1. Recurrent implantation failure:
gamete and embryo factors
Mausumi Das, M.D., and Hananel E. G. Holzer, M.D.
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec,
Canada
Chromosomal abnormalities, sperm DNA damage, zona hardening, inadequate culture conditions, and suboptimal embryo development all play a sig-
nificant role in the etiology of recurrent implantation failure. Evidence suggests that preimplantation genetic screening does not increase implantation or
live birth rates. Comparative genomic hybridization array and analysis of single nucleotide polymorphisms could enable a more comprehensive screening
of chromosomes. Assisted hatching may help to overcome zona hardening in selected cases. Optimal culture conditions and blastocyst transfer could
contribute toward improving implantation and pregnancy rates. Novel embryo assessment and selection procedures, such as time-lapse imaging and
metabolomics, may help in better evaluation of embryo quality and viability and help in selecting embryos with the highest implantation potential.
The safety and efficacy of emerging treatment modalities should be evaluated in prospective randomized clinical trials before being applied in routine
clinical practice. (Fertil SterilÒ 2012;97:1021–7. Ó2012 by American Society for Reproductive Medicine.)
Key Words: Implantation failure, IVF, embryo, oocyte, sperm, chromosome
D
espite the immense strides that depends on the synchronization of var- inversions, and deletions, have been
have been made in the field of ious factors such as the quality of the demonstrated in women with high-
IVF many patients still experi- embryo, optimal culture conditions, order RIF (3). In support of these find-
ence recurrent implantation failure. the receptivity of the endometrium, ings, Stern et al. (4) observed an overall
Besides causing immense distress to and the maternal immune system. The chromosomal abnormality rate of 2.5%
couples who require multiple cycles of aim of this article is to review the estab- (13/514) in patients with RIF. Most
treatment, it significantly increases the lished etiologies affecting embryo de- of these abnormalities were chromo-
cost of the procedure. Recurrent implan- velopment in patients with RIF and to somal translocations (reciprocal and
tation failure (RIF) may be defined as the evaluate recent advances in oocyte Robertsonian). They proposed that bal-
repeated transfer of morphologically and embryo selection, as well as current anced parental translocations may be
good embryos to a normal uterus with- recommended management strategies. implicated in the pathogenesis of im-
out achieving successful implantation plantation failure in IVF, and that ge-
and a clinical pregnancy. Traditionally, ETIOLOGY netic evaluation should be considered
failure to achieve a pregnancy after two Chromosomal abnormalities, inade- as part of the investigation of these cou-
to six IVF cycles, in which more than quate culture conditions, suboptimal ples (4).
10 high-grade embryos were transferred embryo development, zona hardening, Aneuploid embryos have decreased
to the uterus was defined as RIF (1). and improper ET technique all play an ability to undergo successful implanta-
However, in most IVF programs, failure important role in the etiology of RIF. tion and result in a viable pregnancy,
of three cycles in which reasonably but cannot be distinguished from nor-
good embryos were transferred would mal embryos using standard morpho-
warrant investigation (2). In spite of op- Chromosomal Abnormality logical criteria. Data obtained from
timization of treatment protocols and It is now well established that a major embryos and oocytes of patients under-
huge advancements in laboratory tech- cause of repeated implantation failure going preimplantation genetic screen-
nologies, the management of RIF poses after IVF is a high frequency of chromo- ing (PGS) because of advanced age,
a major challenge to clinicians and em- somal aneuploidy. An increased inci- recurrent pregnancy losses, or multiple
bryologists universally. The process of dence of chromosomal abnormalities, failed IVF cycles, support the concept
embryo implantation in the uterus such as translocations, mosaicism, that many embryos and eggs obtained
during IVF are intrinsically abnormal
Received February 2, 2012; accepted February 21, 2012; published online March 15, 2012.
and thus fail to implant (5). Using fluo-
M.D. has nothing to disclose. H.E.G.H. has nothing to disclose. rescence in-situ hybridization (FISH) on
Reprint requests: Hananel E. G. Holzer, M.D., Department of Ob & Gyn, McGill University Heath Cen- blastomeres from biopsied day 3 em-
ter, McGill Reproductive center, 687 Pine Avenue West, Montreal, Quebec H4W 2A6, Canada
(E-mail: hananel.holzer@muhc.mcgill.ca). bryos for chromosomes 13, 16, 18, 21,
22, X, and Y, Pehlivan et al. (6) found
Fertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00
Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc.
that there was a significantly higher
doi:10.1016/j.fertnstert.2012.02.029 rate of chromosomal abnormalities
VOL. 97 NO. 5 / MAY 2012 1021

2. VIEWS AND REVIEWS
(67%) compared with controls (36%) in patients with three or suboptimal components of a culture system that could
more failed IVF attempts. In another study, using comparative lead to impaired embryo development have been described
genomic hybridization (CGH), Voullaire et al. (7) detected (20). These include osmolality testing, pH measurements,
chromosomal abnormalities in 76/126 (60%) single blasto- and sperm bioassay (20). In some instances of RIF, individ-
meres biopsied from embryos before implantation in 20 ualized specific culture conditions may be required for opti-
women with RIF after IVF. The abnormalities detected in their mal embryo development.
study included aneuploidy for one or two chromosomes as Implantation rates and PRs after ET depend on the quality
well as complex chromosomal abnormality. They suggested and developmental potential of embryos selected for transfer.
that the disruption of the normal sequence of chromosome Suboptimal embryo quality has an adverse effect on implan-
replication and segregation in early human embryos, caused tation and PRs. Evidence suggests that ET technique can
either by maternal cytoplasmic factors or mutations in cell influence the success or failure of embryo implantation. Uter-
cycle control genes, may be a common cause of RIF. ine contractions, blood or mucous on the catheter tip, endo-
A higher incidence of sperm chromosomal abnormalities metrial trauma, and expulsion of embryos have all been
in patients with normal karyotype and RIF has also been associated with unsuccessful ETs (21).
reported. Pregnancy rates (PR) and implantation rates were re-
ported to be significantly lower in patients with teratozoosper- MANAGEMENT OPTIONS
mia. Rubio et al. (8) analyzed sperm aneuploidy and diploidy
Various management options have been proposed to over-
rates for chromosomes 13, 18, 21, X, and Y in patients with nor-
come the challenges of chromosomal abnormality and subop-
mal karyotypes using dual and triple-color FISH techniques.
timal embryo development. Table 1 shows the various
They reported an increased incidence of sex chromosome dis-
etiological factors contributing toward defective embryo de-
omies in couples with RIF after intracytoplasmic sperm injec-
velopment and their proposed management strategies.
tion (ICSI). In addition, centrosome anomalies resulting in
chaotic mosaics were most likely of paternal origin (9, 10).
Evidence suggests that sperm DNA damage is associated Chromosomal Abnormality
with lower PRs after IUI and IVF (11). In addition, increased In view of the higher incidence of chromosomal anomalies,
levels of sperm DNA damage have been linked with an parental karyotype is recommended as part of the work-up
increased risk of pregnancy loss after IVF and ICSI (12). There- in RIF (4). Preimplantation genetic screening has also been in-
fore there is considerable evidence to suggest that chromo- creasingly used in the past decade, the rationale being that an
somal abnormalities, both maternal and paternal, play a key increased PR could be achieved by selecting only chromoso-
role in the etiology of repeated implantation failure in IVF. mally normal embryos for transfer. The main biopsy methods
used for PGS include removal of one or two polar bodies from
the unfertilized oocyte or the zygote, removal of one or two
Zona Hardening
blastomeres at the cleavage stage, or removal of several cells
The mammalian oocyte is surrounded by an acellular matrix, at the blastocyst stage (22). Polar body biopsy analyses mater-
the zona pellucida (ZP), which is composed of glycoproteins, nal causes of chromosomal abnormality and is an indirect
carbohydrates, and ZP-specific proteins (13). It plays a role in method of screening for aneuploid embryos. It has, however,
sperm binding, induction of the acrosome reaction, and pro-
motes sperm–egg fusion (14). The zona hardens naturally af-
ter fertilization to prevent polyspermic fertilization, protects
the integrity of the preimplantation embryo, and facilitates TABLE 1
oviductal transport (15). The zona is required during early
Management options for factors affecting embryo development and
cleavage stages to maintain the integrity of the inner cell implantation in recurrent implantation failure.
mass (ICM), but it is usually shed during expansion of the
blastocyst, allowing implantation to occur (16). Upon reach- Management options
ing the blastocyst stage, physical expansion of the embryonic Chromosomal abnormality
mass along with the action of lysins produced by the cleaved Preimplantation genetic screening
Comparative genomic hybridization array
embryo and/or the uterus, all play a role in zona hatching Single nucleotide polymorphisms
(17–19). Failure of the ZP to rupture after blastocyst Zona hardening
expansion, resulting in impaired hatching, could contribute Assisted hatching
to RIF (15). Prolonged exposure of oocytes and embryos to Suboptimal culture
Optimal culture media
artificial culture conditions may also adversely affect the Blastocyst transfer
embryo's ability to undergo normal hatching and could Coculture
impair successful implantation (15). ZIFT
Assessment of embryo quality and viability
Time-lapse imaging—EmbryoScope
Metabolomics
Embryo Culture and ET Technique Proteomics
The use of high quality, standardized culture media is funda- Improving ET technique
Note: ZIFT ¼ zygote intrafallopian transfer.
mental to the success of any IVF program. Inadequate cul-
Das. Recurrent implantation failure. Fertil Steril 2012.
ture conditions could play a role in RIF. Assays to identify
1022 VOL. 97 NO. 5 / MAY 2012

3. Fertility and Sterility®
been suggested that if oocyte maturation to the metaphase II producing a unique DNA fingerprint for each embryo tested
stage is completed just before the polar body biopsy, it may (33). However, a disadvantage of SNP microarrays is a lack
result in damage to the meiotic spindle of the oocyte (23). of diagnostic accuracy at individual SNP loci as well as
Cleavage stage biopsy is the most commonly used method high cost of microarrays and labeling techniques (33). In the
for screening preimplantation embryos for aneuploidy (24). future, PGS-FISH may be replaced by comprehensive proce-
However, cleavage stage embryos have an increased inci- dures such as array CGH and SNP microarrays. However,
dence of mosaicism (22). Biopsy at the blastocyst stage may the efficacy and practicality of these procedures in improving
have a smaller risk of aneuploidy than embryo biopsy at the implantation and live birth rates in patients with RIF will have
cleavage stage, because mosaic embryos have a higher pro- to be determined in well-designed prospective randomized
portion of aneuploid cells on day 2/3 and will not develop controlled trials before they can be widely applied in clinical
to the blastocyst stage (23). practice.
Although initial studies suggested that PGS with FISH
could be used to achieve favorable implantation and PRs in
patients with RIF (6, 25), evidence from recent randomized Assisted Hatching
controlled trials does not support these findings (26, 27). In Elasticity and thinning of the ZP are essential prerequisites for
a prospective randomized controlled trial, Blockeel et al. successful embryo hatching and implantation (15, 37). It has
(26) observed that PGS did not increase the implantation been observed that cleaved embryos with a good prognosis for
rates after IVF-ICSI in women with RIF. In this study, the in- implantation have reduced zona thickness (38). It has been
vestigators analyzed chromosomes 13, 16, 18, 21, 22, X, and suggested that an artificial opening made in the ZP may
Y using FISH on blastomeres of day 3 cleavage stage embryos facilitate the hatching process (39). Cohen et al. (40)
in the study group. There was a significant difference in live observed a higher implantation rate per ET after partial
birth rate between the PGS group (21%) and the control group zona dissection. The implantation window occurs 1–2 days
(39%). The miscarriage rate did not differ between the two earlier in women undergoing ovarian stimulation than in
groups (26, 27). A recent meta-analysis of randomized natural cycles (41). Embryos with artificial gaps in the zona
controlled trials demonstrated that in women with advanced initiate hatching earlier than zona intact embryos,
maternal age as well as women with repeated implantation compensating for the reduced development rate in vitro
failure, PGS significantly lowered live birth rates after (42). It has also been proposed that breaching the integrity
IVF (27). of the zona could enhance the transport of nutrients from
The reasons that have been proposed for the inefficiency the incubating media, which in turn would augment
of PGS are possible damage from the biopsy procedure, failure embryo development and blastocyst formation (43). It could
rate from the technique, limitations of the FISH analysis, and also serve as a channel for a two-way exchange across the
embryo mosaicism (27, 28). In addition, the efficacy of FISH is ZP of metabolites and growth factors (42).
limited because only a few chromosomes can be detected The artificial rupture of the ZP is known as assisted hatch-
simultaneously in a single biopsied cell. The lack of ing and aims to improve implantation and clinical PRs. Var-
usefulness of PGS may be because the tested blastomere is ious techniques have been used to aid zona hatching. These
not representative for the whole embryo (29). The American involve the creation of an opening in the ZP either by me-
Society of Reproductive Medicine (ASRM), the European chanical partial zona dissection (39), chemically by zona dril-
Society of Human Reproduction and Embryology (ESHRE), ling with acid Tyrode (42), chemical zona thinning (44),
and the British Fertility Society have concluded that PGS enzymatic treatment (45), laser-assisted hatching (46, 47),
does not improve the live birth rates in patients with RIF, or by using a piezo-micromanipulator (48).
advanced maternal age, or recurrent pregnancy loss (30–32). The clinical relevance of assisted hatching procedures in
Alternative approaches have been proposed to overcome the management of RIF is controversial. Although some stud-
the limitations of FISH for PGS. These include CGH or the ies have reported that assisted zona hatching improves PRs
analysis of single nucleotide polymorphisms (SNPs) (33, 34). and implantation rates in patients with RIF (49, 50), other
Comparative genomic hybridization is a DNA-based method, investigators have not reported any advantage (46). Recent
which is applicable to cells in any phase of the cell cycle (33). studies seem to suggest that assisted hatching may be of
The CGH microarray enables a more comprehensive screening benefit in selected patients. In a prospective randomized
of chromosomes. Many chromosomal aneuploidies identified study, comparing chemical removal of ZP from day 5
using CGH would not have been detected using FISH for five in vitro cultured human embryos by using acidic Tyrode's
or nine chromosomes (35). Microarrays have an advantage solution versus no removal, the implantation rate per ET
over conventional CGH because the evaluation of fluores- and the clinical PR were significantly higher in the ZP-free
cence ratios is simple, rapid, and easily automated (33). A group (51). Stein et al. (52) reported that assisted hatching
proof-of-principle study concluded that chromosomal aneu- by partial zona dissection resulted in a significant increase
ploidy of the oocyte can be accurately predicted by array in the implantation and clinical PRs in women older than
CGH analysis of both polar bodies (36). 38 years with RIF. Similarly, Petersen et al. (53) observed
Single nucleotide polymorphisms are common polymor- that for patients with repeated implantation failures, the im-
phic DNA sequences found throughout the genome. The plantation rate in those who received laser-thinned embryos
probes used for SNP microarrays provide genotype data in was significantly higher than in those whose embryos were
addition to chromosome copy number information, thereby not laser thinned. Interestingly, this difference was not
VOL. 97 NO. 5 / MAY 2012 1023

4. VIEWS AND REVIEWS
observed in patients with a history of only one previous im- patients, selecting the best embryos by culturing to the blas-
plantation failure. In support of these findings, in a recent tocyst stage assumes even greater significance. In a prospec-
meta-analysis of randomized control trials (five trials with tive randomized study, Levitas et al. (63) reported that in
761 participants), assisted hatching was reported to be associ- patients with RIF with an adequate ovarian response, transfer
ated with a significant improvement in clinical pregnancy of blastocyst stage embryos carries a significantly higher im-
when performed in fresh embryos transferred to women plantation rate compared with ET on days 2–3. The multiple
with RIF (relative risk [RR] ¼ 1.73; 95% confidence interval PR was not significantly different between the two groups
[CI] ¼ 1.37–2.17) (54). No increase was observed in clinical (63). In another study, Guerif et al. (64) also observed that
PRs when performed in fresh embryos transferred to unse- the live birth rates and implantation rates per cycle were
lected or nonpoor prognosis women or to women of advanced higher after blastocyst transfer compared with day 2 ET.
age. Assisted hatching was also related to increased multiple They suggested that improved embryo selection and uterine
PRs in women with previous repeated implantation failure. receptivity may explain the additional benefit of ET at the
However, due to the small sample size of the included studies, blastocyst stage for couples with RIF (64). However, it should
this meta-analysis was not able to draw any conclusions re- be noted that a percentage of fertilized eggs will never reach
garding live birth or miscarriage rates (54). the blastocyst stage. Proper selection of cases suitable for
blastocyst transfer is therefore critical to reduce the number
of cycle cancellations (63).
Embryo Culture
Optimum culture conditions are a prerequisite for satisfactory Stimulation Protocols
embryonic development and lack of these conditions may
contribute to RIF. Various coculture systems have been devel- Variations in ovarian stimulation protocols have been sug-
oped as a means of improving embryo culture conditions. The gested in some studies as a means of improving embryo devel-
main aim is to increase the metabolic chances of the human opment and quality. The use of GnRH antagonist protocols in
embryo to achieve the blastocyst stage because this leads to controlled ovarian hyperstimulation (COH) has been shown to
a high implantation rate and PR. The suggested favorable ef- improve pregnancy outcome in patients with a history of RIF
fects of cocultures include the secretion of embryotrophic fac- with GnRH agonist protocols. The investigators proposed that
tors, such as nutrients and substrates, growth factors and this was most likely due to improvement of the quality of the
cytokines, and the removal of free radicals and potentially blastocysts generated (65). Natural cycle IVF has also been
harmful substances (55). Although multiple cell types have proposed as a means of improving implantation rates in pa-
been used for coculturing embryos, ranging from human re- tients with RIF (66). Despite some personal experience with
productive tissues, such as oviducts (56), endometrium (57), natural cycle IVF and in vitro maturation of oocytes in pa-
sequential oviduct-endometrial coculture (58), and cumulus- tients with RIF, the lack of randomized clinical studies in
granulosa cells (GC) (59–61), homologous endometrial cells this field does not allow any recommendations to be made
appear to be the most promising coculture system (57). with regard to their efficacy.
Using coculture of embryos on homologous endometrial
cells in patients with RIF, Jayot et al. (57) reported an Zygote Intrafallopian Transfer
overall PR of 21% per transfer versus 8% in previous IVF-ET Zygote intrafallopian transfer (ZIFT) allows the early embryo
cycles. Similarly, using autologous endometrial coculture in to grow in the natural tubal environment and physiological
patients with RIF, Spandorfer et al. (62) reported a significant transport of the embryos into the uterine cavity. It also over-
improvement in embryo quality and clinical PRs. However, comes the problem of technically difficult ET because of cer-
the advantage of coculture systems remains controversial. In vical stenosis (2). Although initial nonrandomized studies
addition, most IVF units do not have the necessary personnel implied that ZIFT may be of value in RIF (67), a subsequent
or facilities to perform coculture on a regular basis. meta-analysis of randomized controlled trials failed to dem-
onstrate any benefit for ZIFT (68). In fact, there was a trend
Blastocyst Transfer toward increased risk of ectopic pregnancy (EP) with ZIFT
(68). These findings led to the procedure being abandoned
Embryo transfer at the blastocyst stage has been proposed as by most units.
a strategy to improve implantation rates and PRs in patients
with RIF. Blastocyst transfer is a more physiological approach
as the human embryos usually enter the endometrial cavity 5 ET Technique
days after fertilization, at the morula-blastocyst stage in nat- A meticulous ET technique is of utmost importance in achiev-
ural conception cycles (2). Better embryo selection for transfer ing a successful pregnancy outcome. Studies show that avoid-
and improved endometrial receptivity are obvious advantages ance of blood (69), mucus (70), bacterial contamination,
of this approach. Some clinicians transfer several embryos trauma to the endometrium, touching the fundus, and exces-
after RIF. Culturing embryos to the blastocyst stage helps in sive uterine contractions (71) are all associated with better
selecting embryos with the best implantation potential. PRs and implantation rates after ET. Several techniques
Therefore fewer embryos have to be transferred to achieve have been proposed to optimize the technique of ET. Methods,
a successful pregnancy, thereby decreasing the risk of multi- such as a trial transfer (72), filled bladder (73), ultrasono-
ple pregnancy. With single ET becoming the norm in younger graphic guidance (74), and use of soft catheters, all appear
1024 VOL. 97 NO. 5 / MAY 2012

5. Fertility and Sterility®
to facilitate a successful ET (21), whereas bed rest after ET has viability (83). Newer methods, such as vibrational spectros-
not been shown to be of any benefit (75). copy, both Raman and near infrared, have been used to ana-
lyze spent culture medium from human embryos, measuring
bonds within functional groups of molecules at specific wave-
Cytoplasmic Transfer lengths. Results from initial studies indicate that spectral pro-
Ooplasmic factors play a role in the continued development files reflective of oxidative stress appear to have a good
of the zygote, especially during the early cleavage stage. Co- correlation with pregnancy outcome (84).
hen et al. (76) transferred ooplasm from donor eggs at meta-
phase II stage into developmentally compromised metaphase
II oocytes in patients with multiple implantation failure (76). CONCLUSION
They noted that this led to an improvement in embryo mor- Regardless of the considerable improvement in treatment pro-
phology. Cytoplasmic transfer from fertile donor oocytes or tocols and laboratory technologies, RIF still poses a significant
zygotes into developmentally compromised oocytes from pa- challenge to clinicians and embryologists. Chromosomal ab-
tients with RIF has led to the birth of several healthy babies normalities and suboptimal embryo development play a major
worldwide (77). It has been suggested that this procedure role in the etiology of RIF. Emerging technologies, such as
may correct an imbalance between anti- and pro-apoptotic CGH array and analysis of SNPs could enable a more compre-
factors and/or correction of defective mitochondrial mem- hensive screening of chromosomes. Assisted hatching may
brane potential (78). However, the transferred cytoplasm help to overcome zona hardening in selected patients. Opti-
could contain messenger RNAs, proteins. and mitochondria mal culture conditions and blastocyst transfer may contribute
(77). In addition, it is not known whether the physiology of toward improving the implantation rates and PRs in RIF.
the early embryo is affected. The procedure is still experimen- Novel embryo assessment and selection procedures, such as
tal and will require assessment of ooplasmic anomalies and time-lapse imaging and metabolomics, may help in better
optimization of techniques before it can be applied in clinical evaluation of embryo quality and viability and help in select-
practice. ing embryos with the highest implantation potential. It should
be noted that only those treatment options that are evidence
based should be offered to patients. The safety, efficacy, and
New Methods of Embryo Assessment practicality of new, emerging methods of treatment should
Assessment of embryo quality is critical in selecting the best be evaluated in prospective randomized clinical trials before
embryo(s) to transfer or cryopreserve. As visual assessment being accepted in clinical practice.
of embryo quality using morphological criteria can be subjec-
tive and requires considerable expertise, newer methods of as-
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