbindin and osteopontin
genes) that are involved to implantation and vitamin D and calcium metabolism.29 In early preg-
nancy, there is an upregulation of the production of vitamin D in the endometrium by the blas-
tocyst.30 It is also suggested that the immunomodulatory effects of vitamin D may be linked to its
protective role on abortion.31
Vitamin D insufficiency is associated with miscarriage, as shown by the Odense Child Cohort.32
In this prospective cohort study of 1683 pregnant women, those with higher 25(OH)D concentra-
tions presented a lower adjusted hazard ratio (HR) of first-trimester miscarriage (HR 0.98, 95% CI
0.96–0.99). Another cross-sectional study of 252 women undergoing ICSI has revealed a signifi-
cant positive association of 25(OH)D with clinical pregnancy and endometrium thickness.33 It has
been suggested that there is an increased risk of early spontaneous pregnancy loss when 25(OH)D
Polycystic ovary syndrome 29
concentrations are lower than 20 ng/mL (RR 2.24, 95% CI 1.15–4.37),34 though the data are conflict-
ing.35 A meta-analysis by Chu et al. on the association of vitamin D and live birth in women under-
going ART, although showing an increased likelihood of clinical pregnancy in women replete in
vitamin D in comparison with those with vitamin D insufficiency or deficiency, failed to reveal any
association between miscarriage and vitamin D concentration.36 Another study has detected differ-
ences on the association of vitamin D concentration and reproductive outcomes in different ethnic
groups. According to Rudick et al., vitamin D status is positively associated with favorable reproduc-
tive outcomes after IVF in non-Hispanic white women.37 After adjustment for age and number and
quality of embryos transferred, the odds of pregnancy in this ethnic group were four times higher in
vitamin D replete versus deficient patients. However, no similar observation was made in Hispanic
whites or Asian women.37 It has also been suggested that an excess of serum and follicular fluid vita-
min D concentrations, together with a decrease in follicular fluid glucose levels, may lead to adverse
IVF outcome.38 However, this finding is not always confirmed by other studies and the predictive
role of follicular fluid vitamin D concentrations regarding pregnancy rate after IVF is limited.39
A recent original study by Li et al. has demonstrated that 25(OH)D, TGF-β concentrations, and
VDR expression are significantly decreased in women with recurrent spontaneous abortions when
compared with women with normal pregnancies.40 In the same study, IL-17 and IL-23 concentra-
tions were significantly increased.40 In donor-recipient cycles, clinical pregnancy and live birth
rates were lower in vitamin D deficient recipients in comparison to those with vitamin D suf-
ficiency,41 implying a favorable role of vitamin D at the human endometrium. There is no direct
evidence however on the association of vitamin D status and RIF. Of note, obesity may influence
vitamin D status in pregnant women as lower 25(OH)D concentrations are reported in pregnant
women with higher body mass index (BMI), in comparison with those with normal BMI, and thus,
vitamin D may be the key player in some of the obesity-related adverse pregnancy outcomes.42
POLYCYSTIC OVARY SYNDROME

In women of reproductive age, polycystic ovary syndrome (PCOS) is a highly prevalent endocri-
nopathy, with an incidence that varies between 6% to 10%, based on the National Institutes of Health
(NIH) criteria, and 20% to 22%, based on the American Society of Reproductive Medicine/European
Society of Human Reproduction and Embryology (ASRM/ESHRE) criteria.43,44 Early pregnancy loss
is increased in women with PCOS, up to five-fold, compared with women without PCOS.45
It seems that hyperinsulinemia, together with hyperandrogenism, may alter the endometrial
environment and may have an impact in the increased percentage of early pregnancy loss and infer-
tility in women with PCOS.45 Hyperinsulinemia and insulin resistance, common features of PCOS,
may be risk factors for early pregnancy loss or implantation failure by themselves, as implied by
the favorable action of metformin in the reduction of early pregnancy loss in women with PCOS.46
Indeed, metformin administration throughout pregnancy could lower the risk of early preg-
nancy loss in women with PCOS, as shown by a meta-analysis of six studies (OR 0.19 [0.12–0.28],
I2 = 0%).45 Even in lean women with PCOS, the risk of early miscarriage has been reported to be
increased and the chance of live birth decreased, as compared with controls.47 Animal data do show
a direct favorable action of metformin in implantation.48
Animal and human data demonstrate that uterine receptivity in women with PCOS is impaired.
Li et al. have demonstrated not only high circulating estrogen in DHEA-induced PCOS mice, but
also compromise in the uterine decidualization and changes in the expression of implantation-
related genes.49 Women with PCOS show increased endometrial androgen receptor (AR) expres-
sion, possibly inducing endometrial abnormalities in women with PCOS.50 Oocyte and embryo
quality after IVF is also reported to be compromised in women with PCOS, probably due to
increased oxidative stress in granulosa cells.51 Τhe exact mechanism is unknown, but it seems that
the NADPH oxidase (NOX) pathway may play a role in reactive oxygen species (ROS) production
by granulosa cells in women with PCOS.51 Elevated aggrecan, disintegrin, and metalloproteinase
with thrombospondin motif-1 (ADAMTS-1) concentrations were found in the follicular fluid of
women with PCOS, indicating a potential role of aggrecan in the pathogenesis of PCOS. A positive
predictive effect of ADAMTS-1 on implantation was noted, indicating a potential role as a marker
of high-quality embryos in women with PCOS.52
DIABETES MELLITUS
Women with DM type 1 and 2 have been reported to present pregnancy complications, including
miscarriage. Insulin resistance and hyperinsulinemia have been related to adverse outcomes fol-
lowing IVF, though the exact mechanism is yet to be elucidated.
Diminished oocyte and embryo quality, as well as impaired implantation have been suggested
to play a role.53
Local factors in the endometrium of women with DM may lead to impaired endometrial recep-
tivity and RIF. Insulin receptors are present in the human endometrium and have varying expres-
sion during the different phases of the menstrual cycle, thus implying functionality of the receptor
locally.5 Immune and vascular defects in the endometrium of women with DM during gestation
have been suggested to lead to fetal loss.54 In a hyperinsulinemic mouse model, it was demon-
strated that the endometrium receptivity is adversely affected by maternal hyperinsulinemia.53
The implantation rate is reduced in diabetic mice,55 with uterine environment alterations being
the main reason. The upregulation of lactoferrin (Ltf ) and complement 3 (C3) estrogen-responsive
genes in diabetic mice implies higher estrogen concentrations. The increased estrogen concentra-
tions, in turn, may impair embryo implantation in diabetic animals. Ιn pregnant diabetic rats,
when uterine tissue was examined, immune staining density of CD68, CD45, and CD56 was found
to be decreased, and significant degeneration of uterine tissue was present.56
LEPTIN
Obesity includes a risk factor for recurrent spontaneous abortion, and obese women may experi-
ence various reproductive adverse events. On the other hand, a weight loss that exceeds 10% of
current body weight has been reported to improve female fertility and reduce the spontaneous
abortion rate by 50%.57 It is suggested that obesity has an impact on reproductive ability in various
ways, including worsening of oocyte quality and inducing changes in human endometrium recep-
tivity.57 In women with PCOS, success rates and adjusted odds ratios for all pregnancy outcomes
progressively worsen as BMI increases.58
A recent theory underlines the role of leptin and leptin receptor (OB-R) gene polymorphisms
on successful implantation and ongoing pregnancy. Leptin is a 16-kDa protein secreted mainly by
adipocytes and expressed in human endometrium, suggesting a probable autocrine/paracrine role
during implantation. It is suggested that in human endometrium, OB-R levels are higher during the
implantation window. Women with PCOS or endometriosis have been reported to achieve lower
concentrations of OB-R during implantation in comparison to controls,59 possibly due to dysregu-
lation of ion channel expression. It is reported that there are lower levels of OB-R in patients with
implantation failure in comparison with those with implantation success.60 According to Muller
et al.,61 genetic variability in the leptin receptor gene is associated with recurrent spontaneous abor-
tions. When women with unexplained RIF were compared with fertile women, lower endometrium
expression of leptin and higher endometrium expression of leptin receptors was noted in women
with RIF.4 The presence of OB-R mRNA at all developmental stages of the embryo underlines the
role of leptin at the preimplantation stage.62 It has been suggested that leptin expression by the
embryo influences its own development and its interaction with the endometrium, thus also leading
to implantation and placentation.62,63 As a result, leptin and OB-R gene expression are being studied
as probable new markers for the prediction of implantation failure59 and leptin is now assessed as a
contributor to the preparation of the endometrium for blastocyst implantation.62 Similarly, adipo-
nectin is also suggested to have an impact in human uterine receptivity and embryo implantation.59
Other than that, leptin can regulate the hypothalamic—pituitary—thyroid axis,64 thus also
influencing implantation through thyroid hormone. Studies on pigs have shown that exogenous
leptin can upregulate luteinizing hormone (LH) secretion in early pregnancy, when applied alone,
References 31
and downregulate it, in the presence of gonadotropin-releasing hormone (GnRH).65 It is also

demonstrated that leptin knockout mice are not able to reproduce, but fertility is recovered when
recombinant leptin is administered exogenously.66
In the setting of IVF/ICSI, OS seems to be associated with elevated leptin concentrations,67,68
however evidence is limited. This increase in leptin serum concentrations is suggested to lead to
negative reproductive outcomes.62 Of note, leptin concentrations in peripheral blood and follicular
fluid have also been shown to be increased in obese women undergoing IVF in comparison to non-
obese women.69
CONCLUSIONS
Endocrine disorders have emerged as probable causes of RIF. The fact that different hormones have
a catalytic role in the process of implantation explains the observation of endocrine disorders being
involved in the pathogenesis of RIF. Various hormones interact with the human endometrium,
whereas hormonal receptors are distributed in reproductive tissues, including the endometrium.
However well-designed human studies in women with RIF are limited.
Subclinical hypothyroidism and TAI may compromise the reproductive outcome after IVF.
Indeed, LT4 supplementation in women with subclinical hypothyroidism or in women with TSH
concentrations above 2.5 µIU/mL (high normal concentration) leads to higher delivery and lower
miscarriage rates, though application of this threshold has not been tested in women with RIF. TAI
is also associated with adverse pregnancy outcomes after IVF. The association of vitamin D defi-
ciency and RIF is complex, but vitamin D is present in the human endometrium and it seems that
it has a favorable role regarding endometrium receptivity. Hyperinsulinemia and insulin resistance
by themselves, or as features of PCOS, also affect endometrium receptivity and thus IVF reproduc-
tive outcomes. Leptin and adiponectin are newly recognized key players in the implantation pro-
cedure. Leptin concentrations are generally increased in obese women and may further increase
after IVF, but the endometrium expression of leptin is lower in women with RIF, with endome-
trium expression of leptin receptors being higher. In any case, evidence on leptin concentrations
in women undergoing IVF is limited, not only due to the small number of studies, but also due to
heterogeneity and methodological issues of the studies.
As new evidence on the association of endocrine disorders with RIF is gathered, new therapeutic
opportunities will emerge. Assessment by an endocrinologist of women with RIF is necessary and
may prove helpful in better management of women with RIF, not only for better reproductive out-
comes, but also for the prevention of long-term complications of endocrine diseases.
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Congenital uterine anomalies
and recurrent implantation
5
failure
GRIGORIS F. GRIMBIZIS, SARA Y. BRUCKER, and RUDI CAMPO
INTRODUCTION
The uterus, being one of the two partners (together with the developing embryo) in the achieve-
ment of pregnancy, plays a key role in implantation and subsequent pregnancy outcome. It seems
reasonable that anything that alters normal anatomy of the myometrium and/or endometrial
cavity might have an effect on implantation and the achievement of pregnancy. This seems to be
extremely important in the subgroup of patients having a history of recurrent implantation failure
(RIF), since the possibility of restoring the cause might reverse the negative effect of a potential
uterine factor.
Congenital uterine anomalies (CUA) are common entities resulting from embryological defects
in one or more stages of normal embryological development of the female genital tract.1–3 They
are characterized by a distorted anatomy of the endometrial cavity. The American Society of
Reproductive Medicine (ASRM), formerly the American Fertility Society (AFS), classified the
anomalies into six classes:4 class I, including aplasia and agenesis (formation defect); class II, uni-
cornuate uterus with its subclasses (formation defect); class III, didelphys uterus (fusion defect);
class IV, bicornuate uterus (fusion defect); class V, septate uterus (absorption defect); and class VI,
arcuate uterus (absorption defect), keeping also a place in the classification scheme for T-shaped
uterus as a result of diethylstilbestrol (DES) administration during fetal life. The main restriction
of this system, which received wide acceptance and was used until recently in almost all published
studies, was the absence of definitions; it was only a descriptive presentation of the classes through
only a classification scheme. Moreover, the inclusion of two separate categories for fusion defects,
septate as the clinically important variant and arcuate as the variant with no clinical importance,
without defining the anatomical borders for differential diagnosis between them, creates a huge
confusion in the evaluation of their clinical consequences.3
Recently, the European Society for Human Reproduction and Embryology (ESHRE) and the
European Society for Gynaecological Endoscopy (ESGE) published a new classification of female
genital anomalies, with clear anatomical definitions of the categories, giving the opportunity to
evaluate their clinical impact; uterine, cervical, and vaginal anomalies were classified in indepen-
dent categories.5,6 Furthermore, the two societies published a consensus for their diagnosis as an
additional tool to clinicians and researchers to avoid subjectivity in their management.7,8 Uterine
anomalies were categorized into five classes: class I, dysmorphic uterus (including T-shaped and
infantilis uteri); class II, septate uterus (absorption defects/arcuate deleted); class III, bicorporeal
uterus (fusion defects/including former didelphys and bicornuate uteri); class IV, hemi of unicor-
poreal uterus (formation defect/former unicornuate uterus); and class V, aplastic uterus (formation
defect/including only cases of uterine aplasia).5,6
The aim of the chapter is to critically review whether or not the anatomically distorted endome-
trial cavity in patients with CUA might have an adverse effect on implantation and, consequently,
be a cause of RIF.
35
36 Congenital uterine anomalies and recurrent implantation failure
METHODOLOGICAL COMMENTS
Ideally, the study question could be answered by studies comparing infertile patients with and
without CUA, treated with in vitro fertilization (IVF) and having RIF as the primary outcome;
however, it is not clear yet what is defined as RIF. In the absence of such a definition and, also, of
relevant studies, an alternative study outcome could be implantation rate per se; it is reasonable to
assume that, if CUA impairs implantation, they might be a cause of RIF. In the same line, as the
achievement of pregnancy is the result of a successful implantation, conception rates in patients
with and without CUA could be another indirect study parameter. The prevalence of CUA in the
general and infertile population could also provide indirect evidence for their potential impact on
the achievement of pregnancy and, consequently, on implantation.
It is important to note that most of the available studies have used the ASRM classification sys-
tem. However, the absence of clear definitions for the different CUA categories, as well as of criteria
for the differential diagnosis between them, especially between bicornuate, septate, and arcuate
uteri, represents an important limitation. Furthermore, the use of different diagnostic methods
with variable diagnostic accuracy for categorization of patients in the available studies is an addi-
tional limitation. However, with the increasing availability of high-accuracy diagnostic methods
and clinicians’ awareness, the objectivity of CUA diagnosis has improved in more recent studies,
though still not overcoming the problem of differential diagnosis within the different categories
and, potentially, that of overdiagnosing arcuate uterus.
Taking into account the previous methodological limitations, the impact of CUA on implanta-
tion will be assessed based on:
1. Prevalence of CUA in the general and infertile population; and
2. Conception and implantation rates in patients with CUA.
PREVALENCE IN THE GENERAL AND INFERTILE POPULATION

Historically, the first review examining that issue was published in 2001,9 concluding that there is
no difference in the prevalence of CUA between the general and the infertile populations. However,
the pooled prevalence of CUA in the studied populations was estimated by including all the avail-
able studies independent of the diagnostic method used; this represented an obvious major limita-
tion since, for example, gynecological examination (used as a method in one study) has extremely
low diagnostic accuracy compared to three-dimensional ultrasound (3D US).
Two subsequent reviews tried to address this limitation. Saravelos et al.10 reviewed the accu-
racy of the available methods in diagnosis and differential diagnosis of CUA; endoscopy, as the
historical “gold standard,” hydrosonography (HSG), and 3D US, having an accuracy >90%, were
characterized as high-accuracy methods. However, in studies performed with high-accuracy diag-
nostic methods, a similar pooled CUA prevalence of 6.7% (95% CI 6.0%–7.4%) and 7.3% (95% CI
6.7%–7.9%) in the general and infertile population, respectively, was observed.
Three years later, Chan et al.,11 including additional high accuracy studies, found a CUA
prevalence of 5.5% (95% CI 3.3%–8.5%) and 8.0% (95% CI 5.3%–12.0%) in the general and infer-
tile populations, respectively. Moreover, they observed a statistically higher CUA prevalence of
13.3% (95% CI 8.9%–20.0%) in recurrent aborters and the impressive statistically higher CUA inci-
dence of 24.5% (95% CI 18.3%–32.8%) in women with infertility and recurrent pregnancy losses.11
Although a statistically increased prevalence of CUA in the infertile population compared with the
general one was not shown, there were two clinically interesting points that should not be ignored:
(a) the prevalence of CUA was higher in the infertile population compared with the previous review
and (b) the coexistence of infertility and recurrent pregnancy loss further increases the prevalence
of CUA. Thus, the increasing clinical awareness and the use of more effective techniques in the
diagnosis of CUA seems to gradually alter the available data in the field.
Conception and implantation rates in women with congenital uterine anomalies 37
It should be noted, however, that the degree of anatomical distortion is not the same in all type of
CUA, and this might exert a different effect on implantation. Thus, the distribution of the various
categories of CUA in the different populations is another clinically interesting parameter.
Saravelos et al.,10 based on data from high-accuracy studies, found that the pooled prevalence of
septate uterus was 2% and 3.5% in the general and infertile populations, respectively. Furthermore,
the observed prevalence of CUA excluding arcuate uterus was 2.3% in the general and 5.6% (more
than double) in the infertile population (Figure 5.1). Chan et al.,11 in a similar type of analysis,
found that the pooled prevalence of septate uterus was 2.3% in the general and 3% in the infertile
population. Moreover, the observed prevalence of CUA excluding arcuate uterus was 3.2% in the
general and 5.8% in the infertile population (Figure 5.2). It seems, therefore that the diagnosis of
the “gray zone” arcuate uterus might be responsible for the vast majority of CUA observed in the
general population (Figures 5.1 and 5.2).
Thus, indirect evidence from prevalence studies supports the notion that CUA might have an
adverse effect on woman’s fertility, potentially leading to infertility. Furthermore, it seems that the
more severe the anatomical distortion of the cavity is, the higher the possibility of impaired fertil-
ity potential of a woman; septate uterus and more severe types of CUA appear to be the clinically
important variants that adversely affect fertility.
CONCEPTION AND IMPLANTATION RATES IN WOMEN WITH

CONGENITAL UTERINE ANOMALIES
Direct published evidence on the effect of uterine anomalies on implantation is not available in the
relevant literature. Thus, as pointed out previously, indirect evidence coming from the study of the
conception rates in patients with CUA might mirror a potential effect on implantation; for spon-
taneous pregnancies, this relationship is almost linear, whereas for pregnancies after IVF, embryo
transfer policy is an important variable.
Chan et al.,12 in their systematic review of evidence, found that patients with septate uterus have
∼15% (RR 0.86, 95% CI 0.77–0.96) significantly lower risk of conception compared to the anatomi-
cally normal ones. Conception rates were not different in patients with unicornuate uterus, but this
observation should not be considered as conclusive due to the insufficient sample size.12 Three years
15
Arcuate CUA without arcuate
12.1
10
7.1
5.6
4.9
5
2.3
1.9
0
General Infertile Recurrent aborters
Figure 5.1 Prevalence of arcuate uterus and congenital uterine anomalies without arcuate uterus in
the general and selected populations. (Data from Saravelos SH et al. Hum Reprod Update. 2008;14:415–9.)
30
Arcuate CUA without arcuate
25.6
25
20
15
9.4
10
5.8 6.6
3.9
5 3.2 2.9
1.8
0
General Infertile Recurrent Aborters and
aborters infertile
Figure 5.2 Prevalence of arcuate uterus and congenital uterine anomalies without arcuate uterus in
the general and selected populations. (Data from Chan YY et al. Hum Reprod Update. 2011;17:761–71.)
later, Venetis et al.13 examined again pregnancy rates in both natural and assisted conception cycles
in a larger cohort of meanwhile-published relevant studies. They did not found any statistically
significant difference in natural (RR 0.96, 95% CI 0.89–1.04) and assisted cycles (RR 0.66, 95% CI
0.37–1.19) when reviewed separately; nevertheless, when natural and assisted cycles were analyzed
together, patients with CUA experienced ∼15% (RR 0.86, 95% CI 0.74–1.00) lower risk of pregnancy
compared with those without CUA, confirming the findings of the previous meta-analysis.
In a recent prospective observational study, Prior et al.14 examined the outcome of assisted repro-
duction in patients with CUA compared with normal controls. Patients with arcuate uterus com-
pared with those having normal uterus, had similar clinical pregnancy (43.2% vs. 43.7%, p = 0.78)
and live birth (36.7% vs. 37.2%, p = 0.91) rates; according to them, arcuate uterus was defined as
the presence of a concave fundal indentation at an obtuse angle, thus including patients with any
indentation even as small as 1 mm. On the other hand, patients with real CUA (septate, bicornuate,
didelphys, and unicornuate), characterized by them as “major,” had significantly decreased clinical
pregnancy (28.8% vs. 43.7%, p = 0.048) and live birth (22.2% vs. 37.2%, p = 0.042) rates. Embryo
transfer policy, stage, and number of embryos transferred were similar in all studied groups, indi-
cating that the presence of uterine anatomy distortion in patients with CUA has an adverse effect
on implantation.
The results of hysteroscopic treatment15,16 could offer some additional evidence for the potential
role of these anomalies on patients’ fertility. However, although an increase in conception rates
after surgical correction of the anomaly might be an indirect evidence of an impairment of wom-
an’s fertility by the anomaly, a neutral effect might not, since this could be due to the “trauma” and
healing process following the procedure.
Looking to the effect of septum excision, Venetis et al.,13 in their systematic review of compara-
tive studies, failed to detect any statistically significant increase in the probability of conception
(RR 1.14, 95% CI 0.79–1.65) after hysteroscopic treatment; surgical correction of uterine septa
was followed by ∼60% decrease (RR 0.37, 95% CI 0.25–0.55) in the risk of abortion. However, the
fact that the presence of septum impairs the probability of conception as well as the evolution of
Conclusions and recommended current practice 39
pregnancy combined with the observed effect size in this meta-analysis suggests that further evi-
dence is necessary to properly access the value of hysteroscopic treatment for infertility.
In another type of analysis, Mollo et al.17 compared prospectively two groups of patients with
unexplained infertility: patients with septate and with normal uteri. Conception (38.6% vs. 20.4%,
p = 0.016) and live birth (34.1% vs. 18.9%, p < 0.05) rates after septum excision in the septate group
were significantly higher than those in the normal group. These findings support the notion that
the presence of septum has an adverse effect on fecundity and that, potentially, hysteroscopic treat-
ment has a beneficial effect.
Although some of the existing evidence is coming from prospective studies, it is important to
note that most of them, especially the comparative ones, included in the prementioned systematic
reviews are retrospective studies. Thus, solid conclusions cannot not be drawn yet, but according to
the best available evidence (level C/meta-analysis of retrospective comparative studies), the pres-
ence of CUA might adversely affect implantation as indicated by the decrease in the relative risk of
pregnancy by ∼15%. Furthermore, concerning the achievement of pregnancy, it seems that septum
excision might have a beneficial effect (class IIb recommendation).18,19
POSSIBLE BIOLOGICAL EXPLANATIONS

Two main theories have been proposed to explain the potential association between CUA, implan-
tation, and subsequent pregnancy. According to the first one, impaired implantation is the result of
alterations in vascularization. The hypothesis of an impaired blood supply of the septum and of an
overlying functionally defected endometrium was supported as an explanation of a poorer implan-
tation; thus, when embryos implant on the septum site, pregnancy is less likely to occur.15,20–25
However, the poorer pregnancy outcome in patients with vascularized septa compared with the
avascularized ones,25 the increased density of vessels found in tissue samples from septa by some
investigators,25 and the potential impairment of implantation in other types of anomalies are not
explained with this hypothesis. Thus, it is supported that it is not the decreased vascularization but
the alterations of the vascular “bed” in the different types of anomalies which decreases receptiv-
ity to the invading trophoblast21,22 by impairing later, vascular stages of implantation; once the
implanting embryo overcomes the endometrial barrier, uterine vasculature and stroma could rep-
resent a subsequent important barrier.26
Another pathophysiological explanation for the impaired implantation focuses on the differ-
ences in the structure of the uterine musculature in patients with CUA. Uterine contractility seems
to be an important factor for successful implantation and evolution of pregnancy; altered uterine
anatomy and the resulting alterations of normal uterine contractility could exert a detrimental role
in implantation and early stages of pregnancy.15,21,25,27–29 Decreased uterine volume does not seem
to play a role in implantation but only, potentially, in the evolution of pregnancy and its later stages
for complications from the second trimester onward.15,17
The more sophisticated hypothesis of alterations in the expression of HOX genes at different
age stages was also included in the “armamentarium” of pathophysiological explanations: HOX
genes are important both for normal female genital tract embryogenesis and for development of the
endometrium.30 However, it seems that the accumulation of more epidemiological data on the exact
role of each type of uterine deformity on the reproductive potential of the woman is necessary for
clarifying the contributing factors and the underlying biological mechanisms.
CONCLUSIONS AND RECOMMENDED CURRENT PRACTICE

The original AFS classification,4 being only a descriptive presentation of CUA, provided no defini-
tion for the classes of the system;4 this gave place to the adoption of a wide range of arbitrary criteria
for their differential diagnosis, especially, but not only, between the arcuate and septate category.3
This, as a result, led to a great confusion in the relevant research. Although, ASRM recently tried to
treat this restriction with the adoption of anatomical criteria for the septate group,18 the insistence
in having arcuate uterus as a separate category from the normal one is expected only to continue
the existing research uncertainties.
As the need for further research on the “hot” issue of the reproductive impact of CUA is obvi-
ous, clear categorization of them is crucial. The ESHRE/ESGE classification of female genital
anomalies with the adoption of clear anatomical definitions offers a research platform for study-
ing their clinical impact;5,6,31 the deletion of gray zone anomalies should be considered as crucial
and opens a new era. It is important to note that other groups also accepted the idea to abandon
the gray zone arcuate category, proposing the normal/arcuate category as one entity and the sep-
tate one as the other, despite the fact that their anatomical criteria differ from that of the ESHRE/
ESGE classification.32
However, from the existing evidence, it seems that the presence of an anatomically defected
uterus is associated with a ∼15% decrease in the chance for achieving a pregnancy, either spon-
taneously or with the use of assisted conception, indicating an impaired implantation in women
with CUA. Although it is not yet clear which is the exact impact of each different type of CUA on
implantation and pregnancy outcome, it seems that the more severe the anatomical defect of the
uterus, the more the probability of an impaired implantation and evolution of pregnancy. Although
the contribution of minor defects is questionable, it is accepted that the presence of a septate uterus
and more severe anatomical defects are associated with an adverse effect on the probability of preg-
nancy. Thus, women with repeated implantation failures should have an assessment of the uterine
anatomy, preferably with the use of 3D US, which seems to be the diagnostic method of choice.7,8
In the presence of an anomaly and especially of a septum, hysteroscopic treatment might be
considered as a way of restoring anatomy and the implantation potential of the woman; patients
with RIF are symptomatic women and, based on the existing evidence, it is reasonable to consider
hysteroscopic restoration of the cavity.18,19 Finally, it is important to note that well-designed studies
on that issue remain a necessity.
REFERENCES
1. Acien P, Acien M, Sanchez-Ferrer M. Complex malformations of the female genital tract. New
types and revision of classification. Hum Reprod. 2004;19:2377–84.
2. Acien P, Acien MI. The history of female genital tract malformation classifications and pro-
posal of an updated system. Hum Reprod Update. 2011;17:693–705.
3. Grimbizis GF, Campo R. Congenital malformations of the female genital tract: The need for
a new classification system. Fertil Steril. 2010;94:401–7.
4. American Fertility Society (AFS); Committee for Mullerian Anomalies: Gibbons W, Buttram
VC Jr, Jan Behrman S et al. The American Fertility Society classifications of adnexal adhe-
sions, distal tubal occlusion, tubal occlusion secondary to tubal ligation, tubal pregnancies,
Mullerian anomalies and intrauterine adhesions. Fertil Steril. 1988;49:944–55.
5. Grimbizis GF, Gordts G, Di Spiezio Sardo A et al. The ESHRE/ESGE consensus on the clas-
sification of female genital tract congenital malformations. Hum Reprod. 2013;28:2032–44.
6. Grimbizis GF, Gordts G, Di Spiezio Sardo A et al. The ESHRE/ESGE consensus on the clas-
sification of female genital tract congenital malformations. Gynecol Surg. 2013;10:199–212.
7. Grimbizis GF, Di Spiezio Sardo A, Saravelos SH et al. The Thessaloniki ESHRE/ESGE consen-
sus on diagnosis of female genital anomalies. Hum Reprod. 2016;31:2–7.
8. Grimbizis GF, Di Spiezio Sardo A, Saravelos SH et al. The Thessaloniki ESHRE/ESGE consen-
sus on diagnosis of female genital anomalies. Gynecol Surg. 2016;13:1–16.
9. Grimbizis GF, Camus M, Tarlatzis BC et al. Clinical implications of uterine malformations
and hysteroscopic treatment results. Hum Reprod Update. 2001;7:161–4.
10. Saravelos SH, Cocksedge KA, Li T-C. Prevalence and diagnosis of congenital uterine anomalies
in women with reproductive failure: A critical appraisal. Hum Reprod Update. 2008;14:415–9.
References 41
11. Chan YY, Jayaprakasan K, Zamora J et al. The prevalence of congenital uterine anoma-
lies in unselected and high-risk populations: A systematic review. Hum Reprod Update.
2011;17:761–71.
12. Chan YY, Jayarpakasan K, Tan A et al. Reproductive outcomes in women with congenital
uterine anomalies: A systematic review. Ultrasound Obstet Gynecol. 2011;38:371–82.
13. Venetis C, Papadopoulos S, Campo R et al. Clinical implications of congenital uterine anoma-
lies: A meta-analysis of comparative studies. RBM Online. 2014;26:665–83.
14. Prior M, Richardson A, Asif S et al. Outcome of assisted reproduction in women with con-
genital uterine anomalies: A prospective observational study. Ultrasound Obstet Gynecol.
2018;51:110–7.
15. Grimbizis G, Camus M, Clasen K et al. Hysteroscopic septum resection in patients with
recurrent abortions or infertility. Hum Reprod. 1998;13:1188–93.
16. Saman AM E, Shahin AY, Nasr A et al. Hybrid septate uterus, coexistence of bicornuate and
septate varieties: A genuine report. J Obstet Gynaecol Res. 2012;38:1308–14.
17. Mollo A, De Franciscis P, Colacurci N et al. Hysteroscopic resection of the septum improves
the pregnancy rate of women with unexplained infertility: A prospective controlled trial.
Fertil Steril. 2009;91:2628–31.
18. Pfeifer S, Butts S, Dumesic D et al. Uterine septum: A guideline. Fertil Steril. 2016;106:530–40.
19. National Institute for Health and Care Excellence: Hysteroscopic metroplasty of a uterine
septum for primary infertility (IPG509). nice.org.uk/guidance/ipg509
20. Candiani GB, Fedele L, Zamberletti D et al. Endometrial patterns in malformed uteri. Acta
Eur Fertil. 1983;14:311–8.
21. Dabirashrafi H, Bahadori M, Mohammad K et al. Septate uterus: New idea on the histologic
features of the septum in this abnormal uterus. Am J Obstet Gynecol. 1995;172:105–7.
22. Fedele L, Bianchi S, Marchini M et al. Ultrastructural aspects of endometrium in infertile
women with septate uterus. Fertil Steril. 1996;65:750–2.
23. Leible S, Munoz H, Walton R et al. Uterine artery blood flow velocity waveforms in pregnant
women with müllerian duct anomaly: A biologic model for uteroplacental insufficiency. Am J
Obstet Gynecol. 1998;178:1048–53.
24. Homer HA, Li TC, Cooke ID. The septate uterus: A review of management and reproductive
outcome. Fertil Steril. 2000;73:1–14.
25. Kupesic S. Clinical implications of sonographic detection of uterine anomalies for reproduc-
tive outcome. Ultrasound Obstet Gynecol. 2001;18:387–400.
26. Crocker IP, Wareing M, Ferris GR et al. The effect of vascular origin, oxygen, and tumour
necrosis factor alpha on trophoblast invasion of maternal arteries in vitro. J Pathol.
2005;206:476–85.
27. Pellerito JS, McCarthy SM, Doyle MB et al. Diagnosis of uterine anomalies: Relative accu-
racy of MR imaging, endovaginal sonography, and hysterosalpingography. Radiology.
1992;183:795–800.
28. Rock JA, Murphy AA. Anatomic abnormalities. Clin Obstet Gynecol. 1986;29:886–911.
29. Sparac V, Kupesic S, Ilijas M et al. Histologic architecture and vascularization of hystero-
scopically excised intrauterine septa. J Am Assoc Gynecol Laparosc. 2001;8:111–6.
30. Taylor HS. The role of HOX genes in human implantation. Hum Reprod Update. 2006;75–9.
31. Grimbizis GF, Gordts S, Di Spiezio Sardo A. Reply: Are the ESHRE/ESGE criteria of female
genital anomalies for diagnosis of septate uterus appropriate? (letter to the editor). Hum
Reprod. 2014;29:868–9.
32. Ludwin A, Martins WP, Nastri CO et al. Congenital Uterine Malformation by Experts
(CUME): Better criteria for distinguishing between normal/arcuate and septate uterus?
Ultrasound Obstet Gynecol. 2018;51:101–9.
Immunological causes of
6
Comprehensive insights into
mechanisms and therapeutic
approaches
ARI KIM, WAEL SAAB, NAYOUNG SUNG,
and JOANNE KWAK-KIM
INTRODUCTION
Recurrent implantation failure (RIF), during in vitro fertilization (IVF) and embryo transfer of
fresh or frozen embryos, refers to unsuccessful implantation after repeated transfers of good-
quality embryos.1 Until now, there has been no international consensus for a definition of RIF
regarding a number of failed trials or transferred embryos. However, it is often defined as failure
of embryos to implant in at least three consecutive IVF cycles in which at least four high-grade
embryos are transferred.1 Due to the complexity of the underlying pathophysiology of RIF, vari-
ous etiologies have been reported. Embryonic etiologies include genetic abnormalities, inadequate
culture condition, suboptimal embryo development, zona hardening, improper embryo trans-
fer technique, impaired embryo development in utero, male factor, immunological factors, etc.
Maternal etiologies include endometrial and systemic factors, such as immunological, throm-
bophilic, metabolic, anatomical, and infectious factors, and multifactorial effectors including
endometriosis and hydrosalpinges. With the recent development and the better understanding
of immunology, it is more evident that immune inflammation significantly contributes to the
underlying pathology of RIF.1
Much progress has been made in in vitro embryogenesis and gamete and embryo cryopreserva-
tion technologies. However, little attention has been paid to improve the implantation after embryo
transfer. During the implantation, maternal immune recognition and tolerance to the invading
embryo play a major role. Both local and systemic immune effectors, cytokines, chemokines,
and various immune factors actively contribute to embryonic implantation and maintenance of
pregnancy. Hence, the dysregulated maternal immune responses to invading embryos may result
in RIF, recurrent pregnancy losses (RPL), and second- and third-trimester obstetrical complica-
tions. RIF is a physically, emotionally, and mentally distressing disorder, and patients who suffer
from RIF warrant further investigation, although diagnosis and management of RIF is still an
evolving field. Treatment for RIF should be stratified based on underlying etiologies and person-
alized by individual characteristics. Possible immune etiologies and immunological therapeutic
modalities for RIF are summarized in Figure 6.1. In this review, various immune etiologies for RIF
and therapeutic modalities for these conditions are addressed.
42
Immune etiologies 43
Therapeutic targets Immunosuppressive treatment
Elevated NK cell levels lntralipid Tx.

Elevated NK cytotoxicity
Tacrolimus
Elevated TH1/TH2 immune TNF-α inhibitors
response
Prednisone
(Dexamethasone*) IVIg Tx.
ANA, antibodies to nuclear
antigens
Hydroxychloroquine
Antiphospholipid antibody
lmmunotrophic treatment
Decreased treg cells G-CSF (Filgrastim)
Decreased endometrial CD56+

NK cells and IL-15/Fn14 gene Endometrial
expression ratios scratching
Figure 6.1 Therapeutic targets and immune modulation treatment for recurrent implantation fail-
ure (RIF). Cellular immune etiologies involving Th1, Th2, Treg, and NK cells and autoimmune abnormalities
such as ANA, autoantibodies to nuclear antigens, and antiphospholipid antibodies are the major causes
of RIF. These biomarkers can be potential therapeutic targets for immune modulation treatment includ-
ing immunosuppressive and immunotropic treatment. Dotted lines represent the possible treatment with
limited clinical data.
IMMUNE ETIOLOGIES
Antiphospholipid antibody and antinuclear antibody
The most reliable marker for acquired thrombophilia is antiphospholipid antibodies (aPLs),
including criteria-defined aPLs (such as anticardiolipin antibody (aCL), lupus anticoagulant, and
β2-glycoprotein I antibody) and noncriteria-defined aPLs (such as antiphosphatidylserine antibody
[aPS], antiphosphatidylethanolamine antibody, and other phospholipid antibodies that bind to nega-
tively charged phospholipids nonspecifically).2–4 It has been suggested that maternal thrombophilia,
such as aPL, may disturb blood flow to the endometrium and placenta, which results in altered endo-
metrial receptivity and implantation failure.5 Additionally, aPL may exert their detrimental effects
during pregnancy by directly binding to trophoblasts, then altering trophoblast signaling, prolif-
eration, invasion, and secretion of hormones and cytokines, and by increasing apoptosis.6 Patients
(n = 74) with RIF showed a significantly higher prevalence of IgG-aPS and IgG-aCL as compared to
women with a history of early RPL or controls.7 In a long-range study, women with two and more
IVF failures (48% and 50%, respectively, n = 853) are associated with significantly higher serum
levels of aPLs against phosphatidylinositol and L-phosphatidylserine (p < 0.01).8 When IgG, IgA,
and IgM isotypes of antibodies against seven phospholipids (i.e., cardiolipin, L-phosphatidylserine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, as well as
against beta2-glycoprotein I) were investigated, a quarter of women with RIF had three or more pos-
itive aPLs.8 Therefore, autoimmune activation with positive aPL, including not only criteria-defined
but noncriteria-defined aPLs, is common in women with RIF. Whether the association of aPL with
infertility and RIF is causative needs to be elucidated further.
44 Immunological causes of recurrent implantation failure
The presence of antinuclear antibody (ANA) and anti-centromere antibody was reported to inter-
fere with the oocyte and embryo development significantly, and reduce implantation and pregnancy
rates in patients undergoing IVF treatment.9 The levels of ANA in serum and follicular fluid were
positively correlated, and IVF outcomes were markedly poorer in ANA+ women as compared with
those of ANA− women. In a prospective controlled study of women undergoing IVF or intracyto-
plasmic sperm injection (ICSI) and embryo transfer (ET), pregnancy rate, clinical pregnancy rate,
and implantation rate were significantly lower in follicular fluid ANA+ women (n = 36, 27.8%, 25%,
and 15.9%, respectively) as compared with those of follicular fluid ANA− women (n = 50, 57.8%,
48.4%, and 31.7%, respectively; p = 0.004, 0.022, and 0.009, respectively).10 This effect became worse
with an increased level of serum ANA,10 although a low-titer ANA was reported not to affect IVF
outcome adversely.11 In another prospective controlled study of infertile women undergoing IVF/
ICSI (n = 517), 39.34% of infertile women had ANA+, while 16.13% of controls (n = 186) had ANA+
(p < 0.001).12 In ANA+ women, the implantation rate (16.09% vs. 27.03%) and clinical pregnancy
rate (27.72% vs. 45.03%) were decreased significantly (p < 0.001), while the early miscarriage rate
(21.43% vs. 3.39%) was increased (p < 0.001) as compared with those of ANA− women.12 In RIF
women with a failure of implantation after eight or more good embryos transferred, ANA was posi-
tive in 33.6% (n = 122), which was significantly higher than that of fertile controls (10%, n = 20).13
Natural killer cell pathology

During the implantation period, a complex micromilieu is necessary including various cytokines,
chemokines, and factors produced by the blastocyst and endometrium, which have multidirec-
tional pleiotropic effects for the embryonic development and implantation. Inadequate or overac-
tive immune responses and an unbalanced cytokine network may lead to implantation failure,
pregnancy loss, and obstetrical complications.14,15 T helper (Th) 1, Th2, T regulatory (Treg), Th17,
and natural killer (NK) cells are the major immune effectors affecting human pregnancy. Women
with unexplained infertility had higher proportions of activated NK cells,16 and the percentages
of peripheral blood CD56+ and CD56+CD16+ cells on the day of ET were higher in women with
IVF failure (19.1 ± 0.7% and 12.0 ± 0.6%, respectively) than those with successful implantation
(15.2 ± 1.2% and 9.5 ± 0.9%, respectively) (p < 0.02 and p < 0.05, respectively). In women with RPL
and RIF, the proportion (%) of CD56bright/IFN-γ+/TNF-α+ cells was significantly higher than that
of healthy controls. The TNF-α+/GM-CSF+ expressing CD56bright cell ratio was significantly higher
in women with RPL and RIF than in controls. This finding indicated that NK-1 shift in peripheral
blood NK cells was present in nonpregnant women with RPL and RIF.14 In a prospective study,
the percentage of peripheral blood NK cells out of the total lymphocyte population was higher in
women with idiopathic RIF (13.4 ± 1.2%; range, 2.63–29.01) than in controls (8.4 ± 0.7%; range,
5.72–13.28; p = 0.026). In this study, idiopathic RIF was defined as no abnormalities in karyotyping
of both the man and the woman, hysteroscopy, endometrial culture and microbiological analysis
of endometrial biopsy, sperm fluorescent in situ hybridization (FISH) for chromosomes 21, 18, 13,
X, and Y, and sperm DNA fragmentation. In addition, peripheral blood CD56+ NK cells were posi-
tively correlated with endometrial CD56+ cells (rho = 0.707; p < 0.001). NK cells have been reported
to contribute to trophoblast implantation during early pregnancy via vascular remodeling, control
the embryo implantation, and provide local maternal immunity.17 Hence, dysregulated uterine NK
(uNK) cells may contribute to IVF failures or RPL. In women who aborted compared with those
who delivered, there was an increase in the percentage of CD56dimCD16+ cells and a decrease in
the percentage of CD56brightCD16 – cells in the endometrial tissue.18 Testing for NK cells might be
useful for the diagnosis of immune etiology RIF among women with idiopathic RIF19 and selection
of patients undergoing assisted reproductive technology (ART) cycles, who can be benefit from
adjuvant therapy.20 Indeed, intravenous immunoglobulin G (IVIG) treatment has been reported
to reduce NK cell percentage and cytotoxicity significantly and increase killer inhibitory receptors
while decreasing killer activating receptors,21,22 while significantly improving pregnancy outcome
in women with RPL and IVF failures.20,22
Immune etiologies 45
T cell immunity
The proportion of TNF-α producing CD3+CD4+ cells (p < 0.05), and TNF-α/IL-4 (p < 0.05) and
TNF-α/IL-10 (p < 0.005) producing CD3+CD4+ were significantly higher in women with multi-
ple IVF failures without a history of RPL as compared with those of controls.23 Recently, periph-
eral blood IFN-γ/IL-4, IFN-γ/IL-10, IFN-γ/TGF-β1, IL-6/IL-10, IL-6/TGF-β1, IL-1β/TGF- β1 and
TNF-α/TGF-β1 cytokine ratios have been shown to be significantly higher in women with RIF when
compared to controls.24 The prevalence of dominant Th1 immune responses in peripheral blood
lymphocytes may reflect the systemic contribution of Th1 cytokines to RPL or RIF in IVF cycles,
and the Th1/Th2 ratios may affect clinical outcomes in IVF.23,25 Other T cell subsets, such as Treg
and Th17 cells and their cytokine products have not been studied well despite their importance in
pregnancy. Th17 cells are proinflammatory cells secreting effector cytokines, such as IL-17A, IL-17F,
IL-22, IL-26, TNF-α, and IFN-γ.26 Contrarily, Treg cells suppress Th1- and Th17-mediated immunity
and regulate maternal immune tolerance to the fetus. Previously, we reported that increased Th17
cells and decreased Treg cells in women with RPL as compared with fertile controls,27 and the IL-7/
IL-7R signaling pathway plays a role in upregulating Th17 immunity, meanwhile downregulating
Treg immunity.28 Hence, Th1, Th2, Th17, and Treg cells and their cytokine products could be explored
as biomarkers for early pregnancy prognosis and should be evaluated in the context of RIF as well.29
Endometrial immune responses

Local immune effectors and factors in endometrium may play a role in RIF. Inhibitor of DNA-
binding protein 3 (Id3) is required for angiogenesis and proliferation of Treg cells.30 T lymphocyte-
associated molecule-4 (CTLA-4) is expressed on the surface of Treg and conventional T cells and acts
as a brake to shut down the activation of effector T cells.31 Id3+ and CTLA-4+ cells, but not forehead
box P3 (FOXP3)+ cells, were significantly increased in the endometrium of women with RIF and
RPL as compared with those in controls. There was neither coordinated expression of Id3+ cells and
CD34+ vessels nor colocalization of Id3+ and FOXP3+ cells in the endometrium. Aberrant expres-
sions of Id3 and CTLA-4 in peri-implantation endometrium may suggest a population of women
with RIF may have chronic inflammatory microenvironments in endometrium during the embryo
implantation period.30 Several endometrial biomarkers for the endometrial immune inflammatory
status have been reported. Ratios of the IL-15/fibroblast growth factor-inducible 14 (Fn-14) mRNA
expression in endometrium, together with the uNK cell count, were reported to reflect uNK cell
activation and maturation status. Ratios of the IL-18/TNF-like weak inducer of apoptosis (TWEAK)
mRNA expression in endometrium were reported to be a biomarker of both angiogenesis and the
Th1/Th2 balance. In 81.7% of the RIF patients, at least one of these parameters (CD56+ NK cell
numbers in the endometrium, the ratios of IL-15/Fn-14 mRNA, and IL-18/TWEAK mRNA) was
reported to be dysregulated.32 Based on these data, women with RIF may have dysregulated immu-
notropism and angiogenesis at the implantation site, and further studies are needed to advance cur-
rent knowledge and understanding of immune responses during implantation and early pregnancy.
Genetic polymorphisms affecting immune responses

Genetic polymorphisms have been reported to affect the implantation process after IVF-ET. In the
meta-analysis of five studies for the correlation between HLA-G polymorphism and RIF, a signifi-
cant association was found in the population of Caucasian origin under allele contrast (OR = 1.73,
95% CI 1.20–2.50) and genetic models of +14bp/+14bp versus –14bp/–14bp (OR = 3.09, 95% CI
1.43–6.65). HLA-G 14-bp insertion allele may increase the risk of RIF in Caucasians.33 Additionally,
allelic variations, particularly in exons 3 and 4, and intron 2 of the HLA-G gene have been reported
to be associated with ART failures.34 Further studies with a large sample size of different ethnic
backgrounds are needed to explore the association between HLA-G polymorphism and RIF.
During embryo attachment and implantation, loss of mucin 1 (MUC1), transmembrane mucin
expressed at the apical surface of uterine epithelium, is required. The MUC1 expression is modulated
via dynamic interplay among cytokine-activated transcription factors, nuclear factor κB (NF-κB),
progesterone receptor isoforms, and transcriptional coregulators. Therefore, polymorphisms in the
NF-κB gene may have important consequences in implantation failure and RPL.35 In women with
RIF (n = 209), the allelic and genotypic frequencies of the rs28362491 promoter in the NF-κB gene
were statistically significantly different from those of healthy controls. The frequencies of the del/
del genotype and the rs28362491 del allele were significantly higher in RIF patients than in healthy
controls (p < 0.004). In the haplotype analysis, the A-C haplotype occurred significantly more fre-
quently, and A-G haplotypes occurred less frequently in RIF subjects than healthy controls.36
MicroRNAs regulate gene expression post-transcriptionally and contribute to folliculogenesis
and oocyte maturation. miR-146a is enriched in the oocyte and has been reported to be upregulated
in the plasma and ovarian granulosa cells of premature ovarian failure.37 miR-196a2 is expressed
during oocyte maturation and early bovine embryonic development and regulates the homeobox
gene expression of the newborn bovine ovary during early embryogenesis.38 A case-control study
of 354 Korean women investigating the association of microRNA polymorphisms (miR-146aC>G,
miR-149T>C, miR-196a2T>C, and miR-499A>G) with RIF, demonstrated that the polymor-
phisms in miR-146a and miR-196a2 were related to RIF.39 Collectively, these polymorphisms may
serve as biomarkers for detecting oocyte pathology of RIF via immune mechanisms.
IMMUNOLOGICAL TREATMENT
Glucocorticoid
Prednisolone, which belongs to glucocorticoids, is the most widely prescribed treatment for immu-
nomodulation in patients with infertility and RIF due to its easy application, low cost, and relative
safety in short-term treatment regimes. Prednisolone was reported to be effective in RIF women
with high NK cell levels and/or activity.20,40 In a randomized controlled, prospective study, pred-
nisolone 20 mg was given to women undergoing ICSI treatment. Patients with elevated NK cell
activation marker (CD69+ >1% of total lymphocytes) by flow cytometric analysis were included.
Women with an immunological disease, thrombophilia, or uterine or endometrial abnormali-
ties were excluded. Clinical pregnancy rate of women with prednisolone (n = 58) was 48.3% as
compared with 29.6% in controls (n = 54, p < 0.05, RR 1.63, CI 1.00–2.66).20 Prednisolone induces
maternal immune tolerance by decreasing peripheral blood NK cell levels and activities. It has been
hypothesized that elevated peripheral blood CD56bright NK cells in the failed IVF cycle may account
for reduced decidual recruitment and predict implantation failure.41 One week after ET, women
with failed IVF showed elevated peripheral blood NK (both CD56bright and CD56dim) and NKT-like
cell proportions, increased perforin-containing CD56bright cells, more activated and degranulated
CD56dim NK cells, and enhanced NK cell-activating receptor expression on both cell types and
both NK cell subsets (CD160, NKG2D).42 All these findings reflect unfavorable type 1 changes of
NK and NKT-like cells during this period.
It is suggested that uteroplacental thrombosis and vasoconstriction, resulting from binding
of antibodies to platelet and endothelial membrane phospholipids, may contribute to adverse
reproductive outcomes in women with autoantibodies. Corticosteroids are considered suppress-
ing autoantibodies and thereby may contribute to successful implantation and placentation in
those women.43 In a retrospective study that evaluated the effects of prednisolone in women with
ANA+ who underwent IVF (n = 120), implantation and clinical pregnancy rates in the prednis-
olone-treated ANA+ women were significantly higher than those of untreated ANA+ women.44
The higher pregnancy rate in women with autoantibodies treated with prednisolone was noted in
spite of no significant decrease in autoantibody titers. In a prospective controlled study of ANA+
women (n = 133), 60 women were treated with prednisone 10 mg/day and aspirin 100 mg/day,
starting three months before ovulation induction, and 73 women served as an untreated control
group. Fertilization, implantation, and clinical pregnancy rates of ANA+ women (71%, 27.4%, and
53.3%) with prednisone and aspirin treatment were significantly higher when compared with those
of controls (58.0%, 13.6%, and 30.1%) (p < 0.01, p = 0.002, p = 0.007, respectively). Miscarriage rate
Immunological treatment 47
was significantly lower in the prednisone and aspirin treatment group (9.4%) as compared with
that of controls (36.4%, p = 0.36, implies that the difference is NOT significant).45 In a prospec-
tive controlled trial, 66 women with ANA+ who had poor IVF/ICSI outcomes received a daily
oral dose of prednisone 10 mg and aspirin 100 mg up to three months before the second IVF/
ICSI cycle. Women who were treated with prednisone and aspirin had a significantly higher num-
bers of embryos (6.90 ± 3.24 vs. 4.81 ± 2.93, p = 0.006) and transferred embryos/cycle (2.76 ± 0.58
vs. 2.06 ± 0.44, p = 0.008) than women without any treatment. Pregnancy rate (57.1% vs. 12.5%,
p = 0.006) and implantation rate (27.9% vs. 6.06%, p = 0.013) were significantly higher in women
with prednisone plus low-dose aspirin treatment as compared with those of controls.46 In the meta-
analysis of randomized controlled trials (RCTs), the efficacy of administering glucocorticoid in the
peri-implantation period in subfertile women undergoing IVF or ICSI, but not women with RIF,
failed to show a significant difference in pregnancy rates between the intervention and control
groups. However, subgroup analysis revealed that couples undergoing IVF rather than ICSI showed
a significantly higher pregnancy rate in women who received glucocorticoids (OR 1.50, 95% CI
1.05–2.13, p = 0.02, 6 RCT, 650 women, I2 = 0%). In this analysis, patients with autoantibodies
were excluded from the meta-analysis.47 Hence, the clinical efficacy of glucocorticoid in subfertile
women with autoimmune etiology could not be assessed.
Intravenous immunoglobulin G (IVIG) infusion

IVIG, which is isolated from human plasma and contains more than 95% unmodified immuno-
globulin G, has been established as an effective treatment for autoimmune and inflammatory dis-
eases.48 The suggested mechanism of IVIG in reproduction is a reduction of peripheral NK cell
cytotoxicities, enhancement of regulatory T cells, downregulation of antibody production, and
favoring Th2-mediated immune response by decreasing Th1 immunity.22,43,49 For several decades,
IVIG infusion has been utilized for the prevention of RPL potentially caused by immunological
etiologies.22 However, the efficacy of IVIG treatment for RIF patients has not been well elucidated.50
In a prospective controlled study, 8 of 30 infertile women with a history of three or more RIF or
three or more RPL presented with high numbers of CD3+CD56+ NKT cells (>93.6 cells/µL), which
declined after treatment with IVIG. Women with NK/NKT cell expansion with and without autoim-
munity achieved a significantly higher pregnancy rate with IVIG treatment compared with women
with average numbers of NKT cells and no evidence of autoimmunity (p = 0.018). In this trial, hepa-
rin and aspirin were also given if patients were positive for aCL or aPL. Elevated NKT cell levels alone
were the independent predictor of success on IVIG treatment (p = 0.003). IVIG increased pregnancy
and live birth rates in selected women with RIF who had NK/NKT cell expansion.51 Hence, the
selection of the proper population of IVIG treatment may enhance the treatment outcome. In a
prospective controlled study (n = 188), IVIG therapy in a cohort of women with RIF and elevated
circulating NK (CD3−CD56+CD16+, >12% of lymphocytes) and lack of aPLs significantly improved
the pregnancy rate to 50.5% and implantation rate to 21% compared with 15.0% and 9.3%, respec-
tively, in women not receiving IVIG.52 In another prospective controlled study of women with a his-
tory of RPL or RIF (n = 157) undergoing IVF cycle, 64 (40.8%) women had elevated NK/NKT-like
cells. Expansion of blood NK cells (CD3–CD56+CD16+, CD3–CD56+CD16–, or CD3–CD56–CD16+
lymphocytes) was defined as proportions above 12% and for NKT-like cells (CD3+CD56+CD16+), as
proportions above 10% of total lymphocytes. Among the women with elevated NK/NKT-like cells
and three or more RIF (n = 40), a total of 20 women received IVIG treatment, and 20 did not receive
IVIG treatment (controls). The first IVIG infusion (400 mg/Kg of body weight) was given within
24 hr before the embryo transfer, then 15 days after with the confirmed biochemical pregnancy, and
every three weeks during the first trimester of gestation. After gestational week 13, patients were
given IVIG with a monthly dose of 200 mg/Kg of body weight until 35 weeks of gestation. The clini-
cal pregnancy rate of RIF patients with IVIG treatment was 85% versus 10% in controls (p ≤ 0.0001,
OR 37), and the live birth rate was 75% with IVIG versus 5% in controls (p ≤ 0.0001, OR 34).53
Meta-analysis of three published RCTs of IVIG in IVF failure patients shows a significant increase in
the live birth rate per woman (p = 0.012, number needed to treat for one additional live birth, NNT = 6.0
women). Using the live birth rate per embryo transferred, and adding data from two unpublished
cohort-controlled trials to the meta-analysis, further supports this conclusion (overall p = 0.000015,
NNT = 3.7 women). This study also revealed that properties and scheduling of the IVIG and selec-
tion of patients with abnormal immune test results were relevant variables for these trials.48 However,
one of the RCT studies included in the meta-analysis compared the outcome of women with IVIG,
heparin, and aspirin treatment with the controls who were treated with heparin and aspirin only.54 If
this study is excluded due to the study design of using co-intervention, the difference reported is not
significant. Hence, the data should be carefully interpreted. Recently, another systemic analysis was
reported including two non-RCTs (n = 129). In this analysis of women with elevated NK cell numbers
or activity, outcomes of IVIG treatment were sought. Individual reported outcomes of identified obser-
vational studies using IVIG and the results of pooled data had shown benefit of the respective interven-
tion with a relative risk (RR) of 3.41 (95% CI 1.90–6.11) for clinical pregnancy rates (65.3% for IVIG vs.
19.1% for controls), and an RR of 3.94 (95% CI 2.01–7.69) for live birth rates (58.7% for IVIG vs. 14.9%
for controls).20 This demonstrated that adjuvant therapies, such as IVIG, in women with elevated NK
cell numbers or cytotoxicities seem to confer some benefit on ART outcome. However, well-designed
RCTs with an appropriate population selection by the standardized NK testing are needed to verify
these findings.
Tacrolimus
Tacrolimus inhibits T cell activation by binding to the intracytoplasmic FK-binding protein. Once
the complex is formed, it inactivates calcineurin, which is calcium and calmodulin-dependent phos-
phatase and consequently inhibits transcription factors in activated T cells. Tacrolimus has been
used for the treatment of T cell-associated diseases, such as ulcerative colitis, eczema, atopic derma-
titis and vitiligo, and immunosuppression after transplantation.55 In a prospective cohort study of
42 patients with RIF and elevated peripheral blood Th1/Th2 cell ratios (CD4+IFN-γ+/CD4+IL-4+),
25 patients were treated with tacrolimus (treatment group) and 17 received no treatment (control
group). In the treatment group, tacrolimus was initiated two days before embryo transfer and con-
tinued until the day of the pregnancy test for 16 days. The clinical pregnancy rate of the treatment
group was 64.0%, which was significantly higher than that of the control group (0%) (p < 0.0001). In
the treatment group, the miscarriage rate was 6.3%, and the live birth rate was 60.0% (p < 0.0001).
There were no serious side effects from tacrolimus in the treatment group, and no women developed
obstetrical complications during pregnancy.56 In a recent study from the same investigators, patients
were divided into three groups: the low (CD4+IFN-γ+/CD4+IL-4+ less than 22.8), the middle (22.8 to
less than 28.8), and the high (28.8 or greater) Th1/Th2 groups, and the tacrolimus dose was adjusted
(1–3 mg/day) based on peripheral blood Th1/Th2 ratios (CD4+IFN-γ+/CD4+IL-4+). The clinical preg-
nancy rate of the tacrolimus treatment was 48.8% in the low, 43.9% in the middle, and 33.3% the
high groups, with similar results among these groups. However, the ongoing pregnancy/delivery
rate of the low Th1/Th2 group (46.3%) was significantly higher than that of the high Th1/Th2 group
(21.4%).57 In these trials, no significant side effects were reported. Adverse effects of tacrolimus were
demonstrated in a case study of 100 pregnancies of women with a history of organ transplantation,
in which the incidence of malformations was similar to that reported with other immune suppres-
sants in transplant recipients.58 Further clinical trials are required to verify these results.
Hydroxychloroquine
Hydroxychloroquine, an antimalarial drug, is often used for various autoimmune diseases, par-
ticularly systemic lupus erythematosus. Its use in pregnant patients with antiphospholipid anti-
body syndrome (APS) was reported to improve pregnancy outcomes.59 It has anti-inflammatory,
antithrombotic, and immunoregulatory properties and impairs complement-dependent antigen-
antibody reactions.60 Besides, hydroxychloroquine was reported to restore trophoblast fusion and
differentiation caused by aPL and diminished toll-like receptor 4 (TLR-4) expression.61 Those
immunomodulatory effects led to its use in patients with RIF or RPL; however, there is a lack of
Immunological treatment 49
robust clinical evidence to improve clinical outcome.49 One retrospective study has shown a 78%
live birth rate in a few refractory APS patients after add-on treatment with hydroxychloroquine.59
Hydroxychloroquine reversed the aPL-inhibition of trophoblast IL-6 secretion and partially lim-
ited aPL inhibition of cell migration.62 Therefore, hydroxychloroquine may be beneficial to RIF
patients, particularly with APS. Currently, the clinical data on the use of hydroxychloroquine in
RIF are lacking, and prospective studies are necessary.
TNF-α inhibitors
TNF-α inhibitors (adalimumab, infliximab, golimumab, etc.) are TNF-α blocking biologics to treat
diseases such as rheumatoid arthritis and inflammatory bowel disease. They interact and neutral-
ize TNF-α, which is produced by Th1 and NK cells and induces a cell-mediated immune response.
TNF-α inhibitors were reported to promote trophoblast invasion by reducing proinflammatory
immune responses at the maternal-fetal junction43 and have been used for RIF and RPL. There are
no randomized trials for the efficacy of adalimumab on pregnancy outcomes in patients with RIF,
but a few observational studies in IVF patients have been reported.63,64 In a study evaluating women
with a high TNF-α/IL-10 producing Th cell ratio undergoing IVF or ICSI, the implantation rates
were 59% (50/85), 47% (21/45), 31% (4/13), and 0% (0/9) for the treatment groups using IVIG + anti-
TNF-α, IVIG, anti-TNF-α, and no treatment, respectively. There was a significant improvement
in implantation, clinical pregnancy, and live birth rates for the treatment group with IVIG + anti-
TNF-α when compared with the no treatment group (p = 0.0007, 0.0009, and 0.003, respectively).64
TNF-α blockers can be safely used during the implantation period and pregnancy.65 The US Food
and Drug Administration (FDA) has categorized the medication as pregnancy category B after
evaluating the congenital anomaly rate in pregnant women exposed to the drug for the treatment
of rheumatoid arthritis and inflammatory bowel disease.49 Since data on the actual mechanism
of JAK-STAT pathways in inflammatory obstetric disorders, including embryo implantation, are
scarce, for the time being, therapeutic interventions in this setting should be carefully determined.
Intralipid solution
The infusion of intralipid, which is a sterile, nonpyrogenic fat emulsion, has been reported to improve
pregnancy outcomes in patients with RIF. A number of studies suggest that intralipid has immune
modulatory functions including suppression of NK cell cytotoxicities and proinflammatory cyto-
kine production.66 These studies indicated that intralipid could be a therapeutic option to modulate
abnormal NK cytotoxicity in women with reproductive failure.67 In a retrospective review of preg-
nancy outcomes of 200 women including 162 women with RIF and 38 with RPL, who had elevated
NK cell cytotoxicity, intralipid infusion treatment was as effective as IVIG treatment in the overall
live birth/ongoing pregnancy rate per cycle between women treated with IVIG (56%) and intralipid
(61%, p = NS).68 While there is potential for intralipid therapy as an economically affordable treat-
ment strategy to improve pregnancy outcome, substantial datasets are hard to come by, rendering it
difficult to make a firm recommendation.69 Although generally considered as a safe and easily acces-
sible treatment by clinicians, in 2015, Public Health England became aware of three women who
developed severe sepsis following administration of intravenous intralipid (20%), believed to be as a
result of poor practice in the administration leading to contamination of the product.
Granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is well known to play a significant role in a wide
variety of granulocytic and myelocytic proliferation. G-CSF has been reported to proliferate cho-
rionic villi, increase the Treg cell density, activate dendritic cells, and reduce cytotoxicity of NK
cells.70 With those perspectives, several studies using G-CSF treatment have been conducted. The
results were contradictory, and furthermore, the potential malignoma, congenital fetal malfor-
mation, and fetal hematologic abnormalities could not be excluded.71 In one study, acromegaly,
omphalocele, Down syndrome, Edward a syndrome, and Patau syndrome were observed in RIF
women treated with G-CSF.71 In a recent multicenter randomized controlled trial, 112 infertile
women with repeated IVF failure were evaluated. The efficacy of systemic single-dose subcutane-
ous G-CSF administration on IVF success in implantation rate (18% vs. 7.2%, p = 0.007), chemical
pregnancy (44.6% vs. 19.6%, p = 0.005), and clinical pregnancy (37.5% vs, 14.3%, p = 0.005) rates
was evaluated and shown to be significantly higher in the intervention group compared with the
controls.72 However, in another randomized placebo-controlled clinical trial, the success in the
G-CSF group was not statistically significantly different from the controls. Statistical models look-
ing at treatment effects on clinical pregnancy and implantation rates demonstrated no effect of
G-CSF treatment.3 In a randomized controlled study of normal IVF patients with normal endome-
trial thickness (n = 100), the intrauterine infusion of G-CSF did not improve pregnancy outcome.73
Endometrial scratching
Injuring the endometrium may enhance endometrial receptivity by modulating various local fac-
tors, including proinflammatory cytokines, in the endometrium. It has been proposed to increase
vascularity and lead to increased sustained implantation as well.49 Based on these findings, endome-
trial scratching was recommended in women with low endometrial CD56+ NK cells (<10), decreased
IL-15/Fn14 (<0.3) and IL-18/TWEAK (<0.02) gene expression ratios during a window of implan-
tation.32 In a study of 173 women with RIF undergoing FET cycle, the clinical pregnancy rate per
transfer was significantly higher in women with soft curettage to the endometrium twice (n = 38)
(42.1%, 16/38) compared with women without a curettage (22.2%, 20/90). The crude and adjusted ORs
were 2.55 and 2.49, respectively (95% CI 1.13–5.78, p = 0.03 and 1.01–6.17, p = 0.048, respectively).74
On the other hand, contradictory results were reported in a retrospective study with RIF patients
(n = 208). The clinical pregnancy rate in the subsequent ET cycle following the local endometrial
injury (n = 94) was not different from that of women without the local injury (n = 114).75 In women
with polycystic ovarian syndrome, the local endometrial injury was the most effective treatment to
improve the pregnancy outcome in women suffering RIF with uncompromised ovarian reserve.75
In a recent meta-analysis, the endometrial injury done between day seven of the previous cycle and
day seven of the ET cycle was reported to be associated with an improvement in the live birth and
clinical pregnancy rates in women with more than two failed previous embryo transfers (RR = 1.42,
p = 0.01).76 Inconsistencies in the literature remain, and there is a lack of robust evidence.77 In a recent
scientific impact paper, the Royal College of Obstetricians and Gynecologists suggests that available
evidence points toward a potential benefit of endometrial scratch in women with RIF when done in
the cycle preceding the IVF treatment cycle. However, further prospective randomized studies of suf-
ficient power are required to rule out the clinical value of local endometrial trauma.78
CONCLUSIONS
RIF remains one of the most enigmatic areas of ART, although many efforts have been made to explore
underlying etiologies. In most cases, routine infertility evaluations fail to identify the underlying eti-
ology of RIF. Immunological, thrombophilic, anatomical and genetic evaluations can be considered
for women with RIF. Treatment should be established based on its potential etiologies and focused on
improving the maternal environment to enhance embryonic implantation while optimizing embryo-
genesis. Since a variety of underlying etiologies are present, personalized therapeutic approaches should
be considered. Maternal interventions targeting dysregulated humoral and cellular immunity may
improve pregnancy outcomes in women with RIF. Immunological treatments, including corticosteroid,
IVIG, tacrolimus, hydroxychloroquine, TNF-α inhibitors, endometrial scratch, have been investigated
and reported to demonstrate some beneficial effect. Laboratory studies also have demonstrated the
pharmacological and immunological effects of these treatments in vitro. However, it has been a chal-
lenge to translate the laboratory findings to clinical medicine. Additionally, clinical data for the effi-
cacy of these treatments are limited, partly due to the difficulties in RCT for ART cycles and pregnant
women. Further studies with larger sample sizes and randomized approaches are needed in the future.
Until that time, patients should be thoroughly informed and counseled about the limitation of clinical
data prior to the treatment.
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papers/sip_54.pdff
Acquired uterine conditions,
reproductive surgery, and
7
DIMITRA AIVAZI, ELENI TSAKALIDOU,
and GRIGORIS F. GRIMBIZIS
INTRODUCTION
The uterus plays a key role in the achievement and evolution of pregnancy. Successful implantation
of an invading embryo relies on a normal functioning endometrium supported by a healthy myo-
metrial bed. Factors that might affect normal response to ovarian steroids, anatomy, vasculariza-
tion, and/or motility, altering uterine integrity and function, could act as “barriers” to the early and
late stages of implantation. This is even more significant nowadays, as a result of the trend to delay
parenthood in modern countries: a number of acquired conditions, not present when a woman is
young, are expected to be more frequent with increasing age.
In their presence, surgical treatment aiming to restore the reproductive potential of the patient
represents an important option; however, its application is dependent on the need for histological
documentation of the lesion’s benignity and on the efficacy of the treatment to restore normality.
The uterus consists of two major anatomical entities: myometrium and endometrium. Acquired
conditions of the myometrial layer are fibroids and adenomyosis. Furthermore, endometrial polyps
(including the adenomyotic ones), adhesions, and chronic endometritis represent acquired condi-
tions affecting the endometrium and endometrial cavity. All these uterine conditions might impair
the implantation process, altering the chain of events necessary for the embryo to be accepted and
invade the uterine partner.
The aim of this chapter is to critically evaluate the potential impact of each acquired condition on
implantation and the efficiency of surgical, preferably endoscopic, treatment to restore the repro-
ductive potential of the patient.
METHODOLOGICAL COMMENTS
The initial question for each acquired condition is if it has any association with recurrent
implantation failure (RIF). Apart from the fact that the definition of RIF is still unclear, the
existing evidence on the association between uterine conditions and RIF seems to be either
poor or absent. Thus, another way of examining the potential effect is to review the existing evi-
dence on implantation per se, as it is reasonable to conclude that if a condition impairs implan-
tation, it might be a cause of RIF. Furthermore, the achievement of pregnancy might be used as
an indirect parameter to assess the effect on implantation, as pregnancy is the end result of a
successful implantation. If any effect is present, the existing proposals on the potential biologi-
cal explanation will be reviewed.
Another important clinical dilemma, if impairment is present, is whether treatment could
restore fertility; a review of the existing evidence on that issue will also be provided. Finally, based
on the currently best available evidence, recommendations for the management of uterine acquired
conditions in patients with RIF will be provided.
55
56 Acquired uterine conditions, reproductive surgery, and recurrent implantation failure
FIBROIDS
Uterine fibroids are benign monoclonal tumors arising from smooth muscle myometrial cells.
Even though the prevalence of uterine fibroids among infertile women is high (26%), studies have
reported the presence of uterine fibroids in 12%–25% of women of reproductive age. However, their
true incidence and prevalence remain uncertain, due to the high rate of undiagnosed cases.1–3
Although not necessarily symptomatic, the clinical manifestations of fibroids as well as their
impact on reproductive outcome can vary widely. Different parameters, such as fibroid size and
number, can play a role in their clinical presentation, but it is generally accepted that the most
important parameter is their location in the myometrium and their anatomical relation to the
endometrium and endometrial cavity.
Thus, classical classification of fibroids includes grouping them into submucosal, intramu-
ral, and subserosal fibroids. However, for submucosal fibroids, a further subclassification for the
needs of surgical treatment has been introduced: those being totally intracavitary (type 0), those
being >50% intracavitary (type 1), and those being <50% intracavitary (type 2). Similarly, a more
detailed subclassification of the other types could be also clinically useful. Hence, the International
Federation of Gynaecology and Obstetrics (FIGO), in order to create a uniform classification sys-
tem, focused on the topography, suggested the classification of fibroids into seven types: types 0–2
include the existing submucosal categories based on the percentage of the lesion protruding into
the endometrial cavity; types 3 and 4 include fibroids with primarily intramural location, type 3
being those in contact with the endometrium but not distorting the cavity (involving the inner
myometrium) and type 4 being those lying in the outer myometrium; types 5–7 include subserosal
fibroids, which are further classified according to the proportion of their intramural component,
with type 7 being the pedunculated ones.4 It seems that this classification could be a useful tool in
evaluating clinical, especially reproductive, consequences.5,6
Evidence of association
Although there are not sufficient data in the literature about the correlation of fibroids with RIF,
there is indirect evidence, through their impact on in vitro fertilization (IVF) outcome, that they
are associated with an adverse effect on implantation and hence with RIF. However, as fibroids con-
stitute a heterogeneous entity, their impact on fertility varies according to the fibroid type and size.
Somigliana et al.7 meta-analyzed the available data from 13 studies on the effect of fibroids on the
probability of conception: submucosal types were found to be associated with a ∼70% decrease in
the odds of pregnancy (odds ratio [OR] 0.3, 95% CI 0.1–0.7) and delivery (OR 0.3, 95% CI 0.1–0.8)
rates, intramural ones with a ∼20% decrease in the odds of pregnancy (OR 0.8, 95% CI 0.6–0.9) and
∼30% decrease in delivery (OR 0.7, 95% CI 0.5–0.8) rates. On the other hand, subserosal types had
no effect on the achievement of pregnancy. In a subsequent systematic review and meta-analysis of
18 case-control studies, Pritts et al.8 further confirmed these findings. Submucosal fibroids reduce
the relative probability of pregnancy up to ∼60% (relative risk [RR] 0.36, 95% CI 0.18–0.74) and
those not distorting the intrauterine cavity up to ∼20% (RR 0.78, 95% CI 0.69–0.88). Focusing on
intramural fibroids, they found a significant decrease in clinical pregnancy (RR 0.81, 95% CI 0.70–
0.94) and live birth (RR 0.70, 95% CI 0.58–0.85) rates; interestingly, data from prospective only
studies showed impaired implantation (RR 0.55, 95% CI 0.39–0.78) and live birth (RR 0.46, 95% CI
0.29–0.74) rates. Subserosal fibroids had no impact on the achievement and evolution of pregnancy.
As the detrimental effect of submucosal fibroids and the absence of any effect of the subsero-
sal ones were considered obvious, Sunkara et al.9 conducted another meta-analysis focusing on
the “gray” zone of intramural fibroids. They included 14 retrospective and five prospective case-
control studies having IVF patients with intramural fibroids ranging between 0.7 and 0.5 cm. They
concluded that even though intramural fibroids do not protrude in the intrauterine cavity, they
have a detrimental effect on IVF outcomes, as they decrease the relative probability of pregnancy
by 15% (RR 0.85, 95% CI 0.77–0.94) and live birth rate up to 20% (RR 0.79, 95% CI 0.70–0.88);
the detrimental effect on the achievement of pregnancy was more pronounced when only the five
Fibroids 57
prospective studies were analyzed, with a reduction of the relative probability of live birth rates
up to 40% (RR 0.60, 95% CI 0.41–0.87). However, this study did not evaluate whether the different
subgroups of intramural myomas have a different effect on implantation.9
It should be noted that other potentially important parameters of intramural fibroids, such as
their size, number, and their location in relation to the endometrium, were not taken into account
in all those meta-analyses. Rarely the mean or median fibroid diameter is above 3 cm in studies on
IVF outcome and fibroids.7,9 Actually, the general empirical policy of IVF specialists is to recom-
mend surgical excision of lesions exceeding 5 cm in diameter; thus, the detrimental effect of intra-
mural fibroids that emerges from studies investigating IVF outcomes might be an underestimation
of their true impact.
On the other hand, Oliveira et al.,10 examining the impact of intramural fibroid size on IVF preg-
nancy rates, found that intramural fibroids >4 cm had significantly lower pregnancy rates than
those <4 cm, supporting the notion that size is a potential critical parameter. Furthermore, Yan
et al.11 found significantly lower live birth rates (adjusted OR 0.86, 95% CI 0.74–0.99) in patients
with intramural fibroids with >2.85 cm in their largest diameter when compared with matched
controls without fibroids. Christopoulos et al.,12 in an observational study, found that IVF patients
with myomas not distorting the endometrial cavity had significantly lower live birth rates only if
two or more fibroids were present (OR 0.47, 95% CI 0.26–0.83) and if the lesion was ≥3 cm in diam-
eter in size (OR 0.41, 95% CI 0.19–0.89); no difference was observed in patients with single fibroids
of <30 mm in diameter.
It seems, therefore, that submucosal fibroids of any type negatively affect IVF outcome and
implantation. Intramural fibroids also have a negative impact, especially if there are more than one
and larger than 3–4 cm in diameter; their effect in relation to their proximity to the endometrial
cavity (FIGO type 3 and 4) has still not been investigated. Subserosal fibroids of reasonable size
have no impact on implantation and IVF outcome.
Potential mechanisms/biological explanation of the effect

Several mechanisms have been proposed to explain the negative effect of fibroids on implantation
and fertility. The distortion of normal architecture of the intrauterine cavity has been implicated
in poor implantation outcomes. Additionally, functional changes such as impaired vascularization
and increased contractility result in a hostile environment for blastocysts and impair their invasion
into the endometrium. Thus, histological examination of endometrium reveals gland atrophy and
chronic inflammation, possibly reflecting an endocrine dysregulation and alterations of the local
secretion of inflammatory and vasoactive substances.
Moreover, even though gene expression profiling of leiomyomas revealed upregulation and
downregulation of genes affecting cellular differentiation and proliferation, such as TGF-β3, only a
few alterations were found in genes related to the window of implantation.13–15
Treatment results
Taking into consideration that myomectomy is associated with myometrial (intramural) or endo-
metrial trauma (submucosal), followed by a healing process, whether myomectomy is able to restore
fertility is another important clinical question. The reported pooled spontaneous pregnancy rates
post laparoscopic/abdominal myomectomy in published comprehensive reviews ranged from 49%
(95% CI 46–52)16 to 57% (95% CI 48–65),7 whereas in women with otherwise unexplained infertil-
ity, the pregnancy rate was 61% (95% CI 51–70)7; furthermore, the pooled pregnancy rates post
hysteroscopic myomectomy were 45% (95% CI 40–50).16
However, these were data from observational noncomparative studies; comparative studies
are rare and randomized controlled trials (RCTs) are still an exception. Bulletti et al.17 compared
patients with fibroids who underwent laparoscopic excision (group 1) with patients who were not
treated (group 2) and patients with unexplained infertility without fibroids (group 3). The group of
laparoscopically treated patients had significantly higher delivery rates (42%) than the untreated
group (11%, p < 0.001) and the group without fibroids (25%, p < 0.001). In a subsequent study, the
same investigators allocated IVF patients with at least one intramural-subserosal fibroid >5 cm
to myomectomy or expectant management and found that women who underwent surgery had
higher pregnancy rates than those who did not (25% vs. 12%, respectively; p < 0.01).18 Despite the
absence of randomization, the beneficial effect of fibroid excision appears to be obvious.
In their prospective study, Casini et al.19 examined the pregnancy rates in women with fibroids
who underwent laparoscopic and/or hysteroscopic myomectomy, compared with those who did
not. Women with hysteroscopically treated submucosal fibroids had significantly higher preg-
nancy rates than those who did not undergo treatment (43.3% vs. 27.2%, respectively; p < 0.05);
also, patients with laparoscopically treated submucosal/intramural myomas had significantly
higher pregnancy rates than those who did not undergo treatment (36.4% vs. 15%, respectively;
p < 0.05). This study, obviously, provides good quality evidence on the beneficial effect of surgical
treatment of fibroids. Furthermore, three different groups of researchers reported similar delivery
rates in IVF patients with submucosal fibroids after hysteroscopic myomectomy compared with
controls without fibroids; this indicates that the negative effect of fibroids on IVF is alleviated by
surgery,7,18,19 or at least that myomectomy does not seem to negatively affect the chances of concep-
tion in IVF cycles.
Pritts et al.,8 in their systematic review, found significantly higher pregnancy rates after surgi-
cal excision of submucosal fibroids, although they failed to demonstrate a beneficial effect after
excision of intramural ones due, apparently, to scarcity of data. In a Cochrane systematic review,
Metwally et al.20 reported that the existing RCTs assessing the effect of myomectomy on fertil-
ity did not provide sufficient evidence due to the very low number of studies. On the other hand,
laparoscopic and abdominal myomectomy have the same results on fertility restoration but the
laparoscopic approach has faster postoperative recovery and lower morbidity.21,22
ADENOMYOSIS
Adenomyosis is an acquired, benign gynecological condition of the myometrium. It is character-
ized by the presence of endometrial glands and stroma within the myometrium. The neighboring
muscle cells respond to this intrusion with hypertrophy and hyperplasia. It can be either diffuse,
where endometrial foci are scattered throughout the myometrium or focal, taking the form of
adenomyoma (defined as a circumscribed nodule within the myometrium) or adenomyotic cyst,
covering different zones of the myometrium (inner or outer myometrium). Histologically, it could
range from mostly solid to mostly cystic.
Although histology is the gold standard of diagnosis, as this is mainly based on hysterectomy
specimens, it could not be used for the diagnosis of the disease in infertile patients. Currently, two
noninvasive imaging techniques have proved to be highly accurate for the diagnosis of adenomyo-
sis: ultrasound and magnetic resonance imaging (MRI). Campaneria et al., 22 in their systematic
review, found that ultrasound has a pooled sensitivity of 0.72 (95% CI 0.65–0.79), specificity of 0.81
(95% CI 0.77–0.85), and an area under the curve (AUC) of 0.85. In a more recent review, Andres
et al.23 found that two-dimensional ultrasound (2D US) had a pooled sensitivity of 0.84 (95% CI
0.79–0.86) and a specificity of 0.64 (95% CI 0.64–0.69), whereas 3D US had a pooled sensitivity of
0.85 (95% CI 0.76–0.91) and a specificity of 0.81 (95% CI 0.73–0.88). Considering MRI, Campaneria
et al.22 found a pooled sensitivity of 0.77 (95% CI 0.67–0.85), specificity of 0.89 (95% CI 0.84–0.92),
and an AUC of 0.93. An additional advantage of MRI is its high positive predictive value24 and its
excellent correlation with histological findings.25 Thus, diagnosis of adenomyosis could be based on
the noninvasive ultrasound and MRI evaluation of the uterus.
The recognized clinical manifestations of adenomyosis include abnormal uterine bleeding and pel-
vic pain (dysmenorrhea and/or chronic pelvic pain). The presence of adenomyosis is found to be
associated with infertility and impaired implantation. By considering the pregnancy rate as a means
Adenomyosis 59
to evaluate implantation, Vercellini et al.,26 in a systematic review, examined the impact of adeno-
myosis on IVF results. Either ultrasound or MRI was used to diagnose adenomyosis. They found
that patients with adenomyosis had significantly lower clinical pregnancy rates (RR 0.72, 95% CI
0.55–0.95), although the study was unable to detect a difference in implantation rates. Furthermore,
a limitation of the results reported was the significant heterogeneity among the studies.
Younes and Tulandi,27 in a recent meta-analysis, also examined the impact of adenomyosis on
IVF outcome including, additionally, the effect on implantation; two more studies were included,
one of them being prospective and well designed. They found that patients with adenomyosis have
significantly lower pregnancy (OR 0.73, 95% CI 0.60–0.90) and implantation (OR 0.66, 95% CI 0.49–
0.88) rates compared with those without. They also observed that patients with diffuse adenomyosis
have a tendency for lower pregnancy rates than those with focal disease (OR 1.36, 95% CI 0.67–2.75).
In another study, Mavrelos et al.28 found that IVF patients with ultrasound findings of adenomyo-
sis had significantly decreased clinical pregnancy rates (29.2% vs, 42.6%; p = 0.044; OR 0.68, 95% CI
0.47–1.00) and that the presence of ≥4 ultrasound features was an independent predictor of clinical
pregnancy (OR 0.35, 95% CI 0.15–0.82) as compared with those with no adenomyosis features.
It seems, therefore, that the presence of adenomyosis is a significant factor affecting implantation
and the more severe the disease, the higher the possibility of decreased pregnancy rates.

The biological explanation of the potentially impaired implantation linking adenomyosis with
infertility and decreased pregnancy rate after IVF is not very clear. An abnormal inflammatory
reaction, as indicated by the observed increased density of stromal macrophages, being the result
of myometrial infiltration by endometrial glands as well as the elevated levels of nitrogen and oxy-
gen-free radicals accumulating in the endometrial cavity of women with adenomyosis has been
proposed as potential pathophysiological explanations.29–31 Other biological interpretations for this
effect include local hyperestrogenism changing the estrogen/progesterone balance in the secre-
tory phase and the density of endometrial capillaries; defective expression of implantation markers
and defective decidualization through overexpression of P450 aromatase; hypervascularization;32
impaired utero-tubal transport; and altered uterine contractility due to the destruction of the nor-
mal architecture of the inner and/or outer myometrium.33
Treatment results
The gold standard treatment for symptomatic adenomyosis is hysterectomy, but in cases of women
desiring to preserve their fertility, this is obviously not an option. Uterine-sparing alternatives in the
management of patients with adenomyosis include medical maneuvers aiming to control or tempo-
rarily regress the disease process and surgical solutions aiming to excise the diseased myometrium.
Obviously, from the different medical therapies proposed up until now (oral contraceptive pills,
selective progesterone receptor modulators, progestins, aromatase inhibitors, levonorgestrel-
releasing intrauterine device, danazol, etc.), gonadotropin-releasing hormone agonists (GnRHa)
are the most appropriate for the management of infertile patients with adenomyosis. Vercellini
et al., 26 in their systematic review, reported that patients with adenomyosis who stimulated for
IVF with the long GnRHa protocol had no difference in pregnancy rates compared with those
without the disease (RR 1.05, 95% CI 0.75–1.48) and those stimulated with the short GnRHa
protocol had significantly lower pregnancy rates (RR 0.58, 95% CI 0.38–0.88). In a more recent
systematic review, which included all the meanwhile published studies, Rocha et al. 34 supported
those findings: IVF/intracytoplasmic sperm injection (ICSI) patients with adenomyosis, stimu-
lated with the long GnRHa protocol, when compared with those stimulated with the short GnRHa
protocol, had higher pooled clinical pregnancy (43.3% vs. 31.8%, respectively; p < 0.001) and live
birth/ongoing pregnancy (43% vs. 23.1%; p < 0.05) rates. Based on that, an extra-long protocol has
been proposed for patients with adenomyosis:35 freezing all embryos from the stimulated cycle
and frozen embryo transfer after downregulation with GnRHa up to three months.
Adenomyomectomy represents the conservative surgical therapeutic option for the treatment of
infertility in patients with adenomyosis.36 Due to disease characteristics (infiltration of the myo-
metrium by adenomyotic tissue), any surgical procedure always excises myometrial tissue as well;
thus, adenomyosis surgery is limited by the need to preserve as much as feasible of the myometrial
layer and to avoid defective myometrial reconstruction.36,37 It is noteworthy to mention that surgi-
cal treatment is quite demanding and the risk of uterine rupture in a subsequent pregnancy should
always be taken into account.36,37 At first, Rocha et al.,34 in their systematic review, reported an
overall pooled clinical pregnancy rate after adenomyomectomy of 38.8% and a pooled live birth
rate of 30.4%, both spontaneous and after assisted reproductive technology (ART). However, the
pooled spontaneous clinical pregnancy rate was low (18.2%), explained potentially by mechanical
reasons such as tubal patency. A high proportion of patients needed ART after surgery to achieve
pregnancy. The use of GnRHa after surgery resulted in a significantly higher spontaneous concep-
tion rate and seems, also, to have a beneficial effect on the results of surgery and ART.27,34 It has
also been supported that in infertile patients, the combination of surgery and GnRHa gives better
results than GnRHa administration alone (OR 6.22, 95% CI 2.34–16.54).27
It seems, therefore, that GnRHa might offer a window of improved implantation by temporary
regression of adenomyosis, thus, exerting a beneficial effect on IVF outcome;27,34,38 this might be
the result of the decrease in the size of adenomyotic lesions39 and the positive effect on endometrial
implantations markers.40 Obviously, any beneficial effect of surgical treatment is due to the reduc-
tion of adenomyotic foci and their impact on implantation.34,38
ENDOMETRIAL POLYPS
Endometrial polyps (EPs) are focal, benign overgrowths of endometrium consisting of endometrial
glands, stroma, and blood vessels. Their prevalence in the general female population is estimated
near 10%.41 However, it is reported to be significantly higher in subfertile women undergoing IVF,
approximately 30%, although there is a wide discrepancy in the reported prevalence among dif-
ferent studies, which can be explained by the diversity of diagnostic means used.42–44 Similarly, EP
prevalence appears to be higher in women with recurrent pregnancy loss (RPL), indicating a pos-
sible negative effect of EP on reproductive outcome.45 Furthermore, as there is a causative relation-
ship between estrogen effect and EP growth, the incidence of EPs is higher in obese women and, as
recently shown, in those suffering from endometriosis.44,46
Their most common clinical manifestation is abnormal uterine bleeding, although EPs are often
diagnosed in asymptomatic women during a routine ultrasound examination.47 Their surface, pro-
jecting from functional endometrium, is often asynchronous to the normal and provokes abnormal
uterine bleeding, possibly as a result of disproportion in the expression of estrogen/progesterone
receptors.48–50 Additionally, atypical necrosis of a polyp may be another cause for intermenstrual
bleeding.51
Due to the lack of high-quality prospective studies in the field of IVF in women with endometrial
polyps, the hypothesis of their unfavorable effect on implantation is currently based on studies
comparing IVF outcome in patients with and without polyps. Tiras et al.52 studied IVF patients with
untreated endometrial polyps <14 mm diagnosed during ovarian stimulation and a control group
of IVF patients with normal cavity; they found similar pregnancy rates in both groups, suggesting
that the presence of polyps does not affect implantation. On the other hand, Yang et al.53 compared
IVF patients with endometrial polyps diagnosed incidentally during ovarian stimulation with age-
matched IVF patients having normal cavity. They observed significantly higher clinical pregnancy
rates after hysteroscopic polypectomy followed by frozen embryo transfer than in normal controls
followed by fresh embryo transfer (63% vs. 41%, p = 0.009), indicating that the presence of polyps
interferes with the achievement of pregnancy and potentially implantation despite the obvious bias
Endometrial polyps 61
in the embryo transfer policy. Furthermore, the achievement of pregnancy was similar 1–3 months
postpolypectomy. Elias et al.,54 although they failed to find any difference between IVF patients
having polyps compared to those with normal cavity, observed significantly higher biochemical
pregnancy rates indicating that polyps could interfere with the evolution of pregnancy.
Nevertheless, the role of EP in RIF is indirectly shown by the fact that uterine abnormalities in
women submitted to their first ART attempt range between 11% and 22%,43 but it is significantly
higher in RIF patients reaching 45%, with the most frequently detected defect being EP,55 suggest-
ing a potential correlation between EP and RIF.

The biological explanation of the potential unfavorable effect of EP on reproductive outcome is not
completely understood; hence there are different possible mechanisms that have been proposed.
Mechanical interference affecting sperm transportation, as well as embryo implantation, is in
line with the hypothesis that the location of EP may influence pregnancy rate; Yanaihara et al.
showed that removal of EP located near the utero-tubal junction provides the best results on fertil-
ity compared with other locations.52
Another alteration that may explain the effect of EP on implantation is the elevated levels of gly-
codelin, a glycoprotein that inhibits natural killer (NK) cell activity and sperm-oocyte binding. In
normal ovulatory endometrium, low levels of glycodelin, six days before until five days after ovula-
tion, permit fertilization followed by a rise in order to suppress NK cells and facilitate implantation.
Elevated levels of glycodelin may modify endometrial receptivity when EP are present.53
In addition to glycodelin, alteration in expression of HOXA10 and HOXA11 genes that has been
described in the presence of endometrial polyps may play a vital role in endometrial receptivity.54
Local inflammatory mediators, as well as other factors such as IGFBP-1 have also been proposed to
impair implantation in EP.55,56
Treatment results
Hysteroscopy remains the gold standard for the diagnosis as well as the treatment of endometrial
polyps. Given that the risk of complications after hysteroscopic polyp removal (adhesion forma-
tion, polyp recurrence, and decrease on endometrial thickness due to electrosurgery) is negligible,
a detrimental effect on endometrial receptivity is not expected by hysteroscopy per se.57–59
Looking to the effect of treatment on natural conception, Varasteh et al. found that patients sub-
mitted to polypectomy have significantly higher spontaneous pregnancy rates than those who did
not (78.3% vs. 42.1%, p < 0.05).45 Furthermore, other groups examined the effect of hysteroscopic
polypectomy on clinical pregnancy rates after four cycles of intrauterine insemination (IUI) as
compared to expectant management. Perez-Medina et al., in a well-designed RCT, found 63.4%
cumulative pregnancy rates in the treated group and 28.2% in the expectant management group
(p < 0.05).60 Similarly, Shohayeb and Shaltout observed 41.7% cumulative pregnancy rates in the
treated group and 20% in the expectant management group (p < 0.05), confirming the beneficial
effect of hysteroscopic polypectomy in the achievement of pregnancy.61 In a subsequent study,
Kalampokas et al. also found statistically significant differences in cumulative pregnancy rates
after IUI, favoring hysteroscopic polypectomy (40.7% vs. 22.3%, p < 0.05).62 The change of the
endometrial environment required for embryo implantation seems to be the primary mechanism
of this effect. The positive effect of hysteroscopic polypectomy on IUI results was confirmed in a
Cochrane Review63 based on the results of the Perez-Medina et al. study.60
On the other hand, the results of hysteroscopic removal of endometrial polyps in IVF patients
remain controversial. Lass et al. found 28.6% ongoing pregnancy rates after hysteroscopic removal
of polyps >2 cm compared with 16.3% after expectant management (p = NS).64 Furthermore,
Isicoglu et al.65 and Tiras et al.66 failed to find any difference in the clinical pregnancy rates whether
or not endometrial polyps <1 and 1.5 cm, respectively, were removed. Furthermore, Ghaffari
et al., also, found similar implantation, clinical pregnancy, and live birth rates in IVF patients who
underwent hysteroscopic polypectomy during ovarian stimulation compared with those who did
not.67 But, the absence of any difference should be considered only as the result of treatment during
the same cycle; recovery of endometrium from hysteroscopic trauma needs at least a period of five
days, since in the same publication, no pregnancy was achieved if the interval between treatment
and embryo transfer was shorter. The positive effect could be obtained from the next cycle on; the
achievement of pregnancy is similar 1–3 months postpolypectomy.68
It seems therefore that, although the role of polypectomy in women undergoing IVF remains
unclear, its beneficial result in natural conception and in IUI is undisputable. Furthermore,
Mouhayar et al.111 found that hysteroscopic removal of polyps when performed in IUI and IVF
patients is cost effective, justifying its application also in IVF patients even in the absence of hard
evidence.
INTRAUTERINE ADHESIONS
Intrauterine adhesions (IUA) are defined as the partial replacement of endometrium with fibrotic
tissue together with adherence of the opposite sites.69,70 Although the term Asherman syndrome
(AS) is often used as a synonym of IUA, AS includes those patients with IUA clinically manifested
with amenorrhea, hypomenorrhea, dysmenorrhea, and recently proposed to include infertility,
RPL, and abnormal placentation.69
Adhesion formation might be the result of fibrosis following endometrial injury; infection at the
time of injury is an augmenting factor as well, shifting the process from re-coverage of the trauma
site with endometrium to a healing process with fibrotic tissue. The causes of IUAs and AS may be
classified in two groups. The first one includes those following a gravid condition such as postabor-
tion or postpartum curettage, miscarriage per se without any invasive procedure, postpartum and
postabortion endometritis, and endometrial ischemia, as a consequence of postpartum hemor-
rhage or uterine artery embolization. The second one involves those caused by a nongravid condi-
tion such as surgical hysteroscopic procedures and infections, mainly tuberculosis of the female
reproductive tract.71
Their reported prevalence varies widely depending on the population studied, the diagnostic
procedure and the classification method used.69 Although various classification systems have been
suggested,69,72–77 further validation is needed in order to define their clinical utility.
Although the effect of IUAs and AS on spontaneous pregnancy is very well documented, data
regarding ART results in this group of patients are insufficient; thus, there is mainly indirect evi-
dence that they have a detrimental effect on the achievement of pregnancy in patients undergoing
ART.
Demirol and Gurgan found that IUAs occurred in 8.5% of women with RIF, indicating that
their presence is related to impaired implantation.78 Schenker and Margalioth found that almost
two-thirds of women with AS are infertile, whereas only 45% of patients with IUAs achieved a
pregnancy, but 40% of them have ended in abortion.79 Moreover, 23% of these pregnancies have
been complicated by preterm deliveries and 13% with placenta accreta. Acunzo et al. reported as
low as 18.3% spontaneous live birth rates in patients with IUAs who did not have adhesiolysis,80
and Roy et al. suggested that there is an association between conception rates and the severity of
adhesions.81
The incidence of IUAs among ART patients reaches 16%, suggesting a link between adhesions
and subfertility,82 although others support that it is more possible that the underlying cause of
adhesions also result in subfertility, rather than IUAs as an independent factor.83

Apart from the mechanical obliteration of cervical canal and tubal ostia, the unfavorable effect
of IUAs on fertility may be explained by the replacement of normal endometrium by fibrotic
Chronic endometritis 63
tissue and, hence, although fertilization occurs, endometrium appears to be hostile for implan-
tation. Additionally, impaired vascularization of the residual endometrial tissue and reduction
of progesterone receptors decrease the chance of pregnancy and increase the risk of miscar-
riage.79,84 It is, also, suggested that IUAs interfere with the early stages of implantation, not allow-
ing embryos to attach to the luminal surface of endometrium, and consequently, if present, may
play a key role in RIF.
Treatment results
Although the endpoint of surgical treatment is the restoration of an anatomically normal cavity,
from a functional perspective, it is the reversal of the presenting symptoms—in cases of abnormal
menstruation, to achieve normal menses and in cases of infertility, to achieve a normal functioning
endometrium with a normal implantation potential. It is obvious that, even in cases with a suc-
cessful anatomically restored cavity, the extent of endometrial fibrosis and the level of endometrial
regeneration play key roles in the functional result. Another factor determining the success is also
adhesion recurrence.
Successful anatomical restoration ranges from 44% to 95%.57,71,85–88 However, the success rate is
highly dependent on the severity of adhesions: the more severe the adhesions the lower the chances
of achieving an anatomically normal cavity. Also, adhesion reformation varied widely from 3.5% to
20% for mild ones to almost 60% for severe cases, indicating a significant dependence on the adhe-
sion’s severity and the extent of fibrosis.57,71,73,88 In this respect, referring severe cases for treatment
to centers with expertise might improve the results.71
Concerning reproductive outcome, Acunzo et al. reported that hysteroscopic adhesiolysis could
significantly increase spontaneous live birth rates from 18.3% to 68.6%.80 Similarly, Pace et al., in
women with AS, observed an increase in spontaneous pregnancy rates from 28.7% before to 53.6%
after hysteroscopic surgery.87 Yu et al.,69 in their review, reported a pooled pregnancy rate, both
spontaneous and after ART, of 74% after hysteroscopic treatment of IUA in women wishing preg-
nancy, much higher than the 46% observed in the nontreated cases; however, the pooled conception
rates in infertile treated women was found to be ∼46% and for severe treated cases, the pregnancy
rate was consistently lower, about 33%. Chen et al.,85 in infertile patients with IUA, found an overall
spontaneous pregnancy rate after treatment of 48.2% with a mean time from treatment to concep-
tion of 9.7 ± 3.7 months; pregnancy rates decreased with IUA severity: 60.7% in mild, 53.4% in
moderate, and 25% in severe cases of IUA. Although the existing evidence is not strong, it seems
that hysteroscopic treatment offers better chances of conception in infertile patients, and this is
inversely related to the degree of the existing endometrial damage and fibrosis.
Regarding prevention of IUA reformation, various methods have been suggested and appear to
improve reproductive outcome. Recently, the American Association of Gynecologic Laparoscopists
(AAGL) in collaboration with the European Society for Gynaecological Endoscopy (ESGE) pub-
lished guidelines for prevention of IUA formation.89 It seems that posthysteroscopically, the use of
mechanical (stent or heart-shaped balloons) and semisolid barriers (e.g., hyaluronic acid) as well
as hormonal treatment (estrogens with or without progestin) might reduce recurrence of IUAs.89
Intrauterine devices (IUD) that contain progestin or copper should not be used; if any is used, it
should be inert with a large surface area.89 Stem cell treatment at the moment should not be offered
outside of rigorous research protocols.
CHRONIC ENDOMETRITIS
Chronic endometritis (CE), the chronic inflammation of the endometrial lining, reflects a loss of
balance between the colonization of endometrium by microorganisms and the reaction of the local
immune system, which leads to anatomic and functional alterations and, eventually, to an unfa-
vorable effect upon fertility.90 The clinical manifestation of CE is usually subtle; the majority of
patients are asymptomatic, or they may present with abnormal uterine bleeding, pelvic pain, and/
or dyspareunia and leukorrhea.91
Hysteroscopic findings suggestive of CE are micropolyps, stromal edema, hyperemia, and straw-
berry pattern of endometrium. Although the overall hysteroscopic accuracy seems to be satisfactory
with a high negative predictive value, the positive hysteroscopic findings should always be comple-
mented by endometrial biopsy, which confirms the diagnosis in 45%–90% of cases.92,93 Infiltration of
endometrial stroma by plasma cells in endometrial biopsy specimens is considered fundamental for
the establishment of diagnosis;94 immunohistochemical detection of CD38 and CD138 plasma cells
seems to provide higher sensitivity than the conventional hematoxylin-eosin staining.95
The first suggestion of an association between CE and infertility was reported in 1978 by
Czernobilsky.96 The reported prevalence of histologically confirmed CE in asymptomatic infertile
patients before their first IVF attempt ranged between 3%97 and 15%,90 whereas a higher preva-
lence was found in patients with RIF, ranging between 15% and 60%.90,93,98,99 The prevalence of
CE reported by Romero et al. was as high as 42% in RIF patients undergoing IVF,90 only 15% in
infertile women, and even lower in the general population.94,100 Moreover, Conway et al. found this
incidence even greater, above 60%.99
The observed differences between the prevalence of CE in the general infertile population and in
RIF patients indicates a potential unfavorable effect of this condition on implantation. However, more
data are needed to elucidate the exact role of CE on implantation and the work-up for its diagnosis.

In the majority of cases, common bacteria were found in endometrial cultures of patients with CE:
gram-negative microorganisms in ∼60% and Ureaplasma urealyticum in ∼10%. Chlamydia was
detected in only 2.7% of cases, while genital tuberculosis could be responsible for CE in developing
countries and in special groups of a population (e.g., immune-suppressed women). Those microor-
ganisms, ascending from the lower genital tract, colonize the endometrial cavity and stimulate the
immune system’s reaction in order to restrict bacterial proliferation, such as formation of mucus
plug and activation of epithelial cells, as well as neutrophils, macrophages, and NK cells. Bacteria
form biofilms to resist the immune mechanisms causing chronic inflammation, secretion of cyto-
kines, and further attraction of NK cells.92,101–105
The disturbance of the normal balance of the paracrine milieu in case of CE could create a hostile
environment for blastocyst invasion and successful implantation.106
In addition to infection, gene reprofiling with upregulation of IGFBP1 and downregulation of
IGF1, as well as alteration of the expression of genes mediating the inflammatory process, could
have a detrimental effect on successful implantation.107
Treatment results
Since microorganism infection is the basis of CE, antibiotic treatment is considered fundamental.
Targeted therapy based on endometrial cultures and antibiogram should be given; in the absence
of cultures or negative cultures, blind therapy to cover potential chlamydia and mycoplasma infec-
tions, which are difficult to be isolated and cultured, should be given. Doxycycline as monotherapy,
as well as ofloxacin in combination with metronidazole, has been found to attenuate the effect of
CE on infertility. Doxycycline at a dose of 100 mg twice daily for 14 days had a beneficial result in
70% of patients with CE and a reported clearance of CD138 plasma cells in 96% of RIF patients.98,108
Combined therapy with ofloxacin 400 mg and metronidazole 500 mg twice daily for 14 days
resulted in improvement of histopathological findings in 73% of patients.109 Alternatively, a five-
day course of azithromycin at a dose of 500 mg orally on the first day and 250 mg from the second
to the fifth days, is found to be effective for patients allergic to doxycycline.98,110
Nevertheless, although histopathological and hysteroscopic findings could be cured with anti-
biotic therapy, successful pregnancy as a requested benefit remains the most vital concern of clini-
cians. In the category of patients with RPL having CE, Cicinelli et al. reported spontaneous clinical
Conclusions and current practice recommendations 65
pregnancy rates as high as 74.8% one year after antibiotic therapy as compared with only 24.4% in
the control group of untreated women,92 and McQueen et al., in their prospective study, found an
increase in spontaneous live birth rate per pregnancy from 7% before to 56% after antibiotic treat-
ment.95 However, despite the impressive results in RPL patients, clinical benefit from antibiotic
treatment in women with CE and RIF remains unclear. Thus, in a retrospective study, although
implantation rates increase after therapy, this increase was not statistically significant,100 while
Yang et al. found a significantly higher implantation rate after antibiotic treatment in patients with
RIF and CE diagnosed by hysteroscopy.68 It is also proposed that in this group of patients, hys-
teroscopic removal of bacterial biofilms could improve reproductive outcome, but there is not yet
enough evidence to support this hypothesis.35
CONCLUSIONS AND CURRENT PRACTICE RECOMMENDATIONS

From the reviewed evidence it seems that the uterus plays a significant role in implantation and that
acquired conditions of the myometrium and endometrial cavity could impair implantation poten-
tial of the invading embryo. Although the existing evidence is not always of high quality and there
is still place for further research in this field, it seems that surgery has a place in restoring normality
and improving the chances for increasing implantation rates and the achievement of pregnancy in
ART patients with RIF.
Based on the currently existing evidence and despite the lack of absolute agreement, the follow-
ing best clinical practice points could be recommended:
Myomas
• Submucosal fibroids negatively affect implantation and the achievement of pregnancy; their
hysteroscopic excision improves the probability of successful pregnancy and clinicians should
offer this option.
• Intramural fibroids not distorting endometrial cavity could negatively impair IVF outcome,
especially if they are more than one and/or larger than 3–4 cm in diameter; clinicians should
consider myomectomy, especially for patients with implantation failures; laparoscopic exci-
sion, if feasible, appears to be the preferred surgical approach.
• Subserosal fibroids do not affect fertility; thus, myomectomy does not offer any beneficial effect
and should be performed only as a treatment for other clinical manifestations arising from
fibroid size and location, or in the case of simultaneous surgical excision of another fibroid.
Adenomyosis
• There is still no general consensus on how significant the impact is of adenomyosis on a wom-
an’s implantation potential, if there is any difference between its different varieties and forms,
which is the best treatment approach, and especially which patient is the best candidate for
which treatment and what are the consequences to a future pregnancy.
• Infertile patients with adenomyosis could be stimulated for IVF with the long or extra-long
GnRHa protocol by having a frozen embryo transfer after downregulation for three months
before hormonal preparation of the endometrium.
• Adenomyomectomy could be offered as an alternative after implantation failures, mainly for
focal disease, after careful estimation of the risks and benefits in experienced centers. GnRHa
treatment after surgery seems to have an added value both for spontaneous conception and
IVF outcome.
Polyps
• Hysteroscopic polypectomy could be offered as an option prior to ART based on the clinical
benefits on fertility, the insignificant risk of complications, and the cost-effectiveness of this
option.
• In women diagnosed with endometrial polyps incidentally during ovarian stimulation, embryo
cryopreservation and frozen embryo transfer after polypectomy could be recommended.
Intrauterine adhesions
• Hysteroscopy and hysteroscopic adhesiolysis, current “gold standards” for diagnosis and treat-
ment of IUA, should be offered to women undergoing IVF presenting with the clinical features
and, even more significantly, to patients with RIF.
• After surgery, intrauterine placement of mechanical and semiliquid barriers might prevent ref-
ormation of adhesions while estrogen therapy might enhance the regeneration of endometrium.
Chronic endometritis
• In patients with histologically confirmed CE after hysteroscopic-guided biopsy, targeted ther-
apy, based on antibiogram when the etiology is known, should be given.
• In patients with hysteroscopic findings of CE with negative or no culture, a blind therapy with
doxycycline or ofloxacin and metronidazole should be administered.
• Treatment should be completed by reevaluation of uterine cavity using hysteroscopy and endo-
metrial sampling to confirm recovery; antibiotic therapy should be repeated in the case of
persisting endometritis.
Nevertheless, patient counseling on RIF should be personalized, taking also into consideration
other parameters, such as age, coexisting pathology, and, more importantly, the couple’s wishes.
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Thrombophilia
Diagnosis and management in women
8
with recurrent implantation failure
KAY NEUMANN and GEORG GRIESINGER
WHAT IS THROMBOSIS AND THROMBOPHILIA?

Thrombosis describes the clotting of blood inside the vessels. Thrombophilia is a condition where
the blood has an elevated tendency to form clots (hypercoagulability). The origin of thrombosis
was described in 1856 by the Virchow triad, which includes endothelial injury, hypercoagulabil-
ity, and stasis.1 Clots usually appear in the venous vessels (e.g., deep vein thrombosis), while their
circulation in the blood system can lead to pulmonary embolism. Arterial thrombosis can cause
myocardial infarction, apoplectic stroke or in the case of the antiphospholipid syndrome, habitual
abortion.2 Thrombophilia can either be acquired or inherited (Table 8.1).3–6 The cause of thrombo-
philia determines the risk of thrombosis.
During a women’s reproductive years, lifetime venous thromboembolism (VTE) is a major
cause for morbidity and mortality. Its mean incidence ranges from 2/10,000 during the mid-teens
to 1/1000 at 50 years of age.7 Estrogens enhance the risk for VTE, which explains the four-fold
increase in relative risk with the use of oral contraceptives (7/10,000) and the increased incidence
of VTEs in pregnancy (20/10,000).8 About 30% of patients with an event of VTE will develop reoc-
currence in the next 10 years.8
WHAT IS RECURRENT IMPLANTATION FAILURE?

Recurrent implantation failure (RIF) describes the clinical phenomenon of repeated failure of
implantation after transfer of an in vitro generated embryo. Failure of implantation is usually
defined as non-elevation of serum beta human chorionic gonadotrophin (beta-hCG) increase
about 10–14 days following embryo transfer.
In the literature there is no unanimity regarding the number of failed embryo transfer cycles
necessary for the definition of RIF. Tan et al. in 20059 reported considerable variations in the defi-
nition of RIF and reported a most common definition of ≥3 failed IVF cycles (range 2–6 cycles).
Rinehard in 200710 attempted to find a concordant definition and concluded that each fertility cen-
ter should establish their own definition of RIF based on their own data and postulated a definition
of RIF for his own program by no elevation of beta-hCG within ≥8 transfers of 8 cell stage embryos
or ≥5 transfers of blastocysts.
One problem in the definition of RIF is the lack of a “normal” implantation rate since patients
undergoing IVF represent already a preselected infertile collective. The study of Paulson et al.34
reported stable implantation rates for up to four cycles for recipients in an oocyte donation pro-
gram in which embryos originating from oocytes of fertile women are usually transferred.10
IS THROMBOPHILIA CAUSING RIF?

Implantation of an embryo is believed to depend on sufficient microvascular blood supply. Therefore,
microvascular clotting of vessels of the decidua during implantation due to hypercoagulability has
been discussed to be the cause of RIF in patients with thrombophilia.11,12 Moreover, the existence
72
Is prophylactic anticoagulation using heparins and aspirin effective in the treatment of RIF? 73
Table 8.1 Thrombogenic risk factors and the associated increase in relative risk for thrombosis.
Acquired Increase in relative risk Inherited Increase in relative risk
Immobilization ∼4–10-fold Factor V mutation ∼7-fold (heterozygous)
∼26-fold (homozygous)
Smoking ∼1.17-fold Prothrombin mutation ∼3-fold (heterozygous)
∼28-fold (homozygous)
Oral contraceptives ∼4-fold Protein S deficiency 2–20-fold
Obesity and metabolic ∼2-fold in odds ratio Protein C deficiency 10–20-fold
syndrome
Antiphospholipid ∼3-fold Antithrombin III 10–100-fold
syndrome deficiency
Cancer ∼12-fold Elevated factor VIII ∼4.8-fold
(depending on subtype) level (>150%)
Source: Data from Ay C et al. Haematologica. 2007 March;92(3):374–80; Cheng YJ et al. PLOS Med. 2013;10(9):e1001515;
Lindqvist PG, von Känel R. Thromb Res. 2015 September;136(3):513–8; Zotz RB et al. Hämotherapie 2009;13.
of antiphospholipid antibodies is thought to be associated with thrombosis, habitual abortion, and

pregnancy complications. The exact mechanisms, however, remain unclear.13
A cause-effect relationship is usually proved by an experiment, either in vivo (randomized trial)
or in an in vitro setting. Since it is not technically and ethically feasible to induce thrombophilia
experimentally in women undergoing IVF, only observational data can be explored for associa-
tions, from which cause-effect inferences may (or may not) be drawn.
Several case-control studies investigated whether patients suffering from RIF have a higher prev-
alence of thrombophilia or antiphospholipid antibodies (Table 8.2).
In a meta-analysis and systematic review of Di Nisio et al.,20 a higher prevalence for factor V
Leiden (FVL) mutations (especially for homozygote women, suggesting a “dose effect”) in patients
failing to achieve pregnancy in an IVF program was reported (odds ratio 3.08). This finding is
shown homogeneously by n = 8 studies with a combined sample size of n = 1404 patients (I2 = 0%;
p < 0.0001). For the other investigated inherited thrombophilic factors (prothrombin mutation,
MTHFR, protein C, protein S, antithrombin), Di Nisio et al.20 report no higher prevalence among
the eight included studies.
Furthermore, a higher prevalence of antiphospholipid antibodies for patients with ART failure
was also reported by Di Nisio et al.20 (odds ratio 3.33). However, the 20 included studies combining
n = 3542 patients were significantly heterogeneous (I2 = 75%), and accordingly, this finding should
be taken with caution.
In summary, increased prevalence for thrombophilic factors has been observed in patients
undergoing IVF treatment and recurrently failing to achieve pregnancy. However, numerous fac-
tors are associated with failure in an IVF program, and ideally, a multivariate analysis that also
takes into account confounders such as age, body weight, duration of infertility, type of infertility,
etc. should be performed on large data sets. A true population-based study considering fertile,
subfertile, and treatment-resistant patients would enhance understanding of the prevalences of
thrombophilic factors.
IS PROPHYLACTIC ANTICOAGULATION USING HEPARINS AND ASPIRIN EFFECTIVE IN THE

TREATMENT OF RIF?
Embryo implantation is a dynamic and complex process and depends on the interaction of mul-
tiple signals. Still, many signal cascades are not fully understood.21 Low-molecular-weight heparin
(LMWH) is thought to improve conditions of implantation via prevention of clot formation during
decidualization and placentation.22,23 Additionally, it was shown that LMWH affects expression
74 Thrombophilia
Table 8.2 Observational studies on thrombophilia and RIF.

Prevalence of Prevalence of P-value (prevalence
Participants Definition of thrombophilia thrombophilia thrombophilia in
Author Year with RIF Thrombophilic factor RIF in RIF group in control group RIF vs. control)
Simur et al.14 2008 n = 51 FVL, prothrombin, MTHFR ≥3 IVF failures 62.7% 53.9% 0.37
Azem et al.15 2004 n = 45 Mutations of prothrombin, FVL, MTHFR, ≥4 IVF failures 26.7% (excluding 9.1% (excluding 0.003
protein C, protein S antithrombin III homozygotic homozygotic
deficiencies MTHFR) MTHFR)
Qublan et al.16 2006 n = 90 FVL, MTHFR antiphospholipid syndrome ≥3 IVF failures 68.9% 25.6% and 25% p < 0.01
(depending on
subgroup)
Grandone et al.17 2001 n = 18 (n = 8 FVL, prothrombin mutation, MTHFR, ≥3 IVF failures 27.7% 0% and 6.0% not available
with fetal protein C and S, antithrombin (depending on
loss) subgroup)
Bellver et al.18 2008 n = 26 FVL, prothrombin mutation, MTHFR, ≥2 good- 19.2% 9.4% “no significant
protein C, protein S, antithrombin, APCR quality embryo difference”
transfers
Vaquero et al.19 2006 n = 59 FVL, prothrombin mutation, MTHFR, ≥2 IVF failures 25% 20% p = 0.31
protein C, protein S, antithrombin, APCR
Abbreviations: APCR = activated protein C resistance; MTHFR = methylene tetrahydrofolate reductase; FVL = factor V Leiden; IVF = in vitro fertilization.
Conclusion 75
Heparin Control Odds ratio Odds ratio

Study or subgroup Events Total Events Total Weight M-H, fixed, 95% CI M-H, fixed, 95% CI
Noci 2011 15 73 13 80 41.6% 1.33 [0.59, 3.03]
Qublan 2008 10 42 1 41 3.3% 12.50 [1.52, 102.85]
Urman 2009 26 75 20 75 55.1% 1.46 [0.73, 2.93]
Total (95% CI) 190 196 100.0% 1.77 [1.07, 2.90]

Total events 51 34
Heterogeneity: Chi2 = 4.05, df = 2 (p = 0.13); l2 = 51%
0.01 0.1 1 10 100
Test for overall effect: Z = 2.24 (p = 0.03)
Favors control Favors heparin
Figure 8.1 The systematic review of Akhtar et al.22 shows a forest plot of interventional studies on
LMWH (low-molecular-weight heparin) treatment in ART. (From Akhtar MA et al. Cochrane Database Syst
Rev. 2013 August 17;(8):CD009452, with permission.)
and activities of proteins as well, suggesting a broad influence on physiology.2 Therefore, LMWH
might modulate decidualization in a way that enhances endometrial implantation competence
rather than mere prevention of blood clotting. LMWH does not cross the placenta and is consid-
ered safe in pregnancy.24 The use of LMWH to increase live birth rates in ART patients was investi-
gated by a meta-analysis from Akhtar et al.,22 which included 386 patients from three randomized
controlled trials. Control groups of the included studies received placebo or no treatment.
Pooling of results of all three studies shows evidence for a significant increase in live birth rates
(odds ratio 1.77) for the LMWH-treated group (Figure 8.1). Quality of evidence, however, was rated
very low because of high heterogeneity and sensitivity of the choice of the statistical approach used.
Furthermore, the combined sample size was rather small. It should be concluded that LMWH
treatment in the context of ART is not justified outside clinical studies.
Additionally, acetylsalicylic acid (aspirin) is thought to improve endometrial microcirculation
by inhibition of platelet aggregation via thromboxan.25 It has been used to increase the chance of
implantation after embryo transfer, reduce the risk of abortion and decrease the rate of preterm
preeclampsia.26 It may counteract hypercoagulability by protection of the trophoblast after placen-
tation has been established.27,28
For patients with a history of RIF, interventional studies have investigated whether prophylactic
anticoagulation using LMWH (in combination with aspirin) is effective to increase implantation
and live birth rates (Table 8.3).
Moreover, a retrospective analysis of patients with RIF and thrombophilia who were treated with
LMWH showed no difference in pregnancy rates for patients with LMWH versus no LMWH.30
For patients with history of RIF without thrombophilia two studies found a marginal nonstatisti-
cal significant increase in pregnancy rates for patients receiving LMWH and suggested further
exploration in larger sample sizes,31,32 whereas one study failed to show any advantage for patients
treated with LMWH.33
Furthermore, since in the study of Paulson et al.,34 patients in oocyte donation programs with
a previous history of RIF (n = 70 patients with n = 114 cycles in the oocyte donation program)
achieved similar pregnancy and live birth rates as other patients, oocyte quality seems to be the
most essential factor for the likelihood of embryo implantation rather than the use of LMWH.
CONCLUSION
Thrombophilia has not been shown to cause RIF in patients undergoing IVF treatment, though
observational case-control studies have reported a higher prevalence of thrombophilic factors in
RIF patients compared with fertile patients. Routine screening of IVF patients for thrombophilia is
currently not warranted. The use of LMWH to improve live birth rates in RIF patients has not been
firmly established; however, risk of thrombosis during ovarian stimulation and pregnancy may
necessitate the use of anticoagulants in patients at risk for thrombosis (e.g., patients with homozy-
gous FVL mutation).
76 Thrombophilia
Table 8.3 Interventional studies on thrombophilia and RIF.

Included
Author Year patients Design Outcome Intervention Results Statistical analysis
Qublan et al.29 2008 n = 83 Prospective, Implantation, Enoxaparin Implantation rate: 20.9% (LMWH) vs. Implantation rate:
randomized pregnancy, and 40 mg/day 6.1% (placebo); pregnancy rate: 31% p < 0.001; pregnancy
trial live birth rates (LMWH) vs. 9.6% (placebo); live birth rate: p < 0.05; live birth
rate: 23.8% (LMWH) vs. 2.8% (placebo) rate: p < 0.05
Stern et al.28 2003 n = 143 Randomized Implantation Unfractionated Implantation rate: 6.8% (LMWH+ASS) Relative pregnancy
crossover rate heparin (5000 IU vs. 8.5% (placebo); live birth rate: 6% rate = 0.65 (95% CI
trial b.i.d.) and aspirin (LMWH+ASS) vs. 7% (placebo) 0.33–1.29); live birth rate:
(100 mg daily) 0.60 (95% CI 0.27–1.35)
Abbreviations: RIF
= recurrent implantation failure; ANA
= antinuclear antibody; APA
= antiphospholipid antibodies; ASS
= aspirin; LMWH = low-molecular-weight heparin;
vs. = versus.
References 77
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after oocyte donation: Implications regarding endometrial receptivity. Hum Reprod. 1997
April;12(4):835–9.
Andrological causes of
9
CHRISANTHI MARAKAKI, GEORGIOS A. KANAKIS,
and DIMITRIOS G. GOULIS
INTRODUCTION
Recurrent implantation failure (RIF) is a term used in in vitro fertilization (IVF) or intracyto-
plasmic sperm injection (ICSI) procedures; however, a uniform definition is still lacking. Based
on a systematic review assessing the definitions of RIF used in literature, RIF is the failure of
attachment of the embryo to the lining of the womb after the transfer of a cumulative number of
at least four good-quality embryos, in a minimum of three consecutive fresh or frozen cycles, in
a woman under the age of 40 years.1 RIF is the consequence of embryo- or uterine-related fac-
tors and should be distinguished from IVF failure, which also includes poor response to ovarian
stimulation and absence of good-quality embryos. 2 It has been difficult to determine the exact
prevalence of RIF, because of the various definitions used to describe the condition as well as the
different outcomes employed to assess implantation success (e.g., biochemical vs. clinical preg-
nancy). The probability of one embryo failing to implant is 70%; thus, according to the aforemen-
tioned definition, following the transfer of four embryos, the probabilities of all embryos failing
to implant is 0.704 = 0.24. Successful implantation requires trophoblastic growth, invasion into
the endometrium, and stimulation of vascularization to provide its own blood supply. Multiple
causes of implantation failure have been proposed, concerning principally the female partner
(poor oocyte quality, thrombophilia, autoimmune disorders, and uterine anomalies). Male fac-
tor infertility has also been found to be associated with high order RIF; however, the pathophysi-
ological basis of this association is still poorly understood. It has been suggested that sperm of
poor quality leads to the formation of morphologically high-quality embryos, which may harbor
inherent defects and fail to implant. 3
Investigations concerning the male partner remain limited and often restricted to sperm
analysis. However, it is widely accepted that conventional semen analysis parameters do not
accurately reflect sperm quality. Consequently, several sperm function tests, such as sperm pen-
etration assay, sperm-zona pellucida binding test (hemizona assay), acrosomal reaction test,
hyaluronan binding test, hypo-osmotic swelling test, and magnetic-activated cell sorting, have
been employed in order to further assess and refine sperm quality, with questionable, however,
clinical utility. In an attempt to improve the predictors of successful implantation, spermatozoa
are also assessed for chromosome aneuploidy using fluorescence in situ hybridization (FISH) or,
alternatively, for the degree of single- and double-stranded DNA breaks, known as DNA frag-
mentation (DF) analysis.4
The aim of this review is to discuss and critically appraise established and potential andrological
causes of RIF as well as the current management strategies.
AVAILABLE EVIDENCE ON SPERM QUALITY

Sperm analysis parameters
The routine semen analysis remains a hallmark in the evaluation of the infertile male. Apart from
evaluating sperm concentration, motility, and morphology, indicators of patency and function
79
80 Andrological causes of recurrent implantation failure
of the male genital tract are also assessed, including semen volume, liquefaction time, and pH.
Moreover, biochemical markers reflecting the function of different segments of the genital tract,
such as fructose, zinc, and α-glycosidase, can be measured in the seminal plasma.
There is published evidence that poor semen parameters, morphology in particular, result in low
blastocyst formation rates after IVF, suggesting that sperm can influence human preimplantation
embryo development.5 Loutradi et al. observed a negative relationship between semen quality and
embryo development, even before the activation of the embryonic genome, suggesting that sperm
can affect embryogenesis from a very early stage.6 Moreover, embryo development, pregnancy, and
implantation rates have been found to be affected by the source of sperm (ejaculated, epididymal,
or testicular) and the type of male factor infertility.7
Many studies have also indicated that the direct quantitative assessment of sperm movement
by computer-aided sperm analysis (CASA) reflects the fertilizing ability of human spermatozoa
in vitro, under conditions where the conventional semen profile is of limited diagnostic value. In
a retrospective analysis of the relationship between the CASA estimates and fertilization rates in
IVF cycles, two parameters—namely curvilinear velocity and rapid sperm movement—provided
reliable estimations of the fertilizing ability of human sperm.8 When cryopreserved donor semen is
used, numerous sperm parameters have also been suggested as possible predicting factors of clini-
cal pregnancy rate, such as prefreezing sperm motility, post-thaw sperm motility, forward progres-
sion, inseminating motile count, and total motile sperm counts.9
Abnormal sperm morphology has been described as a useful determinant of male fertility,
in vivo and in vitro, and has been related to increased levels of chromosomal abnormalities and a
high degree of DF.10 However, such defects cannot be predicted by the morphological parameters of
the embryos prior to transfer. Moreover, as suggested by some studies, this is not the case in couples
undergoing ICSI, where teratozoospermia is not that closely related to the potential of embryonic
development and cycle outcomes.11,12
Genetic factors
Sperm chromosomes and DNA fragmentation
It is widely admitted that, in many cases, routine semen analysis only provides clues that may indi-
cate the need to perform additional evaluation. Patients with a clinical background of RIF are at
risk of harboring sperm chromosomal abnormalities (numerical or/and structural), the incidence
of which are higher in oligoasthenoteratozoospermic patients. In particular, sperm aneuploidy and
diploidy rates were assessed in male partners of ICSI couples with normal karyotypes (using dual-
and triple-color FISH techniques for chromosomes 13, 18, 21, X, and Y) and an increased incidence
of sex chromosome disomies was observed in couples with RIF.13
Male chromosomal structural aberrations, such as translocation, inversion, and pericentric
inversion, are also of particular concern.14 The development of locus-specific and subtelomeric
DNA probes has allowed the analysis of chromosome segregation in carriers of inversions (peri-
centric and paracentric), Robertsonian translocations, and reciprocal translocations. These stud-
ies confirmed that carriers of structural chromosomal reorganizations produce chromosomally
unbalanced sperm. If these gametes fertilize an oocyte, the resulting embryos, depending on the
chromosome regions implicated, can give rise to abortions or offspring affected by chromosomal
abnormalities.15 In addition, centrosome anomalies resulting in chaotic mosaics were most likely of
paternal origin.16 It has been also proposed that balanced parental translocations may be implicated
in the pathogenesis of implantation failure in IVF, and that genetic evaluation should be considered
as part of the investigation of these couples.17 On the other hand, another large study by Rubio et al.
analyzed the results of 131 ICSI cycles in couples with repeated abortion or implantation failure, in
which the increased frequency of aneuploidy in spermatozoa did not affect the success of assisted
reproductive techniques (ART).13
Available evidence on sperm quality 81
Multiple studies have reported the negative effect of high sperm DF on reproductive outcomes
in ART.18 Sedó et al. found that sperm DF significantly affected embryo blastulation and implan-
tation in ICSI patients who received donated eggs and showed that sperm DF might compromise
the progression of embryo development, resulting in arrested embryos.19 An updated systematic
review and meta-analysis concluded that there is sufficient evidence in the existing literature
suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF
and/or ICSI treatment. 20 However, current literature does not support the hypothesis that sperm
DF is an important cause of RIF as well, or that sperm DNA integrity testing has value in such
patients. 21
Y chromosome microdeletions
Microdeletions of the various azoospermia factor (AZF) regions on the long arm of the Y chromo-
some are another major genetic factor involved in male infertility.22 Deletions of the AZFa and
AZFb regions are typically associated with azoospermia and absence of sperm during testicular
sperm extraction (TESE) procedures. On the other hand, deletions of the AZFc region result in a
less-severe defect of spermatogenesis, allowing successful TESE or even the presence of sperm in
the ejaculate. It is reported that the clinical outcomes of ICSI for oligozoospermic/TESE patients
with AZFc microdeletions are comparable to those of infertile patients with normal Y chromo-
somes, 23,24 in contrast to previous studies that associated AZFc microdeletions with lower fer-
tilization rate and poorer embryo quality.25 Thus, the correlation between the incidence of AZF
microdeletions and ART results remains controversial and needs to be verified in a large cohort
of infertile men.
Immunological maladaptation
Immunological maladaptation is a putative cause of implantation failure in humans. As maternal
lymphocytes recognize fetal antigens, tolerance is necessary to prevent a graft versus host type
reaction and consequently abortion. Regulatory T (Treg) cells are recruited to abate the stimula-
tion of maternal lymphocytes by fetal antigens. Villous trophoblast cells express several minor
histocompatibility and paternal antigens (H-Y). Seminal plasma is of relevance as it can induce
paternal antigen-specific Treg cells in the female genital tract. Elimination of Treg cells during
implantation or early pregnancy has been associated with implantation failure or fetal resorption
in mice.26
Sperm epigenetics
Oxidative stress
Accumulating evidence suggests that oxidative stress plays an important role in the pathophysiol-
ogy of male factor infertility.27 A surrogate marker of assessing the magnitude of oxidative stress
in the seminal plasma is the level of reactive oxygen species (ROS). The main sources of ROS in
semen are leukocytes and morphologically abnormal sperm, although ROS can also be produced
by precursor germ cells. Under physiologic conditions, sperm requires a small amount of ROS
to regulate certain functions, such as sperm capacitation, acrosome reaction, and sperm-oocyte
fusion.28 However, the excessive production of ROS might have deleterious effects on sperm struc-
ture and functionality. Sperm is particularly susceptible to ROS-induced damage because their
plasma membranes contain large quantities of polyunsaturated fatty acids, whereas their cyto-
plasm contains low concentrations of scavenging enzymes; thus they have a limited capacity to
repair their own DNA.27 A balance between the generation of ROS and the presence of antioxi-
dants in the seminal plasma is required to avoid excessive oxidative stress.29 Moreover, ROS seem
to have an effect on the sperm proteome, and differences in the levels of sperm protein expression
under oxidative stress have been observed. These differences have been attributed to direct effects
of oxidative stress on gene transcription, as well as post-translational oxidation of amino acid resi-
dues that lead to the formation of protein-protein crosslinked aggregates.30
Proteomics
Proteomic analyses have identified several sperm-specific proteins involved in pre- and postfer-
tilization events, but their exact role in regulating embryonic development is still not elucidated.
Proteomic alterations occurring during spermiogenesis have been proposed to alter sperm epigen-
etic signatures, ultimately resulting in abnormal embryo development.31 These proteomic altera-
tions seem to be subtle, not affecting major features of sperm quality, but putatively influencing
sperm genetic chromatin distribution. Specific proteomic studies aimed to investigate the correla-
tion between sperm protein levels and ART results are scarce.
Thus far, only histones and protamines have been validated to be a part of nongenomic paternal
contribution to the embryo. The replacement of most histones by protamines 1 and 2 during sper-
miogenesis facilitates a high order of chromatin packaging necessary for normal sperm function.32
A significant negative correlation between sperm motility and protamine-2 mRNA levels has been
demonstrated.33 It has also been suggested that protamines are not only important for fertiliza-
tion but may additionally have an impact on the correct initiation of gene expression in the early
embryo.34 Moreover, incorrect histone to protamine exchange results in a prolonged presence of
histones, which are known to exhibit a variety of epigenetic modifications that are transmitted
through fertilization.35
The cysteine-rich secretory proteins (CRISPs) are mainly expressed in the male reproductive
tract and have been implicated in many aspects of male germ-cell biology, involving haploid germ-
cell development, epididymal maturation, capacitation, motility, and the actual processes of fertil-
ization.36 Several lines of evidence suggest a role for CRISPs in the interaction between the sperm
and the oocyte at fertilization.37 However, the exact role of each CRISP protein is still under inves-
tigation and their role in RIF remains to be elucidated.
FACTORS THAT AFFECT SPERM QUALITY

Paternal age
Advanced paternal age has been associated with a decrease in sperm quality (particularly motility),
increase in birth defects, epigenetic modifications and DNA mutations, along with chromosomal
aneuploidies.38 Eventually, the incidence of autosomal dominant diseases, such as achondroplasia,
polyposis coli, and Marfan syndrome, is reported to be increased among the offspring of older
men.39 However, the evidence associating paternal age with IVF failure and RIF is equivocal.
According to several studies, advanced paternal age is believed to impact reproductive and fer-
tility outcomes, including a decrease in ART success rate and an increase in the rate of preterm
birth. In particular, age of the male partner ≥35 years has been associated with decreased clinical
pregnancy rates following artificial intrauterine insemination (IUI), even in the presence of normal
semen analysis and favorable characteristics of the female partner.40 Similarly, a significant decline
in IUI pregnancy rates per cycle in men aged ≥45 years have been reported compared to men <30
years (9.3% vs. 12.3%).41 In a prospective study, it was reported that with increasing paternal age,
the live birth rate of IVF or gamete intrafallopian transfer (GIFT) decreased.42 Similarly, assess-
ment of the outcomes of 859 conventional IVF and 1632 ICSI cycles demonstrated that from the
age of 51 years, paternal age negatively affects the rates of blastocyst formation (IVF) and clinical
pregnancy (ICSI).43 Moreover, a study comparing 1495 women who reported spontaneous abor-
tion in pregnancy with 12,633 women reporting live births in their previous pregnancy found
that increasing paternal age is significantly associated with spontaneous abortion, independent of
maternal age and multiple other factors such as maternal diabetes, maternal smoking, social class,
and education.44
Factors that affect sperm quality 83
On the other hand, there are studies that do not associate advanced paternal age (up to 64 years)
with sperm characteristics or its ability to fertilize human eggs.45 Moreover, it has been reported
that if a couple is able to produce and transfer a single thawed euploid embryo, no difference in
IVF pregnancy outcomes is identified with increasing paternal age.46 To add further contradiction,
there are studies claiming that embryo morphology during cleavage is not affected by male age and
that male age is irrelevant for the outcome of ART procedures.47
Lifestyle factors
Environmental threats, including lifestyle-related factors, may impair male reproductive health and
have been linked to the controversial issue of deterioration of semen quality over the last century.
Smoking
Approximately 37% of men of reproductive age smoke cigarettes. Tobacco smoking may affect
sperm development and function, with a negative effect on semen parameters. It has been reported
that exposure to cigarette smoking was associated with reduced sperm count, motility, and mor-
phology.48 Subgroup analyses indicated that the effect size was higher in infertile men than in the
general population and in moderate/heavy smokers than in mild smokers. Moreover, preconcep-
tion paternal smoking has been correlated with increased incidence of several types of cancer and
birth defects in offspring49 suggesting that the smoke-induced genetic or epigenetic changes that
arise in spermatozoa are transmitted to offspring.
Smoking has also been demonstrated to impact sperm DNA methylation patterns in a consistent
manner.50 Even if altered DNA methylation is not directly passed on to offspring, these altera-
tions might influence early embryonic gene expression or adjust reprogramming or early develop-
ment in other ways. Experiments in rodents show that exposure of adult male mice to sidestream
tobacco smoke significantly increases sperm DNA mutations and aberrations in sperm chromatin
structure.51 In addition, smoking also alters sperm microRNA content, greatly increases the abun-
dance of ROS in the seminal plasma, and causes oxidative DNA damage in sperm. Smokers are
at increased risk of sperm defects, such as partially or fully inactive mitochondria and non-intact
acrosomes, in addition to increases in sperm DF and changes to sperm proteome.52
Alcohol
Ethanol can suppress reproductive function and sexual behavior in humans. Some studies suggest
that alcohol exerts direct toxicity to the testes and causes fertility disturbances by reducing sperm
count and motility,53,54 although others did not confirm these findings.55,56 Chronic ingestion of
ethanol may cause defects in spermatogenesis and sperm motility, aberrant testicular and acces-
sory gland morphology, reduced cauda epididymis sperm content, and impaired epididymal sperm
maturation. Occasional alcohol intake does not seem to impair semen quality, whereas both semi-
nal volume and sperm morphology are negatively affected by daily consumption.57
Observations propose a potential association between preconception male alcohol exposure
and altered epigenetic programming in sperm. Animal models of paternal alcohol exposure show
alterations in the control of key enzymes regulating chromatin structure, as well as changes in the
DNA methylation profiles of alcohol-exposed sperm, which have well-characterized impacts on
both fetal and placental growth.58
Genital heat stress

Epidemiological studies assessing occupational exposure to high temperatures underline the role
of genital heat stress as a cause of impaired semen quality and of reversible infertility in men.59
Several sperm traits, such as sperm head features (morphometry and chromatin integrity), which
likely play specific roles in IVF and embryonic development, are reported to be altered as a result of
a thermal insult.60 Other investigators have demonstrated that scrotal heat stress can compromise
the DNA integrity of sperm and this may have clinical implications for patients undergoing IVF
and ICSI.61 Daily habits such as sedentary lifestyle, insulating genital clothing, bicycling, and use
of a sauna are believed to increase scrotal temperature and may exert detrimental effects on sperm
quality; however, scrotal cooling has not been proved to improve semen quality.62
Recreational drug use

Recreational drugs, including marijuana, cocaine, methamphetamines, and opioid narcotics, have
been shown to negatively impact male fertility. A study in Denmark showed adverse associations
between frequent marijuana use (more than once weekly) and semen quality among healthy young
men, the association being even more pronounced among men abusing other recreational drugs as
well.63 Studies on animal models have demonstrated that chronic cocaine administration in male
rats results in seminiferous tubule degeneration, reduced pregnancy rates, and a decrease in mature
spermatogenic forms.64
Obesity
Recent data suggest that reproductive health may also be impacted by obesity. While it is generally
accepted that female body mass index (BMI) impacts fecundity, the relationship of male infertility
with BMI is less clear. Some studies have suggested that elevated male BMI can lead to impaired
sperm production,65,66 whereas others have found no relationship between male BMI and semen
parameters.67,68 Furthermore, adiposity may be better related to sperm production, when assessed
not only by BMI but by assessing waist circumference as well.69 Overweight and obese men have
been shown to have a higher incidence of low ejaculate volume, sperm concentration, and total
sperm count.
There has been growing concern over whether the BMI of male partners is associated with ART
outcomes. Some researchers suggest that increased BMI may cause decreases in embryo quality
and pregnancy rates,70 while reports indicate that hyperinsulinemic men may exhibit a higher per-
centage of poorly compacted DNA in their sperm and less success in IVF.71 In other studies, IVF is
distinguished from ICSI, as the authors demonstrated that overweight status of the male partner
may cause impaired sperm-egg interaction after IVF, but not after ICSI.72,73 Similarly, in a Chinese
study, increased BMI of the male partner was associated with decreased fertility rate after IVF
cycles. However, male BMI was not shown to be significantly associated with late-stage embryonic
development, pregnancy rates, or miscarriage rates, suggesting that specific sperm impairments
can be repaired by postfertilization events in the oocyte.74
Environmental factors
Hormone disruptors
Hormone disruptors, such as anabolic steroids, polychlorinated biphenyls, dioxins, polycyclic aro-
matic hydrocarbons, phthalates, bisphenol A, pesticides, and alkylphenols have been studied for
their impact on male fertility in several species of animals and in humans. Growing evidence sug-
gests that xenoestrogen bisphenol A (BPA) (a widespread environmental contaminant, employed
in the production of certain plastics and epoxy resins) can bind to receptors on sperm and, thus,
alter sperm function. It has been reported that high concentrations of BPA alter sperm function,
fertilization, and embryonic development via regulation and/or phosphorylation of fertility-related
proteins in sperm collected from experimental mice.75
Phthalates are mainly used as plasticizers, substances added to plastics to increase their flex-
ibility, transparency, and longevity. They are used in many consumer products, such as building
materials, toys, food packaging, cosmetics, and medical devices. A review of studies on labora-
tory animals confirmed that phthalates cause diminished sperm count, increase the frequency of
abnormal sperm, and damage DNA in germ cells, especially after chronic exposure and in cases of
exposure of immature animals (both during gestation and post-term). Phthalates may also induce
Factors that affect sperm quality 85
mutations in male gametes, leading to increased pre- and postnatal mortality of the offspring.
Interestingly, phthalates have been associated with diminished quality of semen in the F1 genera-
tion,76 while in males, but not in females, urinary concentrations of selected metabolites of phthal-
ates and phthalate alternatives are associated with poor blastocyst quality.77
Acrylamide and glycidamide (a reactive epoxide metabolite from acrylamide) are industrial
chemicals that are used in several ways, such as the production of polyacrylamides for wastewater
treatment, textiles, paper processing, and cosmetics. Acrylamide is also a product formed in cer-
tain foods prepared at high-temperature frying, baking, or roasting, such as fried potatoes, bakery
products, and coffee, and has been associated with a decrease in sperm count, motility, and mor-
phology. Acrylamide has been shown to induce disruption or breakage of chromosomes, whereas
glycidamide has mutagenic effects.78,79 There no studies on the effects of these compounds on RIF
but low-dose chronic exposure is proposed to cause mutations without affecting the fertilization
capacity of sperm or leading to deaths in the offspring, therefore allowing these mutations to be
inherited.78
Heavy metals
It has been hypothesized that exposure to heavy metals may compromise male reproduction, as
demonstrated by epidemiological and animal studies.80 Cadmium, mercury, lead, and arsenic lev-
els may affect semen quality. When toxic metals were measured in seminal plasma collected from
30 men of IVF couples to evaluate their impact on semen quality and IVF outcomes, a negative
association was suggested between mercury-adjusted cadmium levels and pregnancy rates.81 It has
been also reported that increased seminal plasma lead levels adversely affect the fertility potential
of sperm in IVF.82 However, no specific relevant implications with RIF have been reported yet.
Radiation
Some epidemiological studies suggest a possible link between the use of mobile phones and
decreased semen quality parameters, but the results are not consistent.83,84 The common practice of
storing mobile phones in close proximity to the testes can significantly impair motility, vitality, and
the integrity of sperm DNA by the induction of ROS generation through the exposing of the repro-
ductive system to relatively high levels of radiofrequency electromagnetic radiation (RF-EMR).85
A mechanistic model has been proposed according to which RF-EMR exposure leads to defective
mitochondrial function, which in turn is associated with increased ROS production.
Regarding ionizing radiation, available data originate by experiments conducted on animals.
When mature male mice were exposed to 2 or 4 Gy of 137Cs γ-rays, and their sperm were used for
IVF, structural chromosomal aberrations, aneuploidy, and mosaicism were documented in early
cleavage embryos.86 In the same study, it is proposed that it is reasonable to consider a heritable risk
of mosaicism rather than aneuploidy in embryos derived from sperm after irradiation. An increased
incidence of aneuploid blastomeres (31.6%) was also reported in eight-cell mouse embryos derived
from the sperm of males exposed to 4 Gy γ-rays.87 No direct data exist on RIF.
Genital tract infections

Chronic inflammatory conditions of the genital tract, such as male accessory gland infections
(MAGIs), are frequently encountered in male fertility problems. With regard to their impact on
male reproductive function, epididymitis seems to be more relevant than inflammation/infection
of the prostate and/or seminal vesicles. Chronic epididymitis may result in reduced sperm count
and motility. Besides changes in the conventional sperm parameters, alterations in DNA integrity
have also been observed.88 A reduction in natural and assisted cumulative pregnancy rate and an
increase in miscarriage rate are related to the presence of human papillomavirus (HPV) at the
sperm level. The exact mechanisms by which MAGI are able to impair implantation rates remain
unclear, with increased oxidative stress or a direct effect of microorganisms on sperm being the
most plausible.89,90
Other
Sperm washing procedures
Sperm must be properly prepared for IVF programs in order to control the fertilization rate and
ensure that embryos are of high quality and have appropriate developmental abilities. García-
Herreros et al.91 concluded that different sperm washing-selection methods commonly employed
during the IVF process may lead to alterations in sperm morphometric characteristics, which
might explain some of the variability of implantation rates in IVF.
SUGGESTED MANAGEMENT
Regarding idiopathic male infertility, a recent meta-analysis suggested a significant improvement
in sperm DF and an increase in ART pregnancy rates with the use of antioxidants. The data are less
robust regarding spontaneous conceptions or in couples with a history of recurrent embryo loss.92,93
Multiple randomized trials have investigated antioxidant supplementation for treatment of male
infertility, with several demonstrating a positive effect on semen quality, especially sperm motil-
ity.94 Omega-3 polyunsaturated fatty acid supplementation has been found to improve the semen
profile, while coenzyme Q10 was found to effectively improve sperm kinetic features. However, the
existing data are still debatable and large-scale studies are required focusing on the impact of anti-
oxidants on sperm parameters and their relationship with early embryo development.
Surgical repair has been suggested for men with clinically apparent varicocele, impaired semen
parameters and infertility, while varicocelectomy has been shown to effectively improve sperm
DF.95 In cases of idiopathic male fertility resulting in ICSI cycle failure with ejaculated sperm, an
ICSI cycle with surgically retrieved testicular sperm is an alternative choice that can have marked
improvement in outcome.96 A possible explanation for this observation is that the testis–blood bar-
rier offers more protection from oxidation insults than the epididymal environment.
Despite the detrimental effects that smoking and excessive alcohol consumption have been dem-
onstrated to exert in male fertility, there are no randomized studies showing that semen parameters
or chance of pregnancy improve if the male partner quits smoking or reduces alcohol consump-
tion.97 Nevertheless, with respect to the general positive health effects that such a lifestyle modifi-
cation may have, avoiding smoking and alcohol consumption at least prior to and during the ART
treatment should be encouraged.98
The same values should be taken into consideration when approaching the obese infertile male
patient. There is only one nonrandomized study, indicating that weight reduction leads to improve-
ment of semen parameters,99 which, however, included only severely obese patients (BMI > 35 kg/
m2). Nevertheless, weight loss is expected to improve reproductive hormone balance, while those
patients with metabolic syndrome and hyperinsulinemia may benefit from metformin treatment
and the use of nutritional supplements with antioxidant properties.100 Increased fruit and vegetable
consumption should also be encouraged, as it has been associated with better IVF outcomes.101
There is also evidence for a favorable effect of vitamin D supplementation on semen quality, testos-
terone concentrations, and fertility outcomes.102
Last but not least, for couples who struggle with RIF, it is essential that the male is examined
for either chromosomal aneuploidy or chromosomal structural abnormalities. While it is difficult
to assess the precise risk associated with increased levels of sperm aneuploidy, such information
is crucial in order to counsel the couples about the associated genetic risks and their reproductive
and testing options. In all cases, genetic counseling should include a three-generation pedigree,
looking specifically for a familial history of infertility, recurrent pregnancy loss, birth defects,
intellectual disability, and apparent genetic disease.103 For men with reciprocal chromosomal
translocations, preimplantation genetic diagnosis (PGD) is recommended, as this has increased
the live birth rate from 4.9% to more than 80% in some studies.104 Nevertheless, considering the
significant cost of the procedure, the option of using donor sperm or adopting might be more
appealing for some couples.
References 87
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Diagnostic evaluation of
the couple with recurrent
10
ENGIN TURKGELDI, SULE YILDIZ, and BARIS ATA
INTRODUCTION
Implantation is a complex process that requires a healthy embryo, a receptive endometrium and
their successful interaction. Factors that affect any of these can contribute to recurrent implanta-
tion failure (RIF).
RIF is an ill-defined sign with a multitude of reasons behind it. RIF patients make up a heteroge-
neous group of infertile couples. This is a methodological problem since it is not usually possible to
perform large-scale studies with well-defined groups. Moreover, embryonic aneuploidy, the leading
cause of implantation failure, causes a low signal-to-noise ratio, rendering the detection of other
etiologic factors difficult. Therefore, most studies on RIF are of low quality and often conflict with
each other. One frequently has to rely on studies involving general assisted reproductive technol-
ogy (ART) patients, not necessarily on RIF. Some theories relate to recurrent pregnancy loss rather
than RIF per se. These are major problems while counseling a couple with RIF.
Having said these, the couple suffering from RIF is confused and at times desperate. They need
effective treatment here and now, before high quality evidence becomes available for every contro-
versy. Therefore, it is important to present as evidence-based of an approach as possible for investi-
gating the problem in depth, instead of taking random shots at an indefinite target. The road map
should be clear and discussed with the couple thoroughly from the beginning.
We will discuss possible causes of RIF and their diagnostic evaluation in the following order:
genetic, anatomic, hematological and immunological, endocrine factors, and endometrial recep-
tivity. The chapter will conclude with a proposed algorithm for assessment of a couple suffering
from RIF.
GENETIC FACTORS
Parental karyotypes
Embryonic aneuploidy is the leading cause of miscarriages and implantation failure.1
People with balanced reciprocal translocations or Robertsonian translocations are functionally
and phenotypically normal since they have a normal quantity of genetic material. However, segre-
gation during gametogenesis may result in unbalanced oocytes or sperm and eventually lead to an
increased incidence of chromosomally abnormal embryos.
Chromosomal abnormalities (CA) are more common in men with severe male factor infertility.
Mau-Holzmann reported the frequency of CA to be 13.1% and 4.3% in infertile men with azoosper-
mia and oligozoospermia, respectively.2 Stern et al. compared karyotype results of 293 women and
221 men who did not achieve clinical pregnancy after transfer of ≥10 embryos with 500 infertile
couples referred for in vitro fertilization (IVF).3 Excluding Turner syndrome and Klinefelter syn-
drome, 13 of 514 (2.5%) RIF patients had CA, whereas 13 of 1000 (1.3%) controls had an abnormal
karyotype. Translocations were significantly more prevalent in the RIF group compared with con-
trols, both in females (3/293 vs. 1/500; p < 0.005) and males (4/221 vs. 2/500; p < 0.005). Raziel et al.
93
94 Diagnostic evaluation of the couple with recurrent implantation failure
reported a strikingly high rate of CA of 15.4% in 65 couples with high-order implantation failure,
defined as not achieving a clinical pregnancy after transfer of ≥15 embryos in ≥6 ART cycles.4 De
Sutter et al. performed chromosomal analysis on 317 women and 298 men who had a history of
≥3 consecutive ART failures.5 A total of 13 (2.1%) patients were diagnosed with CA, eight females
(2.5%) and five males (1.7%). The prevalence of CA was increased in the female RIF population
compared with female newborns (0.78%) and normo-ovulatory women starting ART (0.58%). Male
patients with RIF had a similar prevalence of CA when compared with male newborns. It is impor-
tant to note that patients with severe male factor infertility were excluded from the study, so the
prevalence of CA in male RIF patients is likely underestimated in this study.
Apparently, CA are more common in couples with a history of RIF, and they should be offered
karyotype testing. While only 2%–3% of the patients are likely to have abnormal results and no
curative therapy is possible, this information can be helpful when counseling the couple about their
prospects and offering preimplantation genetic diagnosis or gamete/embryo donation to those who
may benefit from these options.
Sperm DNA integrity

Lately, sperm DNA integrity tests have drawn attention as a parameter of male reproductive poten-
tial. Various methods to test DNA integrity are available, such as the sperm chromatin structure
assay (SCSA), the deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay,
the single-cell gel electrophoresis assay (comet assay), and the sperm chromatin dispersion (SCD)
test. These methods do not test for specific DNA sequences. Instead, they give an estimate of the
general condition of DNA.6
The latest meta-analysis on the relationship between sperm DNA integrity and ART outcomes
included six studies and 998 patients.7 Overall, low DNA damage was associated with higher live
birth rates (RR 1.17, 95% CI 1.07–1.28; p = 0.0005). However, it is noteworthy that DNA damage
threshold values used in the included studies varied between 10% and 50%, and the tests used,
as well as the patient populations, were heterogeneous. Moreover, when only the studies that
accounted for female factors were analyzed (n = 202), live birth rates were similar in men with high
and low sperm DNA fragmentation using ICSI (RR 1.08, 95% CI 0.39–2.96).
Bronet et al. prospectively analyzed embryo aneuploidy rates and sperm DNA fragmentation
in 38 couples undergoing preimplantation genetic screening (PGS) due to recurrent miscarriage
(n = 30) and RIF (n = 8).8 There was no correlation between aneuploidy rate and sperm DNA
fragmentation (R 2 = 0.02, p = 0.37; R 2 = 0.04, p = 0.18, for fresh and processed sperm samples,
respectively).
In conclusion, there is inadequate evidence to promote sperm DNA integrity tests in patients
with RIF. Even with the assumption that the test has predictive value, it is not known if use of
antioxidants or other medications improves reproductive outcomes. There is not much to offer to
the patient with a high DNA fragmentation rate except for lifestyle changes such as switching to a
healthy diet or quitting smoking, which should be suggested regardless of sperm DNA fragmenta-
tion status.
ANATOMICAL FACTORS
In a recent meta-analysis, Venetis et al. reported that except for septate uterus (RR 0.86, 95% CI
0.77–0.96; three studies), chance of spontaneous conception was not decreased in women with con-
genital uterine anomalies (CUA), namely arcuate, didelphys, unicornuate, and bicornuate uteri.9
Neither individual nor combined data for different CUA types (RR 0.66, 95% 0.37–1.19; four stud-
ies) showed a significant difference between ART outcomes in women with or without CUA.9
Arcuate uterus is the mildest of congenital uterine anomalies. In their practice committee report,
the American Society of Reproductive Medicine (ASRM) has declared it as a clinically irrelevant
Anatomical factors 95
variant of the normal. Surgical correction of arcuate uterus is not advised to improve ART success
or obstetric outcomes.10
Septate uterus is the most common congenital uterine anomaly with a prevalence around 1%.11
While its role in first- and second-trimester abortions and other adverse obstetric outcomes such as
malpresentation and premature rupture of membranes are shown in a number of studies, its effect
on natural and assisted conception is uncertain.9 In a prospective study, 44 women with uterine
septum and otherwise unexplained infertility, who underwent septum resection, were followed for
a year without any interventions.12 Their pregnancy rates were compared with those of 132 women
with unexplained fertility who were also managed expectantly. Pregnancy rates were 38.6% versus
20.4%, and live birth rates were 34.1% and 18.9% in the septum and the control groups, respec-
tively, signifying a statistically significant benefit. Unfortunately, the available evidence supporting
metroplasty in patients with uterine septum is from nonrandomized studies with small sample
sizes.13 Yet, given its clear adverse effects on obstetrics and controversial effect on ART outcome,
and the observed benefit on miscarriage rates, it is plausible to recommend hysteroscopic resection
to women with RIF.
Major CUA such as uterus didelphys, unicornuate, and bicornuate are usually spotted in patients
with RIF while undergoing fertility treatments. However, these anomalies are associated with
adverse pregnancy outcomes rather than implantation failure.9
Acquired anatomical factors

Endometrial polyps
Three nonrandomized studies showed improved spontaneous pregnancy rates after polypec-
tomy.14–16 In an RCT involving women undergoing intrauterine insemination cycles, hysteroscopic
removal of polyps, with an average diameter of 9 mm, resulted in significantly increased pregnancy
rates.17
However, there is no evidence supporting a positive effect of polypectomy in IVF patients. There
are only retrospective cohort studies. Of 83 patients with endometrial polyps <2 cm in diameter,
49 had their embryos transferred without any intervention, while 34 had embryo transfer after hys-
teroscopic polypectomy. Clinical pregnancy rates were not significantly different.18 Another study
on the relationship between polypectomy and pregnancy rates did not show improved pregnancy
rates after removal of polyps up to 1.5 cm.19
Despite the lack of evidence for patients with a history of RIF, it could be plausible to remove
endometrial polyps in these patients since even a marginal benefit can be what the couple needs.
Fibroids
While most women with fibroids are fertile, fibroids are more common in the infertile popula-
tion.20 Abnormal vascularization, increased uterine contractility, chronic inflammation, altered
HOXA-10 and cytokine expression in the endometrium are proposed mechanisms that may impair
embryo implantation.21,22
A recent meta-analysis reported significantly lower implantation (RR 0.28), clinical pregnancy
(RR 0.36), and live birth rates (RR 0.32) and higher spontaneous abortion rates (RR 1.68) in women
with submucous fibroids.23 On the other hand, the effect of intramural fibroids on fertility and
their management is less clear. Recent studies point to the potential detrimental effect of intra-
mural fibroids on fertility.23 In a meta-analysis on the effect of non-cavity-distorting intramural
fibroids on the outcome of IVF treatment, data from 11 studies including a total of 1626 patient
with fibroids and 2355 without were pooled. There was a relative reduction of 21% in live birth rates
in women with intramural fibroids that do not distort the endometrial cavity (RR 0.79, 95% CI
0.70–0.88 p < 0.0001).24
Casini et al. prospectively evaluated 181 infertile patients with single fibroids <4 cm and no other
causes of infertility.25 They grouped patients according to location of their fibroids, randomized
them into myomectomy and follow-up subgroups, and followed them for spontaneous pregnancy
for a year. Clinical pregnancy rates were significantly improved with surgery in patients with sub-
mucous and submucous-intramural fibroids (43.3% vs. 27.2%, p < 0.05 and 36.4% vs. 15%, p < 0.05,
respectively), while the improvement was not significant in patients with intramural and intra-
mural-subserous fibroids, despite a trend toward benefit (56.5% vs. 40.9% and 35.3% vs. 21.4%,
respectively). Some researchers tried to determine a threshold fibroid diameter above which fertil-
ity is significantly affected. While being far from conclusive, most of them suggest a cutoff value of
3–5 cm.26–30
Nevertheless, there is no solid evidence to advocate myomectomy for these patients since it is not
clear whether it restores fertility.23–25 When the doubtful benefit is compared with the cost, disad-
vantages, and risks of such a major surgery, including the delay in ART treatment and the small
but present risk of uterine rupture during pregnancy and labor, most clinicians proceed without
myomectomy at the initial cycles. However, with each unsuccessful cycle, this option is considered
more seriously to dismiss any factor that will decrease the chance of implantation. In this line of
thinking, patients with RIF should carefully be evaluated for submucous-intramural fibroids, and
their removal can be discussed as a weighty option.
Adenomyosis
In a meta-analysis, clinical pregnancy and live birth rates per cycle were significantly decreased in
women with adenomyosis (OR 0.73, 95% CI 0.6–0.9 and OR 0.59, 95% CI 0.42–0.82, respectively),
while miscarriage rates were increased (OR 2.2, 95% CI 1.53–3.15).31
Mavrelos et al. studied 375 women undergoing IVF and evaluated them using three-dimensional
ultrasound (3D US) between the third and sixth days of their menstrual cycle. Of these, 19.2% were
diagnosed with adenomyosis, and they showed significantly lower clinical pregnancy rates than
those without the disease (29.2% vs. 42.6%; p = 0.044; RR 0.68, 95% CI 0.47–1.0).32 Despite the fact
that patients with adenomyosis were older and had lower ovarian reserve, logistic regression analy-
sis showed adenomyosis to be an independent factor from age and anti-Müllerian hormone (AMH)
in patients who had at least four of the seven ultrasound signs of adenomyosis. Moreover, clinical
pregnancy rates dropped gradually as more severe disease was discovered, further supporting a
causal relationship.
There is no consensus on management during IVF, especially of the diffuse forms. Surgery, espe-
cially in focal disease, and a suppression period with gonadotropin-releasing hormone (GnRH)
agonists are the two treatment options, with unproven effectiveness.
Adenomyosis should be in the checklist of the clinician since now it is considered as a possibly
important factor hindering IVF success.
Two hundred ten women with normal hysterosalpingography (HSG), who had underwent ≥2 failed
IVF cycles in which ≥2 good-quality embryos were transferred, were evaluated with office hyster-
oscopy, and 8.5% of them were found to have flimsy or mild endometrial adhesions, while 2.3%
had cervical adhesions.33 Most patients with intrauterine adhesions benefit from hysteroscopic
treatment, reaching a 63% pregnancy rate in a pool of 1542 patients.34 Still, severe adhesions may
recur and result in lower live birth rates than mild and moderate forms (31.9%, 81.3%, and 66%,
respectively).35
Patients with RIF, especially if they have a history of uterine procedures, should be carefully
evaluated for intrauterine adhesions as they may be easily missed, and their treatment provides
successful results in expert hands, especially in mild and moderate forms.
Hydrosalpinx
A meta-analysis of 5592 IVF patients with tubal factor infertility, of which 1004 had hydrosal-
pinx, showed pregnancy rates of 19.7% and 31.2% (OR 0.64, 95% CI 0.56–0.74) for patients with
Anatomical factors 97
and without hydrosalpinx, respectively.36 More dramatically, a meta-analysis of 5569 cycles that
included a subgroup of 1144 cycles from patients with hydrosalpinx reported 50% lower clinical
pregnancy rates for patients with hydrosalpinx.37 Finally, an RCT by Strandell et al., involving IVF
patients with visible hydrosalpinx in ultrasonography, showed that laparoscopic salpingectomy
doubled the chance of delivery compared with no intervention.38
Patients with RIF should be carefully evaluated for signs of hydrosalpinx in ultrasonography,
especially if they have undergone any abdominal operations or were diagnosed with a sexually
transmitted disease since their last evaluation with ultrasound or HSG. Despite the absence of evi-
dence, occult hydrosalpinx, which is not large enough to be visualized by ultrasonography, can be
expected to hamper chances of ART success. Thus, we recommend ruling out occult hydrosalpinx
in a woman suffering RIF.
Assessment of uterine factor and hydrosalpinx

Despite the fact that patients with RIF have undergone many examinations during their treatment
cycles, a thorough gynecological examination should be done with a fresh, unconditioned look to
rule out any problems that may lower the chance of implantation in RIF patients.
Three-dimensional (3D) transvaginal US has the advantage of obtaining a coronal view and pro-
viding accurate and reproducible information about both external and internal contours of the
uterus, ideally when endometrium is 5 mm or thicker.39 Three-D US showed 100% specificity and
sensitivity for diagnosing CUA in two different studies, and its concordance with specificity and
sensitivity of laparoscopy and hysteroscopy was reported to be 100% and 96%, respectively.40,41
A study of 54 women with infertility comparing 3D US and HSG reported 3D US to have a sensi-
tivity of 100% and HSG 66.7% for correctly diagnosing and grading intrauterine adhesions. HSG
failed in diagnosing lower uterine segment adhesions mostly, mistaking them for complete cavity
obstructions.42
The preferred method of examination should be 3D transvaginal US, a very sensitive and spe-
cific tool that can successfully diagnose almost all of the uterine pathologies. In cases in which
the suspected pathology’s relationship with the endometrial cavity needs further investigation, it
can be combined with saline infusion sonography (SIS) and provide comparable results to hyster-
oscopy, the accepted golden standard. SIS improves diagnostic performance of both 2D and 3D
US.43–46 Sylvestre et al. documented a sensitivity of 100% and positive predictive value (PPV) of
92% for 3D SIS when compared to hysteroscopy.47 El-Sherbiny compared the accuracy of 2D SIS
and 3D SIS in 120 patients undergoing hysteroscopy.48 Sensitivity, specificity, and PPV and nega-
tive predictive values (NPV) of 2D SIS for intrauterine adhesions were 50%, 98.1%, 75%, and 94.6%,
respectively, while they were 75%, 100%, 100%, and 97.3%, for 3D SIS. Interestingly, in another
recent study, 3D SIS diagnosed synechia in only 50% of the hysteroscopically proven cases in a
study with 16 Asherman syndrome patients.49 However, it should be noted that US examinations
are user-dependent and give the best results in experienced hands.
Bermejo et al. evaluated 65 patients with possible CUA using both 3D US and magnetic reso-
nance imaging (MRI) and demonstrated a high degree of concordance between the two modalities
(kappa index = 0.88).50 Likewise, we reported 100% categorical agreement between 3D US and MRI
in not only diagnosis but also measurements of uterine septa.51 As 3D US provides comparable
results, the more expensive and less practical MRI should be reserved for complex congenital uter-
ine pathologies, some complex multiple fibroid cases, cases where adenomyosis diagnosis should
be confirmed, and for some patients who cannot undergo transvaginal US examinations and for
whom transabdominal US is suboptimal.
Since HSG does not provide information about serosal surface and is not 100% sensitive for diag-
nosing endometrial polyps and submucous fibroids, it should be performed whenever hydrosalpinx
is suspected, when other pathology is ruled out with 3D US-SIS. However, when hydrosalpinx is
clearly visible in US, it is not mandatory. Since 3D SIS provides comparable results, hysteroscopy is
used more for treatment and less for diagnostic purposes.
It is interesting to note that accuracy of diagnostic methods for intrauterine adhesions varies
considerably between the studies. This may be due to the fact that it is technically more difficult to
diagnose adhesions, and operator experience and device quality may play a role.52 The 2017 practice
guidelines of the American Association of Gynecological Laparoscopists (AAGL), in collaboration
with European Society for Gynaecological Endoscopy (ESGE), suggest performing hysteroscopy,
if available, since it is the most accurate method for diagnosis of intrauterine adhesions.53 SIS and
HSG are declared as the reasonable alternatives. On the other hand, some authors point out that
3D SIS provides comparable results with hysteroscopy, and therefore it could be used for diagnosis
and hysteroscopy for indeterminate or suspicious cases.52,54,55
Whether diagnostic hysteroscopy benefits the subgroup of RIF patients was investigated in a
multicenter RCT called TROPHY.56 This study recruited 702 women younger than 38 years with
normal ultrasound findings who had 2–4 failed IVF cycles and randomized the women into out-
patient hysteroscopy before IVF or starting IVF right away. Eighty-five women in the hysteroscopy
group were found to have cervical or uterine abnormalities, mostly minor. Still, 15 of the 34 uterine
cavity abnormalities were surgically corrected. The live birth rates of both groups were the same,
29% (RR 1.0, 95% CI 0.79–1.25; p = 0.96). It was concluded that outpatient hysteroscopy before IVF
did not increase the live birth chance of women with a history of 2–4 failed cycles who had normal
ultrasound findings.
Therefore, it seems reasonable to perform a SIS and perform other diagnostic methods when
needed. Hysteroscopy should be reserved for patients who are suspected or known to have uterine
pathologies that can benefit from surgical correction.
HEMATOLOGICAL AND IMMUNOLOGICAL FACTORS

In a survey performed in the United Kingdom, 75% of the 44 participating centers reported testing
for lupus anticoagulant/anticardiolipin antibodies in patients with RIF, making it the most popu-
lar test, surpassing parental karyotype testing and hysteroscopy.57 Congenital thrombophilias was
the fourth most investigated condition with 59%. Despite the widespread use of these tests, the
evidence on the effect of thrombophilias on ART success is conflicting. Moreover, even if there is
causal relationship between the two, whether or not treatment with anticoagulants improves the
outcomes is unknown.
Congenital thrombophilias
Congenital thrombophilias are genetic conditions that predispose the affected person to venous
thromboembolism. The most well-known are factor V Leiden mutation (FVL), prothrombin gene
mutation G20210A (P2), methylene tetrahydrofolate reductase C667T mutation (MTHFR), and
proteins C and S and antithrombin (AT) deficiencies.58 Some of these conditions may be associated
with late pregnancy complications such as preeclampsia, growth restriction, and second- or third-
trimester fetal loss, but not first-trimester loss.59
Most of the studies on the relationship of RIF and congenital thrombophilia are case reports or
cohort studies. A meta-analysis of available case-control and cohort studies did not find any sig-
nificant association between IVF failure and P2 or deficiencies of proteins C or S or AT.60 FVL was
shown to increase the ART failure three times according to the analysis of eight case-control studies
(OR 3.08, 95% CI 1.77–5.36); however, the association was insignificant when the only three cohort
studies on the subject were analyzed (RR 0.62, 95% CI 0.35–1.08).
In a meta-analysis, a subgroup analysis of eight case-control studies on the relationship between
P2 mutation and ART failure did not show any significant difference when homozygote or hetero-
zygote patients were compared with controls (OR 1.48, 95% CI 0.71–3.06).61 Evaluation of seven
studies showed that patients with either homozygote or heterozygote mutations had similar ART
failure rates with normal controls (OR 1.31, 95% CI 0.95–1.80). According to the analysis of three
studies on protein C, protein S, and AT deficiencies, the incidence of these conditions was similar
Hematological and immunological factors 99
in women with and without a history of ART failure (OR 1.68, 95% CI 0.17–16.49), (OR 1.58, 95%
CI 0.45–5.48), (OR 2.09, 95% CI 0.39–11.28), respectively.62–64
In conclusion, available evidence on the effect of congenital thrombophilias on RIF is based on
case-control or cohort studies, and the pooled data implies that routine testing for these conditions
in patients with RIF is not indicated.
Acquired thrombophilias and antiphospholipid antibodies

A variety of medical conditions may result in acquired thrombophilia, such as antiphospholipid
syndrome (APS), paroxysmal nocturnal hemoglobinuria, polycythemia vera, essential thrombocy-
tosis, heparin-induced thrombocytopenia, or increased serum estrogen levels.58 Most either neces-
sitate specific medical attention or are transitory conditions. On the other hand, APS is a chronic
thrombophilic condition that may have been undiagnosed in patients with RIF.
Antiphospholipid antibody (APA) positivity has been associated with RPL by many clinicians
and is perceived as a potential cause of RIF. As mentioned previously, 75% of the fertility centers in
the United Kingdom screen their patients with RIF for APAs, namely lupus anticoagulant, anticar-
diolipin IgM and IgG, and anti-β2 glycoprotein-I IgM and IgG.57 In 2013, the Obstetric Task Force
of the 14th International Congress on Antiphospholipid Antibodies performed a literature review
of the studies examining the relationship between APA and RPL.65 Of the 46 studies identified, 27
reported an association. It was noted that majority of the studies were small in size, used arbitrary
cutoff values for APA positivity, and documented APA positivity on a single occasion. However,
the updated Sapporo criteria require that the APA levels should be above 40 GPL or MPL, or three
standard deviations of healthy controls on ≥2 occasions, at least 12 weeks apart.66 Therefore, the
task force declared the association between APA and RPL to be inconclusive. How reasonable is
applying the same line of thinking to RIF, a different condition than RPL, is questionable.
A meta-analysis by Di Nisio et al. on the effect of APA on ART success yielded conflicting
results.61 When only the case-control studies were examined (20 studies including 3542 patients),
positivity of at least one APA showed a three-fold increase in ART failure risk (OR 3.33, 95% CI
1.77–6.26). However, analysis of nine cohort studies with a total of 1556 patients on the effect of
APA positivity and ART outcome showed no significant difference in pregnancy test results (RR
0.95, 95% CI 0.76–1.19). Clinical pregnancy and live birth rates were similar as well. This discrep-
ancy between the two analyses can be attributed to the heterogeneity of the studies and use of inap-
propriate control groups, since healthy controls with spontaneous pregnancies may not reflect the
effect of infertility or the ART procedures.
In summary, the current evidence on the association of APA positivity and ART outcomes or
RIF is weak due to problems with heterogeneity, methodology, and lack of power. Moreover, even
if there is an association between APA positivity and ART outcomes, more research on treatment
options and their effects is needed. Therefore, screening for APA is highly controversial and, in
our opinion, should not be offered routinely outside a research setting or in the absence of other
indications than RIF.
Other immunologic factors

A variety of immunologic mechanisms, for example, increased peripheric natural killer (NK) cell
count, Th1/Th2 ratio, have been proposed as etiologic factors for RIF. Resultantly, a wide array of
tests and treatments are being performed worldwide. Even though it could be naïve to assume that
the immune system does not play a role in embryo implantation and viability of pregnancy, several
conditions must be met before any immunological test or treatment is offered in clinical practice.
First, a pathologic process hampering implantation has to be clearly defined. Second, a reliable and
reproducible diagnostic algorithm enabling the identification of couples with this particular pro-
cess needs to be developed. Last, an effective treatment proved by credible trials has to be defined.
Unfortunately, none of these are done yet. Thus, we regard it appropriate to offer any immunologic
tests only in the context of properly designed, ethically sound and approved research projects,
without incurring any additional costs to the couples.
ENDOCRINE FACTORS
Thyroid function and autoimmunity
The Endocrine Society suggests testing for thyroid peroxidase antibodies (anti-TPO) if thyroid stimu-
lating hormone (TSH) is above 2.5 mIU/L repeatedly and starts treatment if antibodies are positive.67
The possible role of thyroid autoimmunity (TAI) in ART outcomes has been a subject of debate
as well. Conflicting results from mostly small-sized studies have been published on the relation-
ship between TAI and ART. A 2016 meta-analysis pooled 299 TAI-positive patients and 1931 TAI-
negative patients from nine cohort studies and concluded that women with TAI had a lower live
birth rate after ART compared with those without TAI (OR 0.73, 95% CI 0.54–0.99; p = 0.04).68,69
However, almost simultaneously, Unuane et al. published a large retrospective cohort study com-
paring cumulative delivery rates after six cycles in euthyroid patients with and without TAI.70 Their
study included 333 TAI-positive patients and 2019 TAI-negative patients, both arms enrolling more
patients than those pooled in the aforementioned meta-analysis. With a cumulative delivery rate of
47% in both groups, Unuane concluded that TAI status does not affect cumulative delivery rates in
IVF/ICSI patients. It is noteworthy that the results were similar when a subgroup analysis accord-
ing to TSH thresholds of 2.5 and 5 mIU/L was performed.
RIF patients have a complicated history; therefore it is easy to overlook a simple biochemical test
like TSH. Even if it has been done, it may be dating a few years back, probably to the beginning of
the treatment. Obtaining a current TSH level before starting a new treatment would be prudent
in a patient with RIF, since it is an inexpensive, simple test that is able to detect an easily treatable
condition. Measuring free thyroxine (fT4) levels or thyroid antibodies can be saved for patients
with TSH levels above 2.5 mIU/L.
Blood glucose
As impaired glucose tolerance may result in adverse pregnancy outcomes, they may have a role
in RIF as well. As a practical and relatively cheap screening tool, hemoglobin A1c can be tested
in these patients. Women with risk factors, such as obesity, polycystic ovarian syndrome, glucose
tolerance, or a family history of diabetes mellitus can be offered oral glucose-tolerance tests.
Vitamin D
A recent meta-analysis reported that vitamin D replete ART patients had a significantly higher live
birth rate (seven studies including 2026 patients, OR 1.33, 95% CI 1.08–1.65) and clinical pregnancy
rate (11 studies including 2700 patients, OR 1.46, 95% CI 1.05–2.02).71
The possible mechanism by which vitamin D affects implantation and early pregnancy is unclear.
To the best of our knowledge, no research has been published regarding vitamin D levels in patients
with RIF. Therefore, it is not possible to declare a firm stance on its screening. Yet, in light of the
current research and due to the fact that it is a relatively easy and cheap test, vitamin D levels can
be tested and supplementation can be prescribed if they are below the 30 ng/mL level, which is the
threshold declared by the Endocrine Society.72
ENDOMETRIAL RECEPTIVITY
Only 65% of euploid blastocysts successfully implant.73 This implies that in addition to genetic
defects at the subchromosal level, other factors, possibly endometrial receptivity or its crosstalk
with the embryo, can be dysfunctional.
It is suggested that the window of implantation (WOI) is not constant for all women, and patients
whose WOI has shifted may experience asynchrony between the endometrium and the embryo,
resulting in RIF.74
Conclusions and suggested algorithm 101
A commercial test called endometrial receptivity array (ERA) has been developed to analyze the
endometrial transcriptome in natural or hormone replacement cycles using an array of 238 genes.75
According to the combination of genes transcribed in the sample acquired from the uterus with
a pipelle catheter on a specific date in the cycle, the result is given as receptive, meaning endome-
trium is synchronous with the WOI, or non-receptive, meaning there is discrepancy between the
two. In the latter case, another sample is acquired in the next cycle at the presumed receptive date
until the receptive period is determined. A prospective multicenter trial compared 85 RIF patients
with 25 women with <2 failed cycles. Seventy-four percent of RIF patients and 88.1% of the c ontrols
were reported to have receptive endometrium. When personalized embryo transfers (i.e., the day of
embryo transfer changed in relation to the duration of endometrial progesterone exposure based
on ERA results) were performed in eight women in the non-receptive group, pregnancy rates were
similar to the general IVF population, 50% versus 48%, respectively. The authors concluded that
in RIF patients, ERA provides pregnancy rates comparable to those who had a maximum of one
failed ART cycle.
However, the fact that only a small percentage of patients are found to be non-receptive and
almost all the clinical studies are coming from a single group raises questions about its practicality
and validity.
In a recent independent study from Japan, ERA was performed on 50 patients with RIF and
24% were found non-receptive.76 Pregnancy rates per patient were 58.8% and 50% for the initially
receptive and non-receptive groups, respectively (p = 0.89). Likewise, implantation rates (32.8% vs.
31.6%, respectively) and take-home baby rates (23.7% vs. 16.7%, respectively) showed no statistical
significance.
More recently, Cho et al. reported a case of a 44-year-old woman with two failed donor blasto-
cyst transfers who had four ERA tests in a 4-month period.77 Significant variability was present
in the first three tests, as none of the tests overlapped in the WOI timeframe, despite the fact that
her weight or the hormone replacement protocol were the same in all cycles. The authors con-
clude that consistency and reproducibility of the ERA test should be validated with independent
studies.
An interesting and promising study was performed in 2016 by Koot et al. on 43 RIF patients and
72 controls, consisting of readily conceived women with ICSI due to severe male infertility.69 The
authors suggested that the only problem is not the endocrine shift in WOI, but probably intrin-
sic molecular endometrial defects. Using mid-luteal endometrial biopsy samples, they were able
to identify a 303-gene transcriptome signature predictive of RIF. Sensitivity, specificity, PPV, and
NPV of the test in the signature development set were 90.3%, 94%, 90.3%, and 94%, respectively.
However, in an independent validation set of 34 samples, the transcriptome signature had 58%
sensitivity but 100% PPV for RIF. If confirmed, this could be a very useful tool to inform couples
of their future prospects.
While, the latter is not commercially available yet, we think ERA is more appropriate for the
research setting than routine clinical practice at the moment.
CONCLUSIONS AND SUGGESTED ALGORITHM

Investigating the etiology of RIF somewhat resembles sailing in uncharted waters. The evidence
behind a causal relationship between suggested pathologies and RIF is, in most cases, low qual-
ity. Likewise, effectiveness of proposed treatments is poorly supported by evidence. With all these
uncertainties, a clinician’s duty is to honestly share their professional opinion on controversies,
while carefully avoiding undue despair as well as raising false hope with tests or treatments of
unproven effectiveness. The majority of the possible reasons behind RIF should have been examined
even before starting the first ART cycle; however, after every failed ART cycle, one should always
check whether necessary investigation has been completely done, noting anything that could have
changed the couple’s condition since the latest test has happened in between cycles. A template can
be used as a framework for a clinic’s own protocol for assessment of couples with RIF (Table 10.1).
Table 10.1 Etiologic factors and diagnostic tests for investigation of recurrent implantation failure.
Tests that are unnecessary
in the absence of another
Etiologic factor Proposed test Tests with unproven value indication
Genetic
Maternal Karyotype
Paternal Karyotype Sperm DNA integrity tests
Anatomic
Uterine 3D US with SISa Hysteroscopyb
Tubal Hysterosalpingography
Hematological & • Peripheric NK count • APS screening with
immunological • Th1/Th2 Ratio Sapporo criteriac
• Other immunologic tests • Congenital
thrombophiliad
Endocrine HbA1C
TSH
Vitamin D
Endometrial Endometrial receptivity array
receptivity
Abbreviations: APS, antiphospholipid antibody; SIS, saline infusion sonography.
a Magnetic resonance imaging for complex congenital uterine anomalies or adenomyosis.
b If history or ultrasound imaging suggests intrauterine adhesions.
c In the presence of recurrent pregnancy loss.
d In the presence of personal or family history of thrombosis.
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37. Zeyneloglu HB, Arici A, Olive DL. Adverse effects of hydrosalpinx on pregnancy rates after in
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38. Strandell A, Lindhard A, Waldenstrom U, Thorburn J, Janson PO, Hamberger L. Hydrosalpinx
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41. Ghi T, Casadio P, Kuleva M et al. Accuracy of three-dimensional ultrasound in diagnosis and
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42. Knopman J, Copperman AB. Value of 3D ultrasound in the management of suspected
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43. Saravelos SH, Cocksedge KA, Li TC. Prevalence and diagnosis of congenital uterine
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44. Shokeir T, Abdelshaheed M. Sonohysterography as a first-line evaluation for uterine abnor-
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45. Ludwin A, Pitynski K, Ludwin I, Banas T, Knafel A. Two- and three-dimensional ultraso-
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46. La Torre R, De Felice C, De Angelis C, Coacci F, Mastrone M, Cosmi EV. Transvaginal sono-
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47. Sylvestre C. A prospective study to evaluate the efficacy of two- and three-dimensional sono-
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48. El-Sherbiny W, El-Mazny A, Abou-Salem N, Mostafa WS. The diagnostic accuracy of two- vs
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52. Saravelos SH, Jayaprakasan K, Ojha K, Li TC. Assessment of the uterus with three-dimen-
sional ultrasound in women undergoing ART. Hum Reprod Update. 2017;23(2):188–210.
53. AAGL practice report: Practice guidelines on intrauterine adhesions developed in col-
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54. Ahmadi F, Javam M. Role of 3D sonohysterography in the investigation of uterine synechiae/
asherman’s syndrome: Pictorial assay. J Medical Imaging Radiat Oncol. 2014;58(2):199–202.
55. Zafarani F, Ahmadi F. Evaluation of intrauterine structural pathology by three-dimensional
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56. El-Toukhy T, Campo R, Khalaf Y et al. Hysteroscopy in recurrent in-vitro fertilisation fail-
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Optimizing embryo culture for
11
KATERINA CHATZIMELETIOU
The human preimplantation embryo is exposed to specific nutrients within the reproductive tract.1
Analysis of the fluids within the fallopian tube and the uterus have revealed that glucose levels are
at their lowest (0.5 mM) and pyruvate (0.32 mM) and lactate (10.5 mM) levels at their highest in
the fallopian tube at the time when the fertilized oocyte and early-cleavage-stage embryo is present,
while in the uterus, glucose is present at a much higher level (3.15 mM), whereas pyruvate (0.1 mM)
and lactate (5.87 mM) are significantly lower than in the fallopian tube.1 This nutrient availability
reflects the energy requirements of the developing preimplantation embryo and is mirrored in the
culture media used for in vitro fertilization (IVF).2–5 The early-cleavage-stage embryo is character-
ized by pyruvate uptake, low levels of oxidative metabolism and low oxygen consumption, whereas
at the blastocyst stage, it exhibits both high levels of glycolysis and high oxygen consumption.6–8
Sequential media (cleavage and blastocyst stage media) were therefore developed to maintain the
embryo’s metabolic needs in culture up to day three and from day three through day five postfer-
tilization, respectively. However, single-step continuous media have lately been introduced, aiming
to provide an uninterrupted culture from day one through day five and enabling the embryo to
choose and consume the ingredients it requires at each specific stage of preimplantation develop-
ment.9–14 To date, no statistically significant differences have been observed between sequential and
one-step media with regard to implantation and pregnancy rates as well as among different media
that belong to the one-step or the sequential category.9–14
EXTENDED CULTURE
Extended culture to the blastocyst stage enables better embryo selection and can lead to increased
implantation rates and reduced need for high-order multiple pregnancy (HOMP) reduction.15
A prospective, nonrandomized analysis was performed in 276 IVF patients who failed to conceive
after at least two early embryo transfers of at least two grade 1–2 embryos per cycle.16 For their next
attempt, these couples chose between day-two embryo transfer (D2 group; n = 147) and day-five/
six blastocyst transfer (D5/D6 group; n = 129) before starting their following attempt. The results
showed that the live birth rates per cycle (27.9% vs. 19.7%) and implantation rates per cycle (25.4%
vs. 12.4%) were higher in the D5/D6 group compared with the D2 group.16 To evaluate the effi-
cacy of blastocyst transfer among patients with at least three previous cleavage-stage failed embryo
transfers and to compare implantation and pregnancy rates of blastocysts according to the day of
transfer (day five or day six), Barrenextea et al.,17 performed a retrospective clinical study. They con-
cluded that blastocysts transferred on day five implanted almost five times the rate of those trans-
ferred on day six (23% vs. 5%). Pregnancy rates were triple as high among the 73 day-five patients
compared with the 63 day-six transfer patients (38% vs. 11%).17
HYALURONIC ACID-ENRICHED TRANSFER MEDIUM

Adjunct treatments currently being offered in IVF laboratories, aiming to enhance embryo qual-
ity and implantation potential, include embryo glue and adherence compounds.18–20 EmbryoGlue
is a hyaluronic acid-enriched embryo transfer (ET) medium developed to improve implantation
of embryos in in vitro fertilization-ET cycles (IVF-ET). The exact mechanism of action of the
107
108 Optimizing embryo culture for recurrent implantation failure
hyaluronic acid on implantation is still unclear. Hyaluronic acid has been shown to increase cell–
cell adhesion and cell–matrix adhesion,21 and in turn may play a key role during the initial stages
of apposition and attachment of the blastocyst and endometrium.22 Its functions also include regu-
lation of protein secretion, gene expression, cell proliferation, and differentiation.23,24 Hyaluronic
acid can promote angiogenesis by both its degradation products25 and by interaction with epider-
mal growth factor (EGF),26 while its physical properties include producing a viscous solution that
may facilitate the embryo transfer process and prohibit the expulsion of embryos from the uterus.22
It has also been suggested that it confers some degree of viral protection and anti-immunogenic
properties that inhibit embryo rejection.27
In a prospective case-control study, embryos were transferred into 50 µL of EmbryoGlue for
10 min prior to transfer in the uterus, while in the control group embryos were transferred to
conventional blastocyst culture medium.20 The clinical pregnancy rate in the study group was 7%
higher than the control group, but not statistically significant. A significant difference in clinical
pregnancy rate was, however, observed with the EmbryoGlue in patients with previous IVF fail-
ure (p = 0.04) as 50% of patients with previous IVF failure had successful implantation, but none
of the patients with previous implantation failure, in the control group, achieved a pregnancy.20
Valojerdi et al., 200628 also reported in a prospective, randomized trial that clinical pregnancy rate
in patients with tubal factor, and implantation rate (IR) in patients with the tubal factor infertil-
ity and those with recurrent implantation failure (RIF), increased significantly in patients whose
embryos were treated with EmbryoGlue compared with those in the control group. In particular,
the clinical pregnancy rate (PR) increased significantly in the patients with tubal factor (38% vs.
18.8%, p < 0.05), whereas the implantation rate increased significantly in the patients with the
tubal factor (20.5% vs. 8.9%, p < 0.05) and in patients with recurrent implantation failures (16% vs.
11%, p < 0.05). In the EmbryoGlue group, the rates of live birth/embryonic sac and triplet deliver-
ies increased significantly compared with those in the control group (74.5% vs. 62.5% for the for-
mer and 9.5% vs. 1.4% for the latter, p < 0.05).28
In a retrospective analysis the effect of hyaluronic acid-enriched transfer medium (HETM) on
frozen-thawed embryo transfer (FET), outcomes were assessed.29 HETM was used for 347 cycles
of 342 patients and standard medium for 1374 cycles of 1290 patients. Overall, FET outcomes were
similar between the groups. For patients undergoing their first FET attempt, the IR (24.3% vs.
31.6%, p = 0.042) and clinical PR (34.3% vs. 50.1%, p = 0.004) were lower in the HETM group.
For patients undergoing their second FET attempt, pregnancy outcomes were similar between the
groups. For patients undergoing their third or more FET attempt, HETM was associated with a
higher IR (33.3% vs. 16.4%, p < 0.001) and higher PR (52.2% vs. 27.4%, p < 0.001).29
SUPPLEMENTATION OF CULTURE MEDIUM WITH EPIDERMAL GROWTH FACTOR,

4-HYDROXYESTRADIOL, USE OF MITOCHONDRIAL NUTRIENTS
Poor embryo development and RIF is sometimes attributed to poor/impaired mitochondrial
function and reduced energy availability.30 Injection of mitochondria-rich cytoplasm from donor
oocytes has been suggested to improve oocyte and embryo quality and increase implantation and
pregnancy rates.31 Administration of mitochondrial nutrients like coenzyme Q10 (CoQ10) has
been shown to exert a beneficial effect to the function of oocyte mitochondria and reverse the
oocyte age-related mitochondrial dysfunction and improve embryo development.32–34 In a prospec-
tive, randomized, double-blind, controlled study, the use of CoQ10 improved pregnancy rates in
patients with poor embryo development and RIF.34 In animal studies, supplementation of medium
with prolactin (PRL), epidermal growth factor (EGF), and 4-hydroxyestradiol (4-OH-E2) prior to
embryo transfer has also been shown to improve implantation potential.35
COCULTURE OF EMBRYOS ON HOMOLOGOUS ENDOMETRIAL CELLS

Coculture of embryos on homologous endometrial cells has been performed in order to increase
implantation rates in patients with repeated implantation failures.36–39 A retrospective comparison
References 109
of pregnancy rates between IVF-ET with coculture and standard culture methods was reported
by Jayot et al.38 The authors concluded that the overall pregnancy rate for these patients was 21%
per transfer versus 8% in previous IVF-ET cycles, while a higher percentage (28%) was obtained
for women <39 years of age or on transfer of at least one morula (32.5% pregnancy per trans-
fer). Eyheremendy et al.39 also showed an increase in pregnancy rates from 5% in the conventional
IVF-ET cycles to 57% in the IVF-ET cycles with the endometrial cell coculture.
TIME-LAPSE SYSTEMS
Embryo scoring has been traditionally performed under a light microscope following removal
of embryos from a conventional incubator for quality assessment by an embryologist. In recent
years, time-lapse systems have been introduced, which can provide digital images of embryos at
frequent time intervals, allowing continuous monitoring of embryo development and enabling
the embryologists to evaluate the quality of the embryos without having to physically remove
them from the incubator.40–45 The potential advantages of a time-lapse system (TLS) include (a)
the ability to maintain a stable culture environment, therefore limiting the exposure of embryos to
exogenous stress- changes in gas composition, temperature, and movement and (b) an advanced
scoring system that allows better embryo selection. In a double-blind, controlled study, Rubio
et al.42 examined the effect of embryo incubation in a TLS combined with the use of a multivari-
able scoring system for embryo selection on implantation and ongoing pregnancy rate. In this
study, the patients were randomized to the TLS group and a control group that had their embryos
developed in a conventional standard incubator (SI) and assessed only by conventional morpho-
logic criteria. The results showed that both the implantation rate and the ongoing pregnancy rate
were significantly higher in the TLS group. In particular, the ongoing pregnancy rate was statisti-
cally significantly increased at 51.4% (95% CI 46.7–56.0) for the TLS group compared with 41.7%
(95% CI 36.9–46.5) for the SI group. For pregnancy rate, differences were not statistically signifi-
cant at 61.6% (95% CI 56.9–66.0) versus 56.3% (95% CI 51.4–61.0). The results per transfer were
similar: statistically significant differences in ongoing pregnancy rate of 54.5% (95% CI 49.6–59.2)
versus 45.3% (95% CI 40.3–50.4) and not statistically significant for pregnancy rate at 65.2% (95%
CI 60.6–69.8) versus 61.1% (95% CI 56.2–66.1). Early pregnancy loss was statistically significantly
decreased for the TLS group with 16.6% (95% CI 12.6–21.4) versus 25.8% (95% CI 20.6–31.9). The
implantation rate was statistically significantly increased at 44.9% (95% CI 41.4–48.4) versus 37.1%
(95% CI 33.6–40.7). However, Amstrong et al.,45 concluded that there is insufficient evidence of
differences in live birth, miscarriage, stillbirth, or clinical pregnancy to choose between TLS,
with or without embryo-selection software, and conventional incubation. To date, no randomized
controlled trials have been published evaluating the utility of TLS to improve outcomes among
patients with RIF.
CONCLUSIONS
Any new technology aiming to improve implantation, clinical pregnancy, and live birth rates
should, in most cases, first be tested in an appropriate animal model, then in clinical trials, to
ensure safety, and finally in a randomized controlled trial (RCT) to provide high-quality evi-
dence that the procedure is safe and effective.18 Improved embryo selection and uterine receptiv-
ity may explain the additional benefit of embryo transfer at the blastocyst stage for couples with
repeated implantation failures.10,15–17,46,47 It is difficult, however, to conclude a favorable role of
Hyaluronic acid-enriched media or coculture systems in patients with recurrent implantation
failure.20,28,36,38,39
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37. Michalski SA, Chadchan SB, Jungheim ES, Kommagani R. Isolation of human endometrial
stromal cells for in vitro decidualization. J Vis Exp. 2018 September 1;(139). doi: 10.3791/57684.
38. Jayot S, Parneix I, Verdaguer S, Discamps G, Audebert A, Emperaire JC. Coculture of embryos
on homologous endometrial cells in patients with repeated failures of implantation. Fertil
Steril. 1995 January;63(1):109–14.
39. Eyheremendy V, Raffo FG, Papayannis M, Barnes J, Granados C, Blaquier J. Beneficial effect
of autologous endometrial cell coculture in patients with repeated implantation failure. Fertil
Steril. 2009;93:769–73.
40. Herrero J, Meseguer M. Selection of high potential embryos using time-lapse imaging: The
era of morphokinetics. Fertil Steril. 2013;99:1030–4.
41. Goodman LR, Goldberg J, Falcone T, Austin C, Desai N. Does the addition of time-lapse mor-
phokinetics in the selection of embryos for transfer improve pregnancy rates? A randomized
controlled trial. Fertil Steril. 2016;105:275–85.
42. Rubio I, Galan A, Larreategui Z et al. Clinical validation of embryo culture and selection
by morphokinetic analysis: A randomized, controlled trial of the EmbryoScope. Fertil Steril.
2014;102:1287–94.
43. Meseguer M, Herrero J, Tejera A, Hilligsoe KM, Ramsing NB, Remohi J. The use of morpho-
kinetics as a predictor of embryo implantation. Hum Reprod. 2011;26:2658–71.
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Reprod Genet. 2012;29:1227–39.
Optimizing endometrial
receptivity for patients with
12
The role of progesterone on the day
of triggering final oocyte maturation
CHRISTOS A. VENETIS, JULIA K. BOSDOU,
INTRODUCTION
Recurrent implantation failure (RIF) as a clinical diagnosis refers to a group of women that repeat-
edly fail to conceive after assisted reproductive technology despite the transfer of a number of
embryos. Notwithstanding the obvious heterogeneity in the composition of this diagnostic group,
the common symptom is the inability of multiple embryos to implant.1
Despite the intense research efforts of the scientific community and the great advances that have
been achieved, the exact process of implantation is still largely considered “a black box”. What
seems to be clear is that implantation usually requires not just a capable embryo but also a receptive
endometrium2 and a successful “crosstalk” between the two.3 Therefore, women affected by RIF are
likely to have compromised endometrial receptivity for a number of reasons.1 Understanding the
physiological basis of endometrial receptivity is important and can lead to a better understanding
of why some women have a higher propensity to be affected by RIF than others.
PROGESTERONE AND ENDOMETRIAL RECEPTIVITY

The endometrium is a dynamic, hormonally responsive tissue that undergoes molecular, histo-
pathological, and immunological changes throughout the menstrual cycle.4 The purpose of these
changes is its preparation for the potential implantation of a hatched blastocyst. Histopathological
analysis of luteal phase endometria shows secretory transformation of the glandular epithelium
and stromal edema.5 This is followed by a predecidual reaction around the terminal portion of
the spiral arteries and the luminar epithelium.4 Decidualization, the differentiation of stromal
fibroblasts to “epitheliod” cells, is a key preparatory step for the establishment and maintenance
of pregnancy.6 Furthermore, gene expression studies have identified specific signatures associ-
ated with the transformation of the endometrium to one that has the capacity to permit embryo
implantation.7,8
Importantly, it is universally accepted that exposure of the endometrium to progesterone is fun-
damental for the attainment of a receptive status. Another key observation is the importance of
timing, since the endometrium becomes receptive for only a specific period of time during the mid-
luteal phase. The pioneering work by Psychoyos et al. demonstrated, using electron microscopy,
that under the effect of progesterone, the endometrium undergoes specific changes that lead to the
appearance of pinopodes.9 These structures were suggested to determine the time during a woman’s
cycle when her endometrium is receptive, named the implantation window,10 the duration of which
113
114 Optimizing endometrial receptivity for patients with recurrent implantation failure
was suggested to be no more than 48 hours.2 The same group produced evidence strongly support-
ing that after ovarian stimulation, this implantation window could be shifted, usually arriving 1–2
days earlier. The strongest predictor of the premature changes observed in these endometria was
serum progesterone concentration in the late follicular phase.11,12
PROGESTERONE CONCENTRATION DURING OVARIAN STIMULATION

In women, the main sources of progesterone are (a) the ovary, (b) the adrenals, and (c) the placenta.
Despite the fact that progesterone is considered a hormone that dominates the luteal phase due to
the increased production by the corpus luteum, it is also produced during the follicular phase.
In the early follicular phase, it has been shown that the main source of progesterone is the adre-
nals.13–15 It has also been demonstrated that, during ovarian stimulation with gonadotrophins for
in vitro fertilization (IVF) and especially during the late follicular phase, serum progesterone fre-
quently increases.16,17
This increase in serum progesterone was initially considered to be consistent with premature
luteinization, which requires a luteinizing hormone (LH) rise or surge, which, in turn, stimulates
the theca interna to produce progesterone and/or also luteinizes the existing follicles. However,
through a number of studies, it was clearly shown that even in cases where LH is suppressed
using gonadotrophin releasing hormone (GnRH) analogues or the oral contraceptive pill, a rise
in serum progesterone is still observed.13–15 Although this led initially to the conclusion that
the adrenal glands were the main source of the progesterone rise during the follicular phase,
a subsequent study in which the adrenals were suppressed using dexamethasone strongly sug-
gested that the progesterone rise is actually associated with the exogenous administration of
gonadotrophins.18
Today, the prevailing theory is that this progesterone rise is actually a side effect of ovarian
stimulation and multifollicular growth.19,20 This has been deducted through the analysis of mul-
tiple observational studies that support that the progesterone rise is associated with the serum
estradiol concentration during the late follicular phase as well as the number of oocytes retrieved.17
Hence, for the majority of patients undergoing ovarian stimulation, the increase in the serum
progesterone concentration seems to be a result of the production of smaller quantities of proges-
terone by the granulosa cells of growing follicles. This notion has recently been backed up by in
vitro experiments that suggested that follicle-stimulating hormone (FSH) can actively promote
the synthesis of progesterone by granulosa cells by upregulating the expression and increasing the
enzymatic activity of 3β-hydroxysteroid-dehydrogenase (3β-HSD), which converts pregnenolone
to progesterone.21
THE PROGNOSTIC ROLE OF PROGESTERONE CONCENTRATION ON THE DAY OF TRIGGERING

FINAL OOCYTE MATURATION FOR THE ESTABLISHMENT OF PREGNANCY AFTER IVF
The prognostic role of progesterone concentration during the late follicular phase for the establish-
ment of pregnancy had already been suggested in the first few years of ovarian stimulation. More
specifically, in 1989 during the 45th Annual Meeting of the American Fertility Society, Schoolcraft
et al. reported that a serum concentration of progesterone greater than 0.9 ng/mL on the day of
triggering of final oocyte maturation with human chorionic gonadotrophin (hCG) was associ-
ated with significantly lower pregnancy rates.22 Not surprisingly, this study motivated multiple
researchers from different groups to examine the predictive role of serum progesterone on the day
of hCG for the achievement of pregnancy. A plethora of studies was published within the next 15
years with some confirming a negative association between progesterone elevation and pregnancy
rates23–33 while others rebutted such a finding.34–44 Amidst this controversy, a systematic review
and meta-analysis published in 2007 attempted to provide an answer on the aforementioned clini-
cal question. By systematically reviewing all the relevant studies until 2005, the authors concluded
that there was no evidence to support a detrimental effect of higher progesterone values on the
The prognostic role of progesterone concentration 115
achievement of pregnancy (odds ratio [OR] 0.75, 95% CI 0.53–1.06).19 Following that systematic
review and meta-analysis, a number of large studies were published focusing on the same research
question. In 2010, Bosch et al. published a seminal article in which, by examining more than 4000
cycles, he demonstrated that a serum progesterone concentration higher than 1.5 ng/mL on the
day of hCG was negatively associated with ongoing pregnancy rates (31.0% vs. 19.1%; p < 0.001).45
These researchers also confirmed that progesterone elevation was associated with a higher daily
dose of gonadotrophins, a larger oocyte yield, and a higher serum concentration of estradiol on the
day of hCG.
Finally, in 2013, Venetis et al., performed a comprehensive systematic review and meta-analysis
of all the available relevant studies until August 2012 (n = 63; 55,199 cycles) and provided conclu-
sive evidence of the negative effect of elevated progesterone on the day of triggering final oocyte
maturation for the achievement of pregnancy after a fresh embryo transfer (ET).17 They analyzed
multiple cutoffs used in the original studies: 0.4–0.6, 0.8–1.1, 1.2–1.4, 1.5–1.75, and 1.9–3.0 ng/mL.
The negative effect of progesterone elevation was already present from serum progesterone cutoffs
of 0.8–1.1 ng/mL (Figure 12.1). This effect could be significant since, as it was shown in patients
with serum progesterone ≥1.5 ng/mL (the most widely used cutoff), pregnancy rates are reduced
by approximately 10% (Figure 12.2) as compared with cycles with serum progesterone <1.5 ng/mL.
Furthermore, this systematic review highlighted that progesterone elevation was not as rare as ini-
tially thought and that depending on the cutoff used, it could be affecting 33.5% (95% CI 26.7–41.0)
of the cycles.
(a) 0.4–0.6 ng/mL (b) 0.8–1.1 ng/mL (c) 1.2–1.4 ng/mL Random effects
Random effects
Random effects OR OR
OR
Edelstein et al., 1990 Hofmann et al., 1996
Schoolcraft et al., 1991 Miller et al., 1996
Silverberg et al., 1991 Doldi et al., 1999 Normal
Antoine et al., 1992 hMG Doldi et al., 1999 PCOS
Schoolcraft et al., 1991 Antoine et al., 1992 pFSH Valencia et al., 2002
Silverberg et al., 1991 Check et al., 1993a Bosch et al., 2003
Check et al., 1993b Andersen et al., 2006 HP-hMG
Antoine et al., 1992 hMG Fanchin et al., 1993a Andersen et al., 2006 rFSH
Antoine et al., 1992 pFSH Gonen et al., 1993 Li et al., 2008
Givens et al., 1994 Papanikolaou et.al., 2009 Cleav
Check et al., 1993a Shechter et al., 1994 Segal et al., 2009 PCOS
Check et al., 1994 Seow et al., 2010
Andersen et al., 2011
Harada et al., 1995
Pooled OR: 0.39 Levy et al., 1995
Guijarro Ponce et al., 2011
(95% CI: 0.14–1.08) Penge et al., 2012
Ubaldi et al., 1995
Rezaee et al., 2012
Hofmann et al., 1996 Huang et al., 2012b Long
Ubaldi et al., 1996a Devroey et al., 2012 HP-hMG
Ubaldi et al., 1996b Devroey et al., 2012 rFSH
0.0 1.0 2.0 3.0 Abuzeid et al., 1996
Huang et al., 1996 Pooled OR: 0.67
Miller et al., 1996
Shulman et al., 1996
(95% CI: 0.53–0.84)
Random effects Fanchin et al., 1997b High R
0.0 1.0 2.0 3.0
Fanchin et al., 1997b Intermediate R
OR Moffitt et al., 1997
(d) 1.5–1.75 ng/mL Fanchin et al., 1997b Low R
Check et al., 1993a

Fanchin et al., 1997
Lindheim et al., 1999
(e) 1.9–3.0 ng/mL Random effects
Check et al., 1993b Urman et al., 1999 OR
Miller et al., 1996 Martinez et al., 2004
European Middle East Orgalutran
European Orgalutran Eleno et al., 2006
North American Ganirelix Azem et al., 2008
Berin et al., 2006 Kilicdag et al., 2009 Check et al., 1993a
Engage trial Papanikolaou et.al., 2009 Cleav Check et al., 1993b
Papanikolaou et.al., 2009 Cleav
Papanikolaou et.al., 2009 Blast Papanikolaou et.al., 2009 Blast
Bosch et al., 2010 Agon Saleh et al., 2009 Givens et al., 1994
Bosch et al., 2010 Antagon Yu et al., 2010 Yovel et al., 1995
Ensure trial Wu et al., 2011
Zimerman et al., 2002
Elgindy et al., 2011 Blast
Elgindy et al., 2011 Cleav Huang et al., 2012a Shapiro et al., 2002
Xpect trial
Fatemi et al., 2012 Corifol Pooled OR: 0.79 Li et al., 2008
Keltz et al., 2012
Kyrou et al., 2012 (95% CI: 0.67–0.95) Yu et al., 2010
Lahoud et al., 2012 Huang et al., 2012b Short
Ochsenkun et al., 2012 0.0 1.0 2.0 3.0
Papanikolaou et al., 2012 Agon Ochsenkun et al., 2012
Papanikolaou et al., 2012 Antagon
Xu et al., 2012 Intermediate R Xu et al., 2012 High R
Xu et al., 2012 Low R
Cohen-Bacrie et al., 2012
Pooled OR: 0.64
(95% CI: 0.54–0.76) Pooled OR: 0.68
0.0 1.0 2.0 3.0 (95% Cl: 0.51–0.91)
0.0 1.0 2.0 3.0
Figure 12.1 Forest plots of odds ratios (ORs) for pregnancy achievement in women with progesterone
elevation (PE) when compared with those without PE (all studies analyzed). CI, confidence interval. (From
Venetis CA et al. Hum Reprod Update. 2013;19(5):433–57, with permission.)
(a) 50% (b)

45%
NNH:9.9
40%
APRR: 10.1%
Baseline pregnancy rate
35%
30%
25%
20%
15%
10%
5%
0%
0.0% 2.0% 4.0% 6.0% 8.0% 10.0% 12.0% 14.0% 16.0% 0 10 20 30 40 50 60 70 80 90 100
Absolute pregnancy rate reduction Number-needed-to-harm (NNH)
(c) 40.0%
39.5%
Expected pregancy rate in the population
39.0%
38.5%
38.0%
37.5%
37.0%
36.5%
36.0%
35.5%
PE rate: 17.2%
35.0%
0% 10% 20% 30% 40%
Progesterone elevation rate in the population
Figure 12.2 Graphical representation of the transformation of the OR 0.64 (95% CI 0.54–0.76) to
(a) absolute pregnancy rate reduction with 95% CIs (dotted lines) and (b) Number needed to harm (NNH)
with 95% CIs (dotted lines), according to a range of baseline pregnancy risks. Based on these calculations,
graph (c) depicts the expected pregnancy rate with 95% CIs (dotted lines) in a population with 40% base-
line pregnancy rate (i.e., pregnancy rate in the non progesterone elevation [PE] patients) according to a
range of PE rates in that population. (From Venetis CA et al. Hum Reprod Update. 2013;19(5):433–57, with
permission.)
The same meta-analysis suggested that a detrimental effect of serum progesterone on the day
of hCG was not present when the embryo created during a cycle with progesterone elevation was
transferred to an endometrium that had not been exposed to increased progesterone levels. More
specifically, by synthesizing data from the studies that had been performed in donor-recipient
cycles or in frozen-thawed embryo transfer (FET) cycles, it became evident that an association
was not present between the presence or not of progesterone elevation during the stimulation cycle
and the pregnancy rates in the recipients or after a FET cycle (Figure 12.3). This once again con-
firmed the theory that the effect of elevated progesterone on the day of hCG is exerted through the
advancement of endometrial maturation and the deterioration of endometrial receptivity. This had
been already demonstrated through conventional histopathological analysis in 199746 and it was
also confirmed through gene expression analyses more recently.47,48
Currently, it is widely accepted that serum progesterone concentration has a negative effect on
the achievement of pregnancy after a fresh ET, while it does not appear to affect the chances of
conception after a FET cycle. This negative effect is present regardless of the magnitude of ovar-
ian response, although it has been suggested that better-quality embryos, usually associated with
younger patients with larger oocyte yields, might be able to somehow mitigate it.49
The potential role of progesterone on the day of triggering final oocyte maturation 117
(a) Frozen-thawed ET cycles
i) 0.8–1.1 ng/mL Fixed effects ii) 1.2–1.4 ng/mL Fixed effects iii) 1.5–1.75 ng/mL Fixed effects (iv) 1.9–2.5 ng/mL Fixed effects
OR OR OR OR
Check et al., 1993b Check et al., 1993b

Check et al., 1993b Lahoud et al., 2011
Zimerman et al., 2002
Xu et al., 2012 Low R
Moffitt et al., 1997 Li et al., 2008 Li et al., 2008
Xu et al., 2012 Intermediate R
Wu et al., 2011 Huang et al., 2012b Long Huang et al., 2012b Short
Fatemi et al., 2012 Corifol
Xu et al., 2012 High R
Fatemi et al., 2012 rFSH
Pooled OR: 1.03 Pooled OR: 0.83 Pooled OR: 1.13 Pooled OR: 1.03
(95% Cl: 0.79–1.34) (95% Cl: 0.62–1.11) (95% Cl: 0.97–1.32) (95% Cl: 0.84–1.27)
0.0 1.0 2.0 3.0 0.0 1.0 2.0 3.0 0.0 1.0 2.0 3.0 0.0 1.0 2.0 3.0
(b) Cycles with donated oocytes

Fixed effects Random effects Random effects
i) 0.8–1.1 ng/mL OR ii) 1.2–1.4 ng/mL OR iii) 1.9–2.5 ng/mL OR
Hofmann et al., 1993
Legro et al., 1993
Check et al., 1994
Shulman et al., 1996 Melo et al., 2006 Yovel et al., 1995
Fanchin et al., 1996 Legro et al., 1993 Check et al., 2010
Pooled OR: 1.18 Pooled OR: 1.61 Pooled OR: 0.51

(95% Cl: 0.76–1.84) (95% Cl: 0.64–4.05) (95% Cl: 0.12–2.19)
0.0 2.0 4.0 6.0 8.0 0.0 2.0 4.0 6.0 8.0 0.0 1.0 2.0 3.0
Figure 12.3 Pooled odds ratio (OR) for achievement of pregnancy in women: (a) Forest plots of ORs for
achievement of pregnancy in women undergoing frozen-thawed embryo transfer after a fresh cycle with
or without progesterone elevation (PE) (per PE threshold group) and (b) Forest plots of ORs for achieve-
ment of pregnancy in women undergoing embryo transfer with donated oocytes from women who did
or did not experience progesterone elevation (PE) (per PE threshold group). (From Venetis CA et al. Hum
Reprod Update. 2013;19(5):433–57, with permission.)
THE POTENTIAL ROLE OF PROGESTERONE ON THE DAY OF TRIGGERING FINAL OOCYTE

MATURATION IN WOMEN WITH RIF AFTER IVF
Considering the importance of late follicular serum progesterone for the achievement of pregnancy
after a fresh ET, it would be relevant to explore the degree to which abnormally elevated progester-
one contributes to RIF for some patients.
This research question was examined in a recently published study by Liu et al.50 In this ret-
rospective, observational, single-center study, the authors analyzed 6673 consecutive cycles per-
formed during 2007–2012 that resulted in a fresh ET. These cycles included women ≤40 years of
age and with up to 20 oocytes retrieved. The authors hypothesized that if progesterone elevation
is a significant contributory factor of RIF, its incidence will be higher in cases with increased
numbers of failed IVF cycles. To test this hypothesis, the authors divided their sample in three
groups. Group I included patients who had never had IVF/ET before, group II included those who
had one previous IVF/ET failure and group III included patients with ≥2 previous failed IVF/ET
cycles. The authors defined progesterone elevation as serum progesterone >6 nmol/L, based on
previous studies.47,51,52 Using this cutoff, 1672/6673 (25.1%) cycles exhibited progesterone eleva-
tion. The authors used stepwise regression and identified age, estradiol concentration on the day
of hCG, number of oocytes retrieved, and total dose of FSH as predictors of progesterone eleva-
tion. Subsequently, using random number generators, the authors matched groups I, II, and III
for the aforementioned potential confounders. When evaluating the prevalence of progesterone
elevation between the three different groups, it was evident that women with ≥2 previous failed
Table 12.1 Prevalence of elevated progesterone (>6 nmol/L) on the day of triggering final oocyte
maturation according to the number of previous failed IVF cycles.
Group I Group II Group III
No previous failed 1 previous failed ≥2 previous failed
IVF/ET cycle IVF/ET cycle IVF/ET cycle p
Unmatched sample 1489/6102 150/489 16/82 <0.05
24.4% 30.7% 40.2%
Matched samplea 300/1784 100/315 25/63 <0.001
16.8% 31.7% 39.7%
Poor responsea 49/551 15/90 5/21 <0.05
8.9% 16.7% 23.8%
Moderate responsea 252/1233 85/225 20/42 <0.001
20.4% 37.8% 47.6%
Source: Adapted from Liu L et al. European J Obstet Gynecol Reprod Biol. 2013;171(1):78–83.
a Matched for age, estradiol level on the day of hCG administration, and number of oocytes retrieved.
IVF/ETs had a significantly higher prevalence of elevated progesterone compared with women
with one failed IVF/ET or no failed IVF/ETs. This finding was also present when their sample
was divided into poor responders (≤4 oocytes retrieved) and moderate responders (5–19 oocytes
retrieved) (Table 12.1). Similarly, women with one failed IVF/ET had significantly higher inci-
dence of elevated progesterone compared to women with no failed IVF/ET. This finding was con-
firmed when the analysis was repeated in subgroups of poor (≤4 oocytes retrieved) and moderate
(5–19 oocytes retrieved) responders (Table 12.1).
Based on these results, the authors of this large retrospective analysis concluded that there is
an association of RIF with elevated progesterone on the day of hCG. This implies that certain
patients have a propensity to exhibit elevated progesterone at the end of their ovarian stimula-
tion which in turn reduces the chance of pregnancy and, hence, might lead to multiple implan-
tation failures.
Recently, Venetis et al.16 performed a retrospective study in 1702 IVF antagonist cycles in
a single center, aiming to find baseline predictors of progesterone elevation (>1.5 ng/mL). The
researchers showed that female age, FSH, and serum progesterone on day two of their menstrual
cycle were the main prestimulation predictors of progesterone elevation. In the same study, it was
demonstrated that duration of stimulation, the number of follicles ≥11 mm in mean diameter,
and serum estradiol on the day of triggering final oocyte maturation were also strong predictors
of progesterone elevation, confirming that progesterone elevation is associated with the intensity
of ovarian stimulation. For that reason, the researchers, using multivariable regression analysis,
attempted to find independent predictors of progesterone elevation after controlling for ovarian-
stimulation characteristics. Interestingly, only basal progesterone was an independent predictor.
More specifically, the higher the serum progesterone on day two of the menstrual cycle, the
higher the probability of progesterone elevation (Figure 12.4). Based on our knowledge of the
sources of progesterone during the early menstrual cycle, the most likely explanation is that
these patients have a higher production of progesterone from their adrenals. When this is com-
bined with the production by their developing follicles in the mid and late follicular phase, the
total serum progesterone concentration is increased, and this detrimentally affects the chances
of pregnancy after a fresh ET. This theory seems to be corroborated by the fact that Venetis et al.
found that women with a history of progesterone elevation in a previous cycle had ∼6 times
higher odds of exhibiting progesterone elevation in the current cycle, independent of the inten-
sity of ovarian stimulation.16
Management of progesterone elevation 119
0.4
0.3
Propability of PE
0.2
0.1
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6
Basal progesterone
Figure 12.4 Graphical representation of the association of basal P with the probability of progester-
one elevation (PE) (1.5 ng/mL) on the day of hCG after adjusting for the stimulation characteristics: total
FSH dose, number of follicles ≥11 mm on the day of hCG and E2 on the day of hCG (shaded area represents
95% confidence intervals). (From Venetis CA Hum Reprod (Oxf, Engl). 2016;31(8):1859–65, with permission.)
The findings of these studies do suggest that elevated progesterone on the day of triggering
final oocyte maturation could be contributing significantly to the phenomenon of RIF and that its
proper management could improve the chances of these patients conceiving.
MANAGEMENT OF PROGESTERONE ELEVATION

Mitigation of the negative effects of progesterone elevation can be achieved either with primary
prevention or active management in patients who exhibit progesterone elevation.
Primary prevention of progesterone elevation

Milder ovarian stimulation
As it has been convincingly shown that the probability of progesterone elevation is increased with
a larger oocyte yield,17,45,49,53,54 it has been hypothesized that actively pursuing the development
and retrieval of a smaller number of oocytes would reduce the chance of progesterone elevation.
Although this approach does make sense physiologically, it should be stressed that it has not been
tested in the context of a randomized controlled trial (RCT), and hence the net benefit, if any, of
such a strategy in terms of pregnancy rates is unknown. Mild stimulation has in the past been sug-
gested to have a beneficial effect on pregnancy rates,55 however, more recent data suggest that the
optimal number of oocytes retrieved in terms of live birth rates is around 15.56 Furthermore, other
studies suggest that a larger oocyte yield is associated with more euploid embryos available for
transfer.57 Hence, aiming for a lower number of oocytes might reduce the chance of progesterone
elevation but at the same time could also reduce the number of euploid embryos.
Reducing the steroidogenic drive on the granulosa cells of developing follicles during
the late follicular phase
An alternative strategy that has also been proposed is reducing the steroidogenic drive on the gran-
ulosa cells of the developing follicles around the end of the follicular phase. Since progesterone
elevation is considered to be mainly due to small amounts of progesterone produced by the granu-
losa cells of multiple follicles under the action of gonadotrophins,45 reducing the concentration of
gonadotrophins might have a beneficial effect. This was indirectly shown in a recent RCT in which
it was demonstrated that performing ovarian stimulation in poor responders for the first seven days
with a single dose of 150 µg of corifollitropin alfa (CFA) led to significantly lower serum concentra-
tions of progesterone on day eight of stimulation when compared with patients who were stimu-
lated with a daily dose of 450 IU of follitropin beta despite the fact that the number of developing
follicles was similar between the two groups.58 This could be attributed to the unique pharmacoki-
netic profile of a single CFA injection which is characterized by an initially high concentration of
CFA followed by a gradual decrease, similarly to a step-down protocol. This finding was recently
confirmed in a reanalysis of two large RCTs on the use of CFA (ENGAGE and PURSUE) which
demonstrated that the incidence of progesterone elevation in patients who were triggered after a
single dose of CFA (i.e., on day eight) was significantly lower compared with those who were stimu-
lated with daily recombinant FSH and triggered also on day eight.59
Similarly, it has also been shown that increased presence of LH activity, especially during the
late follicular phase can also lead to an increase in serum progesterone,60–62 most likely through the
potent stimulatory effect of LH activity on granulosa cells of large follicles that have attained LH
receptors. On the basis of these observations, one could hypothesize that reducing the dose of FSH
and/or LH during the last few days of stimulation would reduce the probability of progesterone
elevation. However, it should be stressed that such a strategy has not been properly tested in the
context of an RCT, and hence it has not satisfactorily been shown to be an effective option.
Earlier administration of hCG and oocyte retrieval

This is an approach that involves close monitoring of serum progesterone and administration of
hCG to trigger final oocyte maturation before serum progesterone reaches a level that is detri-
mental for endometrial receptivity. This strategy was initially proposed by Harada et al.,17 who
demonstrated that an earlier administration of hCG was associated with higher implantation rates.
Interestingly, in a study by Kolibianakis et al. which aimed to determine the best timing for trig-
gering final oocyte maturation, it was shown that an hCG administration as soon as at least three
follicles had a mean diameter of 17 mm (early hCG) was associated with higher ongoing pregnancy
rates compared with triggering final oocyte maturation 48 hours later (late hCG).63 This difference
could be explained by the fact that the early hCG group had significantly lower mean progesterone
concentration (1.1 ng/mL) compared with the late hCG group (1.5 ng/mL). Therefore, this proposed
strategy is only supported by observational data, and at the same time, it requires intense monitor-
ing. Finally, this strategy is based on the assumption that there is a single cutoff above which there
is a detrimental effect on endometrial receptivity; however, more recent data are suggesting that
this is not likely to be true.17,49
Corticosteroid administration
Another strategy that has been proposed for prevention of elevated progesterone on the day of hCG
administration involves the administration of corticosteroids in order to suppress the production of
progesterone by the adrenals. In a prospective, controlled study (n = 120), Fanchin et al.18 adminis-
tered dexamethasone 1 mg/day from the time of pituitary suppression until the time of hCG admin-
istration in 60 patients, while the remaining 60 did not receive any dexamethasone. Although the
net increase in serum progesterone between the start and the end of ovarian stimulation was similar
between the two groups, the group that received dexamethasone had a lower mean serum proges-
terone concentration on the day of hCG. Furthermore, this group had a significantly higher ongoing
pregnancy rate compared with the patients who did not receive dexamethasone (35% vs. 17%). The
administration of dexamethasone for the prevention of elevated progesterone was also shown in a
subsequent study by Eldar-Geva et al.14 In this study, none of the patients who received daily dexa-
methasone during ovarian stimulation exhibited elevated progesterone (>3 nmol/L) on the day of
hCG compared with 13.9% of patients who did not receive dexamethasone.
Management of patients with elevated progesterone 121
In summary, based on the data provided by these two studies, corticosteroid administration
during the follicular phase has also been suggested as a way of reducing the likelihood of elevated
progesterone. However, in the absence of randomized data and, most importantly, data regarding
the safety of this strategy for the woman and potentially her fetus, this option should only be used
in the context of properly designed clinical studies.
MANAGEMENT OF PATIENTS WITH ELEVATED PROGESTERONE

Replacement of high-quality embryos
When elevated progesterone does occur, though, the options are limited. Proceeding with a fresh
ET is of course an option, since progesterone elevation has been shown to reduce, but not com-
pletely eliminate, the possibility of a pregnancy.17 In that regard, it has been suggested that replac-
ing embryos with higher implantation potential can somehow reduce the negative effect of elevated
progesterone on pregnancy rates.64 Based on multiple reports, it does appear that younger patients
with more oocytes retrieved and hence better-quality embryos are less affected by the detrimental
effect of elevated progesterone, while cycles with embryos of poor quality are more profoundly
affected.17,49,53,65,66 This means that replacing an embryo of high quality during a cycle with elevated
progesterone will still lead to acceptable pregnancy rates, although most likely these rates will be
lower than what could be achieved in the absence of elevated progesterone.
Freeze-all embryos
Freezing all available embryos and deferring embryo transfer is probably the most popular method
of managing patients with elevated progesterone. This is based on the fact that, as discussed, the
negative effect of elevated progesterone has been shown to be exerted on the endometrium, and it
does not appear to have an effect on the quality of the oocytes and the resulting embryos.17,67 At
the same time, highly efficacious cryopreservation protocols ensure high survival rates68,69 without
compromising the implantation potential of the cryopreserved embryos.70
The freeze-all policy has been suggested previously to be beneficial for women with implantation
failure. Firstly, in 2014, a retrospective study in 269 patients with previous implantation failure of a
blastocyst in a fresh cycle, compared pregnancy rates between those patients who decided to have
another fresh embryo transfer following their next oocyte retrieval (n = 163) and those who opted
to freeze-all embryos and undergo a FET cycle (n = 109).71 Using multivariate regression analysis,
the authors showed that FET is associated with 3.8 times higher odds of live birth compared with
a fresh embryo transfer.
In a more recent prospective study, Magdi et al., compared the freeze-all policy (n = 81) with
fresh embryo transfer (n = 90) in women who had at least three previous failed embryo trans-
fers with ≥4 high-quality embryos transferred in total.72 Patients were assigned to these groups
quasi-randomly using alternate allocation. The mean serum concentration of progesterone on
the day of hCG was not significantly different between the two groups (1.10 vs. 1.07 ng/mL).
However, there was a significant difference (p = 0.005) in the probability of ongoing pregnancy
between women in the freeze-all group (40.7%) and women in the fresh transfer group (21.1%).
Based on these results, the authors concluded that the freeze-all policy seems to be beneficial for
RIF patients.72
Finally, one small RCT (n = 100) has specifically looked into the value of the freeze-all policy in
patients with elevated progesterone (≥6 nmol/L) on the day of trigger. These patients were random-
ized to a fresh day 5/6 transfer (n = 50) or to a freeze-all and subsequent FET (n = 50).73 Patients
who had a fresh embryo transfer had an 18.4% live birth rate per embryo transfer, while those who
froze all their embryos and subsequently had an FET had a live birth rate per embryo transfer of
31.0%. However, this difference in live birth rates, although potentially clinically r elevant, was not
statistically significant, and due to the small sample of this study and other methodological con-
cerns, no robust conclusions could be drawn.
CONCLUSIONS
Elevated progesterone on the day of triggering final oocyte maturation after ovarian stimulation is
not a rare phenomenon and seems to be more frequent in women with RIF after IVF. Identifying
this issue and managing it appropriately can improve the chances of pregnancy for these patients.
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The role of late follicular
phase luteinizing hormone
13
and estradiol on embryo
implantation
Implications for recurrent
JULIA K. BOSDOU, CHRISTOS A. VENETIS,
INTRODUCTION
Endometrial receptivity and embryo quality are two major factors that contribute to implanta-
tion success.1 During ovarian stimulation for in vitro fertilization (IVF), the development of mul-
tiple follicles leads to abnormal hormonal levels.2 This is true for estradiol (E2), which rises up to
10 times or more compared with a natural cycle,3 and for progesterone, the levels of which also
increase as a consequence of multiple follicular development,4–7 as well as for luteinizing hormone
(LH), the levels of which are variably suppressed by the use of gonadotropin-releasing hormone
(GnRH) analogues.8 If the abnormal hormonal environment during ovarian stimulation for IVF
adversely affects the probability of implantation, then this may contribute to implantation fail-
ure. If hormonal levels, however, are not monitored during ovarian stimulation for multifollicular
development, then implantation failure might be falsely perceived as unexplained.
OVARIAN STIMULATION AND EMBRYO IMPLANTATION

During implantation, the endometrium undergoes a transformation period, regulated by ovarian
steroids, during which it acquires appropriate morphological structure and function. Implantation
requires a number of functional molecules such as hormones, cytokines, and growth factors,
which are actively involved in regulating trophoblast differentiation and invasion.9 Progesterone
and estrogen are considered as the primary hormonal modulators of endometrial development,
supporting embryo implantation.9 This is confirmed by the presence of their receptors in stromal
and epithelial cells during implantation.9,10 After binding to their receptors, steroids have been sug-
gested to promote uterine proliferation and differentiation through the production of local cyto-
kines and growth factors.11
During ovarian stimulation for IVF, the steroid receptor profile of the endometrium on the day
of triggering final oocyte maturation has been shown to resemble that of an endometrium that has
already entered the luteal phase.12 Thus, it is possible that ovarian stimulation, by altering the levels
of periovulatory hormones, may affect endometrial receptivity.13
Indeed, histologically abnormal endometrium is present on the day of oocyte retrieval in all
IVF cycles using GnRH analogues and gonadotropins.13,14 This abnormality is demonstrated by
a difference observed between the histological and the chronological dating of the endometrium,
127
128 The role of late follicular phase luteinizing hormone and estradiol on embryo implantation
referred to as an endometrial advancement. It has been shown that endometrial advancement of

more than three days is significantly associated with a diminished probability of pregnancy.13,14
Moreover, gene expression analysis of the endometrium of stimulated cycles on the day of oocyte
retrieval showed the presence of an altered gene expression in women with histologically advanced
endometrial maturation exceeding three days.15
Regarding LH, it is not clear whether there is an association between the probability of preg-
nancy and its variably suppressed levels during ovarian stimulation as a result of pituitary
downregulation.8,16
On the other hand, the effect of the abnormal hormonal environment to the developing follicles
and eventually to oocyte and embryo quality is still a matter of debate.17 The use of exogenous
gonadotropins for ovarian stimulation has been reported to induce chromosomal abnormalities
such as aneuploidy in the oocyte as well as to affect embryo development.18,19 Reducing the intensity
of ovarian stimulation by using milder ovarian stimulation or even natural IVF cycles has been
suggested to reduce the proportion of aneuploidy in embryos.20
In contrast, it has also been suggested that similar aneuploidy rates with no differences in embryo
quality and types of chromosomal abnormalities are present between unstimulated and stimulated
IVF cycles in the same young egg donors.21 Moreover, it has been shown that the degree of exposure
to exogenous gonadotropins in patients with normal response to ovarian stimulation undergoing
IVF is not associated with the likelihood of aneuploidy.22
Overall, it appears that ovarian stimulation may affect embryo implantation either by altering
endometrial receptivity and/or oocyte/embryo quality; however, certain questions remain unan-
swered. It is not clear, for instance, whether an effect of ovarian stimulation is predominantly due
to a single abnormal hormonal level or due to a combination of different abnormal hormonal levels,
at a specific moment or for a certain period during ovarian stimulation.
This chapter will focus on the role of LH and E2 during the late follicular phase of ovarian stimu-
lation for IVF on the probability of implantation and its implications on recurrent implantation
failure (RIF).
ENDOGENOUS LH ON THE LATE FOLLICULAR PHASE AND EMBRYO IMPLANTATION

The role of endogenous LH during ovarian stimulation for IVF has been in the focus of research16,23
well before the introduction of GnRH analogues in assisted reproductive technologies.24,25 GnRH
analogue use increased the interest in this association, since inhibition of premature LH surge by
GnRH agonists or antagonists is associated with suppressed LH concentrations.26,27 The degree of
LH suppression following administration of GnRH analogues varies depending on the type, dose,
and protocol of the analogue used.
On the other hand, it has been suggested that 0.15%–15% of all LH receptors are occupied with LH
concentrations of 1–10 IU/L.28 Since the percentage of LH receptors that need to be occupied for a
maximum steroidogenic response to be yielded in vitro is 15%–20%,29 it is then possible that higher LH
concentrations could result in a more efficient stimulation of LH receptors in the developing follicle.30
Several studies have suggested an adverse effect of deeply suppressed LH levels on the probability
of pregnancy.16,31 However, evidence for absence of such an effect during ovarian stimulation for
IVF is also present.8 In a relevant systematic review, a negative association between low endogenous
LH levels during ovarian stimulation for IVF using GnRH analogues and the probability of ongo-
ing pregnancy beyond 12 weeks was not observed. Similarly, in an individual patient data meta-
analysis of six randomized controlled trials (RCTs), including 1764 patients undergoing ovarian
stimulation with recombinant follicle-stimulating hormone (rFSH) and GnRH antagonist for IVF/
intracytoplasmic sperm injection (ICSI), no association between the endogenous LH concentra-
tions and pregnancy achievement was present.32
In a recent study in patients stimulated with rFSH and GnRH antagonists for IVF, the associa-
tion of serum LH levels during ovarian stimulation with cycle outcome was evaluated. Implantation
rate and clinical pregnancy rate were not significantly associated with serum LH levels.33
Conclusions and research implications for RIF 129
Based on the currently available evidence, it appears that no association can be supported
between low endogenous LH levels and pregnancy achievement in GnRH analogue downregulated
cycles. Thus, its involvement in RIF appears unlikely, while its assessment does not offer useful
information other than the confirmation of adequate pituitary suppression.
ENDOGENOUS E2 ON THE LATE FOLLICULAR PHASE AND EMBRYO IMPLANTATION

The endogenous E2 concentration is the most frequently used marker for assessing follicular devel-
opment, since E2 levels are associated with the number and size of developing follicles.16 However,
due to the fact that E2 levels during ovarian stimulation for IVF are multiple times higher than
those present during a natural cycle, their impact on the probability of pregnancy has been exten-
sively evaluated.
A sustained interest in a potentially adverse effect of high E2 concentration on endometrial
receptivity,34,35 as well as on oocyte/embryo quality is present in the published literature.36–39
A systematic review published in 2004,40 including 3352 patients treated by IVF using GnRH
agonists and gonadotropins, evaluated the above association. Both a positive41,42 and a negative35,43
association between the serum E2 levels on the day of human chorionic gonadotrophin (hCG)
administration and the probability of pregnancy were reported. On the other hand, the majority of
studies44–48 failed to show an association. However, the low quality of the published studies limits
the importance of the results reported.
Since the publication of that systematic review, several relevant retrospective studies have
been published,34,49–55 as well as one prospective study.56 These studies, using different arbitrary
E2 threshold levels in order to classify patients as those with low or high E2 on the day of hCG
administration, supported a positive association,53–55,57 a negative association,58 or no association at
all between Ε2 levels and pregnancy achievement34,37,49–52,56,59–62 (Table 13.1). The variability in the
reported results might be due to the different E2 thresholds used during the evaluation of the above
association. It should also be noted that the number and quality of the available studies still remain
low, not allowing for solid conclusions to be drawn.
Overall, based on the currently available evidence, it appears that the majority of studies sup-
port the absence of an association between E2 levels on the day of triggering and the probability of
pregnancy. Thus, its involvement in RIF appears unlikely, while its assessment does not offer useful
information other than the evaluation of the intensity of ovarian response and the associated risk
for ovarian hyperstimulation syndrome (OHSS).
CONCLUSIONS AND RESEARCH IMPLICATIONS FOR RIF

Based on the currently available evidence, it appears that no association can be supported between
low LH levels or high E2 levels on the late follicular phase of ovarian stimulation for IVF and the
probability of implantation in GnRH agonist or antagonist cycles. However, whether or not the
absence of such an association is also true also for patients with RIF is not clear, since studies
addressing the above question in these specific patients have not been performed.
Nevertheless, since abnormal development of endometrium in the late follicular phase is pres-
ent in all cycles stimulated with GnRH analogues and gonadotropins, strategies that may assist in
increasing endometrial receptivity need to be explored. In this respect, milder stimulation proto-
cols might be an option.63–65 These protocols might be associated with an improved endometrial
receptivity by decreasing the adverse effect of standard ovarian stimulation, albeit with the cost
of producing fewer numbers of embryos for transfer and hence a decreased chance of pregnancy
achievement.
Alternatively, the freeze-all strategy in which all created embryos are frozen for later transfer,
needs to be explored in RIF patients. Apparently, transfer of embryos in the normal endometrium
of a natural or artificially prepared cycle66 bypasses whatever effect of ovarian stimulation on
endometrial receptivity, at the expense of introducing an additional intervention such as embryo
Table 13.1 Association between serum E2 levels (pg/mL) on the day of hCG administration and the
probability of pregnancy per patient reaching hCG.
Association
(per patient
Authors, reaching
year E2 groups/pregnancy rate per patient reaching hCG hCG)
Ng et al. 37
<2724 2724–5448 >5448 Not
19.5 (30/154) 13.95 (12/86) 7.9 (3/38) significant
Saucedo de la <1000 1001–3000 >3001 Not
Llata et al.61 21.7 (33/152) 35.6 (52/146) 25.7 (9/35) significant
Thelmo <1000 1000–2000 2001–3000 3001–4000 Negative
et al.58 OR 0.29, 95% OR 0.43, 95% Not reported OR 0.28, 95%
CI 0.09–0.94 CI 0.18–0.98 CI 0.11–0.71
Ulug et al.57 113–1349 1350–3249 >3250 Positive
32.4 49.9 54.4
(263/810) (1662/3325) (895/1643)
Wu et al.68 ≤2000 2001–3000 3001–4000 4001–5000 >5000 Not
35.4 (46/130) 39.6 (21/53) 30.4 (14/46) 31 (9/29) 25 (4/16) significant
Kong et al.69 <1634 1634–3268 3268–4903 >4903 Not
22.1 (53/240) 21.2 (83/392) 24.8 (57/230) 26.7 (68/255) significant
Kyrou et al. 56
<1142 1142–2446 >2446 Not
23.5 (12/51) 32.4 (34/105) 29.4 (15/51) significant
Farhi et al.59 <1362 1362–2724 >2724 Not
38.3 (41/107) 38.5 (47/122) 47.1 (24/51) significant
Joo et al. 60
<1000 1000–2000 2000–3000 3000–4000 >4000 Not
13.9 (10/72) 13 (13/100) 19.7 13/66 32 (16/50) 19.8 (33/167) significant
Núñez et al.49 <3000 >3000 Not
36.48 (27/74) 23.07 (9/39) significant
Kara et al. 50
<1000 1000–2000 2000–3000 3000–4000 >4000 Not
10 (3/29) 14 (12/88) 14 (6/42) 18 (6/33) 36 (4/11) significant
Imudia et al.51 ≤2991 >2991 Not
49.4 45.5 (136/299) significant
(1331/2696)
Gursoy et al.52 <2000 2000–4000 >4000 Not
24.6 (16/65) 14.5 (11/76) 28.2 (20/71) significant
Taskin et al. 70 Not
AUC 0.532, 95% CI 0.471–0.593 significant
Zavy et al.54 <2000 2000–4000 >4000 Positive
47.2 (58/123) 59 (170/288) 61.5 (48/78)
Prasad et al.53 >1400, AUC 63.4 (p = 0.007) Positive
Foroozanfard <1500 1500–3500 >3500 Positive
et al.55 8.9 (4/45) 11.4 (4/35) 31.2 (15/48)
References 131
freezing. This strategy has been shown to increase the probability of pregnancy in high respond-
ers;67 however, data for patients with RIF are still not available.
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Manipulating the endometrium
Can recurrent implantation failure be
14
managed by inducing endometrial
inflammation or by the use of novel
research therapies?
ANTONIS MAKRIGIANNAKIS and THOMAS VREKOUSSIS
Recurrent implantation failure (RIF) remains a challenge for reproductive medicine. The patients
characterized as RIFs consist of a heterogeneous group mainly due to the fact that RIF definitions
vary. In general, though, RIF patients are the ones that fail to achieve a clinical pregnancy at least
three times after embryonic transfers with good-quality fresh embryos.1
Implantation success is a multifactorial process. However, two contributing components are
considered critical for an embryo to implant and evolve. Embryo quality is an obvious factor for
a successful pregnancy; endometrial receptivity is the other component. It is now clear that endo-
metrial physiology may predict the potential for an ongoing clinical pregnancy. The endometrium,
under the influence of progesterone, is normally transformed to decidua. Decidualization is a pro-
cess featured by aseptic inflammation of the endometrial stroma.2 Several inflammatory agents,
mainly cytokines, trigger chemotaxis and differentiation of several immune cells.3–5 Thus decidua
is characterized by dendritic cells and natural killer cells, as well as other immune populations. This
inflammation is under hormonal regulation, with progesterone playing the major role.6 The mor-
phological outcome of endometrial decidualization is the formation of pinopodes on the luminal
surface of the endometrium.7 This finding has been recognized as the key morphological alteration
that happens during the window of implantation, the time point at which endometrial receptivity
is maximized and synchronized with the maximum infiltrative potential of the blastocyst. The
cytokine profile that prevails during the implantation has been characterized as the Th2 profile.3
Th2 has been correlated to local immune suppression and is considered favorable for implantation
and early fetal development.8 A possible switch from the Th2 to the Th1 profile has been reported
to be found in cases of implantation failures, including spontaneous or recurrent miscarriages.9 To
the same direction, a favorable profile of decidual macrophages has been reported, maintaining
the concept of Th1/2 immunity. The M2 macrophage profile is considered in favor of implantation,
whereas an M1 profile is considered a contributor to implantation failures.10
Taking into account that implantation is a process featured by a specific aseptic inflammation,
an inspired overview tried to link decidual inflammation to local stress. Indeed, stress mediators,
with corticotropin releasing hormone (CRH) being the main representative of this neuropeptide
family, have been reported to be expressed by the endometrium in a timely spatial pattern.11–13
CRH is overexpressed during the secretory phase and, most importantly, at the implantation site.11
Interestingly, CRH has been reported to be expressed by the trophoblast cells of the blastocyst.
This increased CRH concentration, located at the fetal maternal interphase, seems to contribute
to the establishment of local inflammation.5 CRH has, in turn, been reported to induce Fas ligand
135
136 Manipulating the endometrium
(FasL) expression to decidua cells and trophoblasts, mediating in that way the apoptosis of the
local Fas-expressing T-cell population.14 Such an immune modification (the local downregulation
of cellular immunity) has been reported as crucial to achieving maternal immune tolerance of the
semiallogenic fetus.
Recently, the decidualized endometrium was proved to act as a biological selector of embryos
of good quality. It was shown that apart from the window of implantation, a window of selection
follows.15 During this time point, the endometrium selectively rejects embryos recognized as of
bad quality. This mechanism is based on a further crosstalking between the recently implanted
embryo and the decidua, aiming to scrutinize potential embryonic defects and thereafter proceed
to embryonic rejection early enough.16 In that view, the window of selection may indeed explain the
case of “superfertility” during which an increased number of embryos—later reported to fail—may
implant.16
The essential role of inflammation on successful implantation has led to the formulation of
several hypotheses supporting the exogenous trigger of inflammation as a key act in reversing
RIF.
A. Induction of local endometrial injury. Performing local endometrial injury was reported early
in the beginning of the 2000s. Barash et al. had conducted a randomized controlled trial (RCT)
investigating whether local injury (represented by endometrial biopsy) could have an impact
on the reproductive outcome of women with RIF when offered assisted reproductive technol-
ogy (ART).17 The results were very promising since all basic parameters (implantation, clini-
cal pregnancy, and live birth rates) were significantly improved after local endometrial injury.
The impact of this study on ART practice was rather strong. Several clinical groups had started
applying the technique prior to in vitro fertilization (IVF) treatment and further investigated
all related clinical issues that arose during clinical practice. In that view, several reports using
rather weak samples have addressed timing prior to IVF treatment as well as instrumentation to
be used. Initially, the majority of the reports (reviewed by Almog et al.18) converged to the theses
that local injury could be beneficial if applied during the cycle prior to treatment and that either
pipelle or hysteroscopy produce comparable results. The initial enthusiasm was f urther boosted
by basic science reports aiming to delineate the mechanisms through which endometrial injury
modulates endometrial receptivity. It was shown that endometrial injury could induce both
inflammation and endometrial receptivity genes, thus favoring implantation.19–21 Clinical
reports have been summarized in meta-analyses supporting that endometrial injury indeed can
improve the reproductive outcome of women with RIF.22,23 However, new emerging results from
RCTs, as well as recent meta-analyses, put endometrial injury in doubt.24–26 The recent concept,
upon a possible wider acceptance of the method, is rather r eluctant to be recommended in the
frame of lack of data regarding complications.27 Future studies are expected to clarify whether
endometrial injury is to be incorporated in the relevant guidelines.
. Intrauterine administration of immune cells. An alternative to mechanical injury was pro-
B
posed when the intrauterine administration of autologous peripheral blood mononuclear cells
(PBMCs) was investigated as an inflammation inducer. Yoshioka et al. conducted a clinical trial
to delineate a possible impact of autologous PBMC administration prior to embryo transfer in
case of RIF.28 PBMCs were primed with human chorionic gonadotrophin (hCG) and admin-
istered two days prior to blastocyst transfer. The results were also promising since reproduc-
tive outcomes were significantly improved as well. To the same direction, others have modified
the initial protocol in the aim of maximizing the immunomodulatory effect of the PBMCs on
endometrial receptivity. Our group performed a pilot study administering CRH-primed PBMCs
two days prior to blastocyst transfer to women diagnosed with RIF.29 Our results were in line
with that of hCG-primed PBMCs since clinical pregnancy rates were significantly increased.
However, to date, the idea of manipulating autologous immune cells to improve endometrial
receptivity is not as appealing as was initially expected, perhaps due to the need of laboratory
Conclusions 137
involvement, not to mention the reluctance of the pharmaceutical industry to be involved in

biological material manipulation.
. Administration of hCG. The issue of administration of immune cells was bypassed by other
C
researchers that have introduced the concept of cytokine/hormonal intrauterine administration
as endometrial priming agents prior to embryo transfer. The first approach reported was the
administration of hCG. By the time this was suggested, the role of HCG on endometrial recep-
tivity had already been investigated using in vitro and in vivo models.30–32 The results were ini-
tially very promising, since reproductive outcome parameters were significantly improved.33,34
The initial meta-analysis was supportive for hCG as an endometrial receptivity modulator.35
However new emerging data have also introduced an amount of skepticism.36,37 The most recent
meta-analysis has revealed conditional success, showing that, only in certain cases, HCG may
produce favorable results.38
D. The role of granulocyte-colony stimulating factor (G-CSF). Alternatively to hCG, it was proposed
to administer G-CSF as an endometrial receptivity primer. This approach was mainly based
on the chemotactic effect that G-CSF exerts on macrophages, a cellular component essential in
maintaining an inflammatory profile during implantation. Although initial reports were rather
promising, data from randomized trials are rather short. Recent RCTs show controversial
reproductive outcomes with no effect on reproductive outcome in older women.39,40 However,
recent meta-analyses showed that there could be a role for G-CSF in cases of women diagnosed
as RIF.41,42 The results presented in the meta-analyses revealed that G-CSF treatment is superior
to controls in terms of reproductive outcomes.41,42 No significant difference was noted in terms
of endometrial thickness changes.41 Furthermore, no significant difference was found when
comparing the efficacy of G-CSF administration via different routes (vaginal vs. systematic
administration).42
. Other promising agents.
E
a. Atosiban. Recent emerging data have revealed a role for atosiban in improving reproduc-
tive outcomes in women with RIF. The concept was based on the finding that atosiban is
interfering with the oxytocin/prostaglandin F2a system, minimizing uterine contractility
after embryo transfer. Few studies have been published so far and have already been ana-
lyzed via meta-analysis.43 The authors included 1756 cases from 6 studies. Meta-analysis
revealed a significant improvement in implantation and clinical pregnancy rates, findings
that remained significant even when subgroup analysis was focused on repeated implanta-
tion failures. Properly powered randomized trials are needed to strengthen such a finding at
the level of acceptance in clinical practice.
b. Growth hormone (GH). To date, a single RCT exists in the literature comparing reproductive
outcomes with or without GH treatment44 in a donor oocyte program. The study included
105 infertile patients, of which 70 were diagnosed with RIF. RIF patients were randomly
assigned to receive GH or not (n = 35 for GH group and n = 35 for non-GH [negative con-
trol] group), while the remaining 35 non-RIF patients received their first oocyte, acting
as a positive-control group. The results showed that GH treatment significantly improved
implantation, clinical pregnancy, and live birth rates compared with controls.44 New emerg-
ing data are expected to clarify the role of GH in human reproduction.
CONCLUSIONS
The role of inflammation is pivotal in human reproduction. It has been involved in decidualiza-
tion and implantation. This finding being established has triggered several hypotheses aiming
in improving the reproductive outcomes of women with RIF. Although promising, most results
need further verification to be considered established knowledge and thus eligible for applica-
tion in clinical practice. Until such proof exists, RIF is to be treated with the to-date established
protocols.
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The embryo in recurrent
15
Genetic selection and strategies for
improving its implantation potential
KATERINA CHATZIMELETIOU
The causes behind failure of an embryo to implant into the endometrial lining are multiple.
Although in certain cases it may simply be a failure to properly synchronize the timing of transfer
of a chromosomally normal embryo with the patient’s personalized window of implantation,1
embryonic abnormalities and uterine pathologies play undoubtedly important roles. Various
uterine pathologies, including a thin endometrium or altered expression of adhesive molecules,
immunological factors, endometriosis, and hydrosalpinges, have been shown to exert an adverse
effect and reduce endometrial receptivity.2–5 Patient karyotype abnormalities (including balanced
reciprocal translocations, Robertsonian translocations, qh-polymorphisms, inversions, or mosa-
icism) that can result in embryos with chromosomal imbalance and aneuploidies that are incom-
patible with implantation and further development, genetic and epigenetic defects of the male and
female gametes, postzygotic chromosomal abnormalities during cleavage, and early blastocyst
stages as well as zona hardening are among the most common embryonic reasons for failure of
implantation.6–8
CHROMOSOMAL ABNORMALITIES IN EMBRYOS FROM PATIENTS WITH RECURRENT

IMPLANTATION FAILURE (RIF)
The origin of chromosomal abnormalities may vary in embryos from recurrent implantation
failure (RIF) patients, and although meiotic errors account for a large proportion, postzygotic
errors also play a significant role.6,9–15 Voullaire et al.16 detected chromosomal abnormalities in
60% of embryos from women with RIF. The abnormalities included aneuploidy for one or two
chromosomes (25%) as well as more complex chromosomal abnormalities (29%). Small segmental
translocations have also been identified in embryos from patients with RIF using next-generation
sequencing.8 Voulaire et al.16 reported that the incidence of complex chromosome abnormalities
involving three or more chromosomes was independent of maternal age but highly associated with
RIF, suggesting that the pathology underlying complex abnormality is different from that result-
ing in aneuploidy of one to two chromosomes. Chatzimeletiou et al.9 provided the first cytoskel-
etal analysis of human embryos at all stages of preimplantation development and documented the
mechanisms leading to postzygotic chromosomal abnormalities, embryonic arrest, and implan-
tation failure via multipolar spindle formation, binucleation, multinucleation, and chromosome
lagging (Figure 15.1). The combination of meiotic and postzygotic errors can lead to embryonic
arrest and may arise from disruption of the normal sequence of chromosome r eplication and seg-
regation, caused by maternal cytoplasmic factors, mutations in cell-cycle control genes, or sperm-
related factors.9,6,11,12,16,17
141
142 The embryo in recurrent implantation failure
(a) (b) (c)
Figure 15.1 Photomicrographs of embryos from patients with RIF: (a) arrested embryo with
a nucleate cells and two multipolar spindles (arrows), (b) arrested embryo with a tripolar spindle (arrow),
and (c) arrested embryo with binucleate and multinucleate cells. (The embryos were immunostained with
a-tubulin [green] to visualize spindles and DAPI [blue] to visualize DNA.)
SPERM FACTORS AFFECTING EMBRYO QUALITY

Normal embryonic development in vitro has been shown to be subject to strong paternal (sperm-
derived) effects.11,12,18,19 Centriolar defects can lead to failure in fertilization through impairment or
absence of aster formation while centrosomal abnormalities can cause embryonic arrest through
the formation of abnormal spindles and the accumulation of chromosomally abnormal cells that
derive from them.9,10,11 Tesarik et al.20 compared the quality of embryos resulting from sibling
donor oocytes fertilized by spermatozoa from different patients. They reported that fertilization
with spermatozoa from certain individuals repeatedly resulted in the formation of zygotes with
abnormal pronuclear morphology that tended to cleave slowly and had high levels of fragmenta-
tion and uneven blastomeres. These paternal effects are most likely due to a combination of genetic
origin, related to the minor gene activity of the male pronucleus; epigenetic origin, related to the
sperm-derived oocyte-activating factor; and extragenetic origin, involving defective sperm cent-
riolar function.20
Centriolar abnormalities are more frequently observed in immotile or nonprogressively motile
spermatozoa than in normal motile spermatozoa.21 Since the distal centriole gives rise to the axo-
neme during spermiogenesis, it is possible that distal centriolar defects are responsible for prob-
lems associated with sperm motility. Abnormalities in pronuclear apposition and aster formation
have also been observed in vasectomized patients who produce antisperm antibodies against
centriolar structures, and after intracytoplasmic sperm injection (ICSI) with severely abnormal
spermatozoa.22–25
Molecular cytogenetic analysis in embryos from patients with oligoasthenoteratozoospermia
(OAT) and RIF that show slow or arrested development in vitro often reveal patterns of polyploidy
and postzygotic malsegregation of chromosomes that could be explained by abnormal centrosomal
distribution, following cytokinetic failure and defective spindles.11 Delayed division of blastomeres
can theoretically result in tetraploidy, either through formation of an abnormal monopolar spindle
or failure of both karyokinesis and cytokinesis. The tetraploid blastomere produced by failure of
karyokinesis and cytokinesis has two centrosomes that, when duplicated, would produce four. If
there is unequal allocation of these centrosomes on the spindle poles, for example, 3:1, then it is
possible that unequal numbers of spindle fibers will be present and one-sided divisions will ensue,
as most of the chromosomes will be attached and move toward the spindle pole with the greatest
number of centrosomes.11 The presence of four centrosomes in an unbalanced arrangement (3:1),
three distinct foci at one pole of a spindle and one at the other pole, has been previously reported
The role of embryo biopsy and preimplantation genetic screening in RIF 143
by Chatzimeletiou et al.9 following cytoskeletal analysis of human preimplantation embryos, but

can also occur via mechanisms of centrosome splitting in the presence of impaired DNA integrity
and replication.26
Confocal laser scanning microscopy has also highlighted the presence of tripolar and tetrapolar
spindles with the characteristic Y- and cruciform X-shaped organization, at cleavage and early
blastocyst stages, respectively, in human embryos, following labeling with antitubulin antibodies
and a DNA-specific fluorochrome.9 As there are no checkpoints to monitor excess spindle poles,
multipolar spindles progress in the cell cycle and lead to chaotic chromosomal constitutions. This
study provided the first direct evidence of spindle abnormalities throughout human preimplanta-
tion development, and led to the realization that nuclear and chromosomal aberrations are inter-
related through abnormalities in cytokinesis and spindle formation.9
TREATMENT OPTIONS IN COUPLES WITH SEVERE MALE FACTOR AND RIF

Intracytoplasmic morphologically selected sperm injection (IMSI) could be of value to patients
with RIF that have abnormal sperm parameters. Balaban et al. 27 reported that patients with RIF
who had IMSI experienced higher implantation and clinical pregnancy rates compared with
those who had ICSI, (28.9% vs. 19.5% and 54.0% vs. 44.4%, respectively; p ≥ 0.05). El Khattabi
et al. 28 suggested that IMSI is beneficial for patients with severe teratozoospermia at their first
or second attempts, but it does not improve the pregnancy rate in patients with repeated ICSI
failures in the absence of severe male factor. In particular, these authors showed that live birth
rate was significantly higher when IMSI was used in men with teratozoospermia and RIF com-
pared with ICSI (38% [50/132] vs. 20% [25/126]) but was similar between IMSI and ICSI pro-
cedures (21% [19/90] vs. 22% [28/130]) in men with RIF and no severe male factor. Delaroche
et al. 29 reported that the percentage of top quality embryos at cleavage stages was higher in IMSI
compared with IVF/ICSI cycles (89.8% vs. 79.8%; p = 0.009), the mean number of blastocysts
was higher in IMSI cycles (1.5 ± 1.9) than in in vitro fertilization (IVF)/ICSI cycles (1.0 ± 1.2;
p = 0.03), and the clinical pregnancy and live birth rates were higher in IMSI versus IVF/ICSI
cycles.
It has been postulated that spermatozoa are subjected to post-testicular damage during sperm
transport between the seminiferous tubules and epididymis. It is therefore possible that the injec-
tion of damaged spermatozoa may be a cause behind cases of RIF in which no female factor can
be identified. Increased sperm DNA fragmentation and aneuploidy may also lead to embryos
with increased levels of fragmentation and chromosomal abnormalities that are not compatible
with implantation (Figure 15.2).30 Testicular spermatozoa have been isolated and used to fertil-
ize oocytes with promising results regarding embryo quality, implantation, and pregnancy rates.31
However, currently there is limited, low-quality evidence suggesting that a higher probability of
pregnancy might be expected using testicular rather than ejaculated spermatozoa, only in men
with high DNA fragmentation index (DFI) and oligozoospermia.32
Finally, Blazquez et al.33 investigated the efficacy of using donor sperm in addition to oocyte
donation after RIF in improving pregnancy and live birth rates. There was no difference in fertil-
ization rates (75.3% vs. 75.2%, p = 0.97), biochemical pregnancy rates (52.2% vs. 54.1%, p = 0.79),
clinical pregnancy rates (41.2% vs. 45.9%, p = 0.51), ongoing pregnancy rates (38.2% vs. 37.1%,
p = 0.87), and live birth rate (38.2% vs. 35.8%, p = 0.73) between the double donation (DD) and
oocyte donation (OD) groups.
THE ROLE OF EMBRYO BIOPSY AND PREIMPLANTATION GENETIC SCREENING IN RIF

Preimplantation embryo biopsy by removing polar bodies at the zygote stage (day one), a single
blastomere at the cleavage stage (day three), or trophectoderm cells at the blastocyst stage (day five),
in combination with preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), has
paved the way to detect and eliminate chromosomally abnormal embryos, thereby increasing the
chance of implantation and a healthy pregnancy (Figure 15.3). Initial reports using fluorescence
(a) (b)
(c) (d)
Figure 15.2 Photomicrographs showing sperm analysis by FISH and TUNEL in a patient with OAT and
RIF (a–b) and a patient with normal sperm parameters (c–d). (a, c) Sperm hybridized with a probe for chro-
mosomes X-green, Y-red, 18-aqua showing disomy for chromosome Y (a) and normal haploid chromo-
somal constitution (c). (b, d) Photomicrographs showing TUNEL-labeled sperm. Note that in (b) there is
only one normal spermatozoon (blue), while all the remaining are fragmented (red). In contrast, all the
sperm except one (red) are normal in (d).
in situ hybridization (FISH) to screen for a limited number of chromosomes showed no significant
increase in pregnancy rates in RIF patients.34–37
In a pilot study, Greco et al.38 explored whether preimplantation genetic screening (PGS) by
array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst
in patients with RIF can improve clinical results. Three patient groups were compared: (group RIF
PGS) versus (group RIF NO PGS) versus (group NO RIF PGS), and higher implantation and clini-
cal pregnancy rates were observed in the group RIF PGS compared with the group RIF NO PGS.38
Fragouli et al.39 compared the incidence of aneuploidy and the implantation and pregnancy rates
following blastocyst biopsy and polar body (PB) biopsy in patients with RIF. The oocyte and blas-
tocyst aneuploidy rates were 65.5% and 45.2%, respectively, and the implantation and pregnancy
rates for the patients with blastocyst biopsy were higher compared with those who underwent PB
biopsy (58.3% and 69.2% vs. 11.5% and 21.4%, respectively).
Blastocyst biopsy is followed by immediate vitrification as the time needed for the PGS result to
be given exceeds the window of implantation. Vitrification has high survival rates following warm-
ing, and cytoskeletal analysis by confocal laser scanning microscopy has demonstrated that it does
not adversely affect embryonic development and the ability of cells to carry out normal cell divi-
sions, as the vast proportion of spindle/chromosome configurations are normal.40 However, due to
mechanical stress during exposure to cryoprotectants, a significantly higher incidence of spindle
abnormalities is evident in vitrified embryos compared with fresh embryos. The abnormalities
Treatment options in patients with RIF that have embryos with no chromosomal abnormalities 145
(a) (b)
(c) (d)
Figure 15.3 Chromosomal constitution and development on day five of embryos from a patient with
RIF following embryo biopsy on day three and PGS with array CGH. (a) Hatching blastocyst with normal
chromosomal constitution, (b) hatching blastocyst with Monosomy 21 and Trisomy 22, (c) hatching blasto-
cyst with monosomy 6, and (d) arrested embryo with chaotic chromosomal constitution.
include spindles with a focused and an unfocused pole, chromosome bridging, chromosome lag-
ging, and congression failure, reflecting mechanisms that can lead to chromosomal mosaicism in
early human development. The latter can potentially result in implantation failure or miscarriage
depending on the extent and type of the abnormality.40 In cases of biopsied vitrified embryos, a
hole in the zona has already been introduced that can help the embryo to hatch out and implant
following warming and transfer. During standard cryopreservation of embryos, including cases of
“freeze-all” protocols, which are used in order to optimize better the endometrium or to overcome
problems associated with severe ovarian hyperstimulation syndrome, the zona is intact. Assisted
hatching may be beneficial in those cases to minimize embryo entrapment in the zona following
zona hardening imposed after cryopreservation.41–44 Assisted hatching has been previously pro-
posed as a method to overcome zona hardening in fresh embryos of RIF patients.45,46
TREATMENT OPTIONS IN PATIENTS WITH RIF THAT HAVE EMBRYOS WITH NO

CHROMOSOMAL ABNORMALITIES
Stamenov et al.47 evaluated the efficacy of frozen mixed double-embryo transfer (MDET; the simul-
taneous transfer of day-three and day-five embryos) in comparison with frozen blastocyst dou-
ble-embryo transfer (BDET; transfer of two day-five blastocysts) in patients with RIF. The results
revealed significantly higher implantation and clinical pregnancy rates in patients who under-
went MDET than in those who underwent BDET (60.4% vs. 39.3%, p = 0.03 and 52.1% vs. 30.4%,
p = 0.05, respectively). A significantly lower miscarriage rate was observed in the MDET group
(6.9% vs. 10.7%, p = 0.05). In addition, the multiple pregnancy rate was slightly, but not signifi-
cantly, higher in the MDET group (27.1% vs. 25.0%), suggesting that MDET could be regarded as an
alternative, useful approach to improve implantation/pregnancy rates in RIF patients.47 Esfandiari
et al.48 also reported a successful pregnancy following a novel technique of sequential double
transfer of two frozen embryos at the same developmental stage (eight-cell) at five and seven days
after the luteinizing hormone (LH) surge to a patient with seven previous failed assisted reproduc-
tive technology (ART) cycles.
Loutradis et al.49 also compared pregnancy rates in patients with RIF who underwent single or
double-embryo transfer (group A1 underwent embryo transfer on day two and day four after pick-
up, group A2 underwent embryo transfer on day two and day five after pick, group B-control had
a day four or five only transfer without having an additional day two transfer). These authors con-
cluded that double-embryo transfer had beneficial effects on patients with good-quality embryos
and RIF. In particular, if the additional embryo transfer was done on day four, a 38.2% clinical
pregnancy rate and a 50% total pregnancy rate were reported, while if the additional embryo trans-
fer was done on day five, a 60% clinical rate and 60% total pregnancy rate were achieved.
Rahman et al.50 evaluated the efficacy of recombinant follicle stimulating hormone (r-FSH) sup-
plemented by recombinant LH (r-LH) in the late luteal phase of ovarian stimulation in patients
with RIF. The authors concluded that the number of metaphase II oocytes with cytoplasmic matu-
ration, the number of gestational sacs, as well as implantation and pregnancy rates, were signifi-
cantly higher in the r-LH group compared with the r-FSH only group.
Patients who fail to conceive following multiple assisted reproduction cycles often seek treatment
options that include immunological testing and treatment, allogenic lymphocyte therapy, blastocyst
transfer sequential embryo transfer in fresh or frozen cycles, assisted hatching, coculture of embryos
on homologous endometrial cells, PGD-AS, and mitochondrial DNA assessment.3,38,39,51–55 PGD-AS
by array CGH or next generation sequencing (NGS) may be beneficial for RIF patients with no uter-
ine pathologies who have a high incidence of chromosomally abnormal embryos. In those cases, cor-
rect synchronization of the window of implantation with the transfer of the chromosomally normal
embryo in a well-prepared, receptive endometrium may lead to successful implantation.
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15. Mantzouratou A, Mania A, Fragouli E et al. Variable aneuploidy mechanisms in embryos
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16. Voullaire L, Wilton L, McBain J, Callaghan T, Williamson R. Chromosome abnormalities
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17. McCoy RC, Newnham LJ, Ottolini CS et al. Tripolar chromosome segregation drives the
association between maternal genotype at variants spanning PLK4 and aneuploidy in human
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18. Tesarik J. Paternal effects on cell division in the human preimplantation embryo. Reprod
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19. Chatzimeletiou K, Sioga A, Oikonomou L et al. Semen analysis by electron and fluorescence
microscopy in a case of partial hydatidiform mole reveals a high incidence of abnormal mor-
phology, diploidy, and tetraploidy. Fertil Steril. 2011 June;95(7):2430.e1–5.
20. Tesarik J, Mendoza C, Greco E. Paternal effects acting during the first cell cycle of human
preimplantation development after ICSI. Hum Reprod. 2002;17:184–9.
21. Sathananthan AH, Ratnam SS, Ng SC, Tarín JJ, Gianaroli L, Trounson A. The sperm centri-
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February;11(2):345–56.
22. Navara CS, Hewitson LC, Simerly CR et al. The implications of a paternally derived centro-
some during human fertilization: Consequences for reproduction and the treatment of male
factor infertility. Am J Reprod Immunol. 1997;37:39–49.
23. Hewitson L, Simerly C, Schatten G. Inheritance defects of the sperm centrosome in humans
and its possible role in male infertility. Int J Androl. 1997;20(Suppl. 3):325–8.
24. Hewitson L, Simerly C, Schatten G. Cytoskeletal aspects of assisted fertilization. Semin
Reprod Med. 2000;18:151–9.
25. Hewitson L, Simerly CR, Schatten G. Fate of sperm components during assisted reproduction:
Implications for infertility. Human Fertility (Cambridge). 2002;5:110–6.
26. Hut HM, Lemstra W, Blaauw EH et al. Centrosomes split in the presence of impaired DNA
integrity during mitosis. Mol Biol Cell. 2003;14:1993–2004.
27. Balaban B, Yakin K, Alatas C, Oktem O, Isiklar A, Urman B. Clinical outcome of intracy-
toplasmic injection of spermatozoa morphologically selected under high magnification:
A prospective randomized study. Reprod Biomed Online. 2011 May;22(5):472–6.
28. El Khattabi L, Dupont C, Sermondade N et al. Is intracytoplasmic morphologically selected
sperm injection effective in patients with infertility related to teratozoospermia or repeated
implantation failure? Fertil Steril. 2013 July;100(1):62–8.
29. Delaroche L, Yazbeck C, Gout C, Kahn V, Oger P, Rougier N. Intracytoplasmic morphologi-

cally selected sperm injection (IMSI) after repeated IVF or ICSI failures: A prospective com-
parative study. Eur J Obstet Gynecol Reprod Biol. 2013 March;167(1):76–80.
30. Caseiro AL, Regalo A, Pereira E, Esteves T, Fernandes F, Carvalho J. Implication of sperm
chromosomal abnormalities in recurrent abortion and multiple implantation failure. Reprod
Biomed Online. 2015 October;31(4):481–5.
31. Weissman A, Horowitz E, Ravhon A, Nahum H, Golan A, Levran D. Pregnancies and live
births following ICSI with testicular spermatozoa after repeated implantation failure using
ejaculated spermatozoa. Reprod Biomed Online. 2008 November;17(5):605–9.
32. Awaga H, Bosdou JK, Goulis DG et al. Testicular versus ejaculated spermatozoa for ICSI in
patients without azoospermia: A systematic review. RBM Online. 2018. In press.
33. Blázquez A, García D, Rodríguez A, Vassena R, Vernaeve V. Use of donor sperm in addition
to oocyte donation after repeated implantation failure in normozoospermic patients does not
improve live birth rates. Hum Reprod. 2016 November;31(11):2549–53.
34. Caglar GS, Asimakopoulos B, Nikolettos N, Diedrich K, Al-Hasani S. Preimplantation genetic
diagnosis for aneuploidy screening in repeated implantation failure. Reprod Biomed Online.
2005 March;10(3):381–8.
35. Kahraman S, Benkhalifa M, Donmez E, Biricik A, Sertyel S, Findikli N, Berkil H. The results
of aneuploidy screening in 276 couples undergoing assisted reproductive techniques. Prenat
Diagn. 2004 April;24(4):307–11.
36. Munné S. Preimplantation genetic diagnosis and human implantation–a review. Placenta.
2003 October;24(Suppl B):S70–6.
37. Raziel A, Friedler S, Schachter M, Kasterstein E, Strassburger D, Ron-El R. Increased fre-
quency of female partner chromosomal abnormalities in patients with high-order implanta-
tion failure after in vitro fertilization. Fertil Steril. 2002 September;78(3):515–9.
38. Greco E, Bono S, Ruberti A et al. Comparative genomic hybridization selection of blas-
tocysts for repeated implantation failure treatment: A pilot study. Biomed Res Int. 2014;
2014:457913.
39. Fragouli E, Katz-Jaffe M, Alfarawati S et al. Comprehensive chromosome screening of polar
bodies and blastocysts from couples experiencing repeated implantation failure. Fertil Steril.
2010 August;94(3):875–87.
40. Chatzimeletiou K, Morrison EE, Panagiotidis Y et al. Cytoskeletal analysis of human blas-
tocysts by confocal laser scanning microscopy following vitrification. Hum Reprod. 2012
January;27(1):106–13.
41. Vanderzwalmen P, Bertin G, Debauche Ch et al. Vitrification of human blastocysts with
the Hemi-Straw carrier: Application of assisted hatching after thawing. Hum Reprod. 2003
July;18(7):1504–11.
42. Debrock S, Peeraer K, Spiessens C, Willemen D, De Loecker P, D’Hooghe TM. The effect of
modified quarter laser-assisted zona thinning on the implantation rate per embryo in frozen/
vitrified-thawed/warmed embryo transfer cycles: A prospective randomized controlled trial.
Hum Reprod. 2011 August;26(8):1997–2007.
43. Wan CY, Song C, Diao LH, et al. Laser-assisted hatching improves clinical outcomes of vit-
rified-warmed blastocysts developed from low-grade cleavage-stage embryos: A prospective
randomized study. Reprod Biomed Online. 2014 May;28(5):582–9.
44. Zeng M, Su S, Li L. The effect of laser-assisted hatching on pregnancy outcomes of cryopre-
served-thawed embryo transfer: A meta-analysis of randomized controlled trials. Lasers Med
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45. Olivennes F, Vincent Y, Bergere M, Selva J, Frydman R. [Assisted embryo hatching]. Contracept
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47. Stamenov GS, Parvanov DA, Chaushev TA. Mixed double-embryo transfer: A promis-
ing approach for patients with repeated implantation failure. Clin Exp Reprod Med. 2017
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48. Esfandiari N, Coogan-Prewer J, Gotlieb L, Claessens EA, Casper RF. Successful pregnancy
following double-frozen embryo transfer in a patient with repeated implantation failure.
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49. Loutradis D, Drakakis P, Dallianidis K et al. A double embryo transfer on days 2 and 4 or 5
improves pregnancy outcome in patients with good embryos but repeated failures in IVF or
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51. Huang J, Jin N, Qin H et al. Transcriptomic profiles in peripheral blood between women with
unexplained recurrent implantation failure and recurrent miscarriage and the correlation
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52. Gong Q, Zhu Y, Pang N, Ai H, Gong X, La X, Ding J. Increased levels of CCR7(lo)PD-1(hi)
CXCR5+ CD4+ T cells, and associated factors Bcl-6, CXCR5, IL-21 and IL-6 contribute to
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tocyst transfer after implantation failure. Reprod Biomed Online. 2004 December;9(6):630–6.
54. Jayot S, Parneix I, Verdaguer S, Discamps G, Audebert A, Emperaire JC. Coculture of embryos
on homologous endometrial cells in patients with repeated failures of implantation. Fertil
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55. Fragouli E, Spath K, Alfarawati S et al. Altered levels of mitochondrial DNA are associated
with female age, aneuploidy, and provide an independent measure of embryonic implantation
potential. PLOS Genet. 2015 June 3;11(6):e1005241.
Is oocyte donation efficient
in patients with recurrent
16
implantation failure?
GUSTAVO NARDINI CECCHINO and
JUAN ANTONIO GARCÍA-VELASCO
INTRODUCTION
Implantation is a complex process whereby the embryo adheres to the maternal uterine endome-
trium. In the clinical setting, it is assured whenever an intrauterine gestational sac can be sono-
graphically detected, following either a natural conception or an assisted reproductive treatment.
The impossibility of identifying the gestational sac is referred to as implantation failure.
Regarding in vitro fertilization (IVF) treatments, implantation depends on the balance of several
aspects, ultimately related to: gamete and embryo quality, maternal factors, and technical issues.
Curiously, a considerable proportion of infertile couples undergoing multiple IVF attempts are
unable to achieve a successful pregnancy due to recurrent implantation failure (RIF).
The definition of RIF varies widely.1 Andrological causes for RIF are still controversial and basi-
cally linked to sperm quality. In contrast, maternal factors are well known and include not only
age, but also lifestyle, congenital and acquired uterine conditions, altered endometrial receptivity,
and endocrine, immunological, and hematological disorders. Technical issues involve detrimen-
tal effects of ovarian stimulation protocols, inadequate endometrial priming, gamete and embryo
manipulation, nonoptimized culture conditions and poor transfer efficiency. These topics were
extensively discussed in the previous chapters.
Somehow, the figurative question remains unclear: is the seed or the soil responsible for RIF?
IMPORTANCE
In Western countries, there is an increasing trend to postpone parenthood. A comprehensive
systematic review on behalf of the European Society of Human Reproduction and Embryology
(ESHRE) has shown that the main critical reasons for this phenomenon are the ever-increasing
educational level and labor-force participation among women to compensate for a society in which
gender inequity still predominates.2 Also, immature and ineffective social policy incentives lead to
delayed motherhood.
In spite of remarkable advances in the field of assisted reproductive technologies (ART), not all
embryos transferred implant, and these rates significantly decline as women age. In relation to
the fraction of transferred embryos progressing to delivery, available data are even more frustrat-
ing, as reported by the Centers for Disease Control and Prevention (CDC) in their latest National
Summary Report.3
Taken together, this may contribute for the recurrence of implantation failure. Additionally, the
number of couples requiring oocyte donation (OD) to conceive is continuously increasing. As the
percentage of cycles with women’s own eggs declines with age, the use of donated eggs becomes
more prevalent, reaching over 70% of all ART cycles after the age of 44.3 Among all ART treat-
ments, OD shows the best results. Nevertheless, multiple implantation failures may also affect
recipients of OD.4
150
Advances in ovum donation program 151
Importantly, women with RIF experience significantly higher psychological stress levels than
healthy, fertile women. Feelings of social isolation, diminished sexual enjoyment, and need for
parenthood are more frequent among this group of patients. These feelings may vary according to
the supportive care received and the outcomes of consecutive attempts.5
Furthermore, limited knowledge to fully comprehend the components implicated in RIF has
motivated the employment of countless alternative therapies. In general, these interventions lack
rigorous scientific evidence. Besides not being cost effective, they create unrealistic expectations
among patients who are already emotionally vulnerable.
ADVANCES IN OVUM DONATION PROGRAM

In 1983, a successful pregnancy was established for the first time following a rudimentary OD
procedure. Unfortunately, it ended up in a spontaneous abortion after 10 weeks.6 Later in 1984, the
first newborn was achieved using such a technique.7 Ever since, decisive initiatives have supported
the creation of structured OD programs worldwide.
OD was originally applied to overcome cases of primary or secondary ovarian failure. Since
1989, the use has been extended to women with poor response to ART, repeated IVF failure, and
age-related infertility.8 The first clinical trial comparing the results of OD between women over and
under 40 years of age showed similar reproductive outcomes. Moreover, the IVF results of women
≥40 years undergoing OD were notably superior than those presented by women of the same age
that used their own eggs.9
Currently, women from 35 to 44 years old represent the main group of patients treated with OD,
and the mean age is around 40 years.3 Implantation rates and reproductive outcomes are stable
and comparable among OD recipients from different age groups. This suggests that, rather than
the uterus, a poor oocyte competence is strongly linked to aging. Data regarding age and uterine
receptiveness are still controversial. However, the best evidence available exhibited declining rates
of implantation, clinical pregnancy, and live birth, along with increased miscarriage rates only in
OD recipients beyond 45 years.10–12 Figure 16.1 illustrates these facts.
OD accounts for approximately 9.2% of all ART cycles performed in the US.3 The following
graphs show a significant rise concerning egg donation cycles in both the US and Spain over a
10-year period. Noteworthy, the use of frozen gametes is becoming more common due to the
refinements in cryopreservation techniques (Figure 16.2).
(a) (b)
80% 80%
Fresh donor egg cycles Cryopreserved donor egg cycles
60% 60%
40% 40%
20% 20%
0% 0%
25 30 35 40 45 50 25 30 35 40 45 50
Recipients’ age Recipients’ age
Figure 16.1 Clinical outcomes in (a) fresh and (b) frozen donor egg cycles according to recipient age.
It is important to note the stability until the late 40s and the changes after the age of 45. ◊ = clinical
pregnancy rate; X = delivery rate; O = miscarriage rate. (Adapted from Toner JP et al. Fertil Steril. 2002;78(5):
1038–45, with permission.)
152 Is oocyte donation efficient in patients with recurrent implantation failure?
(a)
25,000.00
Egg donation cycles

20,000.00
15,000.00
10,000.00
5,000.00
-
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
Year
Fresh Frozen Total
(b)
20,000.00
Egg donation cycles
16,000.00
12,000.00
8,000.00
4,000.00
-
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
Year
Total
Figure 16.2 Distribution of egg donation cycles over a 10-year period: (a) US; (b) Spain. (Adapted from
[a] National Center for Chronic Disease Prevention and Health Promotion - Division of Reproductive Health.
Assisted Reproductive Technology National Summary Report; [b] Spanish Fertility Society, National
Summary Report, with permission.)
A recent analysis from the CDC’s National ART Surveillance System revealed not only a boost in
the number of donor oocyte cycles carried out in the US last decade, but also a remarkable reduc-
tion in preterm delivery and low birth weight.13 Similarly, it was well demonstrated that the risks
involved in OD are relatively low, with an incidence of serious complications below 1%, such as
moderate or severe ovarian hyperstimulation syndrome, severe infection, ovarian torsion, or any
other complication requiring hospitalization.14
Budak et al. noticed several improvements while evaluating 10,537 OD cycles performed from
1995 to 2005 in the Valencian Institute of Infertility (IVI) clinics in Spain, such as a sequential
increase in both implantation and pregnancy rates (PR), as well as a reduction in rate of multiple
pregnancies due to a progressive decrease in the number of embryos transferred in each cycle.15
Some of these trends are synthesized in Figure 16.3.
In OD programs, a rigorous screening of donors and recipients is highly recommended.16 This
is especially important considering the massive dissemination of oocyte cryobanks. Large studies
validated the use of cryo-stored oocytes in ovum donation programs by confirming its noninferi-
ority in relation to success rates, including obstetric and perinatal outcomes.17–19
Recently, a new approach to assess IVF success has been described.20 The cumulative live birth
rate (CLBR) per total number of embryos transferred gives a more realistic estimation of a couple’s
probability to reach a newborn after an IVF treatment. Despite the increasing number of embryos
transferred, the CLBR dramatically declines with age and reaches a plateau on women older than
40 years using their own eggs.20 Conversely, it has been proved that in an OD program, the number
Oocyte donation X RIF 153
% 70
60
50 Pregnancy %
Clinical PR
40 Ongoing PR
Implantation %
Miscarriage %
30
High order pregnancy %
20
10
0
1995–1997 1998–2000 2001–2003 2004–2005 Years
Figure 16.3 Trends in different reproductive outcomes over time in an OD program. (Adapted from
Budak E et al. Fertil Steril. 2007;88(2):342–9, with permission.)
of embryos needed to achieve a newborn is lower and the CLBR rates are very similar between
women of distinct ages, as shown in Figure 16.4.21
OOCYTE DONATION X RIF

Apparently, poor oocyte quality is the central cause of age-related deterioration of reproductive
capacity. By the age of 40, almost 80% of women’s oocytes are aneuploid.22 Equally, the rate of aneu-
ploid embryos starts to increase rapidly after 37 years and reaches its peak at the age of 44, when
almost 90% of the embryos are aneuploid.23
(a) Cumulative probability of live-birth (%) (b) Cumulative probability of live-birth (%)
100 100 Age

categories
<35 yrs
80 80 35–37 yrs
38–40 yrs
60 60 >=40 yrs
40 40
20 20
0 0
0 5 10 15 20 25 30+ 0 5 10 15 20 25 30
Embryos transferred needed to reach live-birth Embryos transferred needed to reach live-birth
Figure 16.4 CLBR according to the total number of embryos transferred using (a) own eggs and
(b) OD. (Adapted from [a] Garrido N et al. Fertil Steril. 2012;98(2); [b] Geraedts J et al. Hum Reprod. 2011;26(11):
3173–80, with permission.)
These facts certainly contribute for multiple implantation failure and inferior ART outcomes.
Fortunately, the use of preimplantation genetic testing for aneuploidy (PGT-A) improves the
implantation and live birth rates in both women with advanced maternal age and selected RIF
patients whenever transferring chromosomally normal embryos.24,25
In addition, when analyzing the oocyte-to-infant ratio, it is possible to observe tremendous
b
iological wastage.26 Such a biological loss is even greater in patients undergoing PGT-A. A
prospective study including couples with recurrent pregnancy loss, multiple IVF failures, and
advanced maternal age showed that up to 99% of the retrieved oocytes did not produce live births.
In the RIF group, less than 5% of the inseminated oocytes were capable of producing euploid
embryos.27
Human ooplasmic transplantation emerged in the 1990s as an innovative approach to overcome
the detrimental effects of compromised oocytes on embryonic development. Experiments con-
ducted by Cohen et al. in women experiencing RIF confirmed that the injection of donor ooplasm
into patient eggs was able to enhance embryo developmental potential. Indeed, as a result of these
experiments, women with multiple IVF failures got pregnant and gave birth.28 Nonetheless, the US
Food and Drug Administration banned the use of this technique in 2001 due to ethical and biologi-
cal concerns, essentially related to the pattern of mitochondrial DNA transmission and the risk of
mitochondrial disease inheritance.29 Lately, research involving meiotic spindle transfer and other
nuclear genome transfer techniques are being investigated in animal-based models, but it is still too
early to claim any clinical utility.30
It is reasonable to assume that a great proportion of women suffering from RIF systematically
produce impaired oocytes, which often generate poor-quality embryos. So, it is expected that
changing the gamete can lead to better results. Outcomes from OD models support this concept.15,22
Figure 16.5 highlights that regardless of the indication for OD, reproductive results are excellent
even for cases of RIF.
Once more, the oocyte is probably the main factor responsible for the occurrence of RIF, whereas
the endometrial receptivity seems to play a minor role. Almost 75% of RIF patients show a normal
window of implantation (WOI). Though, in some cases, an endometrial receptivity array (ERA)
permits the diagnosis of a displaced WOI and a personalized embryo transfer, which can normalize
pregnancy and implantation rates.31,32
Cobo et al. recently demonstrated that in a well-developed OD program, the overall probability
of achieving a child can be as high as 97.3% until the late forties. It is worth noting that such an ele-
vated CLBR is usually achieved when 25–40 oocytes are consumed, which represents nearly three
or four donation cycles.18 By all means, this certainly endorses the efficiency of the OD irrespective
of the recipient’s age and infertility cause.
Over the last few years, new observations with respect to maternal killer-cell immunoglobulin-
like receptor (KIR) and fetal HLA-C compatibility have shown that it could also influence implan-
tation and live birth rates among patients with RIF, including in OD cycles.33,34 The mechanisms
through which the KIR-HLA matching could influence implantation and live birth rates are dis-
cussed elsewhere.35 It must be recognized that these are preliminary observational data at best, and
randomized clinical trials are still needed. Future studies are urged to prove the clinical utility of
the KIR-HLA matching, especially in RIF.
CONCLUSIONS
Finally, we go back to the initial question: is the seed or the soil responsible for RIF? The best
guess would be both. The key for couples with an adequate control of maternal and technical ele-
ments probably relies on improving embryo potential, which, in some cases, can only be achieved
using OD.
Thus, as most couples struggling with RIF are prone to accept alternative therapies, OD might be
an attractive treatment option to achieve their reproductive goals.
References 155
(a) Cumulative probability of live-birth (%)
100
Indication
80 Age
ART failure
Endometriosis
Low response
60 Ovarian failure
Recurrent miscarriage
Age-censored
ART failure-censored
40 Endometriosis-censored
Low response-censored
Ovarian failure-censored
20 Recurrent miscarriage-
censored
0 5 10 15 20 25 30
Embryos needed to reach newborn
(b) Pregnancy rate

% 100
80 Overall
Advanced age (>40)
Low responder
Premature ovarian failure
Recurrent IVF failure
60 Endometriosis
Poor oocyte quality
Menopause
Genetic factor
Recurrent miscarriage
40
Surgical menopause
20
1 2 3 4 5
Cycle number
Figure 16.5 OD model: (a) CLBR according to the total number of embryos transferred and (b) PR
according to donor cycle number. (Adapted from [a] Geraedts J et al. Hum Reprod. 2011;26(11):3173–80;
[b] Budak E et al. Fertil Steril. 2007;88(2):342–9, with permission.)
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1331–6.e1.
13. Kawwass J, Monsour M, Crawford S et al. Trends and outcomes for donor oocyte cycles in the
United States, 2000–2010. JAMA. 2013;310(22):2426–34.
14. Maxwell K, Cholst I, Rosenwaks Z. The incidence of both serious and minor complications in
young women undergoing oocyte donation. Fertil Steril. 2008;90(6):2165–71.
15. Budak E, Garrido N, Soares SR et al. Improvements achieved in an oocyte donation program
over a 10-year period: Sequential increase in implantation and pregnancy rates and decrease
in high-order multiple pregnancies. Fertil Steril. 2007;88(2):342–9.
16. Sauer M. Ethics, social, legal, counselling Oocyte and embryo donation 2006: Reviewing two
decades of innovation and controversy. Reprod Biomed Online. 2006;12(2):153–62.
17. Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum dona-
tion programme: A prospective, randomized, controlled, clinical trial. Hum Reprod.
2010;25(9):2239–46.
18. Cobo A, Garrido N, Pellicer A, Remohí J. Six years’ experience in ovum donation using vitri-
fied oocytes: Report of cumulative outcomes, impact of storage time, and development of a
predictive model for oocyte survival rate. Fertil Steril. 2015;104(6):1426–34.e8.
19. Cobo A, Serra V, Garrido N, Olmo I, Pellicer A, Remohí J. Obstetric and perinatal outcome of
babies born from vitrified oocytes. Fertil Steril. 2014;102(4):1006–15.e4.
20. Garrido N, Bellver J, Remohí J, Simón C, Pellicer A. Cumulative live-birth rates per total
number of embryos needed to reach newborn in consecutive in vitro fertilization (IVF) cycles:
A new approach to measuring the likelihood of IVF success. Fertil Steril. 2011;96(1):40–6.
21. Garrido N, Bellver J, Remohí J, Alamá P, Pellicer A. Cumulative newborn rates increase with
the total number of transferred embryos according to an analysis of 15,792 ovum donation
cycles. Fertil Steril. 2012;98(2).
22. Geraedts J, Montag M, Magli MC et al. Polar body array CGH for prediction of the status of
the corresponding oocyte. Part I: Clinical results. Hum Reprod. 2011;26(11):3173–80.
23. Franasiak JM, Forman EJ, Hong KH et al. The nature of aneuploidy with increasing age of
the female partner: A review of 15,169 consecutive trophectoderm biopsies evaluated with
comprehensive chromosomal screening. Fertil Steril. 2014;101(3):656–63.
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with preimplantation genetic screening improves implantation and live birth in women age
40 through 43. J Assist Reprod Genet. 2015;32(3):435–44.
25. Rubio C, Bellver J, Rodrigo L et al. Preimplantation genetic screening using fluorescence in
situ hybridization in patients with repetitive implantation failure and advanced maternal age:
Two randomized trials. Fertil Steril. 2013;99(5):1400–7.
26. Inge GB, Brinsden PR, Elder KT. Oocyte number per live birth in IVF: Were Steptoe and
Edwards less wasteful? Hum Reprod. 2005;20(3):588–92.
27. Patrizio P, Bianchi V, Lalioti MD, Gerasimova T, Sakkas D. High rate of biological loss in
assisted reproduction: It is in the seed, not in the soil. Reprod Biomed Online. 2007;14(1):92–5.
28. Cohen J, Scott R, Alikani M et al. Ooplasmic transfer in mature human oocytes. Mol Hum
Reprod. 1998;4(3):269–80.
29. Cree L, Loi P. Mitochondrial replacement: From basic research to assisted reproductive tech-
nology portfolio tool-technicalities and possible risks. Mol Hum Reprod. 2015;21(1):3–10.
30. Costa-Borges NL, Mestres E, Miguel-Escalada I et al. Meiotic spindle transfer overcomes
embryo developmental arrest in compromised oocytes: Proof of concept in the mouse model.
Fertil Steril. 2017 September 1;108(3):e41.
31. Ruiz-Alonso M, Blesa D, Diaz-Gimeno P et al. The endometrial receptivity array for diagno-
sis and personalized embryo transfer as a treatment for patients with repeated implantation
failure. Fertil Steril. 2013;100(3):818–24.
Steril. 2017;108(1):15–8.
33. Alecsandru D, Garrido N, Vicario JL et al. Maternal KIR haplotype influences live birth
rate after double embryo transfer in IVF cycles in patients with recurrent miscarriages and
implantation failure. Hum Reprod. 2014;29(12):2637–43.
34. Alecsandru D, Aparicio P, Aparicio M, García-Velasco J. Maternal killer-cell immunoglobu-
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birth rate. In: ESHRE. 2016.
35. Alecsandru D, García-Velasco JA. Why natural killer cells are not enough: A further under-
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2017;107(6):1273–8.
Sperm donation and recurrent
17
CHRISTOS P. TSAMETIS and DIMITRIOS G. GOULIS
INTRODUCTION
Sperm donation refers to the provision by a male (donor) of his sperm (donor sperm), which has
been manipulated outside the human body with the purpose of achieving a pregnancy in a female
who is not his sexual partner. The donor, typically, intends to have no legal relationship to any
resulting offspring.1 The sperm may be donated privately and directly to the recipient or through
a sperm bank or fertility clinic. Donor sperm is commonly used in artificial insemination, which
refers to the introduction of sperm into the vagina (intravaginal insemination), uterus (intracervi-
cal or intrauterine insemination), or oviduct by a means other than sexual intercourse. Artificial
insemination using sperm from a male other than the patient’s partner is called therapeutic donor
insemination (TDI). Less commonly, donor sperm may be used in in vitro fertilization (IVF) or
intracytoplasmic sperm injection (ICSI) procedures, especially in women who are not good can-
didates for TDI, such as those with subfertility due to tubal disorder, uterine malformation, active
pelvic infection, or uncorrected anovulation.2
TDI is the oldest artificial reproductive technique in use for the treatment of male infertility. In
the US, over 170,000 women were treated with TDI in 1987, while in 1990, it has been estimated that
11,400–23,400 pregnancies were achieved annually by TDI.3 The advent of ICSI has decreased the
indication for sperm donation; nevertheless, recently emerged reproductive choices have expanded
the therapeutic role of TDI. Currently, the main reasons for TDΙ cycles, according to ESHRE Capri
Workshop Group,4 are
• At least three failed IVF or ICSI cycles with partner’s sperm

• Nonobstructive azoospermia (NOA), confirmed by testicular sperm extraction (TESE)
• Women without a male partner, such as single women seeking conception or women in a
female–female relationship (lesbians)
Moreover, TDI should be considered in
• Couples in whom the male partner has severe sperm or seminal fluid abnormalities or ejacula-
tory dysfunction
• Couples in whom the male or both partners are carriers of a heritable disease
• Couples who are serodiscordant for sexually transmissible viral infections
• Couples who are incompatible for red-cell antigens, associated with hemolytic disease of the
newborn, and with a history of a severely affected infant5
Unsuccessful IVF or ICSI treatment could be due to implantation failure. Repeated implanta-
tion failure (RIF) refers to a situation in which implantation has repeatedly failed to reach a stage
recognizable by pelvic ultrasonography; nevertheless, as yet, no universally accepted definition
exists.6 According to a proposed definition, RIF is the failure to achieve clinical pregnancy after
the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles in
a woman under the age of 40 years.6 RIF could be due to embryo quality or endometrial factors
or both. Regarding the relationship between sperm donation and RIF, two clinically important
158
Sperm donation cycles as a treatment option for RIF 159
questions arise: (a) What is the incidence of RIF in TDI, used as primary treatment for infer-
tility? and (b) What is the therapeutic role of TDI in the management of couples with RIF?
Unfortunately, there are no or few data published as yet that can provide a clear answer to either
questions.
FAILURE OF TID CYCLES AS A PRIMARY INFERTILITY TREATMENT

There are no studies so far reporting on the incidence of RIF in sperm-donation cycles. This is prob-
ably due to the fact that no universally accepted definition of RIF is established and the fact that the
number of recipients not achieving a pregnancy is usually not reported.4 Instead, the clinical out-
come is expressed as the pregnancy and delivery rates, which depend upon the method of insemi-
nation used,2 on the semen parameters,7 and on the age8,9 and body weight10 of the female recipient.
One of the main indications for using TDI is NOA of the male partner. The largest amount of
data comes from the French Federation of Centre d’Etudes et de Conservation des Oeufs et du
Sperme humain (CECOS), which has organized one of the largest sperm-donation programs in
Europe. Between 1998 and 2007, 2757 couples have requested treatment with TDI and have been
included in the CECOS Paris-Cochin study. The indication for sperm donation was azoospermia in
69% and other sperm defects in 27% of the couples. The first treatment was intracervical insemina-
tion (ICI) in 26% of couples, intrauterine insemination (IUI) in 68%, IVF in 4%, and ICSI in 2% of
the couples, while the pregnancy and delivery rates were 6% and 4.8%; 14.9% and 12.1%; 27.2% and
16.3%; and 26.1% and 16.8%, respectively.4 Unfortunately, no data regarding the rate of RIF in IVF
or ICSI cycles were published.
Another large retrospective study examined the crude and expected cumulative delivery rates in
364 women aged <40 years undergoing a total of 660 donor ICSI cycles. The main indications were
severe male infertility, including azoospermia (n = 190), requests from lesbian couples (n = 102),
and requests from single women (n = 72). The crude cumulative delivery rate after three ICSI
cycles in the whole population and in women aged 20–29, 30–37, and 38–39 years were 62%, 79%,
61%, and 50%, respectively. It can be assumed that the failure rates after three donor ICSI cycles,
consistent with the definition of RIF, were 38%, 21%, 39%, and 50%, respectively. However, these
numbers do not reflect the actual incidence of RIF, which is probably significantly less, because
failure to deliver a baby is not only due to implantation failure. After the third cycle, the increase in
delivery rates became less pronounced and reached a plateau in the fourth cycle in all age groups.
As expected, this study has shown that failure rates in donor ICSI cycles increased significantly
with increasing female age.9 The same group reported on the cumulative delivery rates in women
aged >40 years undergoing either TDI or IVF/ICSI with donor sperm or switched from TDI to
donor IVF/ICSI. Crude cumulative delivery rates after six IUI cycles and three primary ICSI cycles
(no previous IUI) were similar in both groups (24% vs. 26%).11 An older and smaller (39 cycles
included) study published data regarding the cumulative pregnancy rate (PR) after IVF with frozen
donor sperm. The cumulative PR for one year (three donor IVF cycles) was 44.7%.12 Finally, a recent
study examined the pregnancy rates after TDI or IVF with donor sperm, according to treatment
indications. They found that pregnancy rates associated with the first treatment cycle were higher
in women undergoing TDI due to NOA than those after ICSI failure or without a male partner
(29.1%, 27.6%, and 22.6%, respectively), while in women treated with donor IVF cycles, the respec-
tive figures were 42.1%, 48.7%, and 38.2%. It seems that donor IVF cycles have higher success rates
than TDI, irrespective of treatment indications, and single women have the highest failure rates,
probably due to their advanced age.13
SPERM DONATION CYCLES AS A TREATMENT OPTION FOR RIF

Rationale for the use of donor sperm
According to a recently proposed definition, RIF is expected to result primarily from endometrial/
uterine factors.6 However, given the fact that current methods for the assessment of embryo quality
160 Sperm donation and recurrent implantation failure
are still quite subjective and not always accurate, inevitably there will be a proportion of RIF cases
due to gamete or embryo factors.6,14 The embryo quality depends both on the quality of oocytes and
spermatozoa. Poor-quality sperm may produce poor-quality embryos. It is known that the genetic
and epigenetic integrity of sperm is essential for fertilization, normal embryo development, and
successful implantation.6
Animal and human studies have linked sperm DNA damage to poor embryo development15 and
failure to achieve either spontaneous16 or assisted conception.17,18 A recent meta-analysis has found
a modest but statistically significant negative effect of sperm DNA damage on pregnancy rates
after IVF and/or ICSI treatment.19 Increased DNA fragmentation has also been associated with
increased miscarriage rates20 and recurrent pregnancy loss after IVF or ICSI;21 nevertheless, its
association with RIF has not been elucidated yet, despite a recent study that did not find any effect.22
Moreover, sperm aneuploidy and chromosomal structural aberrations have been implicated in the
pathogenesis of recurrent pregnancy loss and RIF.23–25 A more detailed presentation of the andro-
logical causes of RIF can be found in Chapter 9.
All couples experiencing RIF should undergo a thorough investigation in order to eluci-
date the cause. The investigation plan should include hysteroscopy, hysterosalpingography,
pelvic ultrasonography, assessment of ovarian reserve and function (follicle-stimulating hor-
mone [FSH], anti-Müllerian hormone [AMH], antral follicular count), and parental karyotyp-
ing. Investigations of research value are thrombophilia screen, sperm DNA integrity tests, and
sperm fluorescence in situ hybridization (FISH).6 If the diagnostic work-up reveals no female
factor and the problem lies with the embryo due to impaired semen quality, a number of treat-
ment modalities have been proposed. In the case of increased DNA fragmentation (DF), treat-
ment options include: lifestyle modifications (healthy diet, weight loss, cessation of smoking,
reduced alcohol consumption); antioxidant therapy;26 selection of sperm with low levels of DNA
damage using a number of techniques, such as annexin-V columns or sperm hyaluronic acid
binding;27,28 the implementation of intracytoplasmic morphologically selected sperm injection
(IMSI), which utilizes spermatozoa selected under high-power magnification according to a
predefined set of morphological criteria;29 and ICSI using surgically retrieved testicular sperm
instead of ejaculated sperm. 30,31 However, data regarding the effectiveness of the above-men-
tioned approaches in increasing the implantation and pregnancy rates in couples with RIF are
limited and inconsistent.6
In the case of chromosomal numerical or structural abnormalities in the male partner, genetic
counseling is essential to inform the couple about the associated risks in the offspring and their
reproductive options.32 Preimplantation genetic diagnosis (PGD) is recommended, especially in
cases of reciprocal translocations, as it has been shown in some studies to produce a more favorable
reproductive outcome.33,34 However, pregnancies conceived with a male partner with sperm aneu-
ploidy, even if PGD has been employed, still carry an increased risk of aneuploidy; thus, appropri-
ate prenatal screening and testing should be offered.32
It is logical to assume that the only way to eliminate the impaired semen as a contributing factor
to RIF and to alleviate the genetic risk to the offspring associated with chromosomal abnormali-
ties of the sperm would be to proceed with donor sperm cycles. However, studies comparing the
efficacy of sperm donor cycles to any of the above-mentioned treatment strategies, as the preferred
first-step management option of couples with RIF due to impaired semen quality, are lacking. All
couples with RIF, after undertaking an appropriate diagnostic evaluation, should be informed
about their reproductive options. Sperm donation may be advised when there is
• Absence of a female factor causing RIF
• Clinical or laboratory evidence of impaired sperm or embryo quality (especially chromosomal
aberrations)
• Implantation failure, despite other treatment attempts or if the prognosis of further IVF cycles
is considered poor
Sperm donation cycles as a treatment option for RIF 161
Efficacy of sperm donation cycles after IVF/ICSI failure

There are very few studies, retrospective only, examining the outcomes of sperm donation cycles
after IVF/ICSI failed attempts. All these studies, however, included couples with at least one unsuc-
cessful IVF/ICSI procedure and did not evaluate the reproductive efficacy of sperm donation spe-
cifically in couples with RIF.
The clinical PR per cycle and the cumulative PR per couple after six or seven donor sperm cycles
vary significantly across studies. A multicenter study conducted in France within CECOS included
162 couples treated with TDI after intraconjugal ICSI failure and reported a pregnancy rate per
cycle of 5.8% in women who had obtained poor-quality embryos in previous ICSI cycles and above
19% in those who had obtained good-quality embryos.35 Gorrill et al. analyzed a series of 61 TDI
cycles in 19 couples who had previous failed ICSI attempts performed for severe male factor infer-
tility and reported a PR per cycle of 27.9% and a cumulative PR of 84.2%. Pregnancies occurred
within a mean of 3.2 ± 1.8 cycles.36 The largest study so far included 319 couples, who underwent
a total of 1159 TDI cycles after a total of 1011 unsuccessful intracouple ICSI cycles. The mean PR
per cycle and the cumulative PR after seven TDI cycles were 12% and 43.6%, respectively. Three-
quarters (78.4%) of pregnancies were achieved during the first four cycles of insemination.37 The
variation in the results could be due to differences in the number of couples included in each study
and in population characteristics.
One study13 reported on the pregnancy rate associated with the first treatment cycle of TDI or
IVF using donor sperm (IVF-D) in couples with at least three previous failed ICSI cycles. The indi-
cations for IVF-D were tubal obstruction, moderate/severe endometriosis, and advanced mater-
nal age (≥39 years old). The success rates for IVF-D and TDI were 48.7% and 27.6%, respectively.
The same study reported that previous ICSI failure was the most prevalent indication for sperm
donation cycles, the other ones being NOA and single women seeking fertility. In TDI procedures,
higher pregnancy rates were achieved in patients with azoospermia (29.1%) than in cases of ICSI
failures (27.6%) and single women (22.6%) (p = 0.020), whereas in IVF-D cycles, better results
were obtained in cases of ICSI failure (48.7%) than in NOA (42.1%) and single women (38.2%)
(p < 0.001), whose advanced age tended to be a factor.
Apart from pregnancy rates, live birth rate is considered to be clinically a more useful parameter
in expressing the success rate of an assisted reproductive technology (ART) program, especially the
cumulative live birth rate after a defined number of treatment cycles.38 According to the study by
Gorrill et al., the application of TDI after ICSI failure produced a live birth rate per cycle of 24.6%
and a cumulative live birth rate of 88% after seven cycles of insemination.36 However, these high
rates were not confirmed in more recent studies. Leguy et al. evaluated retrospectively the repro-
ductive outcome of sperm donation cycles in 71 couples who failed to conceive after intracouple
ICSI treatment for male infertility and reported that, after donation, 30 couples (42.2%) succeeded
in being parents.39 Frapsauce et al. reported on the outcome of 175 TDI cycles in 47 couples after
120 failed ICSI cycles for various reasons. The mean live birth rate per cycle and the cumulative
live birth rate after six cycles were 16.6% and 62%, respectively. Overall, a live birth occurred after
3.7 ± 1.8 cycles.40
It can be concluded that TDI is an effective reproductive option for couples with previous failed
IVF/ICSI treatments, as at least half of them can achieve parenthood. The success rates are compa-
rable to those obtained in couples to whom TDI is proposed as an initial treatment.37 However, in
cases of couples experiencing RIF during oocyte donation (OD) cycles with the partner’s normal
semen, switching to donor sperm in subsequent cycles does not seem to improve the reproductive
outcome in terms of either birth rate or live birth rate.41
Prognostic factors affecting the outcome of TDI

It is of great importance for the clinician involved in the management of couples with RIF to be
able to differentiate those who would have a favorable reproductive outcome with TDI. However,
evidence is limited to only a few studies, which suggest that the main factors implicated in the prog-
nosis of TDI cycles are female partner’s age, ovarian response, number of good-quality embryos
obtained in previous IVF/ICSI cycles, semen characteristics, and number of TDI cycles.
Female age: Increasing female partner’s age decreases the success rate of TDI cycles. Hennebiqc
et al. have shown that cumulative PR was 51.7% for women under 34 years of age, 38.1% for women
between 34 and 40 years, and 23.8% for women aged over 40 years (p = 0.010).37 The negative impact
of woman’s age on the reproductive outcome of TDI has also been reported in other studies.8,42,43
Ovarian response: Poor ovarian response during antecedent intracouple ICSI cycles has
been proposed as a negative prognostic factor for the success of subsequent TDI treatment.8,35
In a larger recent trial, 37 although cumulative PR tended to be higher (44.5%) in cases when
>3 oocytes were retrieved in previous intracouple ICSI cycles compared with cases when ≤3
oocytes were obtained (33.5%), no statistical significance was reached (p = 0.353). Moreover,
using a saturated logistic regression model, no significant relationship was found between ovar-
ian response during intracouple ICSI and clinical PR during TDI (odds ratio [OR] 1.44, 95% CI
0.46–4.51; p = 0.115). 37
Embryo quality: The results of studies evaluating the impact of the availability and the num-
ber of good-quality embryos obtained during intracouple ICSI cycles on the success of TDI are
inconsistent. Saias-Magnan and Mandelbaum have demonstrated that the PR during TDI was sig-
nificantly higher (>19% vs. 5.8%), when couples had morphologically good embryos during previ-
ous ICSI cycles compared with those who had only poor-quality embryos.35 Frapsauce et al. have
found higher, although not statistically significant, live birth rate per cycle (20% vs. 13.3%) and
cumulative live birth rate after six cycles of TDI (68% vs. 54.5%) in couples in whom at least one
top-quality embryo (TQE) was obtained in previous intracouple ICSI cycles compared with those
without TQE.40 The same study also reported that couples with ≥1 TQE achieved live births faster
than couples without TQE.40 By contrast, Gorrill et al. demonstrated a high rate of TDI pregnan-
cies irrespective of the embryo quality during previous failed ICSI procedures.36 A French CECOS
study, which analyzed the results of TDI according to embryo quality on day two after ICSI, has
shown that the rate of TQE was similar in couples who achieved live births after TDI cycles and in
those who failed.39 In a recent larger multicenter study, also from CECOS, no significant difference
was detected in pregnancy rate per TDI cycle (10.5% vs. 12.9%; p = 0.278) or cumulative PR (40.7%
vs. 43.6%, p = 0.651) between couples in whom no TQE and couples in whom at least one TQE
was transferred during intraconjugal ICSI procedure.37 However, the authors of the latter study
have underlined that embryo quality was not addressed properly, and the absence of an effect on
subsequent TDI success could be also due to the multicenter design of the study, leading to a bias
in embryo classification.37
Semen characteristics: Couples with male partners diagnosed with azoospermia or cryptozoo-
spermia seem to have better outcomes in TDI performed after repeated ICSI failures compared
with couples with male partners diagnosed with oligoasthenoteratozoospermia or normal semen
parameters. The recent large multicenter study from CECOS demonstrated that clinical pregnancy
rates during TDI were higher when male partners presented with azoospermia or cryptozoosper-
mia compared with those with oligoasthenoteratozoospermia or normal sperm: 14.8% versus
10.1% (p = 0.015) per cycle and 53.1% versus 37.2% (p = 0.005) per couple, respectively. According
to the origin of sperm used in previous failed ICSI cycles, the TDI cumulative PRs were higher
when the sperm was surgically obtained in comparison with ejaculated sperm: 55.6% versus 36.2%,
respectively (p = 0.001).37 It can be assumed that in cases of azoospermia or cryptozoospermia, the
intraconjugal ICSI failure is mainly due to male factors; therefore, the use of donor sperm would
alleviate the problem and allow an embryo with implantation capacity to be obtained. 37 On the
contrary, in an older study with a smaller sample size, azoospermia was found to be a poor prog-
nostic factor for the success of TDI.39
Number of TDI cycles: The probability of achieving a clinical pregnancy with TDI decreases as a
function of increasing number of TDI cycles (OR 0.87, 95% CI 0.78–0.97; p = 0.010). The reported
References 163
mean PR per cycle during the first four TDI cycles was 21.7% and dropped after the fourth cycle to
4.5%.37 This finding has also been reported in other studies.44 It seems that as the number of TDI
cycles increases, the woman’s age also increases, and the negative impact of advanced female age
on reproductive outcomes is well documented.45 Moreover, it could be due to the fact that the good-
prognosis women fall pregnant quicker and this leaves women of poorer prognosis in the higher
order TDI cycles.
Taken together, the above-mentioned evidence indicates that the transition to sperm donation
cycles should be proposed at key moments, taking into account especially the woman’s age and
the sperm characteristics of the male partner in order to achieve the highest probability of preg-
nancy. The best results should be expected when TDI cycles are offered, as a treatment option after
repeated IVF/ICSI failures, to women aged under 34 years whose partners have been diagnosed
with azoospermia or cryptozoospermia.
CONCLUSIONS
RIF is a distressing condition both to the couples and the treating physician. Data regarding the
incidence of RIF among sperm donation cycles performed as initial treatment for infertility are
lacking. On the other hand, sperm donation could be an effective treatment option for couples strug-
gling with repeated IVF/ICSI failures only after an extensive diagnostic evaluation and detailed
consultation with the couple has been performed. Before offering TDI, the clinician should take into
account specific characteristics of the couple, such as the female age and the semen status in order to
maximize the clinical benefit. However, large-scale prospective studies evaluating the reproductive
efficacy of TDI and the factors affecting the outcome specifically in couples with RIF are warranted.
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to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic
sperm injection outcome. Asian J Androl. 2017;19(1):80–90.
20. Robinson L, Gallos ID, Conner SJ et al. The effect of sperm DNA fragmentation on miscar-
riage rates: A systematic review and meta-analysis. Hum Reprod. 2012;27(10):2908–17.
21. Zini A, Boman JM, Belzile E, Ciampi A. Sperm DNA damage is associated with an increased
risk of pregnancy loss after IVF and ICSI: Systematic review and meta-analysis. Hum Reprod.
2008;23(12):2663–8.
23. Rubio C, Gil-Salom M, Simón C et al. Incidence of sperm chromosomal abnormalities
in a risk population: Relationship with sperm quality and ICSI outcome. Hum Reprod.
2001;16(10):2084–92.
25. Burrello N, Vicari E, Shin P et al. Lower sperm aneuploidy frequency is associated with high
pregnancy rates in ICSI programmes. Hum Reprod. 2003;18(7):1371–6.
26. Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of
sperm DNA fragmentation by oral antioxidant treatment. J Androl. 2005;26(3):349–53.
27. Jakab A, Sakkas D, Delpiano E et al. Intracytoplasmic sperm injection: A novel selec-
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2005;84(6):1665–73.
28. Said T, Agarwal A, Grunewald S et al. Selection of nonapoptotic spermatozoa as a new tool
for enhancing assisted reproduction outcomes: An in vitro model. Biol Reprod. 2006;74(3):
530–7.
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morphologically selected sperm injection outcome: A meta-analysis. Reprod Biomed Online.
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37. Hennebicq S, Blagosklonov O, Eustache F et al. Donor sperm insemination after failed intra-
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2017;32(7):1439–49.
Optimizing embryo transfer
technique for recurrent
18
management
FRANK J. BROEKMANS
INTRODUCTION
The enigmatic condition of recurrent implantation failure (RIF) may be much related to condi-
tions that characterize the reproductive process in the human species. With optimal fertility in
the second and third decade of life, a rapid decline is observed in the fourth, resulting in a state of
natural sterility on average in the early forties. The majority of explanatory factors seem to be pres-
ent in the female, as males tend to have uncompromised fertility up to the age of 50, with a slow
decline thereafter. Factors that are believed to highly contribute to the limited fertile lifespan in the
human female are oocyte and subsequent embryo quality, for which the mechanisms are largely
unknown.1–3 Next to embryonic aneuploidy, mostly related to meiotic errors, many other failures
in the oocyte-follicle complex may contribute to the overall low, and with age rapidly declining,
quality of the human embryo.4,5 Still, aging or dysfunction of the endometrium may have a certain
degree of contribution, although oocyte donation studies have excluded a major role at this level.
It is therefore no surprise that failures occurring in the process of assisted reproductive technol-
ogy (ART), after the moment the embryo has been placed in the uterus, are so frequent. From this
knowledge and awareness, it is within expectation that many couples will not achieve a pregnancy
in spite of repeatedly receiving morphologically well-behaving embryos. This is much to the frus-
tration of both couple and physician and elicits a heart-breaking quest into the “why.” Although
part of the answer can be drawn from the aforementioned, the obligation for the doctor is obvious
to rule out any adjustable factor of “substandard” care.
The process of ART called in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)
comprises three distinct phases: the retrieval of gametes, simple for the human male, complex for
the female, where getting eggs may be more important than a strenuous strive for all that can be
squeezed out; the laboratory phase, where some quality characteristics can be observed such as
fertilization rate, embryo development rate, and embryo implantation rate; and finally, the luteal
phase with the embryo transfer, a handling with only indirect and incomplete information on the
correct “arrival” of the embryo, but also the endocrine management of endometrium development
and timing. The subsequent event, implantation, will then unveil itself at the moment of human
chorionic gonadotrophin (hCG) testing, or not.6
It is important to realize that, although each of the aforementioned three phases are crucial
for the ART process, the relative contribution to the final aim, a live birth, is not so clear. Will
the approach in the stimulation and egg retrieval phase make a difference? Recent studies seem
to indicate that, apart from a few basic concepts, any approach will be good enough.7–9 The IVF
laboratory phase must demonstrate that gametes handling leads to fertilization and embryo devel-
opment, where many tools and gadgets on embryo quality and selection may or may not give the
big advances that are frequently hoped for.10–13 Finally, the management of luteal function toward
166
Introduction 167
the endometrium may have been a slightly neglected area, as it has surfaced through the increasing
application of the agonist trigger, where luteal phase insufficiency developed by the elimination of
the powerful hCG stimulus.
For the latter phase, the execution of the embryo transfer must be considered as a distinct factor
of success of the ART process, where knowledge is both present and absent. We know the role of a
smooth procedure, using specific materials and tools such as ultrasound guidance, but the sugges-
tion that the physician performing the transfer could make an important difference has never led
to robust research to sort out this feature. In this chapter, we will review the existing knowledge
on how the embryo transfer procedure can be optimized, and where research could fill up open
holes in our knowledge. It goes without saying, that, if the transfer procedure has been carried out
according to current best standards, recurrent non-implanting of the embryo(s) cannot easily be
explained from the transfer process. Still, rigorous attention for this step may help in minimizing
the rates of non-implantations.
According to a survey performed in 201414 and 2017,15 there may be quite some practice varia-
tion going on in the steps applied in the embryo transfer procedure. When we would define the
common denominator of what we do daily, then we will allow any trained member of the team to
participate in this activity; we tend to clean the upper vagina and the outer surface of the cervix
with sterile water, we either perform a direct transfer with preload or routinely apply a mock trans-
fer to probe the accessibility of the cavity or use an afterload approach after the catheter has been
introduced. Most of us, if not all, will have abdominal ultrasound guidance to ensure the optimal
position of the catheter tip at the moment of push-out, which will be generally in the middle-upper
part of the endometrium echo, at least halfway between fundus and internal os, but not closer than
1.0 cm from the fundus. In cases in which we have trouble accessing the cavity, most physicians
will revert to the use of a malleable stylette catheter and/or a tenaculum to reposition the cervical-
uterine alignment. In such conditions, the embryo is frequently returned to the incubator until the
passage into the cavity has been accomplished. The injection or push-out procedure is typically
executed by mechanical force on the plunger of the attached syringe, mostly by physician, after
which most of us will wait a few seconds, rotate the catheter, and gently pull it back. Then, over 50%
of clinics will have the patient rest supine for 15–30 minutes. With a difficult transfer completed,
many of us will consider dilating the cervix one month prior to a subsequent transfer.
It is beyond doubt that we all have an idea of what the optimal embryo transfer (ET) procedure is.
In fact, it is the one that leads to a healthy live birth born months later. Still, “suboptimal” ET proce-
dures do lead to live births, and the characteristics of the ideal ET procedure may therefore be dif-
ficult to identify. From studies that have inventoried the optimal transfer execution, it has appeared
that up to 50% of real day life transfer procedures may not be classified as optimal. Reasons for this
are various, such as difficult passaging of the internal os, the need to bend and steer the catheter
around a “corner,” the presence of blood at the tip of the catheter after procedure completion, the
need to use a stiff catheter or a tenaculum to steer, and the need to use ultrasound guidance if not
applied routinely.16–18 One could pose the question: what could the effect be if all these supposedly
nonoptimal transfers could be transformed into optimal ones? From studies comparing ongoing
pregnancy rates among these two groups, it has been demonstrated that, while background factors,
such as female age, duration of infertility, and primary versus secondary, and treatment factors,
such as oocyte and embryo number and endometrial thickness, do not differ, the effects on success
are pronounced. From the review by Phillips18 it becomes clear that the reductive effect is ∼25%
(CI [−34%]−[−14%]) for difficult transfers, while the effect of only blood at the catheter after with-
drawal may be as low as 4% (CI [−18%]−[+14%]).
The question then will be how to identify the factors that really help in increasing the rate of
optimal transfers. In the following is presented the scientific evidence on factors or procedures or
approaches that will or may make a difference. On the basis of that, a best (possible) practice can
be identified that helps in getting the best out of the results of the two preceding phases of the ART
process: getting oocytes (and sperm) and creating embryos.
168 Optimizing embryo transfer technique for recurrent implantation failure management
THE PHYSICIAN
Most IVF centers will apply evaluation of certain key performance indicators (KPIs), among
which the success rates of the physician performing the transfer will be present. In many cases
there will be remarkable differences between operators, but a large part of these differences can
be explained by variation in patient (type, duration and cause of infertility, female age) and
treatment (number of oocytes, embryo usage rate) factors. As manual dexterity may vary among
physicians and the smoothness level of an embryo transfer is strongly associated with the pro-
portion of ongoing pregnancies, the question can be how much it matters whether doctor A or B
will do your transfer. Several articles have suggested that the physician indeed is an independent
success factor, provided that the procedure of catheter loading, the type of catheter, and the use
of ultrasound guidance all have been standardized.19,20 However, when correction for patient and
treatment factors was applied, the differences between operators seemed to become reduced, 21–23
or even absent. Especially in the studies by van Weering and Lalwani, patient and treatment
factors seemed to be evenly distributed across physicians, and differences in ongoing pregnancy
rates were not statistically significant. Still, differences in skills and troubleshooting between
operators do exist, and the question is open whether or not these differences are large enough to
affect the overall performance of an ART program. In a high-quality study, comparing the effect
of the type of ET catheter on live birth rates, no overall difference was observed between the two
treatment arms. However, the three experienced physicians that were assigned to perform trans-
fers in this study did perform differently when applying one or the other ET catheter, thereby
indicating that the “physician” factor does exist, albeit coupled to a specific embryo transfer
catheter.24 Final proof for this may come from a study where, in a blinded fashion, patients are
randomized to either undergo the transfer by one of the team members under routine care plan-
ning, or, by a selection of the “best” performing physicians of this team. Such trial design may
imply quite some challenges.
If we cannot exclude the role for the physician next to patient and treatment factors, the real-
istic challenge may be training and frequent supervision. Typically, starting physicians will need
between 25 and 50 supervised transfer procedures to reach levels of performance comparable to
experienced colleagues.25,26 Several studies have indicated that structured training of this proce-
dure will help starting physicians to perform at the same level as experienced physicians within a
short time period.27,28 The implementation of intrauterine inseminations as a training tool has been
shown to reduce the time period necessary to reach an adequate level of performance,28 while only
minimally jeopardizing the interest of the IVF/ICSI patient. Today, simulators for embryo trans-
fer procedures have entered the market and seem to improve the learning curve toward optimal
proficiency in starting physicians in reproductive medicine,29 although direct comparison to more
“classic programs” are currently lacking. As mentioned, even among well-trained and experienced
physicians, real differences in performance may remain present and may urge to install, or at least
research, regular refreshing courses, in the form of peer-to-peer learning.
THE CATHETER
As the smoothness of the transfer procedure is consistently related to the outcome live birth, the
choice of transfer catheter may be an important step in optimization. While soft catheters have the
property of doing the least physical harm, the stiffer catheters have the benefit of steering “poten-
tial” when access to the uterine cavity is difficult. For many years, soft catheters now have the
preference, based on several comparative trials. Still, testing catheters in trials has in fact been a
work in which one catheter is combined with several physicians, under the assumption that the
combination catheter-with-doctor would not affect the results of the comparison. The contribution
of several doctors to the execution of the transfer therefore was not seen as a potential confounder
of the interpretation of results.
Therefore, we tend to embrace the results of large meta-analyses that have appeared over the last
two decades and that have shown an obvious benefit for the soft catheters. The most recent review
The ultrasound 169
Figure 18.1 Meta-analysis of trials demonstrating clinical pregnancy rates comparing soft versus firm
catheters. (From Abou-Setta AM et al. Hum Reprod. 2005;20(11):3114–21, with permission.)
has been done quite some years ago by pooling available comparative studies and demonstrating
that the soft catheter types offered a 30%–40% increase in clinical pregnancy rates compared with
hard catheters.30,31 Therefore, the current image is that soft catheters are the standard of practice
(Figure 18.1). Still, the current preference for the use of abdominal ultrasound guidance of the
transfer procedure may have slightly changed our views. In two studies, the claim was such that
comparing a soft versus firm catheter with accompanied ultrasound (US) guidance did not produce
differences in pregnancy rates.32 Although the reasoning was that navigation could become more
smooth with the stiff catheters using the ultrasound information, the power of the studies was
insufficient to ensure an equipoise between the two approaches.
Comparisons of various soft catheters has never resulted in a systematic review, due to the scar-
city of high-quality comparisons.33 Yet, the large study by Yao revealed that the overall comparison
of two soft catheters, applying the fixed distance technique without US guidance, demonstrated an
equal rate of clinical pregnancy. Interestingly, for two of the three physicians executing all of the
transfers, a significantly higher clinical pregnancy rate was observed using either the one or the
other catheter. This indicates that the combination of physician and transfer catheter can make a
difference in the prospect for the patient. Even if the soft catheters remain the preferred tool, physi-
cians may have a more successful job when more than one type of catheter is available in a center
to choose from and get experienced with.
THE ULTRASOUND
Many of the physicians in reproductive medicine have been trained in performing “blind”
embryo transfer procedures. Roughly two distinct methods existed, the “clinical-touch” and the
“fixed-distance” approaches, in order to deal with positioning of the catheter at the moment of
push-out of the embryo(s). The clinical-touch method implied a sounding procedure until touch-
ing the uterine fundus, with subsequent withdrawal of the catheter by 1 cm and push-out. This
approach was considered as potentially disadvantageous in the hands of some physicians, espe-
cially when using a firm catheter, as the touching could imply harm done to the endometrium lin-
ing, with bleeding and uterine contractions as the result. In contrast, the fixed-distance technique,
with or without prior assessment of uterine sounding depth, and limiting the introduction of the
catheter to ∼6 cm, with subsequent push-out, was proposed as the more atraumatic, and thereby
more successful, way of working, in the hands of any physician.34
The visualization of the process of catheter introduction, as well as the deposition position of
the embryo(s) by using abdominal ultrasound has professionalized the fixed-distance technique.
Ultrasound guiding has thereby become the standard of care, and systematic reviews have pro-
vided a solid basis for this (Figure 18.2). Interestingly, using US guidance in fact implies the use
of two distinct processes: looking at (and possibly guidance of) the entering catheter and defining
the position for push-out but also altering the uterine position by instructing the patient to have a
Study or subgroup UGET CTET Odds ratio Weight Odds ratio
n/N n/N M-H, Fixed, 95% CI M-H, Fixed, 95% CI
1. Live birth
Azmy 2009 181/435 80/418 10.4 % 3.01 (2.21, 4.10)
Drakeley 2008 190/834 187/815 31.8 % 0.99 (0.79, 1.25)
Martins 2004 23/50 17/50 2.0 % 1.65 (0.74, 3.71)
Matorras 2002 56/255 36/260 6.1 % 1.75 (1.11, 2.77)
Subtotal (95% CI) 1574 1543 50.2 % 1.53 (1.29, 1.80)

Total events: 450 (UGET), 320 (CTET)
Heterogeneity: Chi2 = 32.47, df = 3 (p < 0.00001); l2 = 91%
Test for overall effect: Z = 5.03 (p < 0.00001)
2. Ongoing pregnancy
Ammar 2013 9/45 5/45 0.9 % 200 (0.61, 6.52)
Coroleu 2000 85/182 52/180 6.1 % 2.16 ( 1.40, 3.33)
Davar 2007 17/90 11/90 1.9 % 1.67 (0.73, 3.81)
Eskander 2008 68/183 50/190 6.7 % 1.66 (1.07, 2.57)
Garcia-Velasco 2002 100/187 94/187 9.5 % 1.14 (0.76, 1.71)
Kosmas 2007 36/101 38/95 5.5 % 0.83 (0.47, 1.48)
Marconi 2003 22/41 12/42 1.2 % 2.89 (1.17, 7.18)
Tang 2001 94/400 76/400 12.7 % 1.31 (0.93, 1.84)
Weissman 2003 36/160 28/124 5.3 % 1.00 (0.57, 1.74)
Subtotal (95% CI) 1389 1353 49.8 % 1.40 (1.19, 1.66)

Heterogeneity: Chi2 = 13.05, df = 8 (p = 0.11); l2 = 39%
Test for overall effect: Z = 3.95 (p = 0.000079)
Total (95% CI) 2963 2896 100.0 % 1.47 (1.30, 1.65)
Heterogeneity: Chi2 = 45.91, df = 12 (p < 0.00001); l2 = 74%
Test for overall effect: Z = 6.36 (p < 0.00001)
Test for subgroup differences: Chi2 = 0.48, df = 1 (p = 0.49), l2 = 0.0%
0.5 0.7 1 1.5 2

Favors CTET Favors UGET
Figure 18.2 Meta-analysis of trials showing live birth and ongoing pregnancy rates per woman ran-
domized, comparing ultrasound-guided (UGET) versus clinical-touch (CTET) embryo transfer. (From Brown J
et al. Cochrane Database Syst Rev. 2016;2016(3), with permission.)
The loading 171
full urine bladder at the time of embryo transfer. The latter may affect the ease of introduction by
straightening of the uterine cavity. The relative contribution of the two processes has become clear
from a systematic review in which US-guided transfer was compared in cases with bladder-full
against bladder-empty instructions. With a relatively small sample size, the ongoing pregnancy
rate was significantly better in the group with filled bladder,35 but the quality of the two studies
included was considered quite low. The reviews on US-guided embryo transfer (UGET) compared
with any form of blind (clinical) touch have demonstrated a considerable and consistent positive
effect on ongoing pregnancy rate from using the abdominal ultrasound.36,37 The quality of this evi-
dence was considered moderate to low by the authors, but when looking at only those trials that had
low risk of bias, the evidence of an overall benefit for UGET remained. The size of the most likely
effect was a ∼20% increase in live birth rate (OR 1.17, 95% CI 1.01–1.36), while in the ultrasound
guided transfers, only 8% of transfers were reported to have been difficult, compared with 12% in
the clinical-touch group (risk ratio of non-easy transfer 0.69 [0.57, 0.82]).
As the use of abdominal ultrasound guiding necessitates the availability of a well-trained sonog-
rapher, efforts have been put on applying the transvaginal approach in steering both the catheter as
well as the guiding ultrasound by the physician (VGET). The work-up for this varies across studies,
but mostly, after positioning the outer sheath of an empty transfer catheter just passed the internal
os, the speculum is removed, transvaginal probe with sterile cover put in position, and the loaded
inner catheter sheath inserted by the embryologist. The catheter tip is then positioned by the phy-
sician for placement of the embryo at the center of the endometrial cavity. 38–40 Studies that com-
pared the abdominal to the transvaginal ultrasound-guiding approaches have not demonstrated
any benefit for either of the approaches, indicating that the choice will depend on local availability
of sonographer staff and the preference of the physician. Moreover, patients may appreciate the
absence of the need to have a full bladder.
THE LOADING
The transfer of the embryo(s) into the uterine cavity basically takes place by flushing and filling
off the transfer catheter with culture medium, by using a prefilled syringe, and subsequently posi-
tioning of the embryo(s) in the tip of the catheter in a drop of culture medium, by means of nega-
tive pressure created by gentle withdrawal of the syringe plunger. Push-out of the fluid column
containing the embryo(s) will then be established by forward pressure on the plunger. The culture
medium volume containing the embryo(s) will often be separated from the initial culture medium
filling of the catheter and from the very tip by a small column of air, in order to prevent the fluid
column from being lost during the introduction process (air trap). Also, a large volume may result
in embryo expulsion out of the uterine cavity per se, while low volumes may result in implantation
failure due to suboptimal ambient conditions. In most practices, the medium volume is typically
within the range of 15–30 µL. Variation in this “replacing volume” may be quite considerable, prob-
ably depending on the volume and diameter specifications of the various transfer catheters used.
Studies that compare different loading techniques are limited in number and mostly focus on
the loading volume and the air-trap system. In one study, a low (20–25 µL) was compared to a
high replacement volume (40–45 µL), using the three-drop filling method.41,42 The randomized
control trial (RCT), comprising 216 couples, demonstrated a nonsignificant difference in ongo-
ing pregnancy rates in favor of larger volumes (40.0% vs. 33.3%; p = 0.31). This means that larger
studies need to be executed in order to really exclude an important role for this aspect of catheter
loading. The use of the air-trap method versus using fluid only has only been researched in two
small studies with the overall outcome in a formal meta-analysis that one method is not superior
over the other.43
In a large survey (265 centers across the globe) on techniques of catheter loading, it was noted
that the majority of units wash the catheter prior to loading the embryos. Moreover, a slight
preference was reported for the loading by the medium-air-(medium + embryo)-air-medium
approach (42%), although the methods of medium-(medium + embryo) in catheter tip (20%) or
medium-air-(medium + embryo) (15%) are also frequently used.44 As such, the role of the catheter
loading may seem to be a relatively unknown factor in the chances of obtaining a live birth from
the ART cycle. Comparative studies for different techniques are easy to execute and may unveil
possible gaps in our knowledge on these matters.
THE DEPOSITION
Once positioned in the cavity, the forward pressure applied to the syringe plunger will push out the
fluid column with associated air column(s) from the ET catheter, leaving air with fluid combined,
positioned at some distance ahead of the tip of the inner catheter. This air-fluid combination (bub-
ble) is well visible at ultrasonography and remains so for quite some time. It is believed to represent
the position of the embryo(s) in the cavity. Over the past 15 years, studies have addressed the ques-
tion in which part of the cavity the embryo’s may be best left “behind.”
In an initial systematic review covering over 2000 ET procedures,45 it was shown that pregnancy
rates may be similar when the upper and lower halves of the endometrial cavity are compared, and
that push-out with the catheter tip at ∼20 mm from the fundal end of the endometrium may be
superior to the traditional transfer at ∼10 mm. In the period thereafter, however, much ground was
made for the belief that the bubble position after the deposition could best be within a distance of
10 mm from the internal fundus in order to obtain optimal prospects for implantation.42,46–48 This
would imply that the tip of the catheter at push-out may be best placed at a distance of 15–20 mm
from the fundus,47,49 as in most cases the embryo bubble will attain a position ahead of the catheter
tip (Figure 18.3). However, the control on the direct position of the embryo bubble after deposi-
tion may vary depending on push-out speed.46 In an attempt to control this process, the use of an
automated push-out device was tested as to its effects on pregnancy rates. Although the position
of the embryo bubble directly after the push-out could be better controlled, the effects on ongoing
pregnancy rates were unfortunately not convincing.50
More recent studies have noted that the position of the tip of the catheter may not automati-
cally determine the position of the bubble. The action of introducing the catheter will first elicit
an increase in uterine peristaltic wave frequency, with subsequent effects on fluid movement.
Moreover, the intensity of uterine peristalsis was quantitatively correlated with the distance of fluid
movement in the time period of 30 minutes after the embryo transfer.51 The belief that the position
Figure 18.3 (a) The length of uterine cavity, (b) the distance between the end of the fundal
ndometrial surface and the tip of inner catheter, and (c) the distance between fundal endometrial sur-
e
face and air bubbles. (From Cenksoy PO et al. Eur J Obstet Gynecol Reprod Biol. 2014;172(1):46–50, with
permission.)
The mock transfer 173
of the bubble at the moment of push-out is related to the prospects for pregnancy may therefore
be a misconception, as a recent study has revealed that the position of the bubble some 60 minutes
after the transfer may be best related to the outcome pregnancy.52 So, pregnancy rates can be influ-
enced by the change of the bubble position after the ET completion, although the embryo had been
positioned in the “optimal” part of the uterine cavity. Currently, the data on the effect of changes in
the embryo bubble position after ET on pregnancy rates are too scarce to draw firm conclusions.53
Together, the evidence seems to support depositing the embryo(s) as gentle as possible with soft
pressure on the plunger with the tip at a distance of 15–20 mm from the fundus, in order to bring
the embryo(s) close to the fundus. However, the final position of the air bubble may be outside
the control of the physician performing the transfer, as subsequent uterine peristalsis may further
affect this position.
THE MOCK TRANSFER

As the use of ultrasound guidance has gained enormous ground in reproductive medicine today,
the use of a trial or mock transfer may seem to have become an echo of the past. The philoso-
phy behind the trial transfer is that by scouting the ease of entrance into the uterus will improve
both the easiness of the real embryo transfer, as well as the prognosis for a pregnancy occurring.
However, studies have noted that information obtained from the execution of the mock transfer
may not reliably foresee conditions at the real transfer execution. This may be true for the position
of the uterus,54,55 the uterine cavity depth,56 but the predictability of a difficult real transfer from
the information of the trial transfer seems to be quite high (Figure 18.4).56,57 Difficulties with execu-
tion of the transfer may stem from various sources. In the comprehensive study by Larue, it was
demonstrated that the five most common anatomical causes were endocervical crypts, tortuosity
of the cervical canal, internal os contractions, pronounced ante- or retro-version of the uterus, and
a heterogeneous group of anatomical factors (such as a cesarean section-induced niche, cervical
synechiae, and endocervical polyps).
As such potential difficulties can be predicted prior to real transfer, adjustments of the ET pro-
cedure can be applied by, for instance, the choice of the catheter, endovaginal ultrasound guiding.
or straightening of the uterus. Scientific evidence that prior knowledge of a difficult transfer will
improve pregnancy rates has not been delivered, however. In a study by Mansour, 335 couples with
an indication for IVF were randomized into either group A (n = 167), in which a trial transfer was
Easy Difficult Very difficult

100
90 ****
80
% of patients
70
****
60
50
40
30
20 **** *
10 *
**
0
EET DIT diffET vdiffET
Population group
Figure 18.4 Difficulty of mock transfer among the easy embryo transfer (EET) and difficult transfer (DIT)
groups and the difficult ET (diffET) and very difficult ET (vdiffET) subgroups. Comparisons between EET
and DIT groups and comparisons between diffET and vdiffET groups are depicted (*p < 0.05; **p < 0.01;
****p < 0.0001). (From Larue L et al. J Gynecol Obstet Hum Reprod. 2017;46(1):77–86, with permission.)
carried out to base the choice of the catheter upon, or into group B (n = 168), in which patients
started IVF treatment without a prior trial ET. The embryo transfer procedure was only difficult
in 50 cases (29.8%) in group B, while pregnancy rates (22.8%) in the trial transfer group A were
significantly higher compared with group B (13.1%).58 It may be questioned whether these findings
are valid, since this study is the only fully published report on this matter, and the extended use of
ultrasound guidance may have made prior information on the ease of transfer redundant.
THE BEDREST
What is next if the embryo(s) have been deposited gently in the uterine cavity? Sit up, get dressed, and
go home? Or remain as horizontal as possible, do not move, get transported by stretcher to the relax
room, and return home not before at least one hour later? It is important to wonder why bedrest after
the ET would affect the chances of implanting. The assumption here is that reduced physical activ-
ity and the supine position will decrease the risk of expulsion of the embryo(s). Expulsion could be
caused by uterine contractions, or by the mere effects of gravity. The latter seems not realistic. The
weight of the bubble containing the embryo may be far too low to overcome the resistance that will be
caused by the abutting endometrial layers. Moreover, an anteflexed uterus will be positioned almost
in a horizontal position when standing, making the effects of gravity quite irrelevant. The former,
uterine contractions that may push the fluid-air bubble with embryo around, may be a hazard, but
the question will be whether bedrest will reduce such contractility. Indeed, the fluid-air bubbles fre-
quently move after the embryo transfer, even when remaining horizontal, and this may be caused by
contractions. When fluid-air bubble position was recorded immediately, 30 minutes later and when
the patient stood up, after a transfer with the tip at a distance of 10–20 mm from the fundus, a rather
random movement of the bubble within the endometrial area was observed.59 In fact, no signs have
been found that standing upright after ET would remove the bubble from this area.60–62
The final decision on the role for bedrest comes from only a few studies that together reveal that
there is insufficient evidence to support any specific length of time for women to remain recum-
bent, if at all, following embryo transfer.63,64 Prolonged bedrest may even reduce the implantation
rate.65
ANYTHING ELSE?
Among interventions at or around the time of embryo transfer that have aimed at improving the
chance of a live birth, there are many, in addition to those discussed in the previous sections. Below,
a brief description will be given on a series of possible adjuvants that have been known in the litera-
ture, but that may have not gained much ground or may need more thorough research.
As the mock transfer has been considered a scouting maneuver too remote from the actual ET,
the idea of afterloading was proposed, in which an empty catheter is placed just past the internal
os. After this positioning, the inner catheter is replaced by a second inner sheath containing the
embryo. This allows the benefit of a “mock” transfer, with only minimal manipulation that could
lead to traumatizing the embryo, in case of a difficult introduction.66 With such an approach, the
implantation rate per embryo was not significantly different (24.7% vs. 20.5%), but in the afterload
group, the clinical pregnancy rate was higher (52.4% vs. 34.9%; p = 0.06). Interestingly, after this
primary publication, no additional trials were published.
Contamination of the cavity with endocervical microbial flora is believed to affect the pregnancy
rates significantly. Prophylactic antibiotics administered at the time of oocyte retrieval resulted in a
reduction of positive microbiology cultures of embryo catheter tips, with a suggested improvement
in pregnancy rates.67 However, from a review on studies using amoxicillin/clavulanic acid prior to
embryo transfer, no alterations of clinical pregnancy rates were noted.68
Cervical mucus removal before transferring has been recommended to improve rates of live
birth, by preventing a possible blocked passage of the embryo from the catheter tip and by elimi-
nating the transfer of microorganisms into the cavity. However, the actual introduction of the
catheter may become more difficult.69,70 In a review, comprising over 1700 cases in eight RCTs,
References 175
no statistically significant differences in terms of pregnancy, implantation, or live birth rates

were noted.70
Uterine contractility at the time of embryo transfer can be an important success factor, as it may
affect the position of the embryo in the hours after deposition. It is therefore that uterine relaxants
have been tested as possible success modifiers. Nonsteroidal antiinflammatory drugs (NSAIDs)
will reduce the prostaglandin activity and thereby uterine contractility, with possible beneficial
effects on pregnancy rates.71 Atosiban, as a receptor antagonist for vasopressin V1a and oxytocin,
acts on the uterus to suppress uterine contractions. It may be an appropriate tool for women with
RIF, as from two small studies on a total of 290 cases, an improving effect was noted on both
implantation and live birth rates.72
THE BEST PRACTICE

Today, the execution of the embryo transfer is very likely to obtain highly focused attention of the
embryologist and the physician. Among the procedures to be considered as part of the current
golden standard are the use of a soft embryo transfer catheter, abdominal ultrasound-based guid-
ing, and deposition with the purpose to put the embryo bubble within a distance of 10–15 mm from
the fundal ending of the endometrium. Much more to the individual preference of the transferor,
one could apply a mock transfer procedure, afterloading (being a mock and real transfer in one) or
perform a transvaginal “scout” ultrasound.
Applying such approaches and performing the smoothest transfer ever may still not lead to the
desired outcome of a live birth. There, factors of other origin than the transfer execution will play
the major role, albeit that somewhere in the knowledge of the embryo transfer process we may still
be “missing a point.” The only best step in such cases of implantation failure is repeating the ART
procedures with the “knowledge” that the embryo transfer is not likely to be the limiting factor.
Obviously, we could think of many of the approaches that have been discussed in the aforemen-
tioned, for which solid evidence is unfortunately lacking. Specifically, measures like the use of a
short period of bedrest, or the application of uterine relaxants or antibiotics may come into the doc-
tor’s mind. It seems, however, best to adhere to applying measures that have been well researched
and have been confidently shown efficacious.
In contrast, in RIF couples in which a transfer procedure has been shown difficult, it is with-
out doubt a factor that needs correction; choosing the best replacer in the department, applying
a prior trial or mock transfer in order to unravel the smooth way into the uterus, or simply using
the afterload approach are all options to be applied. In fact, creating the transition from a previous
difficult into a current smooth transfer is very likely to positively affect the remaining prospects for
the couple.
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When should patients
abandon treatment?
19
KONSTANTINOS DAFOPOULOS
INTRODUCTION
Recurrent implantation failure (RIF), although not universally accepted, is usually defined as the
failure to achieve a clinical pregnancy after three consecutive in vitro fertilization (IVF) attempts
and transferring one to two high-quality embryos in each cycle.1 RIF is a distressing situation,
which is rather difficult for women to manage, since they have higher levels of stress than fertile
women and are constantly uncertain whether or not they will remain childless despite further IVF
cycles.2 Furthermore, in the absence of public funding of IVF, the couples have to make significant
financial sacrifices to continue their treatment. It has been shown that IVF failure is one of the
major reasons for discontinuation of treatment, and even when its cost is covered by insurance,
dropout rates are as high as 17%–65%.3–7 In such a difficult psychological environment, the couples
need counseling whether they should proceed with further IVF attempts or undergo alternative
treatment options such as gamete donation or surrogacy.
The optimal tool to counsel such patients would be the epidemiological analysis based on large
databases. As yet, data on cumulative clinical pregnancy and live-birth rates following various
treatments in RIF patients are absent.
FEMALE AGE
Although it has been proposed that the diagnosis of RIF is not appropriate for women with poor
prognosis based on age above 40 years,8,9 a large proportion of patients seeking fertility treatment
are of advanced age. In 2013 in Europe, 17.6% of all oocyte aspirations for IVF, 19.2% of all oocyte
aspirations for intracytoplasmic sperm injection (ICSI), and 14.7% of all thaws were performed in
women above 40 years of age. The delivery rates were 10.1% and 7.6% for IVF and ICSI fresh cycles,
respectively, and 10.6% for frozen cycles.10
Counseling this age group should be based not only on the prognosis of further IVF cycles, but
also on the fact that the delay in pregnancy is followed by adverse pregnancy outcomes related to
advanced maternal age. Many epidemiological studies have clearly shown that advanced mater-
nal age, above 35 years, is associated with intrauterine growth restriction, preeclampsia, placen-
tal abruption, preterm birth, gestational diabetes, admission to the intensive care unit, stillbirth,
and cesarean section.11–15 The incidences of most of these adverse pregnancy outcomes have been
found to be similar regardless of whether donated or their own oocytes have been used for IVF
treatment.15
A prospective study in the UK included 156,947 women who received 257,398 IVF ovarian stim-
ulation cycles and calculated the live-birth rates within the first and subsequent cycles and finally
the cumulative live-birth rates using the Kaplan–Meier method.16 The authors performed various
estimates of the cumulative live-birth rates based on assumptions about the cumulative live-birth
rate in women who discontinue IVF if they had continued with further IVF cycles (Figure 19.1).
These included the optimal estimate (assumes that the cumulative live-birth rate in these women
would have been equal to the rate in women who continued to have further IVF cycles), the previ-
ous oocyte yield-adjusted estimate (assumes that in these women, the cumulative live-birth rate
180
Female age 181
Optimal estimate
Age-adjusted estimate
Prognostic-adjusted
80 estimate
Conservative estimate
Cumulative live-birth (%)
60
40
20
1 2 3 4 5 6 7 8 9
Number of cycles
Figure 19.1 Cumulative live-birth rates across all cycles using different estimates.
would have been equal to the rate in women who had the same oocyte yield in the immediately pre-
vious IVF cycle and who continued to have further IVF cycles), the prognostic-adjusted estimate
(assumes that 30% of women who discontinued IVF did so because of poor prognosis and would
have had a live-birth rate of zero if they had continued further IVF cycles), and the conservative
estimate (assumes that in these women the cumulative live-birth rate would have been zero if they
had continued with further IVF cycles). Among all these estimates, the prognostic-adjusted esti-
mate provides the most realistic value. In the age group below 40 years, 133,379 women; in the age
group of 40–42 years, 15,561 women; and in the age group above 42 years, 4420 women, started
treatment cycles. However, in the last group, a low number of cases were recorded to undergo more
than five cycles.
This study showed that, for women aged 40–42, in the sixth cycle the cumulative prognosis-
adjusted live-birth rate was 31.5% (95% CI 29.7–33.3), with no further increase following more IVF
attempts. The live-birth rates were 12.3%, 10%, 8.6%, 7.8%, 5.3%, and 6.9% for cycles one through
six, respectively.
For women older than 42 years, the live-birth rates were 3.7%, 3.3%, 3.3%, 1.2%, and 4.5% for
cycles one through five, respectively. At the fifth cycle, the cumulative prognosis-adjusted live-birth
rate was 10.7% (95% CI 8.2–13.2), while the low number of cases undergoing more than five cycles
could not provide reliable estimates.
For women below 40 years, the live-birth rates were 32.3%, 27.1%, 24.3%, 21.4%, 19%, 17%, 17.3%,
19.1%, and 19.6%, for cycles one through nine, respectively. At the seventh, eighth, and ninth cycle,
the cumulative prognosis-adjusted live-birth rates were 69.8% (95% CI 69.1–70.4), 70.9% (95% CI
70.1–71.6), and 71.6% (95% CI 70.8–72.5), respectively. The estimated natural fecundity rate of the
general population is about 20% per month, and the cumulative rates of conceiving naturally are
45%, 65%, and 85% after 3, 6, and 12 months.17 It is obvious that in younger women undergoing
IVF treatment, their chances for live birth are similar to the natural conception probability with
high-rank IVF cycles, suggesting that in such women it is worth extending the IVF attempts even
up to nine. However, these data originate from a general IVF/ICSI population with repeated IVF
failures, which may not be identical to the RIF population. The “repeated IVF failure” population
182 When should patients abandon treatment?
may include cases with suboptimal embryo quality, poor response to ovarian stimulation, repeated
fertilization failure, and repeated difficult transfers.9 Although the real prevalence of RIF is not
clear, it has been suggested that the frequency of this condition in the general IVF population is at
least below 20%.17,18
ATTEMPTS FOLLOWING THE EVALUATION AND TREATMENT OF RIF

Assumed etiologies for RIF include decreased endometrial receptivity, embryonic defects, and
factors with combined effect.19 With appropriate investigations, the underlying cause for the
repeated failures may be identified and the suggested treatment should be targeted to the abnor-
mality detected. Therefore, in clinical practice, once the diagnosis of RIF is established, usually
meaning that three unsuccessful cycles have already been performed, and the diagnostic evalu-
ation has identified the abnormality or malfunction that contributes to implantation failure, the
couple should be treated properly before the initiation of the next cycle. It has to be stressed that
some of the conditions related to RIF usually are managed before starting the first IVF cycle
rather than expecting two or three failed attempts. These include smoking cessation, weight
loss in obese patients, hydrosalpinx removal, and hysteroscopic removal of endometrial polyps,
submucosal fibroids, and uterine septum. However, some couples may be reluctant to have these
conditions managed before their first IVF cycle and may choose instead to directly undergo IVF
treatment.
Today there are many studies investigating the various etiologies of RIF and that have shown
that following treatment there are significant improvements in implantation in the subsequent IVF
cycle.1,9 However, the levels of evidence for many of these treatments range from high to empirical.
A significant problem, regarding management and counseling couples with RIF, is the absence of
data on cumulative clinical pregnancy and live-birth rates following various treatments. Moreover,
an unanswered question is the cumulative success rate of alternative or combined treatments for
a specific implantation abnormality in subsequent cycles since there are no relevant studies in the
literature.
Another important issue that has to be considered is that RIF may be a condition ranging
between a lower than normal chance of implantation and a definite failure of implantation. Poor
endometrial preparation or even excessive estradiol levels during superovulation may have detri-
mental effects on implantation, which however may be corrected in subsequent cycles.
Therefore, a rational approach in treating RIF couples may be based on the anticipation that after
correction of the underlying cause of RIF, the couples will enter a new treatment strategy assum-
ing that they have probabilities of pregnancy more or less equal to couples undergoing the first
treatment cycle, with similar infertility characteristics but no RIF. Since the prevalence of RIF in
the general infertility population is estimated to be below 20%, it is not expected to exert a rather
strong effect on the estimation of cumulative pregnancy rates in large databases. Based on these
assumptions, the information from appropriate large studies may be used to counsel RIF couples
specifically on the appropriateness of proceeding with further IVF attempts.
The study by Smith et al.16 is a large prospective study that provides important information for
counseling and decision-making. Based on these data, it seems rationale to suggest that women
with three failed cycles below 40 years, after having their underlying condition treated, should
extend the number of subsequent cycles up to nine. Women aged 40–42 years with RIF, should be
counseled that up to six more cycles may be of benefit, but this strategy should be accomplished in
a short period, since the delay is followed by aging-related detrimental effects on both pregnancy
rates and, in the case of success, on pregnancy outcome. For women older than 42 years with RIF,
the live-birth rates per cycle are very low, below 5%, up to fifth cycle, with severe aging-related
adverse effects on both pregnancy rates and on pregnancy outcome. Older women with RIF attrib-
uted to aging oocytes, should be counseled that they should not delay to enter an oocyte dona-
tion program, since even with donated oocytes, they are at high risk for severe adverse pregnancy
outcomes.15
Attempts following the evaluation and treatment of RIF 183
Another approach for counseling the RIF couples may derive from a recent mathematical chal-
lenge of the RIF definition.20 The authors approach was based on the fact that IVF treatment has
a relatively low success rate, nearly 30% in women without RIF.21 They calculated the expected
pregnancy rates per cycle and the cumulative chances of pregnancy after various numbers of
cycles, postulating that RIF has a prevalence between 5% and 25%. It was found that for women
not affected by RIF and for a constant pregnancy rate of 30% per cycle, the chances of pregnancy
decrease gradually with increasing number of treatment cycles. In this population, the pregnancy
rates are 27%, 26%, 25%, 23%, 20%, and 18% at the first, second, third, fourth, fifth, and sixth
cycles, respectively. Furthermore, the cumulative pregnancy rates increase slowly with increasing
number of cycles, being 27%, 46%, 59%, 68%, 75%, and 79% after the first through sixth cycles,
respectively. It was calculated that in this population with no RIF cases included, the false-positive
rate of an RIF diagnosis was 86%, 81%, 75%, 68%, 60%, and 51%, respectively. However, these fig-
ures are dependent on success rate of IVF and on the frequency of RIF in the studying population.
Therefore, when the rate of IVF success is as high as 50%, the rate of false-positive diagnosis of RIF
decreases from 88% to 69% after two cycles, from 87% to 53% after three cycles, and from 83% to
12% after six cycles. Similarly, when the rate of RIF in the population increases up to 25%, the rate
of false-positive diagnosis of RIF decreases from 90% to 60% after two cycles, from 87% to 51% after
three cycles, and from 69% to 26% after six cycles. This mathematical analysis showed that the rate
of false-positive diagnosis of RIF, defined as three failed IVF cycles, is above 50% in all sensitivity
analyses performed, suggesting that RIF diagnosis seems more reliable after six failed cycles. The
authors proposed a possible definition of RIF based on the prognosis of the patient, and in very
good prognosis patients (50% expected rate of success), RIF could be diagnosed after three failed
IVF cycles, or after six failed cycles in a normal IVF population with a 30% expected rate of IVF
success.
In another study,22 the embryo-uterus model was used, which included intercycle correlations
for the embryo and uterine submodels, and estimated these effects in a large, multicenter UK data-
base. This database of 12,480 embryo transfer cycles from 8768 IVF patients had sufficient informa-
tion to allow reliable estimation of these effects.23 Empirical Bayes estimates were used to predict
the prognosis of continuing IVF attempts based on previous cycle failures. The results suggested
that in excess of 10 treatment failures are required to reduce the chances of subsequent success by
half of the initial cycle. Even more cycles are required in single embryo transfer (SET) cycles. In
support of our suggested approach in counseling RIF couples, this study22 implies that a treatment
failure should be considered as statistical misfortune, and the policy of offering patients only a
small number of IVF attempts has weak clinical justification.
The above suggestions are theoretical or based on mathematical models. There are few studies
investigating the cumulative success rate following treatment of specific conditions related to RIF.
In one study, 1174 RIF couples were treated with supportive lymphocyte immunotherapy (LIT)
in Germany.24 After LIT treatment, for a time period of two years, the RIF women had a mean of
1.5 (1–8) oocyte retrievals and a mean of 1.7 (1–10) embryo transfers, resulting in a 28.4% delivery
rate. Some patients had a high number of attempts, with the highest overall amount of oocyte
retrievals leading to birth was 14, and the highest number of transfers was 17. The results showed
that within two years, the birth rate per fresh embryo transfer and per couple showed a tendency,
although not statistically significant, to decline after the third to the seventh IVF cycle. When
frozen embryo transfers were included, the delivery rate per oocyte retrieval was about 20%. The
average birth rate per fresh embryo transfer was 17.3%, per frozen transfer 8.2%, and 18.6% per
oocyte retrieval, cumulating fresh and frozen transfers. The delivery rates were dependent on
female age, and the two-year cumulative delivery rates were, for women below 30 years 39%, from
30 to 39 years 25%–30%, and above 39 years 17%.
In another prospective study in 421 RIF patients,25 it was found that according to the histo-
pathologic/immunohistochemical examinations, 33.7% had chronic endometritis (CE). With the
first-line doxycycline treatment, the histopathologic cure rate was 92.3%, and after the second-line
184 When should patients abandon treatment?
metronidazole/ciprofloxacin treatment, the overall cure rate was 99.1%. In this study, the live-birth
rate in the first ET cycle and the cumulative live-birth rate after three ET cycles following antibiotic
treatment in the cured RIF group were compared with RIF patients that did not have evidence of
CE. For the cured RIF group, the live-birth rate in the first cycle and the cumulative rate were 32.8%
and 38.8%, respectively, while for the RIF patients with no CE, the respective values were 22.1%
and 27.9%, which both were significantly lower. However, the majority of patients in this study
had cryopreserved ET cycles. It is important to underline that in the two groups of RIF patients,
the live-birth rates at the sixth cycle were 32.8% and 22.1%, while in the study of Smith et al.,16 for
women below 40 years of a general infertility population, the live-birth rate at their sixth cycle was
only 17%. Therefore, it seems rational to attempt more cycles in RIF patients similarly to the general
infertile population. Although, there are no relevant data in the literature, such an approach may
be applied in unexplained RIF cases.
The RIF couples, after unsuccessfully completing the appropriate number of cycles, according to
the above approach, should be informed that alternative treatments may be offered as final options
for childbearing. These options include gamete donation and surrogacy. Based on the history of
the repeated reproductive failures and laboratory investigations, if sperm or oocytes are consid-
ered compromised, with no improvement after relevant treatments, then sperm or oocyte donation
should be advised. If, however, there is evidence of severely impaired endometrial receptivity, such
as Asherman syndrome, that has not improved despite pharmacological or surgical treatment, then
surrogacy should be the realistic option for reproductive success.
REFERENCES
1. Laufer N, Simon A. Recurrent implantation failure: Current update and clinical approach to
an ongoing challenge. Fertil Steril. 2012;97:1019–20.
with and without reproductive failure. Int J Gynaecol Obstet. 2014;124:143–7.
3. Land JA, Courtar DA, Evers JLH. Patient dropout in an assisted reproductive technology
program: Implications for pregnancy rates. Fertil Steril. 1997;68:278–81.
4. Olivius K, Friden B, Lundin K, Bergh C. Cumulative probability of live birth after three in
vitro fertilization/intracytoplasmic sperm injection cycles. Fertil Steril. 2002;77:505–10.
5. Sharma V, Allgar V, Rajkhowa M. Factors influencing the cumulative conception rate and
discontinuation of in vitro fertilization treatment for infertility. Fertil Steril. 2002;78:40–6.
6. Olivius C, Friden B, Borg G, Bergh C. Why do couples discontinue in vitro fertilization treat-
ment? A cohort study. Fertil Steril. 2004;81:258–61.
7. Donckers J, Evers JLH, Land JA. The long-term outcome of 946 consecutive couples visiting a
fertility clinic in 2001–2003. Fertil Steril. 2011;96:160–4.
8. Van Voorhis BJ. Clinical practice. In vitro fertilization. N Engl J Med. 2007;356:379–86.
agement. Reprod Biomed Online. 2014;28:14–38.
10. Calhaz-Jorge C, De Geyter C, Kupka MS et al. Assisted reproductive technology in Europe,
2013: Results generated from European registers by ESHRE. Hum Reprod. 2017;32:1957–73.
11. Odibo AO, Nelson D, Stamilio DM et al. Advanced maternal age is an independent risk factor
for intrauterine growth restriction. Am J Perinatol. 2006;23:325–8.
12. Bayrampour H, Heaman M. Advanced maternal age and the risk of cesarean birth: A system-
atic review. Birth. 2010;37:219–26.
13. Lamminpaa R, Vehvilainen-Julkunen K, Gissler M, Heinonen S. Preeclampsia complicated
by advanced maternal age: A registry-based study on primiparous women in Finland 1997–
2008. BMC Pregnancy Childbirth. 2012;12:47.
14. Lean SC, Derricott H, Jones RL, Heazell AEP. Advanced maternal age and adverse pregnancy
outcomes: A systematic review and meta-analysis. PLOS ONE. 2017;12:e0186287.
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15. Storgaard M, Loft A, Bergh C et al. Obstetric and neonatal complications in pregnancies con-
ceived after oocyte donation: A systematic review and meta-analysis. BJOG. 2017;124:561–72.
16. Smith ADAC, Tilling K, Nelson SM, Lawlor DA. Live-birth rate associated with repeat in vitro
fertilization treatment cycles. JAMA. 2015;314:2654–62.
17. Luke B, Brown MB, Wantman E et al. Cumulative birth rates with linked assisted reproduc-
tive technology cycles. N Engl J Med. 2012;366:2483–91.
18. McLernon DJ, Steyerberg EW, Te Velde ER et al. Predicting the chances of a live birth after
one or more complete cycles of in vitro fertilisation: Population based study of linked cycle
data from 113,873 women. BMJ. 2016;355:i5735.
implantation failure following IVF-ET. Hum Reprod. 2006;21:3036–43.
20. Somigliana E, Vigano P, Busnelli A et al. Repeated implantation failure at the crossroad between
statistics, clinics and over-diagnosis. Reprod Biomed Online. 2017;S1472-6483:30567–9.
21. Calhaz-Jorge C, de Geyter C, Kupka MS et al. Assisted reproductive technology in Europe,
2012: Results generated from European registers by ESHRE. Hum Reprod. 2016;31:1638–52.
22. Roberts SA, Stylianou C. The non-independence of treatment outcomes from repeat IVF
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23. Roberts SA, Hirst WM, Brison DR, Vail A. Embryo and uterine influences on IVF outcomes:
An analysis of a UK multi-centre cohort. Hum Reprod. 2010;25:2792–802.
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Prognostic factors and additional interventions. Arch Gynecol Obstet. 2008;278:135–42.
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for chronic endometritis in infertile women with repeated implantation failure. Am J Reprod
Immunol. 2017;78.
Proposed management
of patients with recurrent
20
implantation failure and
directions for future research
CHRISTOS A. VENETIS and EFSTRATIOS M. KOLIBIANAKIS
INTRODUCTION
The purpose of any management algorithm is to guide clinicians in the most appropriate and effica-
cious evidence-based management of patients with a certain condition. Such an algorithm requires
a clearly defined population and a good understanding of the potential etiologies of the condition
as well as their relative prevalence. It is also imperative that good-quality evidence exists to support
the selection of the most effective treatment depending on the underlying cause.
Unfortunately, as outlined in the preceding chapters, recurrent implantation failure (RIF)
remains a loosely defined clinical entity1,2 and high-quality evidence regarding the therapeutic
management of RIF is not currently available. For this reason, in this chapter we will identify the
research priorities around RIF and propose a management algorithm based on the best available
evidence, acknowledging that this evidence is frequently of very low quality.
IS THE PATIENT SUFFERING FROM RECURRENT IMPLANTATION FAILURE?

As clearly outlined in Chapter 1, there is currently significant heterogeneity in the definition of
RIF by different clinicians and researchers.1,2 This creates uncertainty in what RIF truly is, but also
renders the interpretation of the relevant research problematic. Furthermore, during the last l0
years, there have been significant changes in the laboratory practices and the way in vitro fertiliza-
tion (IVF) is practiced, such as implementation of blastocyst culture,3 adoption of single embryo
transfer,4 and replacement of slow freezing by vitrification.5,6 These changes have transformed the
landscape of modern assisted reproduction technology management, greatly improving the success
rate per embryo transfer while reducing the adverse obstetric outcomes associated with multiple
pregnancies.7
The European Society of Human Reproduction and Embryology (ESHRE) Preimplantation
Genetic Diagnosis (PGD) Consortium has suggested that RIF can be diagnosed after the unsuc-
cessful transfer of >3 high-quality embryos or ≥10 embryos in multiple embryo transfers.
However, since a failed implantation is one of the two expected outcomes after an embryo trans-
fer, the other being achievement of pregnancy, one should be cautious when defining RIF in order
to avoid overdiagnosis. Accounting for the probability that RIF might be attributed to pure chance,
could allow for a more objective diagnosis, limiting unnecessary and expensive diagnostic testing
while also avoiding the iatrogenic stress accompanying the attachment of the RIF label to a patient.
If we accept that at least three embryo transfers are required to diagnose RIF and that 15% or more
is a reasonable probability of failure due to chance, then we could estimate the cumulative number
of embryos transferred required to diagnose RIF. For example, assuming that the probability of
implantation of a euploid day five embryo is around 50%, the probability that three consecutive,
single-embryo transfers of day five euploid embryos would fail due to chance is (1–0.5)3 = 0.125
186
Pretreatment phase 187
(i.e., 12.5%). Hence, patients with three or more failed transfers of a single day five euploid embryo
could be diagnosed as RIF patients. On the other hand, if a good-quality day five embryo has 50%
probability of being euploid for a woman who is 35 years old,8 then seven such embryos need to fail
to implant for that probability to be <15% due to pure chance. Hence, these patients would need to
have at least seven embryos transferred over at least three embryo transfers for RIF to be diagnosed.
The selection of 15% as a cutoff for the RIF definition is arbitrary and could be revised in the future
based on appropriate evidence. Furthermore, such a definition means that the diagnostic thresh-
old will be determined by the probability of implantation of individual embryos, which can vary
depending on their developmental stage,8 the age of the woman,9 and the clinic.10
There is an urgent need for the scientific community to agree on the definition of RIF. This is
crucial for any future research in the field. Using a universally accepted definition will allow for
the proper estimation of the prevalence of RIF as well as the identification of relevant risk factors.
Most importantly, it will allow for the proper evaluation of diagnostic and therapeutic modalities
in this population.
PSYCHOSOCIAL SUPPORT
Failed embryo transfers could have a negative impact on the emotional well-being of the couple (or
woman). Although most couples (or women) with multiple failures seem to emotionally adapt over
time,11,12 it is clinically sensible to ensure that these patients are psychosocially supported by the
staff of the fertility clinic and that they are aware that formal counseling services are available in
case they feel the need for them.13
It is also critical to discern between couples or women that have reached a stage in which they
have very low chances of success using assisted reproduction technology using their own oocytes
(i.e., women of very advanced reproductive age or women with premature menopause). These indi-
viduals might benefit from counseling and relevant psychosocial support on discontinuing treat-
ment or pursuing oocyte donation or adoption.
PRETREATMENT PHASE
Lifestyle factors
A number of lifestyle factors have been linked with a negative effect on implantation.14 Studies per-
formed specifically in RIF patients are lacking,15 and more research is required to ascertain the relative
contribution of lifestyle factors in the etiology of RIF and, furthermore, the potential therapeutic value
of such lifestyle modifications in these patients. However, considering the overall health benefits, it is
sensible to recommended that patients optimize their weight, cease smoking, and avoid excessive alco-
hol consumption. Furthermore, avoiding exposure to endocrine chemical disruptors such as bisphenol
A (BPA), phthalates, polychlorinated biphenyls (PCBs), and heavy metals that are known to adversely
affect implantation, should be advised in patients with otherwise unexplained RIF.
Genetics
In addition to naturally occurring aneuploidy due to advanced female age,9 structural chromo-
somal aberrations (such as inversion and translocation) either in the male or female gametes can
lead to recurrent implantation failures or recurrent miscarriages. There is some evidence suggest-
ing higher prevalence of such chromosomal abnormalities in patients with RIF.16 For this reason,
couples with RIF should be karyotyped, although the number of patients that will be affected is
probably small (2%–3%).16
Endocrine disorders
A number of endocrine causes have been implicated in failed IVF cycles, and these are described in
Chapter 4. The evidence originates from studies performed mostly in the general IVF population,
and, hence, the prevalence of these conditions in RIF patients and the degree to which they might
contribute to RIF have not yet been estimated.
188 Proposed management of patients with recurrent implantation failure
Nevertheless, patients presenting with RIF should probably be investigated for hypo- or hyper-
thyroidism and thyroid autoimmunity. In most cases, these would have been investigated at an
early stage of the couple’s fertility journey. RIF patients should also be investigated for diabetes
mellitus, hyperinsulinemia, and insulin resistance, conditions that have also been suggested to
negatively affect endometrial receptivity.
Vitamin D has also been shown to exert a positive role in implantation, and hence, vitamin D
deficiency should be investigated and treated in any patient commencing their fertility journey,
including RIF patients.
As previously discussed, evidence linking these endocrine disorders specifically with the occur-
rence of RIF and subsequent randomized controlled trials (RCTs) demonstrating efficacy of these
interventions are lacking and, thus, urgently warranted. Until such data become available and con-
sidering the overall health benefits associated with treating most of these disorders, it is advisable
for these conditions to be investigated and treated.
Anatomical causes
Some congenital uterine anomalies (e.g., septate uterus, uterus didelphys, bicornuate, and unicornu-
ate uterus) have been linked with a decrease in the probability of pregnancy.17 The association though
with the achievement of pregnancy after IVF is questionable and well-designed prospective studies in
RIF patients have not been performed. Most of these congenital uterine anomalies have been linked to
first or second trimester pregnancy loss and inferior obstetric outcome. For this reason, women with
RIF should be investigated for the presence of congenital or acquired uterine anomalies, preferably
with three-dimensional ultrasound, in the context of a thorough evaluation of the uterine anatomy. If
a uterine septum is identified, then hysteroscopic resection should be considered.
Furthermore, the presence of primarily submucosal fibroids and potentially intramural fibroids
can have a negative impact on implantation,18 and for this reason, surgical excision should be
considered. Similarly, hydrosalpinges, intrauterine adhesions, endometrial polyps, and chronic
endometritis have been shown to significantly reduce the probability of implantation. Appropriate
investigation (ultrasonography and/or hysteroscopy and/or laparoscopy) and treatment is
recommended.
Finally, adenomyosis has also been shown to be associated with reduced implantation rate after
IVF, and for that reason, its presence should be investigated. Although it can be diagnosed using
pelvic ultrasound, MRI of the pelvis is considered more specific. Surgical treatment of focal adeno-
myosis and/or administration of gonadotropin-releasing hormone agonists pretreatment should be
discussed with these patients, especially in the absence of other factors that can explain RIF.
Immunological causes
An immunological cause of RIF has been hypothesized for many years. It is not surprising that there
is number of cohort or case-control studies investigating differences in the immunological profile
of patients with and without RIF. Despite these intense research efforts, our understanding of the
various immunological mechanisms that contribute to implantation remains limited. A number of
immunological parameters (NK cells, ANA, T cells and genetic polymorphisms affecting immu-
nological responses) have been suggested to be associated with RIF, although the evidence is not
robust. Similarly, a number of immunomodulating treatments have been proposed. These include
administration of corticosteroids,19 intravenous immunoglobulin G (IVIG), intralipid, G-CSF, and
endometrial scratching. From these, endometrial scratching appears to have the largest evidence
base with systematic reviews and meta-analyses supporting its therapeutic effect in RIF patients.20
However, a recent well-designed large RCT supports no benefit in patients with ≥2 previous failed
embryo transfers (ETs).21
Other immunomodulating treatments such as corticosteroids, IVIG, and intralipid have a much
weaker evidence base,22,23 and for that reason, their use should be performed in the context of well-
designed RCTs.
Treatment phase 189
Thrombophilia
Although the prevalence of thrombophilia has been reported to be higher in patients with failed
IVF cycles,24 the evidence supporting the use of either low-molecular-weight heparin (LMWH)
or aspirin is lacking.25,26 For this reason, investigation of thrombophilia in RIF patients and sub-
sequent use of LMWH or aspirin is justified only in the context of well-designed clinical studies.
SPERM
Sperm DNA fragmentation
Increased sperm DNA fragmentation has been suggested to impact negatively on the outcome of
IVF.27 Nevertheless, there is a paucity of data regarding its contribution in the prevalence of RIF.28
Furthermore, the potential therapeutic effect of antioxidants is still highly debated since the studies
suggesting a potential beneficial effect are small and of very low quality.29
Apparently, there is an urgent need for a proper assessment of the prevalence of high sperm DNA
fragmentation in RIF patients. Equally, RCTs are required to prove whether or not administration
of antioxidants or utilization of other laboratory techniques (such as intracytoplasmic morphologi-
cal sperm injection, IMSI) can improve the chance of IVF success in these patients.
TREATMENT PHASE
Optimizing endometrial receptivity
There is a large body of evidence showing the detrimental effect of elevated serum progesterone on
endometrial receptivity following a stimulated IVF cycle.30,31 On the other hand, currently avail-
able evidence does not support an association between serum estradiol or LH with pregnancy rates
after embryo transfer.32 However, these associations have rarely been investigated in RIF patients.
Considering that these factors are not relevant in the frozen-thawed cycle and that almost all
patients today undergo routinely frozen-thawed ET (FET) cycles, it is unlikely that they have a sub-
stantial contribution to the etiopathogenesis of RIF. Nevertheless, for patients who are repeatedly
failing fresh IVF cycles and have not yet been exposed to FETs, freezing all embryos and deferring
the ET for a naturally or artificially prepared endometrium could be explored.
Genetically testing the embryos

Preimplantation genetic testing of the embryos for aneuploidy (PGT-A) embryos can be an impor-
tant diagnostic and therapeutic tool in RIF patients. Although specific data in RIF patients have
not been produced, it can be assumed that PGT-A can ensure that a common cause of implantation
failure, such as naturally occurring embryo aneuploidy due to advanced female age, is addressed.
It can also help diagnose cases with unexpectedly high incidences of embryonic aneuploidy since it
has been shown that even if the parental karyotypes are normal and the patients are not of advanced
reproductive age, oocyte- or sperm-specific defects can lead to the creation of aneuploid embryos
in an abnormally high percentage of cases.33 However, the prevalence of this phenomenon in RIF
patients has not been assessed and is expected to be low.
Finally, ensuring through PGT-A (using a trophectoderm biopsy) that the embryo transferred is
euploid will likely increase the probability of implantation per embryo transfer and allow clinicians
to focus on other potential etiologies of RIF.
Optimizing embryo transfer technique

It is unlikely that poor ET technique is the sole reason for RIF. However, in these patients, it is
important to ensure that the ET technique is optimal and does not compromise the implantation
potential of the embryo. Although there are no studies performed specifically on RIF patients,
available evidence suggests that embryo transfer should be performed using soft catheters,34 under
ultrasound guidance,35 and placing the embryo about 15–20 mm from the uterine fundus.36
WHEN EVERYTHING ELSE FAILS

Oocyte and sperm donation/surrogacy
As previously discussed, RIF can be expected in patients who, either due to advanced female age
or other genetic reasons (e.g., chromosomal aberrations), produce a very high proportion of geneti-
cally abnormal oocytes that lead to embryos with very limited implantation potential.
(a) Is the patient truly a RIF

or just unlucky?
Consider:
Female age,
# of embryos transferred
Embryo quality/developmental stage
Optimize lifestyle
Weight Psychosocial support
Smoking
Alcohol Discuss the availability of counselling
Endocrine chemical disruptors
Uterus
3D U/S ± Hysteroscopy
Genetics for Endocrine disorders
Before treatment
Parental karyotypes Thyroid
Submucosal fibroids and
(chromosomal Diabetes mellitus –
intramural fibroids ≥4 cm
aberrations) insulin resistance
Endometrial polyps Vitamin D
Hydrosalpinges
Adenomyosis
Hysteroscopic resection of
Genetic counselling Uterine septum Endocrinology referral
PGT-A Submucosal fibroids
vitamin D
Oocyte or sperm Intrauterine adhesions
Endometrial polyp supplementation
donation
Laparoscopic excision of
Hydrosalpinges
Adenomyoma
Intramural fibroids >4cm
Antibiotic treatment of
Only in the context of well designed research
Thrombophilia Immunological causes Sperm DNA integrity

Investigations Treatments Investigations Treatments Investigations Treatments
APS LMWH NK cells Glucocorticoids Sperm Antioxidants
antibodies Aspirin KIR/HLA IVIG DNA IMSI
FVL, MTHFR, Folate/B6,B12 ANA Intralipid
Fragmentation
prothrombin, TH2/TH1 G-CSF
ATIII, Protein C Endometrial
and S scratching
Figure 20.1 Proposed management algorithm for patients with RIF: (a) before treatment.
When everything else fails 191
(b)
Endometrial receptivity Embryo euploidy Embryo transfer
During treatment
Investigations Investigations
Serum progesterone at the Optimize ET technique
end of the follicular phase Trophectoderm biopsy and
Soft catheter
NGS testing
Treatments U/S guidance
Replace embryo 15–20 mm
Consider freeze-all if high Treatments
from fundus
progesterone or no FET PGT-A
(c)
When everything else fails
Embryo Uterus/endometrium Treatment discontinuation
After treatment
Oocyte donation Surrogacy Child-free life
Sperm donation Adoption/fostering
Embryo donation
Offer counselling Offer counselling Offer counselling
Figure 20.1 (Continued) Proposed management algorithm for patients with RIF: (b) during treat-
ment, and (c) after treatment.
In these cases, and when PGT-A is not indicated or effective, oocyte donation could represent an
alternative option. Needless to emphasize, no RCTs are currently available on this strategy, and hence
its efficacy is mostly based on retrospective data from oocyte donation cycles.
On the other hand, the quality of the sperm could also be a major contributor in the quality of
the resulting embryos. Hence, despite the absence of high-quality data from RCTs, sperm donation
could be argued as an option in cases in which the sperm are believed to compromise the quality of
the embryos. Unfortunately, the specific indications for which sperm donation is most effective are
not currently clear, and this should be the focus of future research.
Finally, in cases where the cause of RIF is suspected to be the maternal interface and this either
cannot be effectively treated (e.g., severe Asherman Syndrome) or clearly identified, then surrogacy
might be indicated and should be discussed with the patients.
Treatment discontinuation
Despite adequate clinical management, some patients will continue experiencing RIF. The pos-
sibility of treatment discontinuation has to be discussed with these patients, especially when
the probability of pregnancy achievement in future cycles is expected to be very low. There is
currently no specific cutoff, neither in terms of the number of failed cycles already performed
nor in terms of future prognosis (e.g., <5% of success in a future cycle), that has been universally
adopted as the starting point for initiating the discussion around treatment discontinuation.
Cost-effectiveness analyses could in the future also aid in the construction of such decision-
making models by taking into account society’s willingness to pay for a new live birth in patients
with RIF. Until such studies become available, the duty of care dictates that the clinician ensures
that patients understand that IVF is not always successful and treatment discontinuation is the
conclusion of the fertility journey for a proportion of couples.
For the management algorithms proposed, see Figure 20.1.
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Index
A AT, see Antithrombin
AAGL, see American Association of Gynecologic Atosiban, 137
Laparoscopists AUC, see Area under the curve
aCL, see Anticardiolipin antibody Autoimmunity, 100
Acquired anatomical factors, 95; see also Diagnostic AZF, see Azoospermia factor
evaluation of RIF Azoospermia factor (AZF), 81
adenomyosis, 96
endometrial polyps, 95 B
fibroids, 95–96 BDET, see Blastocyst double-embryo transfer
hydrosalpinx, 96–97 beta-hCG, see Beta human chorionic gonadotrophin
intrauterine adhesions, 96 Beta human chorionic gonadotrophin (beta-hCG), 72
uterine factor and hydrosalpinx, 97–98 Bisphenol A (BPA), 20, 84, 187; see also Endocrine disruptive
Acquired thrombophilias, 99 chemicals
Acquired uterine conditions, 55; see also Adenomyosis; Blastocyst biopsy, 144; see also Embryo
Chronic endometritis; Endometrial polyps; Blastocyst double-embryo transfer (BDET), 145; see also
Fibroids; Intrauterine adhesions Embryo
methodological comments, 55 Blood glucose, 100
practice recommendations, 65 BMI, see Body mass index
Acrylamide, 85 Body mass index (BMI), 15
ADAMTS-1, see Aggrecan, disintegrin, and metalloproteinase BPA, see Bisphenol A
with thrombospondin motif-1
Adenomyosis, 58, 65, 96 C
adenomyomectomy, 60 CA, see Chromosomal abnormalities
biological explanation of effect, 59 Caffeine, 16–17
evidence of association, 58–59 CASA, see Computer-aided sperm analysis
treatment results, 59–60 Catheter, 168–169; see also Embryo transfer technique
AFS, see American Fertility Society CDC, see Centers for Disease Control and Prevention
Aggrecan, disintegrin, and metalloproteinase with CE, see Chronic endometritis
thrombospondin motif-1 (ADAMTS-1), 29 CECOS, see Centre d’Etudes et de Conservation des Oeufs et
Alcohol, 17–18 du Sperme humain
American Association of Gynecologic Laparoscopists Centers for Disease Control and Prevention (CDC), 150
(AAGL), 63, 98 Centre d’Etudes et de Conservation des Oeufs et du Sperme
American Fertility Society (AFS), 35 humain (CECOS), 159, 161, 162
American Society of Reproductive Medicine (ASRM), 29, Centriolar defects, 142; see also Embryo
35, 94 CFA, see Corifollitropin alfa
AMH, see Anti-Müllerian hormone Chromosomal abnormalities (CA), 80, 93; see also Embryo
ANA, see Antinuclear antibody in embryos from patients with recurrent implantation
Androgen receptor (AR), 29 failure, 141
Andrological causes of RIF, 79, 150 treatment options, 145–146
Anticardiolipin antibody (aCL), 43 Chronic endometritis (CE), 63, 66, 183
Anti-Müllerian hormone (AMH), 96, 160 biological explanation of effect, 64
Antinuclear antibody (ANA), 43–44 evidence of association, 64
Antiphosphatidylserine (aPS), 43 treatment results, 64–65
Antiphospholipid antibodies (aPLs), 43 Chronic epididymitis, 85
Antiphospholipid antibody (APA), 43–44, 99 CI, see Confidence interval
Antiphospholipid syndrome (APS), 48, 99 CLBR, see Cumulative live birth rate
Antithrombin (AT), 98 Coenzyme Q10 (CoQ10), 108
anti-TPO, see Thyroid peroxidase antibodies Computer-aided sperm analysis (CASA), 80
APA, see Antiphospholipid antibody Confidence interval (CI), 28
aPLs, see Antiphospholipid antibodies Congenital thrombophilias, 98–99
aPS, see Antiphosphatidylserine Congenital uterine anomalies (CUA), 35, 94, 188; see also
APS, see Antiphospholipid syndrome Diagnostic evaluation of RIF
AR, see Androgen receptor with and without arcuate uterus, 37, 38
Area under the curve (AUC), 58 biological explanations, 39
array CGH, see Array comparative genomic hybridization conception and implantation rates, 37–39
Array comparative genomic hybridization (array CGH), 144 ESHRE/ESGE classification of female genital
ART, see Assisted reproductive technology anomalies, 40
Artificial insemination, 158; see also Sperm donation methodological comments, 36
AS, see Asherman syndrome prevalence in general and infertile population, 36–37
Asherman syndrome (AS), 62 recommended current practice, 39–40
ASRM, see American Society of Reproductive Medicine septate uterus, 95
Assisted reproductive technology (ART), 1, 44, 166 CoQ10, see Coenzyme Q10
non-ART, 11 Corifollitropin alfa (CFA), 120
success rate, 161 Corticosteroid administration, 120–121; see also Progesterone
195
196 Index
Corticotrophin releasing hormone (CRH), 19, 135 molecular cytogenetic analysis, 142
Counseling, 180 from patients with RIF, 142
female age, 180–182 replacement of high-quality, 121
following RIF, 182–184 sperm analysis, 144
CRH, see Corticotrophin releasing hormone sperm factors on embryo quality, 142–143
CRISPs, see Cysteine-rich secretory proteins treatment options in severe male factor, 143
CTLA-4, see Cytotoxic T lymphocyte-associated molecule-4 treatment options when no chromosomal abnormalities,
CUA, see Congenital uterine anomalies 145–146
Cumulative live birth rate (CLBR), 152; see also Oocyte -uterus model, 183
donation Embryo culture optimization, 107
Cysteine-rich secretory proteins (CRISPs), 81 coculture on homologous endometrial cells, 108–109
Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), 45 culture medium supplementation, 108
embryo scoring, 109
D extended culture, 107
DD, see Double donation hyaluronic acid-enriched transfer medium, 107–108
DDT, see Dichloro-diphenyl-trichloroethane sequential media, 107
Decidua, 135 time-lapse systems, 109
Decidualization, 135 Embryo implantation, 73, 127–128; see also Late follicular
Deoxynucleotidyl transferase-mediated dUTP nick end phase and embryo implantation
labeling (TUNEL), 94 Embryonic aneuploidy, 93
DES, see Diethylstilbestrol Embryo quality, 127
DF, see DNA fragmentation Embryo scoring, 109
DFI, see DNA fragmentation index Embryo transfer (ET), 44, 107, 115, 167, 188; see also Embryo
Diabetes mellitus (DM), 27, 30 culture optimization
Diagnostic evaluation of RIF, 93; see also Recurrent Embryo transfer technique, 166
implantation failure bedrest, 174
acquired anatomical factors, 95 best practice, 175
acquired thrombophilias and antiphospholipid catheter, 168–169
antibodies, 99 cervical mucus removal, 174–175
adenomyosis, 96 contamination of cavity, 174
algorithm, 101 deposition, 172–173
anatomical factors, 94 distance between endometrium and air bubbles, 172
assessment of uterine factor and hydrosalpinx, 97–98 distance between endometrium and catheter tip, 172
blood glucose, 100 fixed-distance technique, 170
congenital thrombophilias, 98–99 loading, 171–172
congenital uterine anomalies, 94–95 meta-analysis of trials, 169, 170
endocrine factors, 100 mock transfer, 173–174
endometrial polyps, 95 optimizing, 189
endometrial receptivity, 100–101 physician, 168
etiologic factors and diagnostic tests, 102 practice variation, 167
fibroids, 95–96 ultrasound, 169–171
genetic factors, 93 uterine cavity length, 172
hematological and immunological factors, 98 uterine contractility, 175
hydrosalpinx, 96–97 Endocrine causes of RIF, 27
immunologic factors, 99–100 diabetes mellitus, 30
intrauterine adhesions, 96 leptin, 30–31
parental karyotypes, 93–94 polycystic ovary syndrome, 29–30
sperm DNA integrity, 94 thyroid disease, 27–28
thyroid function and autoimmunity, 100 vitamin D deficiency, 28–29
vitamin D, 100 Endocrine disorders, 187
Dichloro-diphenyl-trichloroethane (DDT), 20 anatomical causes, 188
Diethylstilbestrol (DES), 35 immunological causes, 188
DM, see Diabetes mellitus thrombophilia, 189
DNA fragmentation (DF), 79, 160 Endocrine disruptive chemicals (EDCs), 20, 187
DNA fragmentation index (DFI), 143 BPA and phthalates, 20
Donor sperm, 158; see also Sperm donation heavy metals, 21
rationale for use of, 159–160 PCBs, 21
Double donation (DD), 143 pesticides, 20
Doxycycline, 64 Endocrine factors, 100; see also Diagnostic evaluation of RIF
blood glucose, 100
E thyroid function and autoimmunity, 100
E2, see Estradiol vitamin D, 100
EDCs, see Endocrine disruptive chemicals Endocrine Society, 100
EGF, see Epidermal growth factor Endometrial polyps (EPs), 60, 65–66, 95
Embryo, 141; see also Progesterone biological explanation of effect, 61
blastocyst biopsy, 144 evidence of association, 60–61
centriolar defects, 142 treatment results, 61–62
chromosomal abnormalities, 141 Endometrial receptivity, 2, 100–101, 113–114, 122, 127; see
chromosomal constitution and development, 145 also Diagnostic evaluation of RIF; Progesterone
embryo biopsy and genetic screening, 143–145 optimizing, 189
genetically testing, 189 Endometrial receptivity array (ERA), 101, 154
Index 197
Endometrium, 1, 113, 135 Glycidamide, 85

benign overgrowths of, 60 GnRH, see Gonadotropin-releasing hormone
decidualization, 135–136 GnRHa, see Gonadotropin-releasing hormone agonists
and DM, 30 Gonadotropin-releasing hormone (GnRH), 31, 96, 127
dysfunction of, 166 Gonadotropin-releasing hormone agonists (GnRHa), 59
hormonal receptors, 27 Granulocyte colony-stimulating factor (G-CSF), 49–50, 137
immune responses, 45 Growth hormone (GH), 137
during implantation, 127
intrauterine adhesions, 62 H
manipulating, 135–137 Hazard ratio (HR), 28
scratching, 50 hCG, see Human chorionic gonadotrophin
THRs and TSHRs, 27 Heavy metals, 21; see also Endocrine disruptive chemicals
vitamin D metabolizing enzymes, 28 HETM, see Hyaluronic acid-enriched transfer medium
Enzyme CYP2R1, 28 High-order multiple pregnancy (HOMP), 107
Epidermal growth factor (EGF), 108 HOMP, see High-order multiple pregnancy
EPs, see Endometrial polyps HPV, see Human papillomavirus
ER, see Estrogen receptor HR, see Hazard ratio
ERA, see Endometrial receptivity array 3β-HSD, see 3β-hydroxysteroid-dehydrogenase
ESGE, see European Society for Gynaecological Endoscopy HSG, see Hydrosonography; Hysterosalpingography
ESHRE, see European Society for Human Reproduction and Human chorionic gonadotrophin (hCG), 1, 27, 114, 129, 166;
Embryology see also Progesterone
Estradiol (E2), 127; see also Late follicular phase and embryo administration, 120
implantation Human ooplasmic transplantation, 154; see also Oocyte
endogenous concentration, 129 donation
Estrogen, 127 Human papillomavirus (HPV), 85
Estrogen receptor (ER), 27 Human preimplantation embryo, 107
ET, see Embryo transfer Hyaluronic acid, 63, 108
European Society for Gynaecological Endoscopy (ESGE), Hyaluronic acid-enriched transfer medium (HETM),
35, 63, 98 107–108; see also Embryo culture optimization
European Society for Human Reproduction and Embryology Hydrosalpinx, 2, 96–97
(ESHRE), 29, 35, 150, 186 assessment of, 97–98
Extended culture, 107; see also Embryo culture optimization Hydrosonography (HSG), 36
Hydroxychloroquine, 48–49
F 4-hydroxyestradiol (4-OH-E2), 108
Factor-inducible 14 (Fn-14), 45 3β-hydroxysteroid-dehydrogenase (3β-HSD), 114
Factor V Leiden (FVL), 73 Hysterosalpingography (HSG), 96
mutation, 98
FasL, see Fas ligand I
Fas ligand (FasL), 136 ICI, see Intracervical insemination
FDA, see US Food and Drug Administration ICSI, see Intracytoplasmic sperm injection
Female age, success and failure rates, 180–182 Id3, see Inhibitor of DNA-binding protein 3
FertiCalm, 12 Immunological causes of RIF, 42; see also Diagnostic
FET, see Frozen-thawed embryo transfer evaluation of RIF
Fibroids, 56, 95–96 acquired thrombophilias and antiphospholipid
biological explanation of effect, 57 antibodies, 99
classification of, 56 antiphospholipid and antinuclear
evidence of association, 56–57 antibodies, 43–44
myomas, 65 congenital thrombophilias, 98–99
treatment results, 57–58 endometrial immune responses, 45
FIGO, see International Federation of Gynaecology and endometrial scratching, 50
Obstetrics genetic polymorphisms on immune responses, 45–46
FISH, see Fluorescent in situ hybridization glucocorticoid, 46–47
Fixed-distance technique, 170; see also Embryo transfer granulocyte colony-stimulating factor, 49–50
technique hydroxychloroquine, 48–49
Fluorescent in situ hybridization (FISH), 44, 79, 160 immune etiologies, 43
Fn-14, see Factor-inducible 14 immunological treatment, 46
Follicle-stimulating hormone (FSH), 114, 160 intralipid solution, 49
Forehead box P3 (FOXP3)+, 45 IVIG infusion, 47–48
Freeze-all embryos, 121; see also Progesterone natural killer cell pathology, 44
Frozen-thawed embryo transfer (FET), 108, 116, 189; see also tacrolimus, 48
Embryo culture optimization T cell immunity, 45
FSH, see Follicle-stimulating hormone therapeutic targets and immune modulation, 43
FVL, see Factor V Leiden TNF-α inhibitors, 49
Immunological factors, 98, 99–100
G Immunomodulating treatments, 188
Gamete intrafallopian transfer (GIFT), 81 Implantation, 1; see also Recurrent implantation failure
G-CSF, see Granulocyte colony-stimulating factor contributing components, 135
Genetic polymorphisms affecting immune responses, 45–46 failure, 1
GH, see Growth hormone functional molecules, 127
GIFT, see Gamete intrafallopian transfer inflammation on successful, 136
Glucocorticoid, 46–47 Implantation rate (IR), 108
198 Index
IMSI, see Intracytoplasmic morphologically selected sperm Luteinizing hormone (LH), 18, 30, 127; see also Late follicular
injection phase and embryo implantation
Inflammatory agents, 135 in ovarian stimulation, 128–129
Inhibitor of DNA-binding protein 3 (Id3), 45 Lymphocyte immunotherapy (LIT), 183
International Federation of Gynaecology and Obstetrics
(FIGO), 56 M
Intracervical insemination (ICI), 159; see also Recurrent MAGIs, see Male accessory gland infections
implantation failure; Sperm donation Magnetic resonance imaging (MRI), 58, 97
Intracytoplasmic morphologically selected sperm injection Major depressive disorder (MDD), 9
(IMSI), 143, 160; see also Recurrent implantation Male accessory gland infections (MAGIs), 85
failure MDD, see Major depressive disorder
Intracytoplasmic sperm injection (ICSI), 3, 17, 27, 44, 59, 128, MDET, see Mixed double-embryo transfer
158, 166; see also Recurrent implantation failure Methimazole-induced (MMI), 27
Intralipid solution, 49 Methylene tetrahydrofolate reductase C667T mutation
Intrauterine adhesions (IUA), 62, 66, 96 (MTHFR), 98
biological explanation of effect, 62–63 MicroRNAs, 46
evidence of association, 62 Mixed double-embryo transfer (MDET), 145; see also
treatment results, 63 Embryo
Intrauterine insemination (IUI), 28, 61, 159; see also Sperm MMI, see Methimazole-induced
donation Mock transfer, 173–174; see also Embryo transfer
Intravenous immunoglobulin G (IVIG), 44, 188 technique
infusion, 47–48 MRI, see Magnetic resonance imaging
In vitro fertilization (IVF), 1, 9, 27, 36, 150, 158, 166 MTHFR, see Methylene tetrahydrofolate reductase C667T
failures, 9 mutation
role of progesterone on oocyte maturation, 114–116, 117–119 MUC1, see Mucin 1
In vitro fertilization-ET cycles (IVF-ET), 107; see also Embryo Mucin 1 (MUC1), 45
culture optimization Myomas, 65
IR, see Implantation rate
IUA, see Intrauterine adhesions N
IUI, see Intrauterine insemination NADPH oxidase (NOX), 29
IVF, see In vitro fertilization National Institutes of Health (NIH), 29
IVF-D, see IVF using donor sperm Natural killer cells (NK cells), 19, 44
IVF-ET, see In vitro fertilization-ET cycles Negative predictive values (NPV), 97
IVF using donor sperm (IVF-D), 161; see also Sperm donation Next generation sequencing (NGS), 146
IVI, see Valencian Institute of Infertility NF-κB, see Nuclear factor κB
IVIG, see Intravenous immunoglobulin G NGS, see Next generation sequencing
NIH, see National Institutes of Health
K NK cells, see Natural killer cells
Key performance indicators (KPIs), 168 NOA, see Nonobstructive azoospermia
Killer-cell immunoglobulin-like receptor (KIR), 154 Nonobstructive azoospermia (NOA), 158
KIR, see Killer-cell immunoglobulin-like receptor Nonsteroidal antiinflammatory drugs (NSAIDs), 175
KPIs, see Key performance indicators NOX, see NADPH oxidase
NPV, see Negative predictive values
L NSAIDs, see Nonsteroidal antiinflammatory drugs
Late follicular phase and embryo implantation, 127 Nuclear factor κB (NF-κB), 45
endogenous E2 on, 129
endogenous LH on, 128–129 O
ovarian stimulation and embryo implantation, 127–128 OAT, see Oligoasthenoteratozoospermia
research implications for RIF, 129–131 Obesity, 15–16, 30, 84
serum E2 levels and pregnancy probability, 130 chronic oxidative stress, 14
LBR, see Live birth rate endocrine disorders, 27
Leptin, 30–31 -related hyperinsulinemia, 15
Leukemia inhibitory factor (LIF), 27 OD, see Oocyte donation
Leukemia inhibitory factor receptor (LIFR), 27 Odds ratio (OR), 28, 56
LH, see Luteinizing hormone 4-OH-E2, see 4-hydroxyestradiol
LIF, see Leukemia inhibitory factor OHSS, see Ovarian hyperstimulation syndrome
Lifestyle factors and RIF, 14, 21–22 Oligoasthenoteratozoospermia (OAT), 142
alcohol, 17–18 Oocyte donation (OD), 143, 161, 190–191
caffeine, 16–17 CLBR, 153
endocrine disruptive chemicals, 20–21 egg donation cycles over 10-year period, 152
obesity, 15–16 importance, 150–151
periodontal disease, 19–20 model, 155
psychological stress, 18–19 outcomes in fresh and frozen donor egg cycles, 151
smoking, 17 program advancements, 151–153
LIFR, see Leukemia inhibitory factor receptor trends in reproductive outcomes, 153
Lipotoxicity, 15 X RIF, 153–154
LIT, see Lymphocyte immunotherapy Oocyte-follicle complex failures, 166
Live birth rate (LBR), 28 Oocyte pick-up (OPU), 3
LMWH, see Low-molecular-weight heparin OPU, see Oocyte pick-up
Low-molecular-weight heparin (LMWH), 73, 75, 189; see also OR, see Odds ratio
Thrombophilia OS, see Ovarian stimulation
Index 199
Ovarian hyperstimulation syndrome (OHSS), 129 in oocyte maturation, 117–119

Ovarian stimulation (OS), 27, 119, 127–128; see also Late pooled OR for pregnancy achievement, 117
follicular phase and embryo implantation; prevalence of elevated, 118
Progesterone role of, 114–116
endogenous LH during, 128–129 sources of, 114
steroidogenic drive reduction, 119–120
P Progesterone elevation (PE), 115
Parental karyotypes, 93–94; see also Diagnostic evaluation embryo transfer with or without, 117
of RIF management, 119
Patient karyotype abnormalities, 141 prevention of, 119
Patient management, 186 probability, 119
algorithm, 190–191 Prolactin (PRL), 108
diagnostic threshold, 186–187 Psychological consequences of RIF, 9, 12; see also Recurrent
embryo genetic testing, 189 implantation failure
endocrine disorders, 187–189 alternate interventions, 12
genetics, 187 emotional distress, 10–11
lifestyle factors, 187 FertiCalm, 12
oocyte and sperm donation/surrogacy, 190–191 post IVF syndrome, 9
optimizing embryo transfer technique, 189 psychological interventions, 11–12
optimizing endometrial receptivity, 189 Psychological stress, 18–19
pretreatment phase, 187
psychosocial support, 187 R
sperm DNA fragmentation, 189 Radiofrequency electromagnetic radiation (RF-EMR), 85
treatment discontinuation, 191 Randomized control trial (RCT), 47, 57, 109, 128, 171
treatment phase, 189 RCT, see Randomized control trial
PB, see Polar body Reactive oxygen species (ROS), 14, 29, 81
PBMCs, see Peripheral blood mononuclear cells Recombinant follicle-stimulating hormone (rFSH), 128, 146
PCBs, see Polychlorinated biphenyls Recombinant LH (r-LH), 146
PCOS, see Polycystic ovarian syndrome Recreational drugs, 84
PE, see Progesterone elevation Recurrent implantation failure (RIF), 1, 3–5, 55, 158, 180;
Periodontal disease, 19–20 see also Diagnostic evaluation of RIF; Endocrine
Peripheral blood mononuclear cells (PBMCs), 136 causes of RIF; Immunological causes of RIF;
Personalized embryo transfers, 101 Psychological consequences of RIF
Pesticides, 20; see also Endocrine disruptive chemicals andrological causes of RIF, 79, 150
PGD, see Preimplantation Genetic Diagnosis counseling for couple, 180
PGD-AS, see Preimplantation genetic diagnosis for diagnostic threshold, 186–187
aneuploidy screening embryo, 2
PGS, see Preimplantation genetic screening embryo transfers, 186
PGT-A, see Preimplantation genetic testing for aneuploidy etiologies assumed, 182
Phthalates, 20, 84; see also Endocrine disruptive chemicals factors in, 2, 14
Polar body (PB), 144 failures in oocyte-follicle complex, 166
Polychlorinated biphenyls (PCBs), 21, 187; see also Endocrine hydrosalpinges, 2
disruptive chemicals immunological factors/thrombophilia, 2
Polycystic ovarian syndrome (PCOS), 15, 27, 29–30 implantation failure, 1
Positive predictive value (PPV), 97 live-birth rate across all cycles, 181
Post IVF syndrome, 9 maternal age, 3
PPV, see Positive predictive value number of unsuccessful IVF/ICSI cycles, 3
PR, see Pregnancy rate; Progesterone quality and number of embryos transferred, 2–3
Prednisolone, 46 reversing, 136–137
Pregnancy rate (PR), 108, 152, 159; see also Oocyte studies, 4–5
donation success and failure rates, 180–184
Preimplantation embryo biopsy, 143–145; see also Embryo successful implantation, 1
Preimplantation Genetic Diagnosis (PGD), 3, 86, uterine pathologies and endometrial receptivity, 2
160, 186 variables used in definition of, 2
Preimplantation genetic diagnosis for aneuploidy screening Recurrent pregnancy losses (RPL), 42, 10, 60
(PGD-AS), 143 Regulatory T cells (Treg cells), 81
Preimplantation genetic screening (PGS), 94, 144 Relative risk (RR), 28, 56
Preimplantation genetic testing for aneuploidy (PGT-A), Repeated implantation failure, see Recurrent implantation
154, 189 failure
PRL, see Prolactin Reproductive surgery, 55; see also Adenomyosis; Chronic
Progesterone (PR), 27, 113, 127 endometritis; Endometrial polyps; Fibroids;
association of basal P with probability of PE, 119 Intrauterine adhesions
concentration during ovarian stimulation, 114 current practice recommendations, 65
corticosteroid administration, 120–121 methodological comments, 55
earlier administration of HCG and oocyte retrieval, 120 RF-EMR, see Radiofrequency electromagnetic radiation
embryo replacement, 121 rFSH, see Recombinant follicle-stimulating hormone
and endometrial receptivity, 113–114 RIF, see Recurrent implantation failure
forest plots of ORs for pregnancy achievement, 115 r-LH, see Recombinant LH
freeze-all embryos, 121 ROS, see Reactive oxygen species
management of elevated, 121 RPL, see Recurrent pregnancy losses
milder ovarian stimulation, 119 RR, see Relative risk
200 Index
S embryo quality, 162

Saline infusion sonography (SIS), 97 failure as primary infertility treatment, 159
SCD, see Sperm chromatin dispersion female partner’s age, 162
SCSA, see Sperm chromatin structure assay number of cycles, 162–163
Septate uterus, 95 prognostic factors affecting outcome of, 161–163
Sequential media, 107; see also Embryo culture semen characteristics, 162
optimization Three-dimensional ultrasound (3D US), 36, 96
SET, see Single embryo transfer 3D US, see Three-dimensional ultrasound
SI, see Standard incubator Thrombophilia, 72, 75, 189; see also Endocrine disorders
Single embryo transfer (SET), 183 cause-effect relationship, 73
SIS, see Saline infusion sonography interventional studies, 76
Smoking, 17 low-molecular-weight heparin, 73, 75
Sperm, 189; see also Diagnostic evaluation of RIF observational studies, 74
analysis by FISH and TUNEL, 144 prophylactic anticoagulation in RIF treatment, 73, 75
DNA fragmentation, 189 recurrent implantation failure, 72
DNA integrity, 94 thrombogenic risk factors, 73
factors affecting embryo quality, 142–143 Thrombosis, 72; see also Thrombophilia
Sperm chromatin dispersion (SCD), 94 Thyroid
Sperm chromatin structure assay (SCSA), 94 disease, 27–28
Sperm donation, 158, 190–191 function, 100
artificial insemination, 158 Thyroid autoimmunity (TAI), 27, 100
CECOS, 159 Thyroid-binding globulin (TBG), 28
cycle efficacy, 161 Thyroid hormone (TΗRs), 27
cycles as RIF treatment, 159 Thyroid peroxidase antibodies (anti-TPO), 100
donor sperm, 158 Thyroid stimulating hormone (TSH), 27, 100
failure of TDI cycles, 159 Thyroid stimulating hormone receptors (TSHRs), 27
prognostic factors on TDI outcome, 161–163 Time-lapse system (TLS), 109
rationale for donor sperm, 159–160 TLR-4, see Toll-like receptor 4
Sperm epigenetics, 81 TLS, see Time-lapse system
oxidative stress, 81–82 TNF-like weak inducer of apoptosis (TWEAK), 45
proteomics, 82 TNF-α inhibitors, 49
Sperm quality, 79 Toll-like receptor 4 (TLR-4), 48
abnormal sperm morphology, 80 Top-quality embryo (TQE), 162
alcohol, 83 TQE, see Top-quality embryo
DNA fragmentation, 80–81 Treg cells, see Regulatory T cells
environmental factors, 84 TROPHY, 98
evidence on, 79 TSH, see Thyroid stimulating hormone
genetic factors, 80 TSHRs, see Thyroid stimulating hormone receptors
genital heat stress, 83–84 TUNEL, see Deoxynucleotidyl transferase-mediated dUTP
genital tract infections, 85 nick end labeling
heavy metals, 85 TWEAK, see TNF-like weak inducer of apoptosis
hormone disruptors, 84–85 Two-dimensional ultrasound (2D US), 58
immunological maladaptation, 81 2D US, see Two-dimensional ultrasound
lifestyle factors, 83 TΗRs, see Thyroid hormone
management, 86
obesity, 84 U
oxidative stress, 81–82 UGET, see Ultrasound-guided embryo transfer
paternal age, 82–83 Ultrasound (US), 169–171; see also Embryo transfer technique
proteomics, 82 3D US, 36, 96
radiation, 85 2D US, 58
recreational drug use, 84 guiding, 170
smoking, 83 Ultrasound-guided embryo transfer (UGET), 171; see also
sperm analysis parameters, 79–80 Embryo transfer technique
sperm epigenetics, 81 uNK, see Uterine NK
sperm washing procedures, 86 US, see Ultrasound
Y chromosome microdeletions, 81 US Food and Drug Administration (FDA), 49
Standard incubator (SI), 109 Uterine; see also Embryo transfer technique
Structural chromosomal aberrations, 187 anomalies, 35
contractility, 39, 175
T fibroids, 56
Tacrolimus, 48 pathologies and endometrial receptivity, 2
TAI, see Thyroid autoimmunity sparing alternatives in patient management with
TBG, see Thyroid-binding globulin adenomyosis, 59
T cell immunity, 45 Uterine NK (uNK), 44
TDI, see Therapeutic donor insemination Uterus, 35, 55
TESE, see Testicular sperm extraction anatomically defected, 40
Testicular sperm extraction (TESE), 81, 158; see also Sperm arcuate, 37, 94
donation embryo-uterus model, 183
Therapeutic donor insemination (TDI), 158; see also Sperm septate, 37, 95
donation
Index 201
V W
Valencian Institute of Infertility (IVI), 152 Weight-reduction strategies, 16
VDR, see Vitamin D receptor Window of implantation (WOI), 100, 154
Venous thromboembolism (VTE), 72; see also Thrombophilia WOI, see Window of implantation
Vitamin D, 100
deficiency, 28–29
Vitamin D receptor (VDR), 28 Y
VTE, see Venous thromboembolism Y chromosome microdeletions, 81

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Recurrent Implantation Failure

Recurrent Implantation Failure

Christos A. Venetis, MD, MSc(Res),

© 2020 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-138-05578-0 (Hardback)

Library of Congress Cataloging-in-Publication Data

Names: Kolibianakis, Efstratios M., editor. | Venetis, Christos A., editor.

Visit the Taylor & Francis Web site at

1 What is recurrent implantation failure? 1

19 When should patients abandon treatment? 180

Dimitra Aivazi Jeremy J. Cottle

Georgios A. Kanakis Kay Neumann

Engin Turkgeldi Christos A. Venetis

FACTORS CONTRIBUTING TO RIF

Uterine pathologies and endometrial receptivity

VARIABLES USED IN THE DEFINITION OF RIF

Number of unsuccessful IVF/ICSI cycles

Number of unsuccessful cycles alone

THE PSYCHOLOGICAL IMPACT OF RECURRENT FAILURE

IVF FAILURE = EMOTIONAL DISTRESS?

PSYCHOLOGICAL INTERVENTIONS TO REDUCE DISTRESS

ALTERNATIVE FORMS OF INTERVENTIONS

THE PSYCHOLOGICAL IMPLICATIONS OF RECURRENT IMPLANTATION FAILURE

ENDOCRINE DISRUPTIVE CHEMICALS

BPA and phthalates

POLYCYSTIC OVARY SYNDROME

and downregulate it, in the presence of gonadotropin-releasing hormone (GnRH).65 It is also

8. Aghajanova L, Stavreus-Evers A, Lindeberg M, Landgren BM, Sparre LS, Hovatta O. Thyroid-

29. Weisman Y, Harell A, Edelstein S, David M, Spirer Z, Golander A. 1 alpha, 25-dihydroxyvi-

PREVALENCE IN THE GENERAL AND INFERTILE POPULATION

CONCEPTION AND IMPLANTATION RATES IN WOMEN WITH

POSSIBLE BIOLOGICAL EXPLANATIONS

CONCLUSIONS AND RECOMMENDED CURRENT PRACTICE

Therapeutic targets Immunosuppressive treatment

Elevated NK cell levels lntralipid Tx.

Decreased treg cells G-CSF (Filgrastim)

Decreased endometrial CD56+

Natural killer cell pathology

Endometrial immune responses

Genetic polymorphisms affecting immune responses

Intravenous immunoglobulin G (IVIG) infusion

Granulocyte colony-stimulating factor

21. Ahmadi M, Ghaebi M, Abdolmohammadi-Vahid S et al. NK cell frequency and cytotoxicity

Potential mechanisms/biological explanation of the effect

Potential mechanisms/biological explanation of the effect

Potential mechanisms/biological explanation of the effect

Potential mechanisms/biological explanation of the effect

Potential mechanisms/biological explanation of the effect

CONCLUSIONS AND CURRENT PRACTICE RECOMMENDATIONS

57. Preutthipan S, Linasmita V. Reproductive outcome following hysteroscopic lysis of intrauter-

WHAT IS THROMBOSIS AND THROMBOPHILIA?

WHAT IS RECURRENT IMPLANTATION FAILURE?

IS THROMBOPHILIA CAUSING RIF?

of antiphospholipid antibodies is thought to be associated with thrombosis, habitual abortion, and

IS PROPHYLACTIC ANTICOAGULATION USING HEPARINS AND ASPIRIN EFFECTIVE IN THE

Table 8.2 Observational studies on thrombophilia and RIF.

Heparin Control Odds ratio Odds ratio

Total (95% CI) 190 196 100.0% 1.77 [1.07, 2.90]

Table 8.3 Interventional studies on thrombophilia and RIF.

AVAILABLE EVIDENCE ON SPERM QUALITY

FACTORS THAT AFFECT SPERM QUALITY

Genital heat stress