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Recurrent implantation failure: gamete and embryo factors
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 significant 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 have been made in the field of IVF many patients still experience recurrent implantation failure. Besides causing immense distress to couples who require multiple cycles of treatment, it significantly increases the cost of the procedure. Recurrent implantation failure (RIF) may be defined as the repeated transfer of morphologically good embryos to a normal uterus without achieving successful implantation and a clinical pregnancy. Traditionally, failure to achieve a pregnancy after two to six IVF cycles, in which more than 10 high-grade embryos were transferred to the uterus was defined as RIF (1). However, in most IVF programs, failure of three cycles in which reasonably good embryos were transferred would warrant investigation (2). In spite of optimization of treatment protocols and huge advancements in laboratory technologies, the management of RIF poses a major challenge to clinicians and embryologists universally. The process of embryo implantation in the uterus
depends on the synchronization of various factors such as the quality of the embryo, optimal culture conditions, the receptivity of the endometrium, and the maternal immune system. The aim of this article is to review the established etiologies affecting embryo development in patients with RIF and to evaluate recent advances in oocyte and embryo selection, as well as current recommended management strategies.
ETIOLOGY Chromosomal abnormalities, inadequate culture conditions, suboptimal embryo development, zona hardening, and improper ET technique all play an important role in the etiology of RIF.
Chromosomal Abnormality It is now well established that a major cause of repeated implantation failure after IVF is a high frequency of chromosomal aneuploidy. An increased incidence of chromosomal abnormalities, such as translocations, mosaicism,
Received February 2, 2012; accepted February 21, 2012; published online March 15, 2012. M.D. has nothing to disclose. H.E.G.H. has nothing to disclose. Reprint requests: Hananel E. G. Holzer, M.D., Department of Ob & Gyn, McGill University Heath Center, McGill Reproductive center, 687 Pine Avenue West, Montreal, Quebec H4W 2A6, Canada (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.029 VOL. 97 NO. 5 / MAY 2012
inversions, and deletions, have been demonstrated in women with highorder RIF (3). In support of these findings, Stern et al. (4) observed an overall chromosomal abnormality rate of 2.5% (13/514) in patients with RIF. Most of these abnormalities were chromosomal translocations (reciprocal and Robertsonian). They 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 (4). Aneuploid embryos have decreased ability to undergo successful implantation and result in a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological criteria. Data obtained from embryos and oocytes of patients undergoing preimplantation genetic screening (PGS) because of advanced age, recurrent pregnancy losses, or multiple failed IVF cycles, support the concept that many embryos and eggs obtained during IVF are intrinsically abnormal and thus fail to implant (5). Using fluorescence in-situ hybridization (FISH) on blastomeres from biopsied day 3 embryos for chromosomes 13, 16, 18, 21, 22, X, and Y, Pehlivan et al. (6) found that there was a significantly higher rate of chromosomal abnormalities 1021
VIEWS AND REVIEWS (67%) compared with controls (36%) in patients with three or more failed IVF attempts. In another study, using comparative genomic hybridization (CGH), Voullaire et al. (7) detected chromosomal abnormalities in 76/126 (60%) single blastomeres biopsied from embryos before implantation in 20 women with RIF after IVF. The abnormalities detected in their study included aneuploidy for one or two chromosomes as well as complex chromosomal abnormality. They suggested that the disruption of the normal sequence of chromosome replication and segregation in early human embryos, caused either by maternal cytoplasmic factors or mutations in cell cycle control genes, may be a common cause of RIF. A higher incidence of sperm chromosomal abnormalities in patients with normal karyotype and RIF has also been reported. Pregnancy rates (PR) and implantation rates were reported to be significantly lower in patients with teratozoospermia. Rubio et al. (8) analyzed sperm aneuploidy and diploidy rates for chromosomes 13, 18, 21, X, and Y in patients with normal karyotypes using dual and triple-color FISH techniques. They reported an increased incidence of sex chromosome disomies in couples with RIF after intracytoplasmic sperm injection (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 with lower PRs after IUI and IVF (11). In addition, increased levels of sperm DNA damage have been linked with an increased risk of pregnancy loss after IVF and ICSI (12). Therefore there is considerable evidence to suggest that chromosomal abnormalities, both maternal and paternal, play a key role in the etiology of repeated implantation failure in IVF.
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 in sperm binding, induction of the acrosome reaction, and promotes sperm–egg fusion (14). The zona hardens naturally after fertilization to prevent polyspermic fertilization, protects the integrity of the preimplantation embryo, and facilitates oviductal transport (15). The zona is required during early cleavage stages to maintain the integrity of the inner cell mass (ICM), but it is usually shed during expansion of the blastocyst, allowing implantation to occur (16). Upon reaching the blastocyst stage, physical expansion of the embryonic mass along with the action of lysins produced by the cleaved embryo and/or the uterus, all play a role in zona hatching (17–19). Failure of the ZP to rupture after blastocyst expansion, resulting in impaired hatching, could contribute to RIF (15). Prolonged exposure of oocytes and embryos to artificial culture conditions may also adversely affect the embryo's ability to undergo normal hatching and could impair successful implantation (15).
Embryo Culture and ET Technique The use of high quality, standardized culture media is fundamental to the success of any IVF program. Inadequate culture conditions could play a role in RIF. Assays to identify 1022
suboptimal components of a culture system that could lead to impaired embryo development have been described (20). These include osmolality testing, pH measurements, and sperm bioassay (20). In some instances of RIF, individualized specific culture conditions may be required for optimal embryo development. Implantation rates and PRs after ET depend on the quality and developmental potential of embryos selected for transfer. Suboptimal embryo quality has an adverse effect on implantation and PRs. Evidence suggests that ET technique can influence the success or failure of embryo implantation. Uterine contractions, blood or mucous on the catheter tip, endometrial trauma, and expulsion of embryos have all been associated with unsuccessful ETs (21).
MANAGEMENT OPTIONS Various management options have been proposed to overcome the challenges of chromosomal abnormality and suboptimal embryo development. Table 1 shows the various etiological factors contributing toward defective embryo development 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-up in RIF (4). Preimplantation genetic screening has also been increasingly used in the past decade, the rationale being that an increased PR could be achieved by selecting only chromosomally 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 blastomeres at the cleavage stage, or removal of several cells at the blastocyst stage (22). Polar body biopsy analyses maternal causes of chromosomal abnormality and is an indirect method of screening for aneuploid embryos. It has, however,
TABLE 1 Management options for factors affecting embryo development and implantation in recurrent implantation failure. Management options Chromosomal abnormality Preimplantation genetic screening Comparative genomic hybridization array Single nucleotide polymorphisms Zona hardening Assisted hatching Suboptimal culture Optimal culture media Blastocyst transfer Coculture ZIFT Assessment of embryo quality and viability Time-lapse imaging—EmbryoScope Metabolomics Proteomics Improving ET technique Note: ZIFT ¼ zygote intrafallopian transfer. Das. Recurrent implantation failure. Fertil Steril 2012.
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Fertility and Sterility® been suggested that if oocyte maturation to the metaphase II stage is completed just before the polar body biopsy, it may result in damage to the meiotic spindle of the oocyte (23). Cleavage stage biopsy is the most commonly used method for screening preimplantation embryos for aneuploidy (24). However, cleavage stage embryos have an increased incidence of mosaicism (22). Biopsy at the blastocyst stage may have a smaller risk of aneuploidy than embryo biopsy at the cleavage stage, because mosaic embryos have a higher proportion of aneuploid cells on day 2/3 and will not develop to the blastocyst stage (23). 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 controlled trials does not support these findings (26, 27). In a prospective randomized controlled trial, Blockeel et al. (26) observed that PGS did not increase the implantation rates after IVF-ICSI in women with RIF. In this study, the investigators analyzed chromosomes 13, 16, 18, 21, 22, X, and Y using FISH on blastomeres of day 3 cleavage stage embryos in the study group. There was a significant difference in live birth rate between the PGS group (21%) and the control group (39%). The miscarriage rate did not differ between the two groups (26, 27). A recent meta-analysis of randomized controlled trials demonstrated that in women with advanced maternal age as well as women with repeated implantation failure, PGS significantly lowered live birth rates after IVF (27). The reasons that have been proposed for the inefficiency of PGS are possible damage from the biopsy procedure, failure rate from the technique, limitations of the FISH analysis, and embryo mosaicism (27, 28). In addition, the efficacy of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. The lack of usefulness of PGS may be because the tested blastomere is not representative for the whole embryo (29). The American Society of Reproductive Medicine (ASRM), the European Society of Human Reproduction and Embryology (ESHRE), and the British Fertility Society have concluded that PGS does 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 overcome the limitations of FISH for PGS. These include CGH or the analysis 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 CGH microarray enables a more comprehensive screening of chromosomes. Many chromosomal aneuploidies identified using CGH would not have been detected using FISH for five or nine chromosomes (35). Microarrays have an advantage over conventional CGH because the evaluation of fluorescence ratios is simple, rapid, and easily automated (33). A proof-of-principle study concluded that chromosomal aneuploidy of the oocyte can be accurately predicted by array CGH analysis of both polar bodies (36). Single nucleotide polymorphisms are common polymorphic DNA sequences found throughout the genome. The probes used for SNP microarrays provide genotype data in addition to chromosome copy number information, thereby VOL. 97 NO. 5 / MAY 2012
producing a unique DNA fingerprint for each embryo tested (33). However, a disadvantage of SNP microarrays is a lack of diagnostic accuracy at individual SNP loci as well as high cost of microarrays and labeling techniques (33). In the future, PGS-FISH may be replaced by comprehensive procedures such as array CGH and SNP microarrays. However, the efficacy and practicality of these procedures in improving implantation and live birth rates in patients with RIF will have to be determined in well-designed prospective randomized controlled trials before they can be widely applied in clinical practice.
Assisted Hatching Elasticity and thinning of the ZP are essential prerequisites for successful embryo hatching and implantation (15, 37). It has been observed that cleaved embryos with a good prognosis for implantation have reduced zona thickness (38). It has been suggested that an artificial opening made in the ZP may facilitate the hatching process (39). Cohen et al. (40) observed a higher implantation rate per ET after partial zona dissection. The implantation window occurs 1–2 days earlier in women undergoing ovarian stimulation than in natural cycles (41). Embryos with artificial gaps in the zona initiate hatching earlier than zona intact embryos, compensating for the reduced development rate in vitro (42). It has also been proposed that breaching the integrity of the zona could enhance the transport of nutrients from the incubating media, which in turn would augment embryo development and blastocyst formation (43). It could also serve as a channel for a two-way exchange across the ZP of metabolites and growth factors (42). The artificial rupture of the ZP is known as assisted hatching and aims to improve implantation and clinical PRs. Various techniques have been used to aid zona hatching. These involve the creation of an opening in the ZP either by mechanical partial zona dissection (39), chemically by zona drilling 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 in the management of RIF is controversial. Although some studies have reported that assisted zona hatching improves PRs and implantation rates in patients with RIF (49, 50), other investigators have not reported any advantage (46). Recent studies seem to suggest that assisted hatching may be of benefit in selected patients. In a prospective randomized study, comparing chemical removal of ZP from day 5 in vitro cultured human embryos by using acidic Tyrode's solution versus no removal, the implantation rate per ET and the clinical PR were significantly higher in the ZP-free group (51). Stein et al. (52) reported that assisted hatching by partial zona dissection resulted in a significant increase in the implantation and clinical PRs in women older than 38 years with RIF. Similarly, Petersen et al. (53) observed that for patients with repeated implantation failures, the implantation rate in those who received laser-thinned embryos was significantly higher than in those whose embryos were not laser thinned. Interestingly, this difference was not 1023
VIEWS AND REVIEWS observed in patients with a history of only one previous implantation failure. In support of these findings, in a recent meta-analysis of randomized control trials (five trials with 761 participants), assisted hatching was reported to be associated with a significant improvement in clinical pregnancy when performed in fresh embryos transferred to women with RIF (relative risk [RR] ¼ 1.73; 95% confidence interval [CI] ¼ 1.37–2.17) (54). No increase was observed in clinical PRs when performed in fresh embryos transferred to unselected or nonpoor prognosis women or to women of advanced age. Assisted hatching was also related to increased multiple PRs 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 regarding live birth or miscarriage rates (54).
Embryo Culture Optimum culture conditions are a prerequisite for satisfactory embryonic development and lack of these conditions may contribute to RIF. Various coculture systems have been developed as a means of improving embryo culture conditions. The main aim is to increase the metabolic chances of the human embryo to achieve the blastocyst stage because this leads to a high implantation rate and PR. The suggested favorable effects of cocultures include the secretion of embryotrophic factors, such as nutrients and substrates, growth factors and cytokines, and the removal of free radicals and potentially harmful substances (55). Although multiple cell types have been used for coculturing embryos, ranging from human reproductive tissues, such as oviducts (56), endometrium (57), sequential oviduct-endometrial coculture (58), and cumulusgranulosa cells (GC) (59–61), homologous endometrial cells appear to be the most promising coculture system (57). Using coculture of embryos on homologous endometrial cells in patients with RIF, Jayot et al. (57) reported an overall PR of 21% per transfer versus 8% in previous IVF-ET cycles. Similarly, using autologous endometrial coculture in patients with RIF, Spandorfer et al. (62) reported a significant improvement in embryo quality and clinical PRs. However, the advantage of coculture systems remains controversial. In addition, most IVF units do not have the necessary personnel or facilities to perform coculture on a regular basis.
Blastocyst Transfer Embryo transfer at the blastocyst stage has been proposed as 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 days after fertilization, at the morula-blastocyst stage in natural conception cycles (2). Better embryo selection for transfer and improved endometrial receptivity are obvious advantages of this approach. Some clinicians transfer several embryos after RIF. Culturing embryos to the blastocyst stage helps in selecting embryos with the best implantation potential. Therefore fewer embryos have to be transferred to achieve a successful pregnancy, thereby decreasing the risk of multiple pregnancy. With single ET becoming the norm in younger 1024
patients, selecting the best embryos by culturing to the blastocyst stage assumes even greater significance. In a prospective randomized study, Levitas et al. (63) reported that in patients with RIF with an adequate ovarian response, transfer of blastocyst stage embryos carries a significantly higher implantation rate compared with ET on days 2–3. The multiple PR was not significantly different between the two groups (63). In another study, Guerif et al. (64) also observed that the live birth rates and implantation rates per cycle were higher after blastocyst transfer compared with day 2 ET. They suggested that improved embryo selection and uterine receptivity may explain the additional benefit of ET at the blastocyst stage for couples with RIF (64). However, it should be noted that a percentage of fertilized eggs will never reach the blastocyst stage. Proper selection of cases suitable for blastocyst transfer is therefore critical to reduce the number of cycle cancellations (63).
Stimulation Protocols Variations in ovarian stimulation protocols have been suggested in some studies as a means of improving embryo development and quality. The use of GnRH antagonist protocols in controlled ovarian hyperstimulation (COH) has been shown to improve pregnancy outcome in patients with a history of RIF with GnRH agonist protocols. The investigators proposed that this was most likely due to improvement of the quality of the blastocysts generated (65). Natural cycle IVF has also been proposed as a means of improving implantation rates in patients with RIF (66). Despite some personal experience with natural cycle IVF and in vitro maturation of oocytes in patients with RIF, the lack of randomized clinical studies in this field does not allow any recommendations to be made with regard to their efficacy.
Zygote Intrafallopian Transfer Zygote intrafallopian transfer (ZIFT) allows the early embryo to grow in the natural tubal environment and physiological transport of the embryos into the uterine cavity. It also overcomes the problem of technically difficult ET because of cervical stenosis (2). Although initial nonrandomized studies implied that ZIFT may be of value in RIF (67), a subsequent meta-analysis of randomized controlled trials failed to demonstrate any benefit for ZIFT (68). In fact, there was a trend toward increased risk of ectopic pregnancy (EP) with ZIFT (68). These findings led to the procedure being abandoned by most units.
ET Technique A meticulous ET technique is of utmost importance in achieving a successful pregnancy outcome. Studies show that avoidance of blood (69), mucus (70), bacterial contamination, trauma to the endometrium, touching the fundus, and excessive uterine contractions (71) are all associated with better PRs and implantation rates after ET. Several techniques have been proposed to optimize the technique of ET. Methods, such as a trial transfer (72), filled bladder (73), ultrasonographic guidance (74), and use of soft catheters, all appear VOL. 97 NO. 5 / MAY 2012
Fertility and Sterility® to facilitate a successful ET (21), whereas bed rest after ET has not been shown to be of any benefit (75).
Cytoplasmic Transfer Ooplasmic factors play a role in the continued development of the zygote, especially during the early cleavage stage. Cohen et al. (76) transferred ooplasm from donor eggs at metaphase II stage into developmentally compromised metaphase II oocytes in patients with multiple implantation failure (76). They noted that this led to an improvement in embryo morphology. Cytoplasmic transfer from fertile donor oocytes or zygotes into developmentally compromised oocytes from patients with RIF has led to the birth of several healthy babies worldwide (77). It has been suggested that this procedure may correct an imbalance between anti- and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential (78). However, the transferred cytoplasm could contain messenger RNAs, proteins. and mitochondria (77). In addition, it is not known whether the physiology of the early embryo is affected. The procedure is still experimental and will require assessment of ooplasmic anomalies and optimization of techniques before it can be applied in clinical practice.
New Methods of Embryo Assessment Assessment of embryo quality is critical in selecting the best embryo(s) to transfer or cryopreserve. As visual assessment of embryo quality using morphological criteria can be subjective and requires considerable expertise, newer methods of assessing embryo quality and viability are being developed. Emerging techniques such as time-lapse imaging may lead to better assessment of embryo quality and help in selecting embryos with the highest implantation potential. It has been suggested that time-lapse observations using an incubator with an integrated optical microscope may minimize the changes in the culturing environment by integrating the culture, observation, and time-lapse recording of cells into one system. The removal of embryos from the incubator for intermittent observation can therefore be avoided while enabling the continuous monitoring of embryo development (79). There is evidence that time-lapse monitoring in the EmbryoScope (Unisense FertiliTech) does not impair embryo quality while allowing for morphological and spatial analysis of embryo development (80). However, besides being more expensive than standard incubators, the culture preparation procedure is more time consuming compared with conventional culture methods (81). Different approaches are also being developed to test the culture environment of a developing embryo to gain important information regarding its viability. Metabolomic analysis of follicular fluid (FF) can provide valuable information about individual oocyte maturation and developmental potential. Various methods have been described, which include measurement of oxygen (81), pyruvate, and glucose consumption by the embryo in the culture medium (82). Amino acid turnover, which appears to be correlated to blastocyst development, can be measured as an indication of embryo VOL. 97 NO. 5 / MAY 2012
viability (83). Newer methods, such as vibrational spectroscopy, both Raman and near infrared, have been used to analyze spent culture medium from human embryos, measuring bonds within functional groups of molecules at specific wavelengths. Results from initial studies indicate that spectral profiles reflective of oxidative stress appear to have a good correlation with pregnancy outcome (84).
CONCLUSION Regardless of the considerable improvement in treatment protocols and laboratory technologies, RIF still poses a significant challenge to clinicians and embryologists. Chromosomal abnormalities and suboptimal embryo development play a major role in the etiology of RIF. Emerging technologies, such as CGH array and analysis of SNPs could enable a more comprehensive screening of chromosomes. Assisted hatching may help to overcome zona hardening in selected patients. Optimal culture conditions and blastocyst transfer may contribute toward improving the implantation rates and PRs in RIF. 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. It should be noted that only those treatment options that are evidence based should be offered to patients. The safety, efficacy, and practicality of new, emerging methods of treatment should be evaluated in prospective randomized clinical trials before being accepted in clinical practice.
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Habana AE, Palter SF. Is tubal embryo transfer of any value? A meta-analysis and comparison with the Society for Assisted Reproductive Technology database. Fertil Steril 2001;76:286–93. Goudas VT, Hammitt DG, Damario MA, Session DR, Singh AP, Dumesic DA. Blood on the embryo transfer catheter is associated with decreased rates of embryo implantation and clinical pregnancy with the use of in vitro fertilization–embryo transfer. Fertil Steril 1998;70:878–82. Egbase PE, al-Sharhan M, al-Othman S, al-Mutawa M, Udo EE, Grudzinskas JG. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in-vitro fertilization and embryo transfer. Hum Reprod 1996;11:1687–9. Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer— can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998;13:1540–6. Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization. Fertil Steril 1990;54:678–81. Sundstrom P, Wramsby H, Persson PH, Liedholm P. Filled bladder simplifies human embryo transfer. Br J Obstet Gynaecol 1984;91:506–7. Hurley VA, Osborn JC, Leoni MA, Leeton J. Ultrasound-guided embryo transfer: a controlled trial. Fertil Steril 1991;55:559–62. Botta G, Grudzinskas G. Is a prolonged bed rest following embryo transfer useful? Hum Reprod 1997;12:2489–92. Cohen J, Scott R, Alikani M, Schimmel T, Munne S, Levron J, et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 1998;4:269–80. Barritt JA, Willadsen S, Brenner C, Cohen J. Cytoplasmic transfer in assisted reproduction. Hum Reprod Update 2001;7:428–35. Levy R, Elder K, Menezo Y. Cytoplasmic transfer in oocytes: biochemical aspects. Hum Reprod Update 2004;10:241–50. Nakahara T, Iwase A, Goto M, Harata T, Suzuki M, Ienaga M, et al. Evaluation of the safety of time-lapse observations for human embryos. J Assist Reprod Genet 2010;27:93–6. Cruz M, Gadea B, Garrido N, Pedersen KS, Martinez M, Perez-Cano I, et al. Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging. J Assist Reprod Genet 2011;28:569–73. Nel-Themaat L, Nagy ZP. A review of the promises and pitfalls of oocyte and embryo metabolomics. Placenta 2011;32(Suppl 3):S257–63. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001;76:1175–80. Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002;17:999–1005. Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and nearinfrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007;88:1350–7.
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Chromosomal abnormalities, sperm DNA damage, zona hardening, inadequate culture conditions, and suboptimal embryo development all play a significant 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 have been made in the field of IVF many patients still experience recurrent implantation failure. Besides causing immense distress to couples who require multiple cycles of treatment, it significantly increases the cost of the procedure. Recurrent implantation failure (RIF) may be defined as the repeated transfer of morphologically good embryos to a normal uterus without achieving successful implantation and a clinical pregnancy. Traditionally, failure to achieve a pregnancy after two to six IVF cycles, in which more than 10 high-grade embryos were transferred to the uterus was defined as RIF (1). However, in most IVF programs, failure of three cycles in which reasonably good embryos were transferred would warrant investigation (2). In spite of optimization of treatment protocols and huge advancements in laboratory technologies, the management of RIF poses a major challenge to clinicians and embryologists universally. The process of embryo implantation in the uterus
depends on the synchronization of various factors such as the quality of the embryo, optimal culture conditions, the receptivity of the endometrium, and the maternal immune system. The aim of this article is to review the established etiologies affecting embryo development in patients with RIF and to evaluate recent advances in oocyte and embryo selection, as well as current recommended management strategies.
ETIOLOGY Chromosomal abnormalities, inadequate culture conditions, suboptimal embryo development, zona hardening, and improper ET technique all play an important role in the etiology of RIF.
Chromosomal Abnormality It is now well established that a major cause of repeated implantation failure after IVF is a high frequency of chromosomal aneuploidy. An increased incidence of chromosomal abnormalities, such as translocations, mosaicism,
Received February 2, 2012; accepted February 21, 2012; published online March 15, 2012. M.D. has nothing to disclose. H.E.G.H. has nothing to disclose. Reprint requests: Hananel E. G. Holzer, M.D., Department of Ob & Gyn, McGill University Heath Center, McGill Reproductive center, 687 Pine Avenue West, Montreal, Quebec H4W 2A6, Canada (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.02.029 VOL. 97 NO. 5 / MAY 2012
inversions, and deletions, have been demonstrated in women with highorder RIF (3). In support of these findings, Stern et al. (4) observed an overall chromosomal abnormality rate of 2.5% (13/514) in patients with RIF. Most of these abnormalities were chromosomal translocations (reciprocal and Robertsonian). They 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 (4). Aneuploid embryos have decreased ability to undergo successful implantation and result in a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological criteria. Data obtained from embryos and oocytes of patients undergoing preimplantation genetic screening (PGS) because of advanced age, recurrent pregnancy losses, or multiple failed IVF cycles, support the concept that many embryos and eggs obtained during IVF are intrinsically abnormal and thus fail to implant (5). Using fluorescence in-situ hybridization (FISH) on blastomeres from biopsied day 3 embryos for chromosomes 13, 16, 18, 21, 22, X, and Y, Pehlivan et al. (6) found that there was a significantly higher rate of chromosomal abnormalities 1021
VIEWS AND REVIEWS (67%) compared with controls (36%) in patients with three or more failed IVF attempts. In another study, using comparative genomic hybridization (CGH), Voullaire et al. (7) detected chromosomal abnormalities in 76/126 (60%) single blastomeres biopsied from embryos before implantation in 20 women with RIF after IVF. The abnormalities detected in their study included aneuploidy for one or two chromosomes as well as complex chromosomal abnormality. They suggested that the disruption of the normal sequence of chromosome replication and segregation in early human embryos, caused either by maternal cytoplasmic factors or mutations in cell cycle control genes, may be a common cause of RIF. A higher incidence of sperm chromosomal abnormalities in patients with normal karyotype and RIF has also been reported. Pregnancy rates (PR) and implantation rates were reported to be significantly lower in patients with teratozoospermia. Rubio et al. (8) analyzed sperm aneuploidy and diploidy rates for chromosomes 13, 18, 21, X, and Y in patients with normal karyotypes using dual and triple-color FISH techniques. They reported an increased incidence of sex chromosome disomies in couples with RIF after intracytoplasmic sperm injection (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 with lower PRs after IUI and IVF (11). In addition, increased levels of sperm DNA damage have been linked with an increased risk of pregnancy loss after IVF and ICSI (12). Therefore there is considerable evidence to suggest that chromosomal abnormalities, both maternal and paternal, play a key role in the etiology of repeated implantation failure in IVF.
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 in sperm binding, induction of the acrosome reaction, and promotes sperm–egg fusion (14). The zona hardens naturally after fertilization to prevent polyspermic fertilization, protects the integrity of the preimplantation embryo, and facilitates oviductal transport (15). The zona is required during early cleavage stages to maintain the integrity of the inner cell mass (ICM), but it is usually shed during expansion of the blastocyst, allowing implantation to occur (16). Upon reaching the blastocyst stage, physical expansion of the embryonic mass along with the action of lysins produced by the cleaved embryo and/or the uterus, all play a role in zona hatching (17–19). Failure of the ZP to rupture after blastocyst expansion, resulting in impaired hatching, could contribute to RIF (15). Prolonged exposure of oocytes and embryos to artificial culture conditions may also adversely affect the embryo's ability to undergo normal hatching and could impair successful implantation (15).
Embryo Culture and ET Technique The use of high quality, standardized culture media is fundamental to the success of any IVF program. Inadequate culture conditions could play a role in RIF. Assays to identify 1022
suboptimal components of a culture system that could lead to impaired embryo development have been described (20). These include osmolality testing, pH measurements, and sperm bioassay (20). In some instances of RIF, individualized specific culture conditions may be required for optimal embryo development. Implantation rates and PRs after ET depend on the quality and developmental potential of embryos selected for transfer. Suboptimal embryo quality has an adverse effect on implantation and PRs. Evidence suggests that ET technique can influence the success or failure of embryo implantation. Uterine contractions, blood or mucous on the catheter tip, endometrial trauma, and expulsion of embryos have all been associated with unsuccessful ETs (21).
MANAGEMENT OPTIONS Various management options have been proposed to overcome the challenges of chromosomal abnormality and suboptimal embryo development. Table 1 shows the various etiological factors contributing toward defective embryo development 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-up in RIF (4). Preimplantation genetic screening has also been increasingly used in the past decade, the rationale being that an increased PR could be achieved by selecting only chromosomally 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 blastomeres at the cleavage stage, or removal of several cells at the blastocyst stage (22). Polar body biopsy analyses maternal causes of chromosomal abnormality and is an indirect method of screening for aneuploid embryos. It has, however,
TABLE 1 Management options for factors affecting embryo development and implantation in recurrent implantation failure. Management options Chromosomal abnormality Preimplantation genetic screening Comparative genomic hybridization array Single nucleotide polymorphisms Zona hardening Assisted hatching Suboptimal culture Optimal culture media Blastocyst transfer Coculture ZIFT Assessment of embryo quality and viability Time-lapse imaging—EmbryoScope Metabolomics Proteomics Improving ET technique Note: ZIFT ¼ zygote intrafallopian transfer. Das. Recurrent implantation failure. Fertil Steril 2012.
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Fertility and Sterility® been suggested that if oocyte maturation to the metaphase II stage is completed just before the polar body biopsy, it may result in damage to the meiotic spindle of the oocyte (23). Cleavage stage biopsy is the most commonly used method for screening preimplantation embryos for aneuploidy (24). However, cleavage stage embryos have an increased incidence of mosaicism (22). Biopsy at the blastocyst stage may have a smaller risk of aneuploidy than embryo biopsy at the cleavage stage, because mosaic embryos have a higher proportion of aneuploid cells on day 2/3 and will not develop to the blastocyst stage (23). 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 controlled trials does not support these findings (26, 27). In a prospective randomized controlled trial, Blockeel et al. (26) observed that PGS did not increase the implantation rates after IVF-ICSI in women with RIF. In this study, the investigators analyzed chromosomes 13, 16, 18, 21, 22, X, and Y using FISH on blastomeres of day 3 cleavage stage embryos in the study group. There was a significant difference in live birth rate between the PGS group (21%) and the control group (39%). The miscarriage rate did not differ between the two groups (26, 27). A recent meta-analysis of randomized controlled trials demonstrated that in women with advanced maternal age as well as women with repeated implantation failure, PGS significantly lowered live birth rates after IVF (27). The reasons that have been proposed for the inefficiency of PGS are possible damage from the biopsy procedure, failure rate from the technique, limitations of the FISH analysis, and embryo mosaicism (27, 28). In addition, the efficacy of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. The lack of usefulness of PGS may be because the tested blastomere is not representative for the whole embryo (29). The American Society of Reproductive Medicine (ASRM), the European Society of Human Reproduction and Embryology (ESHRE), and the British Fertility Society have concluded that PGS does 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 overcome the limitations of FISH for PGS. These include CGH or the analysis 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 CGH microarray enables a more comprehensive screening of chromosomes. Many chromosomal aneuploidies identified using CGH would not have been detected using FISH for five or nine chromosomes (35). Microarrays have an advantage over conventional CGH because the evaluation of fluorescence ratios is simple, rapid, and easily automated (33). A proof-of-principle study concluded that chromosomal aneuploidy of the oocyte can be accurately predicted by array CGH analysis of both polar bodies (36). Single nucleotide polymorphisms are common polymorphic DNA sequences found throughout the genome. The probes used for SNP microarrays provide genotype data in addition to chromosome copy number information, thereby VOL. 97 NO. 5 / MAY 2012
producing a unique DNA fingerprint for each embryo tested (33). However, a disadvantage of SNP microarrays is a lack of diagnostic accuracy at individual SNP loci as well as high cost of microarrays and labeling techniques (33). In the future, PGS-FISH may be replaced by comprehensive procedures such as array CGH and SNP microarrays. However, the efficacy and practicality of these procedures in improving implantation and live birth rates in patients with RIF will have to be determined in well-designed prospective randomized controlled trials before they can be widely applied in clinical practice.
Assisted Hatching Elasticity and thinning of the ZP are essential prerequisites for successful embryo hatching and implantation (15, 37). It has been observed that cleaved embryos with a good prognosis for implantation have reduced zona thickness (38). It has been suggested that an artificial opening made in the ZP may facilitate the hatching process (39). Cohen et al. (40) observed a higher implantation rate per ET after partial zona dissection. The implantation window occurs 1–2 days earlier in women undergoing ovarian stimulation than in natural cycles (41). Embryos with artificial gaps in the zona initiate hatching earlier than zona intact embryos, compensating for the reduced development rate in vitro (42). It has also been proposed that breaching the integrity of the zona could enhance the transport of nutrients from the incubating media, which in turn would augment embryo development and blastocyst formation (43). It could also serve as a channel for a two-way exchange across the ZP of metabolites and growth factors (42). The artificial rupture of the ZP is known as assisted hatching and aims to improve implantation and clinical PRs. Various techniques have been used to aid zona hatching. These involve the creation of an opening in the ZP either by mechanical partial zona dissection (39), chemically by zona drilling 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 in the management of RIF is controversial. Although some studies have reported that assisted zona hatching improves PRs and implantation rates in patients with RIF (49, 50), other investigators have not reported any advantage (46). Recent studies seem to suggest that assisted hatching may be of benefit in selected patients. In a prospective randomized study, comparing chemical removal of ZP from day 5 in vitro cultured human embryos by using acidic Tyrode's solution versus no removal, the implantation rate per ET and the clinical PR were significantly higher in the ZP-free group (51). Stein et al. (52) reported that assisted hatching by partial zona dissection resulted in a significant increase in the implantation and clinical PRs in women older than 38 years with RIF. Similarly, Petersen et al. (53) observed that for patients with repeated implantation failures, the implantation rate in those who received laser-thinned embryos was significantly higher than in those whose embryos were not laser thinned. Interestingly, this difference was not 1023
VIEWS AND REVIEWS observed in patients with a history of only one previous implantation failure. In support of these findings, in a recent meta-analysis of randomized control trials (five trials with 761 participants), assisted hatching was reported to be associated with a significant improvement in clinical pregnancy when performed in fresh embryos transferred to women with RIF (relative risk [RR] ¼ 1.73; 95% confidence interval [CI] ¼ 1.37–2.17) (54). No increase was observed in clinical PRs when performed in fresh embryos transferred to unselected or nonpoor prognosis women or to women of advanced age. Assisted hatching was also related to increased multiple PRs 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 regarding live birth or miscarriage rates (54).
Embryo Culture Optimum culture conditions are a prerequisite for satisfactory embryonic development and lack of these conditions may contribute to RIF. Various coculture systems have been developed as a means of improving embryo culture conditions. The main aim is to increase the metabolic chances of the human embryo to achieve the blastocyst stage because this leads to a high implantation rate and PR. The suggested favorable effects of cocultures include the secretion of embryotrophic factors, such as nutrients and substrates, growth factors and cytokines, and the removal of free radicals and potentially harmful substances (55). Although multiple cell types have been used for coculturing embryos, ranging from human reproductive tissues, such as oviducts (56), endometrium (57), sequential oviduct-endometrial coculture (58), and cumulusgranulosa cells (GC) (59–61), homologous endometrial cells appear to be the most promising coculture system (57). Using coculture of embryos on homologous endometrial cells in patients with RIF, Jayot et al. (57) reported an overall PR of 21% per transfer versus 8% in previous IVF-ET cycles. Similarly, using autologous endometrial coculture in patients with RIF, Spandorfer et al. (62) reported a significant improvement in embryo quality and clinical PRs. However, the advantage of coculture systems remains controversial. In addition, most IVF units do not have the necessary personnel or facilities to perform coculture on a regular basis.
Blastocyst Transfer Embryo transfer at the blastocyst stage has been proposed as 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 days after fertilization, at the morula-blastocyst stage in natural conception cycles (2). Better embryo selection for transfer and improved endometrial receptivity are obvious advantages of this approach. Some clinicians transfer several embryos after RIF. Culturing embryos to the blastocyst stage helps in selecting embryos with the best implantation potential. Therefore fewer embryos have to be transferred to achieve a successful pregnancy, thereby decreasing the risk of multiple pregnancy. With single ET becoming the norm in younger 1024
patients, selecting the best embryos by culturing to the blastocyst stage assumes even greater significance. In a prospective randomized study, Levitas et al. (63) reported that in patients with RIF with an adequate ovarian response, transfer of blastocyst stage embryos carries a significantly higher implantation rate compared with ET on days 2–3. The multiple PR was not significantly different between the two groups (63). In another study, Guerif et al. (64) also observed that the live birth rates and implantation rates per cycle were higher after blastocyst transfer compared with day 2 ET. They suggested that improved embryo selection and uterine receptivity may explain the additional benefit of ET at the blastocyst stage for couples with RIF (64). However, it should be noted that a percentage of fertilized eggs will never reach the blastocyst stage. Proper selection of cases suitable for blastocyst transfer is therefore critical to reduce the number of cycle cancellations (63).
Stimulation Protocols Variations in ovarian stimulation protocols have been suggested in some studies as a means of improving embryo development and quality. The use of GnRH antagonist protocols in controlled ovarian hyperstimulation (COH) has been shown to improve pregnancy outcome in patients with a history of RIF with GnRH agonist protocols. The investigators proposed that this was most likely due to improvement of the quality of the blastocysts generated (65). Natural cycle IVF has also been proposed as a means of improving implantation rates in patients with RIF (66). Despite some personal experience with natural cycle IVF and in vitro maturation of oocytes in patients with RIF, the lack of randomized clinical studies in this field does not allow any recommendations to be made with regard to their efficacy.
Zygote Intrafallopian Transfer Zygote intrafallopian transfer (ZIFT) allows the early embryo to grow in the natural tubal environment and physiological transport of the embryos into the uterine cavity. It also overcomes the problem of technically difficult ET because of cervical stenosis (2). Although initial nonrandomized studies implied that ZIFT may be of value in RIF (67), a subsequent meta-analysis of randomized controlled trials failed to demonstrate any benefit for ZIFT (68). In fact, there was a trend toward increased risk of ectopic pregnancy (EP) with ZIFT (68). These findings led to the procedure being abandoned by most units.
ET Technique A meticulous ET technique is of utmost importance in achieving a successful pregnancy outcome. Studies show that avoidance of blood (69), mucus (70), bacterial contamination, trauma to the endometrium, touching the fundus, and excessive uterine contractions (71) are all associated with better PRs and implantation rates after ET. Several techniques have been proposed to optimize the technique of ET. Methods, such as a trial transfer (72), filled bladder (73), ultrasonographic guidance (74), and use of soft catheters, all appear VOL. 97 NO. 5 / MAY 2012
Fertility and Sterility® to facilitate a successful ET (21), whereas bed rest after ET has not been shown to be of any benefit (75).
Cytoplasmic Transfer Ooplasmic factors play a role in the continued development of the zygote, especially during the early cleavage stage. Cohen et al. (76) transferred ooplasm from donor eggs at metaphase II stage into developmentally compromised metaphase II oocytes in patients with multiple implantation failure (76). They noted that this led to an improvement in embryo morphology. Cytoplasmic transfer from fertile donor oocytes or zygotes into developmentally compromised oocytes from patients with RIF has led to the birth of several healthy babies worldwide (77). It has been suggested that this procedure may correct an imbalance between anti- and pro-apoptotic factors and/or correction of defective mitochondrial membrane potential (78). However, the transferred cytoplasm could contain messenger RNAs, proteins. and mitochondria (77). In addition, it is not known whether the physiology of the early embryo is affected. The procedure is still experimental and will require assessment of ooplasmic anomalies and optimization of techniques before it can be applied in clinical practice.
New Methods of Embryo Assessment Assessment of embryo quality is critical in selecting the best embryo(s) to transfer or cryopreserve. As visual assessment of embryo quality using morphological criteria can be subjective and requires considerable expertise, newer methods of assessing embryo quality and viability are being developed. Emerging techniques such as time-lapse imaging may lead to better assessment of embryo quality and help in selecting embryos with the highest implantation potential. It has been suggested that time-lapse observations using an incubator with an integrated optical microscope may minimize the changes in the culturing environment by integrating the culture, observation, and time-lapse recording of cells into one system. The removal of embryos from the incubator for intermittent observation can therefore be avoided while enabling the continuous monitoring of embryo development (79). There is evidence that time-lapse monitoring in the EmbryoScope (Unisense FertiliTech) does not impair embryo quality while allowing for morphological and spatial analysis of embryo development (80). However, besides being more expensive than standard incubators, the culture preparation procedure is more time consuming compared with conventional culture methods (81). Different approaches are also being developed to test the culture environment of a developing embryo to gain important information regarding its viability. Metabolomic analysis of follicular fluid (FF) can provide valuable information about individual oocyte maturation and developmental potential. Various methods have been described, which include measurement of oxygen (81), pyruvate, and glucose consumption by the embryo in the culture medium (82). Amino acid turnover, which appears to be correlated to blastocyst development, can be measured as an indication of embryo VOL. 97 NO. 5 / MAY 2012
viability (83). Newer methods, such as vibrational spectroscopy, both Raman and near infrared, have been used to analyze spent culture medium from human embryos, measuring bonds within functional groups of molecules at specific wavelengths. Results from initial studies indicate that spectral profiles reflective of oxidative stress appear to have a good correlation with pregnancy outcome (84).
CONCLUSION Regardless of the considerable improvement in treatment protocols and laboratory technologies, RIF still poses a significant challenge to clinicians and embryologists. Chromosomal abnormalities and suboptimal embryo development play a major role in the etiology of RIF. Emerging technologies, such as CGH array and analysis of SNPs could enable a more comprehensive screening of chromosomes. Assisted hatching may help to overcome zona hardening in selected patients. Optimal culture conditions and blastocyst transfer may contribute toward improving the implantation rates and PRs in RIF. 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. It should be noted that only those treatment options that are evidence based should be offered to patients. The safety, efficacy, and practicality of new, emerging methods of treatment should be evaluated in prospective randomized clinical trials before being accepted in clinical practice.
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