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Recurrent implantation failure:gamete and embryo factorsMausumi 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,CanadaChromosomal 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 orlive birth rates. Comparative genomic hybridization array and analysis of single nucleotide polymorphisms could enable a more comprehensive screeningof chromosomes. Assisted hatching may help to overcome zona hardening in selected cases. Optimal culture conditions and blastocyst transfer couldcontribute toward improving implantation and pregnancy rates. Novel embryo assessment and selection procedures, such as time-lapse imaging andmetabolomics, 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 routineclinical practice. (Fertil SterilÒ 2012;97:1021–7. Ó2012 by American Society for Reproductive Medicine.)Key Words: Implantation failure, IVF, embryo, oocyte, sperm, chromosomeD 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 overallBesides 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. Mosttreatment, 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 andtation 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 begood 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 consideredfailure 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 decreasedto 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 fromtimization 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 multiplebryologists universally. The process of dence of chromosomal abnormalities, failed IVF cycles, support the conceptembryo implantation in the uterus such as translocations, mosaicism, that many embryos and eggs obtained during IVF are intrinsically abnormalReceived 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) onReprint 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: email@example.com). bryos for chromosomes 13, 16, 18, 21, 22, X, and Y, Pehlivan et al. (6) foundFertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. that there was a signiﬁcantly higherdoi:10.1016/j.fertnstert.2012.02.029 rate of chromosomal abnormalitiesVOL. 97 NO. 5 / MAY 2012 1021
VIEWS AND REVIEWS(67%) compared with controls (36%) in patients with three or suboptimal components of a culture system that couldmore failed IVF attempts. In another study, using comparative lead to impaired embryo development have been describedgenomic 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 qualitywell 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 caneither 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 beenin 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 OPTIONSmia. 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 variousThey 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 inchaotic mosaics were most likely of paternal origin (9, 10). Evidence suggests that sperm DNA damage is associated Chromosomal Abnormalitywith 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-upincreased 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 ansomal 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 twoZona Hardening blastomeres at the cleavage stage, or removal of several cellsThe 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 indirectcarbohydrates, 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, protectsthe integrity of the preimplantation embryo, and facilitates TABLE 1oviductal transport (15). The zona is required during early Management options for factors affecting embryo development andcleavage stages to maintain the integrity of the inner cell implantation in recurrent implantation failure.mass (ICM), but it is usually shed during expansion of theblastocyst, allowing implantation to occur (16). Upon reach- Management optionsing the blastocyst stage, physical expansion of the embryonic Chromosomal abnormalitymass along with the action of lysins produced by the cleaved Preimplantation genetic screening Comparative genomic hybridization arrayembryo 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 hardeningexpansion, resulting in impaired hatching, could contribute Assisted hatchingto RIF (15). Prolonged exposure of oocytes and embryos to Suboptimal culture Optimal culture mediaartiﬁcial culture conditions may also adversely affect the Blastocyst transferembryos ability to undergo normal hatching and could Cocultureimpair successful implantation (15). ZIFT Assessment of embryo quality and viability Time-lapse imaging—EmbryoScope MetabolomicsEmbryo Culture and ET Technique ProteomicsThe 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 identify1022 VOL. 97 NO. 5 / MAY 2012
Fertility and Sterility®been suggested that if oocyte maturation to the metaphase II producing a unique DNA ﬁngerprint for each embryo testedstage is completed just before the polar body biopsy, it may (33). However, a disadvantage of SNP microarrays is a lackresult in damage to the meiotic spindle of the oocyte (23). of diagnostic accuracy at individual SNP loci as well asCleavage stage biopsy is the most commonly used method high cost of microarrays and labeling techniques (33). In thefor 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 improvinghave a smaller risk of aneuploidy than embryo biopsy at the implantation and live birth rates in patients with RIF will havecleavage stage, because mosaic embryos have a higher pro- to be determined in well-designed prospective randomizedportion of aneuploid cells on day 2/3 and will not develop controlled trials before they can be widely applied in clinicalto the blastocyst stage (23). practice. Although initial studies suggested that PGS with FISHcould be used to achieve favorable implantation and PRs inpatients with RIF (6, 25), evidence from recent randomized Assisted Hatchingcontrolled trials does not support these ﬁndings (26, 27). In Elasticity and thinning of the ZP are essential prerequisites fora 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 forrates after IVF-ICSI in women with RIF. In this study, the in- implantation have reduced zona thickness (38). It has beenvestigators analyzed chromosomes 13, 16, 18, 21, 22, X, and suggested that an artiﬁcial opening made in the ZP mayY 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 partialbirth 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 ingroups (26, 27). A recent meta-analysis of randomized natural cycles (41). Embryos with artiﬁcial gaps in the zonacontrolled 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 vitrofailure, PGS signiﬁcantly lowered live birth rates after (42). It has also been proposed that breaching the integrityIVF (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 augmentof PGS are possible damage from the biopsy procedure, failure embryo development and blastocyst formation (43). It couldrate from the technique, limitations of the FISH analysis, and also serve as a channel for a two-way exchange across theembryo 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. Thesenot 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 PRsanalysis of single nucleotide polymorphisms (SNPs) (33, 34). and implantation rates in patients with RIF (49, 50), otherComparative genomic hybridization is a DNA-based method, investigators have not reported any advantage (46). Recentwhich is applicable to cells in any phase of the cell cycle (33). studies seem to suggest that assisted hatching may be ofThe CGH microarray enables a more comprehensive screening beneﬁt in selected patients. In a prospective randomizedof chromosomes. Many chromosomal aneuploidies identiﬁed study, comparing chemical removal of ZP from day 5using CGH would not have been detected using FISH for ﬁve in vitro cultured human embryos by using acidic Tyrodesor nine chromosomes (35). Microarrays have an advantage solution versus no removal, the implantation rate per ETover conventional CGH because the evaluation of ﬂuores- and the clinical PR were signiﬁcantly higher in the ZP-freecence ratios is simple, rapid, and easily automated (33). A group (51). Stein et al. (52) reported that assisted hatchingproof-of-principle study concluded that chromosomal aneu- by partial zona dissection resulted in a signiﬁcant increaseploidy of the oocyte can be accurately predicted by array in the implantation and clinical PRs in women older thanCGH 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 embryosprobes used for SNP microarrays provide genotype data in was signiﬁcantly higher than in those whose embryos wereaddition to chromosome copy number information, thereby not laser thinned. Interestingly, this difference was notVOL. 97 NO. 5 / MAY 2012 1023
VIEWS AND REVIEWSobserved 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 in761 participants), assisted hatching was reported to be associ- patients with RIF with an adequate ovarian response, transferated 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 multiplewith 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 thatPRs when performed in fresh embryos transferred to unse- the live birth rates and implantation rates per cycle werelected 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 uterinePRs in women with previous repeated implantation failure. receptivity may explain the additional beneﬁt of ET at theHowever, due to the small sample size of the included studies, blastocyst stage for couples with RIF (64). However, it shouldthis meta-analysis was not able to draw any conclusions re- be noted that a percentage of fertilized eggs will never reachgarding 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 CultureOptimum culture conditions are a prerequisite for satisfactory Stimulation Protocolsembryonic development and lack of these conditions maycontribute 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 inembryo to achieve the blastocyst stage because this leads to controlled ovarian hyperstimulation (COH) has been shown toa high implantation rate and PR. The suggested favorable ef- improve pregnancy outcome in patients with a history of RIFfects of cocultures include the secretion of embryotrophic fac- with GnRH agonist protocols. The investigators proposed thattors, such as nutrients and substrates, growth factors and this was most likely due to improvement of the quality of thecytokines, and the removal of free radicals and potentially blastocysts generated (65). Natural cycle IVF has also beenharmful 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 withproductive 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 ingranulosa cells (GC) (59–61), homologous endometrial cells this ﬁeld does not allow any recommendations to be madeappear to be the most promising coculture system (57). with regard to their efﬁcacy.Using coculture of embryos on homologous endometrialcells in patients with RIF, Jayot et al. (57) reported an Zygote Intrafallopian Transferoverall PR of 21% per transfer versus 8% in previous IVF-ET Zygote intrafallopian transfer (ZIFT) allows the early embryocycles. Similarly, using autologous endometrial coculture in to grow in the natural tubal environment and physiologicalpatients 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 studiesaddition, most IVF units do not have the necessary personnel implied that ZIFT may be of value in RIF (67), a subsequentor 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 trendBlastocyst Transfer toward increased risk of ectopic pregnancy (EP) with ZIFT (68). These ﬁndings led to the procedure being abandonedEmbryo transfer at the blastocyst stage has been proposed as by most units.a strategy to improve implantation rates and PRs in patientswith RIF. Blastocyst transfer is a more physiological approachas the human embryos usually enter the endometrial cavity 5 ET Techniquedays 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 betterselecting embryos with the best implantation potential. PRs and implantation rates after ET. Several techniquesTherefore 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 appear1024 VOL. 97 NO. 5 / MAY 2012
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 goodof 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 metaphaseII oocytes in patients with multiple implantation failure (76). CONCLUSIONThey 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 signiﬁcantzygotes 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 majorworldwide (77). It has been suggested that this procedure role in the etiology of RIF. Emerging technologies, such asmay 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 maybrane 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 astal and will require assessment of ooplasmic anomalies and time-lapse imaging and metabolomics, may help in betteroptimization 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, andNew Methods of Embryo Assessment practicality of new, emerging methods of treatment shouldAssessment of embryo quality is critical in selecting the best be evaluated in prospective randomized clinical trials beforeembryo(s) to transfer or cryopreserve. 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