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 orlive birth rates. Comparative genomic hybridization array and analysis of single nucleotide polymorphisms could enable amore 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 Steril� 2012;97:1021–7. �2012 by American Society for Reproductive Medicine.)Key Words: Implantation failure, IVF, embryo, oocyte, sperm, chromosome

D espite the immense strides thathave been made in the field ofIVF many patients still experi-

ence recurrent implantation failure.Besides causing immense distress tocouples who require multiple cycles oftreatment, it significantly increases thecost of the procedure. Recurrent implan-tation failure (RIF)may be defined as therepeated transfer of morphologicallygood embryos to a normal uterus with-out achieving successful implantationand a clinical pregnancy. Traditionally,failure to achieve a pregnancy after twoto six IVF cycles, in which more than10 high-grade embryoswere transferredto the uterus was defined as RIF (1).However, in most IVF programs, failureof three cycles in which reasonablygood embryos were transferred wouldwarrant investigation (2). In spite of op-timization of treatment protocols andhuge advancements in laboratory tech-nologies, the management of RIF posesa major challenge to clinicians and em-bryologists universally. The process ofembryo implantation in the uterus

Received February 2, 2012; accepted February 21, 20M.D. has nothing to disclose. H.E.G.H. has nothing tReprint requests: Hananel E. G. Holzer, M.D., Depart

ter, McGill Reproductive center, 687 Pine Aven(E-mail: hananel.holzer@muhc.mcgill.ca).

Fertility and Sterility® Vol. 97, No. 5, May 2012 0015Copyright ©2012 American Society for Reproductivedoi:10.1016/j.fertnstert.2012.02.029

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depends on the synchronization of var-ious factors such as the quality of theembryo, optimal culture conditions,the receptivity of the endometrium,and the maternal immune system. Theaim of this article is to review the estab-lished etiologies affecting embryo de-velopment in patients with RIF and toevaluate recent advances in oocyteand embryo selection, as well as currentrecommended management strategies.

ETIOLOGYChromosomal abnormalities, inade-quate culture conditions, suboptimalembryo development, zona hardening,and improper ET technique all play animportant role in the etiology of RIF.

Chromosomal Abnormality

It is now well established that a majorcause of repeated implantation failureafter IVF is a high frequency of chromo-somal aneuploidy. An increased inci-dence of chromosomal abnormalities,such as translocations, mosaicism,

12; published online March 15, 2012.o disclose.ment of Ob & Gyn, McGill University Heath Cen-ue West, Montreal, Quebec H4W 2A6, Canada

-0282/$36.00Medicine, Published by Elsevier Inc.

inversions, and deletions, have beendemonstrated in women with high-order RIF (3). In support of these find-ings, Stern et al. (4) observed an overallchromosomal abnormality rate of 2.5%(13/514) in patients with RIF. Mostof these abnormalities were chromo-somal translocations (reciprocal andRobertsonian). They proposed that bal-anced parental translocations may beimplicated in the pathogenesis of im-plantation failure in IVF, and that ge-netic evaluation should be consideredas part of the investigation of these cou-ples (4).

Aneuploid embryos have decreasedability to undergo successful implanta-tion and result in a viable pregnancy,but cannot be distinguished from nor-mal embryos using standard morpho-logical criteria. Data obtained fromembryos and oocytes of patients under-going preimplantation genetic screen-ing (PGS) because of advanced age,recurrent pregnancy losses, or multiplefailed IVF cycles, support the conceptthat many embryos and eggs obtainedduring IVF are intrinsically abnormaland thus fail to implant (5). Using fluo-rescence in-situ hybridization (FISH) onblastomeres from biopsied day 3 em-bryos for chromosomes 13, 16, 18, 21,22, X, and Y, Pehlivan et al. (6) foundthat there was a significantly higherrate of chromosomal abnormalities

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(67%) compared with controls (36%) in patients with three ormore failed IVF attempts. In another study, using comparativegenomic hybridization (CGH), Voullaire et al. (7) detectedchromosomal abnormalities in 76/126 (60%) single blasto-meres biopsied from embryos before implantation in 20women with RIF after IVF. The abnormalities detected in theirstudy included aneuploidy for one or two chromosomes aswell as complex chromosomal abnormality. They suggestedthat the disruption of the normal sequence of chromosomereplication and segregation in early human embryos, causedeither by maternal cytoplasmic factors or mutations in cellcycle control genes, may be a common cause of RIF.

A higher incidence of sperm chromosomal abnormalitiesin patients with normal karyotype and RIF has also beenreported. Pregnancy rates (PR) and implantation rates were re-ported to be significantly lower in patients with teratozoosper-mia. Rubio et al. (8) analyzed sperm aneuploidy and diploidyrates for chromosomes 13, 18, 21,X, andY inpatientswithnor-mal karyotypes using dual and triple-color FISH techniques.They reported an increased incidence of sex chromosome dis-omies in couples with RIF after intracytoplasmic sperm injec-tion (ICSI). In addition, centrosome anomalies resulting inchaotic mosaics were most likely of paternal origin (9, 10).

Evidence suggests that sperm DNA damage is associatedwith lower PRs after IUI and IVF (11). In addition, increasedlevels of sperm DNA damage have been linked with anincreased risk of pregnancy loss after IVF and ICSI (12). There-fore there is considerable evidence to suggest that chromo-somal abnormalities, both maternal and paternal, play a keyrole in the etiology of repeated implantation failure in IVF.

TABLE 1

Management options for factors affecting embryo development andimplantation in recurrent implantation failure.

Management options

Chromosomal abnormalityPreimplantation genetic screeningComparative genomic hybridization arraySingle nucleotide polymorphisms

Zona hardeningAssisted hatching

Suboptimal cultureOptimal culture mediaBlastocyst transferCocultureZIFT

Zona Hardening

The mammalian oocyte is surrounded by an acellular matrix,the zona pellucida (ZP), which is composed of glycoproteins,carbohydrates, and ZP-specific proteins (13). It plays a role insperm 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 facilitatesoviductal transport (15). The zona is required during earlycleavage stages to maintain the integrity of the inner cellmass (ICM), but it is usually shed during expansion of theblastocyst, allowing implantation to occur (16). Upon reach-ing the blastocyst stage, physical expansion of the embryonicmass along with the action of lysins produced by the cleavedembryo and/or the uterus, all play a role in zona hatching(17–19). Failure of the ZP to rupture after blastocystexpansion, resulting in impaired hatching, could contributeto RIF (15). Prolonged exposure of oocytes and embryos toartificial culture conditions may also adversely affect theembryo's ability to undergo normal hatching and couldimpair successful implantation (15).

Assessment of embryo quality and viabilityTime-lapse imaging—EmbryoScopeMetabolomicsProteomics

Improving ET techniqueNote: ZIFT ¼ zygote intrafallopian transfer.

Das. Recurrent implantation failure. Fertil Steril 2012.

Embryo Culture and ET Technique

The use of high quality, standardized culture media is funda-mental to the success of any IVF program. Inadequate cul-ture conditions could play a role in RIF. Assays to identify

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suboptimal components of a culture system that couldlead to impaired embryo development have been described(20). These include osmolality testing, pH measurements,and sperm bioassay (20). In some instances of RIF, individ-ualized specific culture conditions may be required for opti-mal embryo development.

Implantation rates and PRs after ET depend on the qualityand developmental potential of embryos selected for transfer.Suboptimal embryo quality has an adverse effect on implan-tation and PRs. Evidence suggests that ET technique caninfluence the success or failure of embryo implantation. Uter-ine contractions, blood or mucous on the catheter tip, endo-metrial trauma, and expulsion of embryos have all beenassociated with unsuccessful ETs (21).

MANAGEMENT OPTIONSVarious management options have been proposed to over-come the challenges of chromosomal abnormality and subop-timal embryo development. Table 1 shows the variousetiological factors contributing toward defective embryo de-velopment and their proposed management strategies.

Chromosomal Abnormality

In view of the higher incidence of chromosomal anomalies,parental karyotype is recommended as part of the work-upin RIF (4). Preimplantation genetic screening has also been in-creasingly used in the past decade, the rationale being that anincreased PR could be achieved by selecting only chromoso-mally normal embryos for transfer. The main biopsy methodsused for PGS include removal of one or two polar bodies fromthe unfertilized oocyte or the zygote, removal of one or twoblastomeres at the cleavage stage, or removal of several cellsat the blastocyst stage (22). Polar body biopsy analyses mater-nal causes of chromosomal abnormality and is an indirectmethod of screening for aneuploid embryos. It has, however,

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been suggested that if oocyte maturation to the metaphase IIstage is completed just before the polar body biopsy, it mayresult in damage to the meiotic spindle of the oocyte (23).Cleavage stage biopsy is the most commonly used methodfor screening preimplantation embryos for aneuploidy (24).However, cleavage stage embryos have an increased inci-dence of mosaicism (22). Biopsy at the blastocyst stage mayhave a smaller risk of aneuploidy than embryo biopsy at thecleavage stage, because mosaic embryos have a higher pro-portion of aneuploid cells on day 2/3 and will not developto the blastocyst stage (23).

Although initial studies suggested that PGS with FISHcould be used to achieve favorable implantation and PRs inpatients with RIF (6, 25), evidence from recent randomizedcontrolled trials does not support these findings (26, 27). Ina prospective randomized controlled trial, Blockeel et al.(26) observed that PGS did not increase the implantationrates after IVF-ICSI in women with RIF. In this study, the in-vestigators analyzed chromosomes 13, 16, 18, 21, 22, X, andY using FISH on blastomeres of day 3 cleavage stage embryosin the study group. There was a significant difference in livebirth rate between the PGS group (21%) and the control group(39%). The miscarriage rate did not differ between the twogroups (26, 27). A recent meta-analysis of randomizedcontrolled trials demonstrated that in women with advancedmaternal age as well as women with repeated implantationfailure, PGS significantly lowered live birth rates afterIVF (27).

The reasons that have been proposed for the inefficiencyof PGS are possible damage from the biopsy procedure, failurerate from the technique, limitations of the FISH analysis, andembryo mosaicism (27, 28). In addition, the efficacy of FISH islimited because only a few chromosomes can be detectedsimultaneously in a single biopsied cell. The lack ofusefulness of PGS may be because the tested blastomere isnot representative for the whole embryo (29). The AmericanSociety of Reproductive Medicine (ASRM), the EuropeanSociety of Human Reproduction and Embryology (ESHRE),and the British Fertility Society have concluded that PGSdoes not improve the live birth rates in patients with RIF,advanced maternal age, or recurrent pregnancy loss (30–32).

Alternative approaches have been proposed to overcomethe limitations of FISH for PGS. These include CGH or theanalysis of single nucleotide polymorphisms (SNPs) (33, 34).Comparative genomic hybridization is a DNA-based method,which is applicable to cells in any phase of the cell cycle (33).The CGHmicroarray enables a more comprehensive screeningof chromosomes. Many chromosomal aneuploidies identifiedusing CGH would not have been detected using FISH for fiveor nine chromosomes (35). Microarrays have an advantageover conventional CGH because the evaluation of fluores-cence ratios is simple, rapid, and easily automated (33). Aproof-of-principle study concluded that chromosomal aneu-ploidy of the oocyte can be accurately predicted by arrayCGH analysis of both polar bodies (36).

Single nucleotide polymorphisms are common polymor-phic DNA sequences found throughout the genome. Theprobes used for SNP microarrays provide genotype data inaddition to chromosome copy number information, thereby

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producing a unique DNA fingerprint for each embryo tested(33). However, a disadvantage of SNP microarrays is a lackof diagnostic accuracy at individual SNP loci as well ashigh cost of microarrays and labeling techniques (33). In thefuture, PGS-FISH may be replaced by comprehensive proce-dures such as array CGH and SNP microarrays. However,the efficacy and practicality of these procedures in improvingimplantation and live birth rates in patients with RIF will haveto be determined in well-designed prospective randomizedcontrolled trials before they can be widely applied in clinicalpractice.

Assisted Hatching

Elasticity and thinning of the ZP are essential prerequisites forsuccessful embryo hatching and implantation (15, 37). It hasbeen observed that cleaved embryos with a good prognosis forimplantation have reduced zona thickness (38). It has beensuggested that an artificial opening made in the ZP mayfacilitate the hatching process (39). Cohen et al. (40)observed a higher implantation rate per ET after partialzona dissection. The implantation window occurs 1–2 daysearlier in women undergoing ovarian stimulation than innatural cycles (41). Embryos with artificial gaps in the zonainitiate hatching earlier than zona intact embryos,compensating for the reduced development rate in vitro(42). It has also been proposed that breaching the integrityof the zona could enhance the transport of nutrients fromthe incubating media, which in turn would augmentembryo development and blastocyst formation (43). It couldalso serve as a channel for a two-way exchange across theZP of metabolites and growth factors (42).

The artificial rupture of the ZP is known as assisted hatch-ing and aims to improve implantation and clinical PRs. Var-ious techniques have been used to aid zona hatching. Theseinvolve the creation of an opening in the ZP either by me-chanical partial zona dissection (39), chemically by zona dril-ling with acid Tyrode (42), chemical zona thinning (44),enzymatic treatment (45), laser-assisted hatching (46, 47),or by using a piezo-micromanipulator (48).

The clinical relevance of assisted hatching procedures inthe management of RIF is controversial. Although some stud-ies have reported that assisted zona hatching improves PRsand implantation rates in patients with RIF (49, 50), otherinvestigators have not reported any advantage (46). Recentstudies seem to suggest that assisted hatching may be ofbenefit in selected patients. In a prospective randomizedstudy, comparing chemical removal of ZP from day 5in vitro cultured human embryos by using acidic Tyrode'ssolution versus no removal, the implantation rate per ETand the clinical PR were significantly higher in the ZP-freegroup (51). Stein et al. (52) reported that assisted hatchingby partial zona dissection resulted in a significant increasein the implantation and clinical PRs in women older than38 years with RIF. Similarly, Petersen et al. (53) observedthat for patients with repeated implantation failures, the im-plantation rate in those who received laser-thinned embryoswas significantly higher than in those whose embryos werenot laser thinned. Interestingly, this difference was not

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observed in patients with a history of only one previous im-plantation failure. In support of these findings, in a recentmeta-analysis of randomized control trials (five trials with761 participants), assisted hatching was reported to be associ-ated with a significant improvement in clinical pregnancywhen performed in fresh embryos transferred to womenwith RIF (relative risk [RR] ¼ 1.73; 95% confidence interval[CI] ¼ 1.37–2.17) (54). No increase was observed in clinicalPRs when performed in fresh embryos transferred to unse-lected or nonpoor prognosis women or to women of advancedage. Assisted hatching was also related to increased multiplePRs in women with previous repeated implantation failure.However, due to the small sample size of the included studies,this meta-analysis was not able to draw any conclusions re-garding live birth or miscarriage rates (54).

Embryo Culture

Optimum culture conditions are a prerequisite for satisfactoryembryonic development and lack of these conditions maycontribute to RIF. Various coculture systems have been devel-oped as a means of improving embryo culture conditions. Themain aim is to increase the metabolic chances of the humanembryo to achieve the blastocyst stage because this leads toa high implantation rate and PR. The suggested favorable ef-fects of cocultures include the secretion of embryotrophic fac-tors, such as nutrients and substrates, growth factors andcytokines, and the removal of free radicals and potentiallyharmful substances (55). Although multiple cell types havebeen used for coculturing embryos, ranging from human re-productive tissues, such as oviducts (56), endometrium (57),sequential oviduct-endometrial coculture (58), and cumulus-granulosa cells (GC) (59–61), homologous endometrial cellsappear to be the most promising coculture system (57).Using coculture of embryos on homologous endometrialcells in patients with RIF, Jayot et al. (57) reported anoverall PR of 21% per transfer versus 8% in previous IVF-ETcycles. Similarly, using autologous endometrial coculture inpatients with RIF, Spandorfer et al. (62) reported a significantimprovement in embryo quality and clinical PRs. However,the advantage of coculture systems remains controversial. Inaddition, most IVF units do not have the necessary personnelor facilities to perform coculture on a regular basis.

Blastocyst Transfer

Embryo transfer at the blastocyst stage has been proposed asa 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 5days after fertilization, at the morula-blastocyst stage in nat-ural conception cycles (2). Better embryo selection for transferand improved endometrial receptivity are obvious advantagesof this approach. Some clinicians transfer several embryosafter RIF. Culturing embryos to the blastocyst stage helps inselecting embryos with the best implantation potential.Therefore fewer embryos have to be transferred to achievea successful pregnancy, thereby decreasing the risk of multi-ple pregnancy. With single ET becoming the norm in younger

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patients, selecting the best embryos by culturing to the blas-tocyst stage assumes even greater significance. In a prospec-tive randomized study, Levitas et al. (63) reported that inpatients with RIF with an adequate ovarian response, transferof blastocyst stage embryos carries a significantly higher im-plantation rate compared with ET on days 2–3. The multiplePR was not significantly different between the two groups(63). In another study, Guerif et al. (64) also observed thatthe live birth rates and implantation rates per cycle werehigher after blastocyst transfer compared with day 2 ET.They suggested that improved embryo selection and uterinereceptivity may explain the additional benefit of ET at theblastocyst stage for couples with RIF (64). However, it shouldbe noted that a percentage of fertilized eggs will never reachthe blastocyst stage. Proper selection of cases suitable forblastocyst transfer is therefore critical to reduce the numberof cycle cancellations (63).

Stimulation Protocols

Variations in ovarian stimulation protocols have been sug-gested in some studies as ameans of improving embryo devel-opment and quality. The use of GnRH antagonist protocols incontrolled ovarian hyperstimulation (COH) has been shown toimprove pregnancy outcome in patients with a history of RIFwith GnRH agonist protocols. The investigators proposed thatthis was most likely due to improvement of the quality of theblastocysts generated (65). Natural cycle IVF has also beenproposed as a means of improving implantation rates in pa-tients with RIF (66). Despite some personal experience withnatural cycle IVF and in vitro maturation of oocytes in pa-tients with RIF, the lack of randomized clinical studies inthis field does not allow any recommendations to be madewith regard to their efficacy.

Zygote Intrafallopian Transfer

Zygote intrafallopian transfer (ZIFT) allows the early embryoto grow in the natural tubal environment and physiologicaltransport of the embryos into the uterine cavity. It also over-comes the problem of technically difficult ET because of cer-vical stenosis (2). Although initial nonrandomized studiesimplied that ZIFT may be of value in RIF (67), a subsequentmeta-analysis of randomized controlled trials failed to dem-onstrate any benefit for ZIFT (68). In fact, there was a trendtoward increased risk of ectopic pregnancy (EP) with ZIFT(68). These findings led to the procedure being abandonedby most units.

ET Technique

Ameticulous ET technique is of utmost importance in achiev-ing a successful pregnancy outcome. Studies show that avoid-ance of blood (69), mucus (70), bacterial contamination,trauma to the endometrium, touching the fundus, and exces-sive uterine contractions (71) are all associated with betterPRs and implantation rates after ET. Several techniqueshave been proposed to optimize the technique of ET. Methods,such as a trial transfer (72), filled bladder (73), ultrasono-graphic guidance (74), and use of soft catheters, all appear

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to facilitate a successful ET (21), whereas bed rest after ET hasnot been shown to be of any benefit (75).

Cytoplasmic Transfer

Ooplasmic factors play a role in the continued developmentof the zygote, especially during the early cleavage stage. Co-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).They noted that this led to an improvement in embryo mor-phology. Cytoplasmic transfer from fertile donor oocytes orzygotes into developmentally compromised oocytes from pa-tients with RIF has led to the birth of several healthy babiesworldwide (77). It has been suggested that this proceduremay correct an imbalance between anti- and pro-apoptoticfactors and/or correction of defective mitochondrial mem-brane potential (78). However, the transferred cytoplasmcould contain messenger RNAs, proteins. and mitochondria(77). In addition, it is not known whether the physiology ofthe early embryo is affected. The procedure is still experimen-tal and will require assessment of ooplasmic anomalies andoptimization of techniques before it can be applied in clinicalpractice.

New Methods of Embryo Assessment

Assessment of embryo quality is critical in selecting the bestembryo(s) to transfer or cryopreserve. As visual assessmentof embryo quality using morphological criteria can be subjec-tive and requires considerable expertise, newer methods of as-sessing embryo quality and viability are being developed.Emerging techniques such as time-lapse imaging may leadto better assessment of embryo quality and help in selectingembryos with the highest implantation potential. It hasbeen suggested that time-lapse observations using an incuba-tor with an integrated optical microscope may minimize thechanges in the culturing environment by integrating the cul-ture, observation, and time-lapse recording of cells into onesystem. The removal of embryos from the incubator for inter-mittent observation can therefore be avoided while enablingthe continuous monitoring of embryo development (79).There is evidence that time-lapse monitoring in the Embryo-Scope (Unisense FertiliTech) does not impair embryo qualitywhile allowing for morphological and spatial analysis of em-bryo development (80). However, besides being more expen-sive than standard incubators, the culture preparationprocedure is more time consuming compared with conven-tional culture methods (81).

Different approaches are also being developed to test theculture environment of a developing embryo to gain impor-tant information regarding its viability. Metabolomic analysisof follicular fluid (FF) can provide valuable information aboutindividual oocyte maturation and developmental potential.Various methods have been described, which include mea-surement of oxygen (81), pyruvate, and glucose consumptionby the embryo in the culture medium (82). Amino acidturnover, which appears to be correlated to blastocystdevelopment, can be measured as an indication of embryo

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viability (83). Newer methods, such as vibrational spectros-copy, both Raman and near infrared, have been used to ana-lyze spent culture medium from human embryos, measuringbonds within functional groups of molecules at specific wave-lengths. Results from initial studies indicate that spectral pro-files reflective of oxidative stress appear to have a goodcorrelation with pregnancy outcome (84).

CONCLUSIONRegardless of the considerable improvement in treatment pro-tocols and laboratory technologies, RIF still poses a significantchallenge to clinicians and embryologists. Chromosomal ab-normalities and suboptimal embryo development play amajorrole in the etiology of RIF. Emerging technologies, such asCGH array and analysis of SNPs could enable a more compre-hensive screening of chromosomes. Assisted hatching mayhelp to overcome zona hardening in selected patients. Opti-mal culture conditions and blastocyst transfer may contributetoward improving the implantation rates and PRs in RIF.Novel embryo assessment and selection procedures, such astime-lapse imaging and metabolomics, may help in betterevaluation of embryo quality and viability and help in select-ing embryos with the highest implantation potential. It shouldbe noted that only those treatment options that are evidencebased should be offered to patients. The safety, efficacy, andpracticality of new, emerging methods of treatment shouldbe evaluated in prospective randomized clinical trials beforebeing accepted in clinical practice.

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