Evidence-Based Management of Infertile Couples with Repeated Implantation Failure Following IVF

200 Current Women’s Health Reviews, 2010, 6, 200-218 Evidence-Based Management of Infertile Implantation Failure Following IVF Couples with Repeated Kim D. Ly1, Nabil Aziz2, Joelle Safi1 and Ashok Agarwal1,* 1 Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; 2Liverpool Women’s Hospital and The University of Liverpool, Liverpool, UK Abstract: Embryo implantation depends on both embryo quality and the endometrial environment. Implantation failure has a complex, variable pathophysiology and is detrimental to the outcome of in vitro fertilization (IVF). Thus, patients with multiple implantation failure require an individualized approach to diagnosing and managing treatment options for future IVF cycles. These options should be based on concrete, unambiguous, consistent scientific evidence with randomized, controlled trials. We review and discuss 14 treatment options: (i) blastocyst transfer, (ii) assisted hatching, (iii) co-culture, (iv) preimplantation genetic screening, (v) hysteroscopy, (vi) sildenafil, (vii) salpingectomy for tubal disease, (viii) oocyte donation, (ix) transfer of six or more embryos, (x), intratubal embryo transfer, (xi) natural cycle IVF, (xii) antiphospholipid antibodies (APA) testing and treatment, (xiii) allogenic lymphocyte therapy, and (xiv) IV immunoglobin therapy. The approaches were evaluated based on available information from studies, expert opinions, consensus, etc. We conclude that blastocyst transfer, assisted hatching, salpingectomy for tubal disease, and hysteroscopy in IVF procedures are clinically effective. This review serves as a summary of current treatment options for clinicians to counsel patients and manage their expectations based on strong and reliable evidence. Keywords: Repeat implantation failure, in vitro fertilization, endometrial receptivity, blastocyst transfer, assisted hatching, hysteroscopy, salpingectomy. INTRODUCTION The world's first baby conceived by in vitro fertilization (IVF) was Louise Joy Brown in 1978. Today, more than 3 million children worldwide have been born as a result of IVF. The numbers continue to increase as science discovers ways to overcome barriers for various subgroups of infertile patients. However, despite the improvements in IVF technology and methods, many couples experience multiple IVF failures. After each failed IVF attempt, pregnancy rates in subsequent attempts decrease by as much as 57% with the most remarkable decrease after the third attempt [1, 2]. The main causes of multiple IVF failures include: (i) poor response to ovarian stimulation, (ii) repeated fertilization failure, (iii) repeated difficult transfers, and (iv) repeated implantation failures (RIF). Implantation failure is a major limiting step for IVF [3]. The process of embryo implantation is described as having three phases: 1. Apposition: “unstable adhesion” of the transferred embryo to the surface of the uterine lining. 2. Attachment (adhesion): “stable adhesion,” believed to involve signaling back and forth between the embryo and the lining. 3. Penetration (invasion): invasion of the trophectoderm cells from the embryo through the surface of the lining deeper into *Address correspondence to this author at the Center for Reproductive Medicine Cleveland Clinic 9500 Euclid Avenue, Desk A19.1 Cleveland, OH 44195 USA; Tel: 216-444-9485; Fax: 216-445-6049; E-mail: agarwaa@ccf.org 1573-4048/10 $55.00+.00 the stroma of the uterine lining, forming a vascular connection to the mother. The etiological causes of implantation failure include embryo quality; endometrial receptivity; immunological factors; uterine, tubal and peritoneal factors; and culture media [3]. Poor response to superovulation and chromosomal aneuploidy due to advanced maternal age negatively affect embryo quality; suboptimal embryos are less likely to implant. The disruption in prostaglandin synthesis is one of many factors that decrease endometrial receptivity in some patients prone to RIF [4]. Other cellular and adhesion pathways affected by abnormal gene expression in the endometrium have been observed to be linked to RIF [5]. The cultured endometrial cells of RIF patients were found to have different gene expressions than those found in the cultured endometrial cells of women who miscarried or had an ongoing pregnancy [6]. From this observation, the differential gene expression of RIF patients is assumed to negatively affect critical signaling pathways important for the development of adhesion molecules in the embryo-endometrium bond and may be linked to implantation failures. Immunological factors such as antiphospholipid antibodies (APA), abnormal expression of endometrial natural killer cells, cytokines [3], local and systemic immune factors, anti-sperm antibodies, and anti-thyroid antibodies [7] have been found in significant amounts among RIF patients and have been reported to affect implantation. APA interferes with the normal function of blood vessels by either causing narrowing/irregularity of the blood vessels (vasculopathy) or by causing the develop© 2010 Bentham Science Publishers Ltd. Evidence-Based Management of Infertile Couples ment of blood clots in the blood vessels (thrombosis). In the majority of cases, failed implantation appears to be related to the quality of embryos transferred rather than to the endometrial receptivity. Part of the evidence stems from the significantly higher implantation rates found in egg donation programs, even in couples that have failed IVF repeatedly using their own eggs. In this paper, we focus on the options supported by clinical evidence to improve implantation and pregnancy rates for couples with multiple IVF failures. Evidence-based medicine has three levels of recommendation. Level A recommendations are based on good and consistent scientific evidence with randomized, controlled trials. At level B, recommendations are based on limited or inconsistent scientific evidence with clinical controlled trials, cohort, etc. Level C includes recommendations based primarily on consensus and expert opinion. Clinicians would benefit from knowing to which category a treatment option belongs to be able to counsel patients and manage their expectations for future IVF cycles. We will examine 14 approaches to repeated implantation failure in IVF: (i) blastocyst transfer, (ii) assisted hatching, (iii) co-culture, (iv) preimplantation genetic screening, (v) hysteroscopy, (vi) sildenafil, (vii) salpingectomy for tubal disease, (viii) oocyte donation, (ix) transfer of six or more embryos, (x), intratubal embryo transfer, (xi) natural cycle IVF, (xii) APA testing and treatment, (xiii) allogenic lymphocyte therapy, and (xiv) IV immunoglobin therapy. (See also Table 1). Table 1. Fourteen Possible Approaches to the Management of Infertile Couples with Repeat Implantation Failure Blastocyst transfer Assisted hatching (AH) Co-culture Preimplantation genetic screening (PGS) Hysteroscopy Sildenafil Salpingectomy for tubal disease Oocyte donation Transfer of six or more embryos Intra-tubal embryo transfer Natural cycle IVF Antiphospholipid antibodies (APA) testing and treatment Allogenic lymphocyte therapy I.V. immunoglobin therapy Current Women’s Health Reviews, 2010, Vol. 6, No. 3 201 and these advances have enabled researchers to culture zygotes to the blastocyst stage prior to implantation in the uterus. The original intent of blastocyst transfer was to select the healthiest embryos for transfer. Thus, a single or the fewest possible embryos may be transferred per cycle to reduce high order pregnancies, which are associated with very real, serious risks to mother and baby. Several advantages of culture and transfer of blastocysts make this option appealing for RIF patients. Self-selection of blastocysts increases endometrial receptivity. The trophectoderm cells of the blastocyst cross-talk with the endometrium and develop the ability to attach to the lining of the endometrial lining of the uterus, both of which are likely to improve implantation success. However, blastocyst culture and transfer usually is recommended for patients with a good prognosis--those who are younger in age with > 6 oocytes available from an uneventful ovarian stimulation. Sequential media, a two-part culture medium, commences with the embryo initially grown in media rich with pyruvate to Day 3. The embryo is then transferred to the second media, rich with glucose, on Day 3 to support embryo growth from the eight-cell stage to the blastocyst stage. A study by Weissman et al. observed a significant improvement in pregnancy rates from blastocyst culture and transfer to patients who were young, did not have multiple IVF failures, who produced multiple oocytes, and whose zygotes developed into good quality cleavage-stage embryos [1]. However, for patients in the general population with a single failed IVF attempt, pregnancy and implantation rates were observed to significantly decrease with subsequent IVF attempts employing blastocyst culture and transfer strategy [12]. In patients with multiple implantation failures, Cruz et al. reported a positive correlation between blastocysts and improved implantation and pregnancy rates for RIF patients in a non-randomized population [13]. However, a prospective, randomized control study by Levitas et al. that specifically examined the effects of blastocyst transfer for RIF patients compared with Day 2 embryo transfer found that blastocyst transfer was beneficial only in patients with an acceptable response to ovarian stimulation [14]. However, there was no difference in the incidence of multiple pregnancies between the groups. Blastocyst transfer on Day 5 for RIF patients permits the selectivity of a higher quality embryo after embryonic genomic activation has occurred [15]. It may also decrease the rate of ectopic pregnancy because of the larger diameter size of the Day 5 embryo; because the blastocyst normally resides in the uterus, an improvement to receptivity is also expected. ASSISTED HATCHING (AH) Embryo transfer occurs either during the cleavage stage on Day 2/Day 3 or during the blastocyst stage (the embryo has inner cell mass, trophectoderm layer, and blastocoele) on Day 5/Day 6. Several studies have demonstrated higher rates of implantation and pregnancy with blastocyst culture and transfer compared with Day 3 cleavage stages [8-11]. Differences in embryo development during IVF treatment compared with in vivo are well-documented. One observed difference is the hardening of the zona pellucida (ZP), which prevents the blastocyst from escaping the ZP during hatching. The culture medium, which differs in its available nutritive source from the in vivo environment, and the cryopreservation method have been implicated as possible causes of implantation failure [16]. Recent advances in culture technology have been made due to a better understanding of early embryo metabolism, Assisted hatching was developed as a workaround to the hardened ZP of in vitro embryos. It involves thinning or cre- BLASTOCYST TRANSFER 202 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 ating a hole in the ZP, either of which can be performed by a mechanical (glass pipette), chemical (acidic Tyrodes’s or enzymes), or laser method (contact and non-contact) with equal efficacy. The industry standard today includes the use of laser-assisted hatching for rapid and precise drilling. AH is not a benign procedure since the time between the zona drilling and embryo transfer is a vulnerable period for the blastocyst as it is exposed to foreign elements, undefined antibodies, or surveillance cells in the endometrial environment. However, blastocysts are believed to be more prepared for exposure to uterine lymphocytes and immune cells at this stage [17]. In addition, an increased incidence of monozygotic twinning may occur during the ablation procedure, which in some reports has caused herniation and division of the inner cell mass into two [18]. This may result from (i) a too-narrow opening of the ZP, trapping the blastomere in a figure-eight formation, thus causing a subdivision of the blastocyst to form twins or (ii) a premature hatching of the blastocyst from a too-large opening combined with loosening of the tight junctions between blastomeres to cause division of the inner cell mass to form twins [18]. A Cochrane review of AH that included 28 randomized control trials from 24 publications or abstracts shows an increase in clinical pregnancy rates for women with RIF but suggests additional research is needed [19]. There is no evidence supporting AH after one failed IVF attempt, but women with multiple implantation failures benefited most from AH [19]. The review’s validity, coupled with multiple good quality, randomized, controlled studies and strong recommendations, suggest that AH does improve implantation rates for women with multiple implantation failures (3+) due to hardening of the ZP, occurring mainly from cultured IVF treatment or methods of embryo cryopreservation. CO-CULTURE Co-culture refers to the placement of human and nonhuman live cell types (feeder cells) alongside the embryo during in vitro culture. It has been suggested that co-culture improves embryo growth and development by improving in vitro parameters--it removes toxic substances such as heavy metals and ammonium and free radicals [20]. Endometrial (Simon, Mercader et al. 1999, Weichselbaum, Paltieli et al. 2002) and granulose [21-23] cells are commonly used in co-culture. Studies have demonstrated conclusively the existence of cross-talk between endometrial cells and embryonic cells, resulting in a paracrine release of molecules believed to improve implantation [20]. The presence of uterine epithelial cells, the first cells in contact between maternal and fetal cells, may initiate a signaling cascade for cells from the blastocyst to become depolarized in preparation for endometrial attachment, thus improving embryo competence for implantation [24]. The heterogeneous co-culture cell lines that have been utilized include human and bovine oviductal cells [25-30], bovine uterine epithelial cells [31, 32], African Green Monkey kidney cells (Vero cells) [28, 33-35], ovarian cancer cells [36], buffalo rat liver cells [37], and human skin fibroblasts [38]. One randomized study using conventional media compared the various types of co-culture cell lines and found that granulosa cells and bovine oviductal uterine epithelial Ly et al. cells were associated with a higher percentage of early embryos developing to the eight-cell stage. In some countries, including the United States, nonhuman cell types have been banned by the Food and Drug Administration due to concerns regarding disease transmission from the cell types to the developing embryo or mother. Consequently, the use of autologous endometrial cells has gained in popularity [39]. Several different human cell types are now available, including cumulus cells, luteinized granulosa cells, fallopian ampullary epithelial cells (FAEC), and endometrial epithelial cells [40]. No randomized controlled studies are available to compare and determine which of the human cell types provides maximum benefit. In a study by Weichselbaum et al., very poor quality embryos, in which more than 50% of embryo volume had fragmentation and the blastomeres were of unequal size with grainy to dark cytoplasm, were rescued from cleavage arrest and degeneration when co-cultured with fallopian ampullary epithelial cells. In addition, blastocyst formation increased by 56%, suggesting that co-culture may help embryos overcome developmental incompetence [40]. Eyheremendy et al. used the best embryos from women with multiple implantation failure to co-culture with monolayers of autologous endometrial cells [41]. Of the 68 patients who failed to become pregnant after multiple IVF treatments, 39 become pregnant, 19 attained a live birth, and 10 remained pregnant beyond 12 weeks. The study also suggested that an endometrial biopsy should be performed about 7 days after ovulation and that the co-culture medium contain both stromal and glandular cells in the monolayer to improve implantation rates [41]. In 2008, Desai et al. reported for the first time the success of a human endometrial culture system in a clinical environment, thus supporting the two previously mentioned studies [42]. Interestingly, although the benefits of co-culture media have been demonstrated over the years, its prevalence is not widespread, perhaps because it remains a labor-intensive and unproven technique [41]. Several limitations must be overcome to realize its benefit. The most serious limitation is that supplementation of human or non-human live cells with the culture media results in an uncontrolled and undefined alteration to the conditions of the media with unknown growth factors in unknown concentrations [43]. The introduction of sequential media in the past decade has slowly replaced not only non-human but also human cell types because of its safety and ease of use [44]. Additional research is needed in the area of co-culture, with special attention to the type of cells and type of culture media (conventional or sequential media), to support its use prior to its widespread adoption in clinical settings. PREIMPLANTATION GENETIC SCREENING Chromosomal abnormalities have been widely reported to be a major cause of early spontaneous abortions in as much as 60% of the general population [45]. In couples with multiple implantation failures undergoing fertility treatment, the frequency of chromosomal abnormalities appears to be higher regardless of maternal age [46-49]. Specifically, embryonic aneuploidy in patients with multiple implantation failures was 54-57% compared with 35% in a control group Evidence-Based Management of Infertile Couples [50, 51]. Thus, preimplantation genetic screening (PGS) could be utilized as an appropriate means for selecting normal embryos to improve implantation and pregnancy rates and, ultimately, live birth rates in couples with RIF. However, recent studies present conflicting data supporting the use of PGS for couples with RIF. The European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium Steering Committee reports the use of PGS as having little impact on improving the pregnancy rate for women with RIF [52]. In addition, Gianaroli et al. showed that PGS did not increase implantation or pregnancy rates per embryo transfer in RIF patients [53]. Other data from ESHRE’s review, ESHRE PGD Consortium IX, demonstrated that 57 IVF centers performed 748 IVF cycles with oocyte retrieval for RIF, which resulted in a 27% clinical pregnancy rate, 24% implantation rate, and an 11% delivery rate between January and December 2006 [54]. For comparison, the 27% clinical pregnancy rate for PGS in RIF patients is better than the 18% clinical pregnancy rate in the total patient population undergoing PGS for various reasons. This latest data collection from 2006 highlighted RIF as a main indication for PGS in fertility centers despite the ongoing debate regarding its efficacy [54]. In a retrospective study of 121 first PGS for RIF, multivariate logistic regression analysis was utilized to generate a predictive model [55]. The model demonstrated that to have a 90% probability of having an embryo transfer after PGS, the patient should have at least 10 mature oocytes, eight normally fertilized oocytes, and six embryos for biopsy on Day 3. The cause of aneuploidy among RIF patients differs from other cases of random failed implantation. One main characteristic of chromosomal abnormality found in preimplantation embryos of couples with multiple implantation failures is the low probability of meiotic errors resulting in chromosomal abnormalities [56, 57]. Two studies by Mantzouratou et al. and Voullaire et al. have shown that meiotic errors are an unlikely cause in this group of patients and that chromosomal abnormalities are reflective of an inefficiency in mitotic division due to abnormal cell cycle regulation by the embryo. This may explain the lower rate of success in the management of fertility for couples with RIF in studies using polar body analysis. In 2009, Fragouli et al. released a study comparing results of PGS between polar body I, polar body II and blastocyst stages in which the aneuploidy rates were 36.5%, 45.8%, and 45.2% after meiosis I, meiosis II, and mitosis, respectively [48]. Though the numbers appear to indicate that aneuploidy rates are higher after meiosis II, errors from meiosis I carried into meiosis II should be considered. The higher aneuploidy rate at the blastocyst stage may be explained by the fact that the embryo is more vulnerable to developmental arrest between the four-cell and eight-cell stage as it switches from maternal to embryo gene expression [58]. Maximum embryonic gene expression has been shown not to occur until the blastocyst stage. Therefore, the disturbed immature cell cycle regulation increases the likelihood of chromosomal abnormalities, which persists to the blastocyst stage, thus reducing the likelihood of successful implantation. These observations suggest that future studies Current Women’s Health Reviews, 2010, Vol. 6, No. 3 203 should focus on understanding the embryonic role in RIF by sampling the blastocyst stage where maximum embryonic gene expression occurs [59]. The occurrence and pathology underlying complex chromosomal abnormality (three or more whole chromosome imbalance) was another characteristic abnormality found in patients with RIF in a recent study using array comparative genomic hybridization (CGH) analysis of chromosomal material in Day 3 cleavage-stage embryos. Chromosomal breakage and failure of mitotic cell cycle checkpoints to detect abnormalities has been suggested as the cause of mosaic complex chromosomal abnormalities in nearly 30% of RIF patients [57, 60]. This study was further supported by the use of CGH analysis of blastocysts [48]. Abnormal replication and segregation of chromosomes during early embryo development of RIF patients is likely caused by maternal cytoplasmic factors or mutation in the cell cycle control genes [61]. Thus, the use of PGS in identifying abnormal embryos in advance to improve implantation is restricted. Fluorescent in situ hybridization (FISH) is the most commonly used method today to detect chromosomal aneuploidy. However, FISH techniques can only analyze a small subset of the chromosomes, usually the most commonly involved in aneuploidy. Embryonic mosaicism (multiple germ cell lines in one embryo) further complicates analysis of test results in terms of reliability since only one blastomere, representing the entire embryo, is removed for testing. According to one study, 38% of embryos were incorrectly graded as normal using a five-probe set on blastomeres [57]. In another study using a nine-probe set, 25% of embryos were misdiagnosed [62]. In the most recent study, which used a 12-probe set, 19% of embryos were incorrectly graded as normal [48]. The acceptable error rates from the studies suggest FISH is able to reliably detect aneuploidy in mosaic embryos and further implies that mosaic embryos have a sufficiently high ratio of abnormal-to-normal blastomeres for cleavage-stage biopsy to serve as clinically useful. Despite several recent advances in diagnostic methods, including whole genome amplification with comparative genomic hybridization and the use of microarrays to overcome the limitations in FISH, identifying complex chromosomal abnormalities has limited success, is labor-intensive, and costly [65]. Currently, convincing evidence for the wide use of PGS in RIF is insufficient. The technique did not improve implantation rates for RIF patients [53, 63, 64]. Moreover, some normal embryos might be lost due to the error rate. Furthermore, with the advent of less invasive methods for predicting better quality embryos such as metabolomics and proteomics, PGS may become a less popular option. It is not likely to happen anytime soon because no studies to date have identified the significant metabolites or proteins involved in early development that are unique to embryos with a low implantation success rate. The role of PGS in the management of RIF patients remains unresolved. HYSTEROSCOPY Hysteroscopy is an invasive diagnostic procedure that is used to visualize uterine pathologies, including submucous fibroids, polyps, intrauterine adhesions, and uterine malfor- 204 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 Ly et al. mations (i.e. septate uterus) that may be associated with infertility. Studies have shown the procedure to be more sensitive, specific and accurate than pelvic ultrasound in evaluating uterine pathology for patients with RIF [65]. Currently, hysteroscopy has not been adopted as part of the routine workup for infertility but rather as a secondary investigative measure to evaluate patients after three or more implantation failures [66, 67]. plasminogen activator inhibitor 1(PAI-1), and (iii) vascular endothelial growth factor (VEGF) – 1154. The study found a link between one or more of the genes and patients with repeat implantation failure. A significantly higher correlation existed for RIF patients than the control group. More studies are needed to confirm the findings, which could facilitate the clinician's ability to identify and counsel patients prone to implantation failure [72]. Women with RIF have been shown to have a higher incidence of uterine pathologies. In the most recent observational study of 1475 women with RIF, an abnormality was found in the uterus of 36.6% of these women (16.7% endometrial polyps, 12.5% endometrial adhesions, 1.5% endocervical adhesions, 4.3% endometritis, 0.9% uterine septa, and 0.8% submucous fibroids) [68]. The study reported that 22.2% of the population had a prior ultrasound screening with a false negative result and subsequent hysteroscopy intrauterine pathology (endometrial adhesions in 12.5%, endometritis in 4.3%, endometrial polyps in 3.3%, endocervical adhesions in 1.5%, uterine septa in 0.5%, and submucuos myomas in 0.1%). The same study also compared the use and non-use of hysteroscopy for women with RIF in a new IVF attempt and showed a significant increase in the implantation rate and pregnancy rate in the former group [68]. It strongly suggests the use of hysteroscopy for women after two failed IVF attempts for two reasons: (i) a significant number of uterine pathologies are undetected by ultrasound and (ii) the significant success rate of ongoing pregnancy after hysteroscopy [68]. For women with endometrial polyps, a hysteroscopic polypectomy (surgical removal of polyps) may be more efficacious than nonintervening hysteroscopy [69]. Several studies have focused on increasing endometrium thickness with sildenafil therapy for women with RIF. In 2000, Sher and Fisch applied sildenafil vaginally in women with RIF and a thin endometrium to increase blood flow for endometrial growth. They hypothesized that a thicker endometrium (> 8 mm) may improve implantation and pregnancy rates [73]. The preliminary study showed that three of the four women had a successful pregnancy outcome after sildenafil treatment during the proliferative phase. Two years later, Sher and Fisch released a follow-up study on 105 patients who were given sildenafil; 70% developed endometrial thickness  9 mm with the remaining patients (30%) being
9 mm. Of the patients who developed endometrial thickness  9 mm, 45% had a significant implantation rate [74]. The study does not explain why the majority (55%) of women treated with sildenafil experienced optimal endometrial growth but subsequent IVF failure. However, it highlighted the importance of endometrium quality [75]. A novel approach to the use hysteroscopy for evaluating endometrial cell integrity has been described [70]. The technique involves the application of chemical agents to highlight cellular-level or mucosal anomalies of the endometrium (chromohysteroscopy). Preliminary studies on a small patient population have shown promising results for women with RIF [70]. SILDENAFIL (VIAGRA) Sildenafil (Viagra®, Pfizer, New York, N.Y.), most notable for the treatment of male erectile dysfunction, is a vasodilator used to improve vascular supply [71]. Endometrial receptivity is believed to improve with increased blood flow in the uterine arteries of women with thin endometrium to increase the thickness of the endometrium, the portion of the uterus associated with successful implantation. Both the quality and quantity of endometrial tissue are important to consider in a strategy aimed at improving the endometrial environment for a successful pregnancy. A recent case control study by Goodman et al. investigated three gene polymorphisms that have been linked to patients with repeat implantation failures in prior individual studies [72]. Successful implantation depends on the blastocyst’s ability to infiltrate the endometrium and develop a sustaining blood supply, which requires the following genes to produce the necessary proteins for digesting the endometrial cellular matrix, regulate cell growth, and induce angiogenesis: (i) p53 codon 7 tumor suppressor factor, (ii) In a case report on two patients with Asherman’s syndrome, sildenafil improved endometrial thickness but did so by a much smaller margin. The endometrium of one woman increased from 6.5 mm to 8.9 mm, and another woman’s increased from 5.0 mm to 6.6 mm. Despite suboptimal endometrial measurements of
9 mm, both women had healthy offspring [76]. In a study by Paulus et al., the data did not support Sher and Fisch’s findings or those of Zinger et al. In 10 women with at least one IVF failure, sildenafil increased endometrial thickness, but only three of 10 patients had a successful pregnancy. However, the heterogeneous and small patient population may have affected the results [77]. Natural killer cell activity levels, which have been reported as a predictor for recurrent miscarriages, may also be involved in RIF [78-80]. An increase in both peripheral blood natural killer cells and endometrial natural killer cells appears to be associated with lower pregnancy rates in patients with recurrent miscarriages. Upon activation with nitric oxide, the natural killer cells release cytokines such as tumor necrosis factor-, which has been implicated as a cause of implantation failure [81]. One study extended the hypothesis to suggest that repeated implantation failures may be caused by high levels of peripheral natural killer cell activities. It showed that sildenafil lowered peripheral blood natural killer cell activity, thereby improving the local endometrial immunological environment in women with multiple IVF failure. However, more randomized control studies must be done to confirm these findings. It also will be important to determine the appropriate period to prescribe sildenafil therapy [75]. In a pilot study, the amount of NO released by the embryo in vitro correlated with implantation success. It was reported that higher quality embryos producing an elevated Evidence-Based Management of Infertile Couples amount of NO in culture media had a higher success rate than the controls [82]. Thus, the use of sildenafil to block the breakdown of cyclic guanosine monophosphate (cGMP) causes an accumulation of NO. As a result of increased NO, radial uterine blood vessels dilate to increase the vasculature and perpetuate endometrial growth. However, as NO induces natural killer cells to produce cytokines that can cause implantation failure, it has been recommended that sildenafil not be used five or more days prior to embryo transfer [74]. On the other hand, one study using mouse embryos reported that higher concentrations of NO in vitro and in vivo resulted in lower implantation rates in a dose-dependent manner [81]. The same higher concentration of NO (1 mM) inhibited implantation in vitro as it did for mouse embryos. Cytostatic and cytotoxic effects resulting from an extended production period of NO in reproductive tissues to protect against infection, immunological reactions, and pathological conditions (e.g. endometriosis, reproductive tract infections) is a suggested cause for the lower implantation rates found in the mouse embryo study [81]. The effects of sildenafil therapy on the endometrium and nearby environments should be better understood before it is widely adopted in IVF clinics for RIF patients. Natural killer cells and NO play important roles in the female reproductive tract. Alterations in NO-synthase (an enzyme that converts the nitrogen in L-arginine to NO in the presence of NADPH and dioxygen) and/or NO production of these tissues could directly affect the development of human embryos, especially during the early stages of pregnancy. SALPINGECTOMY FOR TUBAL DISEASE Of all the different tubal pathologies, hydrosalpinx (distension of the fingered portion of the fallopian tube due to an abnormal accumulation of fluid or water most likely resulting from an inflammatory response) has the most detrimental effect on implantation [83]. Generally, hydrosalpinx is caused by abortion, pelvic inflammatory disease, endometriosis, previous operations, a history of tuberculosis and peritonitis, or an unknown reason [84]. It usually occurs on both sides but can occur exclusively on one side. Interestingly, a one-sided hydrosalpinx will usually correspond with an abnormal fallopian tube on the opposite side. For women with infertility problems, removing the fallopian tubes is a major decision. Nonetheless, hydrosalpingectomy has been reported as a promising option for a subgroup of RIF women with severe tubal factor infertility [85, 86]. The main theoretical reasons for its use as a treatment option are (i) the embryotoxic effect of the fluid in the region by leakage of Table 2. Current Women’s Health Reviews, 2010, Vol. 6, No. 3 205 hydrosalpinx fluid, resulting in either endometrial alterations that are hostile to embryo implantation and development [87], (ii) the mechanical wash out of the embryo [88] before it has a chance to implant, (iii) altered endometrial receptivity due to variations in the levels of certain biomarkers such as LIF [89] and
v3 integrin [90, 91], (iv) release of intrauterine cytokines, prostaglandins, leukotrienes, and other inflammatory compounds directly to the endometrium or via the circulatory or lymphatic system [92, 93], or (v) chronic endometriosis caused mainly by asymptomatic Chlamydia trachomatis [94]. Thus, some studies support preventative salpingectomy for infertile patients meeting two criteria: (i) large hydrosalpinges visible by ultrasound and (ii) bilateral hydrosalpinges [85, 86]. The emphasis on the severity of the tubal disease (see Table 2 for hydrosalpinx scoring system) for salpingectomy as a treatment option is important since it limits or removes chances for a natural conception and is likely to result in disruptions to ovarian blood flow and nerve supply and reduce ovarian reserves [95] -- all of which are important for follicle production, hormone production, and the number and quality of the ova [84]. An additional criterion for irreversibility of the tubal condition should also be included so that patients have the option to maintain their fallopian tubes. A study by Dechaud et al. reported significantly higher implantation rates per transfer among women <41 years of age who had experienced RIF after laparoscopic bilateral hydrosalpingectomy compared with those who did not [96]. The pregnancy rate was also higher for the group with salpingectomy than the group without (23.5% and 9.9 %, respectively, P value = 0.01). Bilateral salpingectomy also was reported to not only increase implantation rates but also decrease the time to pregnancy. A group of women who underwent salpingectomy became pregnant within three IVF attempts as opposed to a group who did not undergo salpingectomy in which some patients required as many as 11 IVF attempts to become pregnant [96]. Furthermore, a recent Cochrane study for the surgical treatment of tubal diseases performed a meta-analysis on five randomized trials and found a significant increase in clinical pregnancy rates and ongoing pregnancy rates for patients with hydrosalpinges who underwent salpingectomy and IVF (for the first time) as compared with those who did not undergo surgical intervention [97]; thus, salpingectomy should be considered for all patients with ultrasound-visible hydrosalpinges. In contrast, there is an argument in preference of tubal microsurgery, which is a less invasive procedure that pre- Hydrosalpinx Scoring System (Puttemans and Brosens 1996) I. Simple hydrosalpinx represented by a thin-walled translucent hydrosalpinx with flattened and separated mucosal folds in a single lumen but without mucosal adhesions. II. Hydrosalpinx foliculans represented by a thin-walled hydrosalpinx with mucosal adhesions which can be focal or extensive. As a result the tubal lumen may become divided into locules by agglutinated folds forming compartments or pseudoglandular spaces. III. Thick-walled hydrosalpinx represented by an ampullary wall >2 mm thick and absent mucosal folds or some fibrotic fold remnants at most; tubal distension is less marked and the amount of invisible intralumina fluid is less abundant.
Source: Puttemans PJ, Brosens IA. Salpingectomy improves in-vitro fertilization outcome in patients with a hydrosalpinx: blind victimization of the fallopian tube?. Hum Reprod 1996; 11(10): 2079-81. 206 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 serves the fallopian tubes, for women with less severe tubal pathology [84]. The higher cost and greater time commitment make it a less popular option among clinicians. Also, no studies have been done to compare efficacy, recovery, psychological factors, risks, invasiveness of tubal microsurgery (a surgical procedure to repair and open the fallopian tubes that is sometimes referred to as tuboplasty) versus hydrosalpingectomy [98]. Perhaps a randomized controlled study to compare salpingectomy and tubal microsurgery for RIF patients with various severity of hydrosalpinx could determine the optimal procedure in the appropriate patient population. Additional randomized controlled studies are needed to clarify whether unilateral or bilateral salpingectomy is more appropriate and necessary than tubal surgery to minimize unnecessary removal of healthy fallopian tubes [97]. OOCYTE DONATION In the past two decades, oocyte donation has become a valuable and effective therapeutic option for infertile patients who choose assisted reproductive technology (ART) and has become an acceptable indication for patients with multiple implantation failures [99]. Egg donors are prescreened for medical and physical history of infectious disease, genetic disorders, polycystic ovarian syndrome, serious malformations (resulting in severe functional or cosmetic handicap such as spina bifida or heart malformations), mental health, and uterine pathologies (for accessibility to oocyte retrieval) and then selected based on age and phenotypic match to the recipient [99, 100]. The screening process also extends to the oocyte recipients (medical, physical, psychological, and social) and their partners (for infectious disease and paternal factors) to identify other possible sources that may adversely affect the outcome. Three groups of patients, those with (i) repeat implantation failure and no other indications, (ii) RIF in combination with advanced age, or (iii) RIF in patients with balanced translocations in homologous chromosomes, may benefit Table 3. I. II. Ly et al. from oocyte donation [100-102] to overcome factors that have been implicated in implantation failure such as genotype and age of the oocyte. [100]. Two committees, the American Society for Reproductive Medicine (ASRM) and Society for Assisted Reproductive Technology (SART) published the 2008 Guideline for Gamete and Embryo Donation, which provides the most recent recommendations and information from the U.S. Center for Disease Control (CDC), U.S. Food and Drug Administration (FDA), and American Association of Tissue Banks (AATB) for optimal screening and testing of oocyte donors and recipients (see Table 3). The guideline specifically identifies women with “multiple previous failed attempts to conceive via ART” as an indication for oocyte donation [99]. More recently, the advancement of cryopreservation methods and technologies for freezing and storing of oocytes has increased the number of available unused oocytes [103]. Patients undergoing controlled ovarian stimulation to cryopreserve their oocytes prior to treatment for illnesses that pose a serious threat to their future fertility (e.g., cancer) have the option to donate their extra oocytes for research or a donor bank once they no longer need the oocytes. Accordingly, RIF patients have a choice between using fresh and cryopreserved oocytes. Although the latter option is available and supported by the ASRM as a fertility preservation strategy, there is not enough evidence to support its safety and efficacy at this time [104]. For the first time, a recent prospective, randomized study comparing fertilization and embryo development rates and ongoing pregnancy rates found no difference between fresh oocytes and vitrified oocytes fertilized via intracytoplasmic sperm injection and then developed in vitro [105]. Further clinical studies are needed to clarify the long-term safety concerns to support the use of cryopreserved oocytes. TRANSFER OF SIX OR MORE EMBRYOS A definitive answer to the question of what constitutes the proper number of embryos for transfer in IVF has eluded ASRM and SART Guidelines for Evaluating the Oocyte Recipient Provide psychological counseling by a mental health professional and further psychological consultation if necessary prior to consent. Obtain medical physical examination and reproductive history to detect reproductive abnormalities. Provide treatment as appropriate prior to use of donor oocytes. III. Complete a general physical exam and pelvic exam. IV. Assess the uterine cavity with hysterosalpingography or similar device to detect any significant uterine abnormality. V. Other recommended tests including: a. Blood type, RH factor, and antibody screen b. Rubella and varicella titers c. HIV-1 and HIV-2 d. Serologic test for syphilis e. Hepatitis B surface antigen f. Hepatitis B core antibody (IgG and IgM) g. Hepatitis C antibody h. Cervical cultures or similar tests for Neisseria gonorrhoeae and Chlamydia trachomatis
Source: Guidelines for gamete and embryo donation: a Practice Committee report. Fertil Steril (2008); 90(5 Suppl): S30-44. Evidence-Based Management of Infertile Couples Current Women’s Health Reviews, 2010, Vol. 6, No. 3 clinicians and scientists in the field for the last decade. In 1995, Azem et al. reported a significant increase in pregnancy rates with the transfer of six or more embryos in comparison with five in women with repeated implantation failure (at least four prior failed IVF-ET attempts) [106]. No other published evidence has demonstrated improved pregnancy or live birth rates after the transfer of more embryos in subsequent IVF-ET cycles than the number transferred in previous failed cycles. Despite this lack of evidence, the transfer of greater numbers of embryos than the recommended guidelines in women with multiple fresh IVF-ET failures is commonly performed in practice. This has also been extended to women predicted to have a poor conception prognosis based on indicators such as embryo quality, also with little supportive evidence regarding efficacy [107]. strictions by considering the transfer of more embryos than recommended only “in exceptional cases when women with poor prognoses have had multiple failed fresh IVF-ET cycles” with a level of evidence/recommendation III-C [111]. The most recent Cochrane systematic review on the number of embryos to transfer in IVF compares pregnancy rates and chances of multiple pregnancies following single versus double, three and four embryo transfer in fresh IVF treatments with various results, reflecting the experience of selected young women in a single fresh cycle of IVF/ICSI. As such, data concerning older women and women with previous multiple failed IVF attempts have yet to be assessed [112]. IVF practices will most likely be modified as scientific advances allow a more accurate assessment of the implantation potential of a given embryo, which will most likely include the determination of the embryonic genome and the metabolic and proteomic fingerprints of viable versus nonviable embryos using microarray technology. On the other hand, women undergoing IVF with multiple embryo transfer face an increased risk of higher-order multiple pregnancies (HOMP) with their known medical, social, and economic consequences. The first guidelines on the number of embryos to transfer were issued by the ASRM in 1996 [108]. These guidelines were revised four times since then, with a subsequent reduction in HOMP. As recently as 2003, the triplet rate for IVF in women younger than 40 was approximately 6%. In 2007, it was less than 2%, and this decrease is directly related to the decrease in the number of embryos transferred per cycle [109]. INTRATUBAL EMBRYO TRANSFER Embryo transfer is a vital step of IVF treatments. Therefore, in patients with repeated implantation failure, clinicians frequently focus on the transfer procedures to enhance embryonic implantation following IVF [113, 114]. Tubal transfer of embryos or zygotes has been widely utilized as part of the arsenal in the treatment of difficult cases of repeated IVF failure [115, 116] although it has never been proven to be beneficial beyond any doubt. In the latest ASRM guidelines, issued in November 2009 [110] (see Table 4) the maximum recommended number of embryos to transfer is five, and this number only applies to women aged 41-42. For all patients with one or two previous failed fresh IVF cycles, the guidelines also recommend transfer of one supplementary embryo (in comparison with the standard recommended number according to age group, see Table 4), after proper counseling regarding the risk of HOMP and justification in the patient’s medical records [110]. This only brings the number of embryos to transfer to six in the 41-42 age group if previous failed IVF attempts are documented. For all other age groups, the numbers are even more limited (as low as 1-2 transferred embryos for women younger than 35). The Society of Obstetricians and Gynecologists of Canada (SOGC) goes even further in their reTable 4. Zygote intrafallopian transfer (ZIFT) has been hypothesized to have many advantages over transcervical embryo transfer (ET), mainly that it provides a “natural” growth milieu for zygotes under physiological regulation with numerous growth factors and cytokines from the tubal fluid, which helps these zygotes attach to the uterus with greater synchronization, thus enhancing implantation potential [117]. After all, natural Day 1/Day 2 embryos belong in the fallopian tubes and not the uterus. Environment or in vitro culture systems play a crucial role in the early development stages of the embryo, and a suboptimal environment may have adverse effects. The ZIFT technique also prevents spillage of embryos after transcervical ET and solves the problem of Recommended Limits on the Numbers of Embryos to Transfer Age Prognosis 207 < 37 yrs 35-37 yrs 38-40 yrs 42-42 yrs Favorable* 1-2 2 3 5 All others 2 3 4 5 Favorable* 1 2 2 3 All others 2 2 3 3 Cleavage-stage embryos Blastocysts *Favorable = first cycle of IVF, good embryo quality, excess embryos available for cryopreservation, or previous successful IVF cycle. 208 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 technically difficult ET in patients with cervical stenosis [61]. Nowadays, the environmental advantage of tubal transfer seems limited since laboratory conditions have improved along with the composition of culture media over the last two decades. Although initial retrospective reports of ZIFT showed higher pregnancy rates than with intrauterine ET [118-120], other investigators have found that the main value of ZIFT is limited to RIF cases [115]. ZIFT was reported to be a valid alternative to standard ET in most subgroups of patients with either first or multiple failed attempts at IVF [114, 115]. It was demonstrated that in patients with tubal factor and a confirmed patency of one fallopian tube, ZIFT can be applied successfully as a treatment for RIF; the pregnancy rates and implantation rates for all ZIFT cycles in RIF patients were 35.1% and 11.1% - significantly higher PRs and IRs as compared to standard transcervical ET [121]. This study concluded that ZIFT should be recommended to IVF patients with a mild form of tubal factor and proved patency of one tube, whereas in severe forms, salpingectomy remains the recommended treatment. The interest in intratubal transfer was greatly diminished after the results of a meta-analysis and a randomized, controlled trial that failed to demonstrate any advantage for ZIFT over standard IVF/ET [122]. Furthermore, a study by Aslan et al. including 229 patients with RIF showed comparable outcomes following ZIFT and transcervical ET [123] . However, these studies are not readily comparable due to different methods and selection criteria. It is likely that a patient’s age, number of previous IVF attempts and etiology of infertility may influence the results [123]. A number of drawbacks to the use of ZIFT exist, such as the need for general anesthesia, laparoscopy, and heavy medical equipment, which increases the medical risks to the patients, as well as the expenses related to the need for operative conditions [123]. An association between tubal embryo transfer and the increased risk of ectopic gestation has been inconsistently reported [122, 123]. ZIFT is normally performed in the pronuclear stage, one day after egg retrieval. For practical reasons, ZIFT is sometimes deferred by one day, and cleavage-stage embryos are transferred two days after egg retrieval--a procedure termed “embryo intra-Fallopian transfer,” or EIFT [114]. In a recent retrospective study by Weissman et al., ZIFT and EIFT transfers had comparable outcomes in regards to implantation and pregnancy rates as well as in all other study parameters such as miscarriage rates, ectopic pregnancy rates, and multiple pregnancy rates, which were found to be unacceptably high [124]. In conclusion, RIF patients are a heterogeneous group with unclear and various pathophysiological diagnoses. The potentially high efficiency of ZIFT in RIF patients is only partially understood, with a commonly accepted interpretation being that ZIFT embryos possibly are protected from expulsion from the uterine cavity by junctional zone contractions as well as from the introduction of cervical microorganisms into the uterine cavity by the transfer catheter [116, 125]. Meanwhile, and despite the scant available evi- Ly et al. dence, ZIFT continues to be proposed in clinical practice exclusively to RIF patients [124]. NATURAL CYCLE IVF Despite the fact that the first IVF/ET baby was born in 1978 after a natural unstimulated cycle, this technique soon was practically abandoned, mainly because of the very high cancellation rates. Controlled pharmacological ovarian hyperstimulation became the standard treatment in IVF cycles of high- and normo-responder patients [126]. However, natural cycles have regained attention in poor-responder patients, where only very few follicles can be recruited and very few oocytes can be retrieved after controlled ovarian hyperstimulation. In these patients, natural IVF cycles may offer comparable outcomes (comparable number of follicles) [127128], reduced side effects such as multiple pregnancy and ovarian hyperstimulation syndrome, and may represent a more cost-effective and patient-friendly alternative [129]. A recent retrospective study of 500 consecutive natural cycles demonstrated that IVF in natural cycles is an affordable and valid alternative in poor-responder patients [129], in accordance with the data reported in an earlier meta-analysis [130]. Controversial data exist as to the efficacy of cycles with minimal stimulation(i.e. GnRH antagonist plus mild gonadotropin stimulation), and there are currently no studies in the literature comparing natural versus minimal stimulation cycles [129]. Moreover, to address the specific issue of implantation failure, a better understanding of the key determinants of successful implantation is warranted, one of which is endometrial receptivity. The endometrium is receptive to blastocyst implantation during a limited spatio-temporal window, called “the implantation window” [131]. In humans, this period begins 6–10 days after the LH surge and lasts for 48 h [132, 133]. Successful implantation depends on synchronization between the developmental stages of the embryo itself and the complex endocrine environment [134, 135]. There is evidence in the literature suggesting that controlled ovarian hyperstimulation (COH) can alter endometrial receptivity [136, 137]. Supraphysiologic doses of hormones can cause asynchronism between the embryo and endometrium and altered concentrations of growth and adhesion factors, causing implantation failure [138, 139]. Ledee-Bataille et al. conducted a study in which endometrial CD56 bright cells (uterine natural killers or uNK) were immunostained. They found elevated numbers of endometrial NK cells in RIF patients after COH cycles and significantly lowered numbers after natural cycles [140]. Data suggest that uNK may be directly or indirectly involved in controlling the early steps of the implantation process, in part because of their role in vascular remodeling, specifically spiral uterine arteries [141]. Furthermore, on the molecular biology level, alterations in endometrial gene expression have been reported with the use of gonadotropins in stimulated cycles [142]. More recently, Haouzi et al., using DNA microarrays of endometrial biopsies, identified for the first time five genes that are up-regulated during the implantation window and proposed them as new biomarkers for exploration of endometrial receptiveness, including during a natural Evidence-Based Management of Infertile Couples cycle. This novel strategy could prove useful in patients with poor implantation after IVF or ICSI [143]. In conclusion, in light of the many potential advantages of natural cycle IVF, and with the many improvements in laboratory conditions and fertilization techniques such as ICSI, it seems worthwhile to re-evaluate the place of natural cycle IVF in the arsenal of fertility treatments, especially in RIF patients. A randomized, controlled trial, comparing natural cycle IVF with current standard practice, is justified. ANTIPHOSPHOLIPID ANTIBODY (APA) TESTING AND TREATMENT Antiphospholipid antibodies (APAs) [144] are acquired immunoglobulins or monoclonal antibodies (IgG, IgM, and/or IgA) directed against negatively charged membrane phospholipids, which were characterized as thrombophilic factors because of their association with slow progressive thrombosis and infarction in the placenta, leading to uteroplacental insufficiency [145]. With regard to implantation and pregnancy, it is more appropriate to classify APAs as autoimmune factors because of the complex nature of their interactions [7]. APAs have been shown experimentally to block in vitro trophoblast migration, invasion, and syncytialization, to reduce trophoblast production of the vital hormone human chorionic gonadotrophin [144] and activate complement on the trophoblast surface inducing an inflammatory response [146]. Increased concentration of APA in the follicular fluid, which was once thought to be an explanation for the direct effect of APA on the implanting embryo [147], is still debatable. A recent study demonstrated that this increased concentration had no adverse effect on the reproductive outcome of women undergoing IVF [148]; another study found a significant relationship between follicular fluid APA concentrations and fertilization rates in IVF failure patients [149]. APAs associated with reproductive failure are lupus anticoagulant (LCA), anti-cardiolipin (ACL), anti-phosphatidyl serine (APS), anti-phosphatidyl inositol (API), antiphosphatidyl glycerol (APG), anti-phosphatidyl ethanolamine (APE), and phosphatidic acid (PA) [150]. Not only do APAs bind to their direct antigens, they also bind to plasma-bound proteins and co-factors such as
2 glycoprotein I (
2GPI), the most studied anti-phospholipid protein, and prothrombin, which is also important in this role [151]. There is evidence that the presence of
2GPI on trophoblast and decidual cell membranes might explain the clinical association between recurrent fetal loss and
2GPI-dependent APA and the pathogenic role for these antibodies at the same time [152]. Antinuclear antibodies (ANA) also may be associated with reproductive failure, but they were shown to be positive in 9% of normal fertile women and appear to lack specificity in low titre [153]. Even after 20 years of investigation, the role of APA remains elusive when it comes to assisted reproduction, mainly because most of the groups reported an increased prevalence of APA in infertile patients [154, 155], but the evidence is much less definite with respect to IVF outcome [156, 157]. An explanation for the conflicting evidence might be related to differences in antibodies tested. Some groups solely tested for ACL, LCA, or ANA [154], while others evaluated a Current Women’s Health Reviews, 2010, Vol. 6, No. 3 209 more comprehensive range of APA [156]. Coulam et al. found a 22% prevalence of seven APAs in women experiencing implantation failure after IVF/ET. Only 4% of women with positive antibodies would have been detected if only ACL were tested and only 14% if APS were added to ACA [156]. Unfortunately, with the exception of ACL, no universally accepted standard for the determination of APA concentrations exists, which adds to the confusion. Actually, the most broadly used clinical assays for these antibodies test for ACL antibodies using enzyme linked immunosorbent assay (ELISA) and lupus anticoagulant (LA) [156]. It is noteworthy here to emphasize the fact that APA can be found in low concentrations in as many as 16% of “normal” controls, i.e. healthy fertile women [153]. The association of antiphospholipids with RIF has been shown in some early studies [145, 153, 154, 156, 158], but large prospective studies failed to reveal an association [155, 157, 159, 160]. A meta-analysis considered the effect of APAs on the likelihood of IVF success and concluded that testing for APAs was unjustified in patients undergoing IVF [161]. However, these results did not close the debate because of the heterogeneity of the cohort studies, the populations included, because the RIF group of patients was not addressed specifically [162]. A strong association was demonstrated between antibodies to the cofactor
2 glycoprotein 1 and IVF implantation failure [153]. Antibodies to annexinV, which acts as an inhibitor of phospholipid-dependent coagulation and may be necessary for trophoblast differentiation, were found to have a significantly greater incidence (8.3%) in women with RIF than in controls (1.1%) [163]. Other findings by Geva et al. suggest that although APAs may be important in recurrent fetal loss and spontaneous abortions, neither the serum concentration nor the number of positive APAs appear to have significance in recurrent implantation failure, cumulative pregnancy, or live birth rates [164]. According to the ASRM (2008), no association is present between APA abnormalities and IVF success, there is no indication for the assessment of APA in couples undergoing IVF, and therapy is not justified on the basis of existing data [165]. Despite the uncertainty concerning the pathophysiology of APAs in reproductive failure, their presumed thrombotic effects have led to the widespread use of heparin and aspirin for women with RIF [145]. Heparin is thought to protect the trophoblast from injury by inhibiting the binding of phospholipids with antibodies, thus promoting implantation and placentation [166]. Only a very few randomized, placebocontrolled studies evaluating the benefits of heparin and aspirin for APA-positive women with RIF have been undertaken. While some evidence exists that treatment with unfractionated heparin and low-dose aspirin can improve live birth rates [167], other studies have shown that neither implantation nor pregnancy rates are improved with heparin and aspirin [168, 169]. A recent randomized, placebocontrolled cross-over study in patients with strictly defined RIF did not show any benefit of heparin and low-dose aspirin in patients seropositive for at least one antiphospholipid (APA), antinuclear (ANA), or beta(2) glycoprotein I autoantibody, when the outcome measured was ongoing pregnancy or implantation rates [169]. 210 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 The most recent Cochrane review by Empson et al. examined the outcomes of all treatments to maintain pregnancy in women with prior miscarriages and positive APA. The results found that only unfractionated heparin combined with aspirin appeared to reduce pregnancy losses (by 54%) when compared with aspirin alone. However, these results were only based on two small trials, one of which lacked satisfactory allocation concealment. Low molecular weight heparin (LMWH) combined with aspirin had no statistically significant effect when compared with aspirin or intravenous immunoglobulin (IVIg) [170]. Aspirin alone had no significant effect on any of the outcomes examined; corticosteroids did not show any benefit but demonstrated increased adverse outcomes [170]. IVIg did not significantly differ from prednisone or aspirin in outcomes [171] and was shown to have lower live births rates than LMWH plus aspirin [172, 173]. The beneficial effect of IVIg has only been proved only in uncontrolled studies [174]. To date, the combined use of low-dose aspirin and heparin is considered standard therapy for women seropositive for APAs, despite the lack of adequate, prospective, randomized, placebo-controlled studies addressing the RIF group of patients specifically. Caution must be exercised in recommending any given treatment. In conclusion, in women with RIF, no consensus exists regarding testing for APA, assays to be used, auto-antibodies to test, definitions of patient groups or therapy for seropositive patients [175]. Patients must, therefore, be counseled prior to starting any treatment that no clear evidence of benefit for anticoagulation exists [116]. ROLE OF IMMUNE MECHANISMS IN RECURRENT PREGNANCY LOSS AND RIF Background The immune system of a patient with a successful pregnancy has been considered a paradox since Medawar in 1953 described the embryo as a semi-allogen, but one that is protected from allogenic recognition by antigenic immaturity, possibly explained by non-classical class I HLA molecules. Since then, numerous studies have supported the theory of alloimmune causes as an explanation for miscarriages and implantation failure. The hypothesis is that the absence of such alloimmunoprotective mechanisms would result in alloimmune-mediated miscarriage [176]. Moreover, Wegmann et al. suggested that successful pregnancy might result from the predominance of T helper 2 (Th2) cytokines over T helper (Th1) cytokines [177]. In spite of the central role attributed to immunology in reproductive failure and the intense debates on its scope, no appropriate diagnostic strategy has been established to date [176]. Genetic and immunological factors interact with each other in a complex network of antibodies, adhesion molecules, metalloproteinases, natural killer cells, and cytokines [150]. Other factors influencing reproduction and implantation include human leukocyte antigen expression, antisperm antibodies, integrins, leukemia inhibitory factor, cytokines, antiphospholipid antibodies, endometrial adhesion factors, mucin-l, and uterine natural killers [116, 150]. Ly et al. NATURAL KILLER CELLS A difference is thought to exist between the uterine and peripheral natural killer (NK) cells of women with recurrent pregnancy loss (RPL) compared with controls. Higher numbers of uterine NK cells have been found in the preimplantation endometrium of women with RIF [178]. However, this abnormality was only part of a composite range of immune and vascular abnormalities found in the endometrium of RIF patients [178, 179]. The uNK from nonpregnant RPL patients who exhibit lower CD56 expression (classified as CD16+ CD56dim) are more frequent than CD16+CD56bright, as opposed to fertile controls [180]. Non-pregnant RPL patients also show evidence of increased numbers of activated NK cells in peripheral blood mononuclear cells [181]. Kwak et al. also observed the up-regulated expression of CD56＋, CD56+-CD16＋, and CD19＋cells in peripheral blood lymphocytes in pregnant women with RPL [182]. Moreover, Aoki et al. also reported that high preconceptional NK cell activity was associated with higher abortion rates in the next pregnancy [80]. Studies of immune factors investigated at the time of miscarriage showed that deficiency of CD56 bright natural killer cells in the decidua and high natural killer cell cytotoxicity in peripheral blood monocyte cells (PBMCs) increase the risk of euploid miscarriage [183-185]. CYTOKINES Strong evidence exists that locally secreted cytokines control the implantation process and can cause implantation failure [186, 187]. The Th1 cytokines include IL-2, IFN and TNF
, and the Th2 cytokines include IL-4, IL-5 and IL-10. Evidence suggests that the mean of the Th1:Th2 ratio in patients with RPL and in patients with multiple implantation failure after IVF-embryo transfer [188] is significantly higher than in normal fertile women. This predominance of Th1 cytokines was demonstrated to exist in endometrial cells as well as peripheral blood mononuclear cells before pregnancy [187, 189, 190] and at the time of miscarriage in decidual cells [191]. However, there are significant discrepancies in the results of the different studies, as some suggest that Th1 cytokines production was higher in normal women than in RPL patients in early pregnancy [192], and others even found that the production of Th1 and Th2 cytokines was similar in RPL patients who subsequently had successful or failed pregnancies [193]. Th1 dominance may well be a result of the miscarriage rather than a cause, and much more basic knowledge is needed about the complex cytokine networks in pregnancy and the correlation between cytokine production in peripheral mononuclear cells and decidual lymphocytes [194] before tests measuring cytokines can be introduced in clinical practice. With further research and newly discovered cytokines, it is now clear that acceptance of the Thl:Th2 paradigm as a single explanation for implantation failure would be an overly simplistic approach to a very complex mechanism [195]. Other cytokines, particularly leukemia inhibitory factor (LIF), recently have been shown to play a role in women with RIF [196]. Mannosebinding lectin, a constituent of the innate immune system that modulates cytokine production by monocytes [197], was shown to have significantly lower levels in women with RPL [198-200]. Evidence-Based Management of Infertile Couples TREATMENT WITH GLOBULINS (IVIg) INTRAVENOUS Current Women’s Health Reviews, 2010, Vol. 6, No. 3 IMMUNO- The mechanism of action of IVIg, a fractionated blood product, is multifactorial [201]. It is involved in a number of processes, including modulation of T cells, B cells, NK cells, monocytes, and macrophages; down-regulation of antibody production; inhibition of antibody function; and modulation of complement activation [202]. Immunoglobulins develop their suppressive activity in vitro through the CD200 tolerance-signaling molecule, which is released from the surface of subsets of blood mononuclear leukocytes and may bind to IVIg [203]. CD200 is known to promote generation of regulatory T cells in mice [204]. A more recent report suggests IVIg suppresses NK activity, specifically the CD56 bright subset of NK cells found at the feto-maternal surface [205]. Additionally, one underlying mechanism may be the restoration of Th1/Th2 balance with dominant Th2 [201]. Highdose IVIgs nearly always have been combined with corticosteroids or anti-thrombotics, so that their precise efficacy cannot be readily estimated and is practically hard to assess [201]. A review of the literature concerning IVIg treatments yields conflicting results. A meta-analysis of six trials by Daya et al. demonstrated a lack of clinical efficacy of IVIg on live birth rates [206], and a prospective, randomized, double-blinded, and placebo-controlled study by Stephenson et al. observed no differences between the IVIg-treated and the placebo groups [207]. A prospective, randomized, double-blinded, and placebo-controlled study by Coulam et al. demonstrated the efficacy of IVIg treatment in increasing the percentage of live births among women experiencing unexplained RIF [208]. In a randomized study by Triolo et al., IVIg was less efficacious than low-dose aspirin and low molecular weight heparin in increasing live births rate [172]. On the other hand, a randomized controlled trial by Christiansen et al. showed no improvement in live birth rates with IVIg compared with placebo, but thye suggested a possible beneficial role of IVIg in women with secondary recurrent miscarriage [209]. This effect was confirmed by the review of Hutton et al., although the data concerning primary recurrent miscarriage was inconclusive [210]. A meta-analysis of randomized and cohort controlled trials of IVIg in RIF patients showed a significant increase in the live birth rate per woman (p=0.012) and number needed to treat for one additional live birth = 6) [211]. Relevant variables appeared to be selection of patients with abnormal immune test results and properties of IVIg preparations, as different biological preparations vary significantly in their ability to suppress NK activity in vitro. Another variable is the scheduling of IVIg treatment, as it is argued that pre-conception treatment is better in both primary recurrent miscarriage patients and in IVF failure patients [211, 212]. An observational pilot study found that elevated numbers of NK cytotoxic CD16+ CD56+ cells are independent predictors of treatment success, and that IVIg ameliorated the numbers of these NK cells in RIF [213]. Another observational study by Winger et al. studied for the first time a subset of IVF patients with elevated Th1/Th2 cytokines and showed an improvement of implantation and live birth rates for the groups treated with IVIg and both IVIg and TNF-alpha inhibitors as compared with the 211 no-treatment group [214]. However, the most current Cochrane review shows no significant increase in the overall pregnancy success rate over placebo or no treatment [215]. In conclusion, IVIg, an expensive treatment with possible side effects, is widely used off-label in the treatment of early reproductive failure. Systematic reviews have generated inconclusive results so evidence concerning its efficacy is controversial. Rigorous randomized, controlled trials studying the efficacy of the treatment on various subgroups of RIF patients and additional measurements of CD200-dependent IVIg effects should be undertaken to solve the current controversy [211]. ALLOGENIC LYMPHOCYTE THERAPY During pregnancy, the paternal human leukocyte antigen (HLA) is recognized by the maternal immune system, which induces production of several alloantibodies. These alloantibodies include anti-paternal cytotoxic antibodies (APCA), anti-idiotypic antibodies (Ab2), and mixed lymphocyte reaction blocking antibodies (MLR-Bf). Once expressed, they may coat the trophoblast to render it undetectable by the maternal immune response system [216]. A reduction in or the absence of these alloantibodies during pregnancy may cause fetal loss [201, 217, 218]. As has been shown repeatedly, increased sharing of HLA may prohibit the mother from producing these alloantibodies, leading to an increased tendency toward repeated fetal loss [201]. Maternal immunomodulation via transfusion of paternal leukocytes (lymphocytes) prior to conception has been proposed as a solution to RIF [219-221], while the use of third party donor white cells or trophoblast membranes transfusions have been largely abandoned because of doubts about efficacy [201]. Allogenic lymphocyte isoimmunization (ALT) also has been proposed to solve the Th1/Th2 paradigm by shifting the balance towards Th2 cytokines, thus enhancing the implantation process [181, 188]. The first randomized, controlled study using ALT with paternal PBMCs showed a 24% increase in successful pregnancy rates [222]. However, subsequent trials provided conflicting results due to variations in cell numbers, number of injections, routes of administration, and types of placebo. These differences make comparisons and meta-analyses difficult to realize [209]. A subsequent intention-to-treat metaanalysis by the Recurrent Miscarriage Immunotherapy Trialists Group showed an increased live birth rate of approximately 9–10% [223]. The number needed to treat for an additional live birth was limited to three to four women with primary recurrent pregnancy losses who were seronegative for autoantibodies (ANA and ACL) [219]. Since then, the beneficial effects of ALT in RIF patients has been demonstrated primarily by non-randomized studies. In a retrospective, non-randomized review of 686 couples referred for ALT, Kling et al. found a temporary beneficial effect lasting for six months after immunization; this effect seemed to be most pronounced in couples who failed three or more cycles of IVF-embryo transfer [224]. Despite the lack of randomized trials for RIF, a large randomized trial in RSA patients failed to show any beneficial effect of ALT [225]. Pooling the results of the randomized and non-randomized studies, Pandey et al. showed that 67% of RSA patients who re- 212 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 ceived paternal lymphocyte immunotherapy had successful pregnancy outcomes in comparison to 36% success in women with RSA in the control group who received either autologous lymphocytes or no therapy [220]. In a double-blind, placebo-controlled trial in women with unexplained RSA, immunization with paternal lymphocytes proved to be beneficial over autologous (maternal) lymphocyte therapy [221]. On the other hand, a Cochrane metaanalysis of relevant trials [215] concluded that paternal and third-party ALT provide no significant beneficial effect over placebo in preventing miscarriages. The results of this metaanalysis are extremely controversial because it included a large negative trial using immunization with paternal lymphocytes stored overnight [225]. This factor impairs the protective anti-abortive effect of the procedure and causes loss in surface CD200, at least in mice [226]. The results of the Ober study, despite the debate over its design, decisively influenced the issues of the most current Cochrane review cited above [215], as well as the US FDA regulation, [227] which states that administration of such cells as allogenic lymphocytes or cellular products in humans should only be performed as part of a clinical research project and then only if an Investigational New Drug application is in effect. Moreover, concerns over possible adverse effects of LIT have been raised. These include transfusion-related problems, autoimmune disorders, graft-versus-host reaction, and transmission of infection such as hepatitis B virus or HIV [176], or even cancer and gestational pathology [228]. Adverse neonatal outcomes are rare, but a case of neonatal alloimmune thrombocytopenia and intracranial hemorrhage in an infant whose mother received immunizations of paternal mononuclear cells has been reported [229]. However, a prospective study by Kling et al., with follow-up after 2-3 years, showed that the acute side effects of intradermal ALT were comparable to those reported after intradermal vaccination for infectious diseases and that specific risks for anaphylaxis, autoimmune, or graft- versus- host disease were not significant [228]. In conclusion, proposing ALT to RIF patients in the absence of standard and broadly applicable diagnostic tests of immune-mediated pregnancy losses, of reliable methods for judging the immunization effects, and of unified protocols of immunization should await further randomized, controlled trials based on adequate patient selection and more complete knowledge of the underlying pathophysiology of the assumed alloimmune causes of recurrent miscarriage. CONCLUSIONS Randomized, controlled trials have shown that blastocyst transfer, assisted hatching, salpingectomy for tubal disease, and hysteroscopy in IVF procedures are beneficial for improving treatment outcome in patients with repeated implantation failure. Studies also demonstrate that treatment with aspirin and heparin with IVIg does not have a clear impact on treatment outcome. Allogenic lymphocyte therapy, ZIFT/EIFT, co-cultures, sildenafil, use of donor oocytes, transfer of six embryos, natural IVF, and PGS await further clinical assessment. The management of RIF should be individualized because the pathophysiology is so variable and often complex. Ly et al. EXPERT COMMENTARY Having to endure not one but two or more failed rounds of IVF is painfully frustrating to the patient as well as to the clinicians and technicians involved. We discuss 14 current options that are supported by varying degrees of scientific evidence. Clinicians would benefit from knowing which treatment options are proven in order to counsel patients effectively and manage their expectations for future IVF cycles. Unfortunately, even the more promising ones are successful only to a certain subgroup of patients with specific conditions. Our understanding of the mechanism behind embryo implantation remains poorly understood, which hampers our ability to find a solution. To date, strong evidence supports the use of blastocyst transfer, assisted hatching, salpingectomy for tubal disease, and hysteroscopy in the management of couples with previous implantation failures and clearly dismisses the use of aspirin and heparin with IVIg. Other treatment options such as allogenic lymphocyte therapy, ZIFT/EIFT, co-cultures, sildenafil, use of donor oocytes, transfer of six embryos, natural IVF and PGS are controversial, and their efficacy remains to be elucidated. FIVE-YEAR VIEW Endometrial receptivity is a vital component for embryo implantation. A better understanding of the critical success factors required for successful implantation is recognized. What is the optimal condition of the embryo? What is the optimal condition of the endometrium? Recent genetic research focused on cultured endometrial cells from RIF women has demonstrated a difference in transcriptional activity during the implantation window. A possible disruption in genetic expression of specific genes that regulate the cell cycle has been implicated. Further research is needed to fully understand the genes involved in the defunct pathway of endometrial cells during the implantation window, thus resulting in implantation failure. KEY POINTS • Implantation failure and embryo quality are major limiting steps for IVF treatment. • Etiological sources of implantation failure include embryo quality; endometrial receptivity; immunological factors; uterine, tubal, and peritoneal factors; and culture media. The treatment options discussed in the article are intended to address the particular causes and improve implantation rates. • Differences in the local environment of the endometrium of RIF patients compared with other infertile patients have been found. These differences (i.e. gene expression) possibly affect cross-talks between the embryo and the endometrium and thus implantation. • Blastocyst transfer, assisted hatching, salpingectomy for tubal disease, and hysteroscopy are highly recommended treatment options based on good, consistent scientific evidence with randomized, controlled studies. Evidence-Based Management of Infertile Couples REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Weissman A, Biran G, Nahum H, Glezerman M, Levran D. Blastocyst culture and transfer: lessons from an unselected, difficult IVF population. Reprod Biomed Online 2008; 17(2): 2208. Martin-Johnston MK, Uhler ML, Grotjan HE, Lifchez AS, Nani JM, Beltsos AN. Lower chance of pregnancy with repeated cycles with in vitro fertilization. J Reprod Med 2009; 54(2): 67-72. Ola, Bolarinde; Li, Tin-Chiu Implantation failure following in-vitro fertilization. Curr Opin Obstet Gynecol 2006; 18(4): 440-5. Achache H, Tsafrir A, Prus D, Reich R, Revel A. Defective endometrial prostaglandin synthesis identified in patients with repeated implantation failure undergoing in vitro fertilization. Fertil Steril 2009. [Epub ahead of print] Koler M, Achache H, Tsafrir A, Smith Y, Revel A, Reich R. Disrupted gene pattern in patients with repeated in vitro fertilization (IVF) failure. Hum Reprod 2009; 24(10): 2541-8. Bersinger NA, Wunder DM, Birkhäuser MH, Mueller MD. Gene expression in cultured endometrium from women with different outcomes following IVF. Mol Hum Reprod 2008; 14(8): 47584. Cline AM, Kutteh WH.Is there a role of autoimmunity in implantation failure after in-vitro fertilization? Curr Opin Obstet Gynecol 2009; 21(3): 291-5. Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil Steril 2000; 73(1): 126-9. Wilson M, Hartke K, Kiehl M, Rodgers J, Brabec C, Lyles R. Integration of blastocyst transfer for all patients. Fertil Steril 2002; 77(4): 693-6. Levron J, Shulman A, Bider D, Seidman D, Levin T, Dor J. A prospective randomized study comparing day 3 with blastocyststage embryo transfer. Fertil Steril 2002; 77(6): 1300-1. Papanikolaou EG, D'haeseleer E, Verheyen G, et al. Live birth rate is significantly higher after blastocyst transfer than after cleavagestage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum Reprod 2005; 20(11): 3198-203. Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril 2001; 76(3): 53842. Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR.Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72(2): 218-20. Levitas E, Lunenfeld E, Har-Vardi I, et al. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2-3 embryo transfer cycles: a prospective, randomized study. Fertil Steril 2004; 81(3): 567-71. Barrenetxea G, López de Larruzea A, Ganzabal T, Jiménez R, Carbonero K, Mandiola M.Blastocyst culture after repeated failure of cleavage-stage embryo transfers: a comparison of day 5 and day 6 transfers. Fertil Steril 2005; 83(1): 49-53. Gabrielsen A, Agerholm I, Toft B, et al. Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A randomized prospective study. Hum Reprod 2004; 19(10): 225862. Germond M, Primi MP, Senn A. Hatching: how to select the clinical indications. Ann N Y Acad Sci 2004; 1034: 145-51. Sheen TC, Chen SR, Au HK, Chien YY, Wu KY, Tzeng CR. Herniated blastomere following chemically assisted hatching may result in monozygotic twins. Fertil Steril 2001; 75(2): 442-4. Das S, Blake D, Farquhar C, Seif MM. Assisted hatching on assisted conception (IVF and ICSI). Cochrane Database Syst Rev 2009; (2): CD001894. Simón C, Mercader A, Garcia-Velasco J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999; 84(8): 2638-46. Carrell DT, Peterson CM, Jones KP, et al. A simplified coculture system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization. J Assist Reprod Genet 1999; 16(7): 344-9. Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] 213 Fabbri R, Porcu E, Marsella T, et al. Human embryo development and pregnancies in an homologous granulosa cell coculture system. J Assist Reprod Genet 2000; 17(1): 1-12. Dirnfeld M, Goldman S, Gonen Y, Koifman M, Calderon I, Abramovici H. A simplified coculture system with luteinized granulosa cells improves embryo quality and implantation rates: a controlled study. Fertil Steril 1997; 67(1): 120-2. Escribá MJ, Escobedo-Lucea C, Mercader A, de los Santos MJ, Pellicer A, Remohí J.Ultrastructure of preimplantation genetic diagnosis-derived human blastocysts grown in a coculture system after vitrification. Fertil Steril 2006; 86(3): 664-71. Bongso A, Ng SC, Ratnam S. Co-cultures: their relevance to assisted reproduction. Hum Reprod 1990; 5(8): 893-900. Bongso A, Soon-Chye N, Sathananthan H, Lian NP, Rauff M, Ratnam S. alImproved quality of human embryos when co-cultured with human ampullary cells. Hum Reprod 1989; 4(6): 706-13. Morgan K, Wiemer K, Steuerwald N, Hoffman D, Maxson W, Godke R.Use of videocinematography to assess morphological qualities of conventionally cultured and cocultured embryos. Hum Reprod 1995; 10(9): 2371-6. Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Fiorentino A, D'Errico A. Human embryo co-culture: results of a randomized prospective study. Int J Fertil Menopausal Stud 1995; 40(5): 254-9. Tucker MJ, Morton PC, Wright G, et al. Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates? Hum Reprod 1996; 11(11): 2434-7. Wiemer KE, Garrisi J, Steuerwald N, et al. Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients. Hum Reprod 1996; 11(11): 2429-33. Wiemer KE, Cohen J, Amborski GF, et al. In-vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells. Hum Reprod 1989; 4(5): 595-600. Wiemer KE, Cohen J, Wiker SR, Malter HE, Wright G, Godke RA. Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation. Fertil Steril 1989; 52(3): 503-8. Van Blerkom J. Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells. Hum Reprod 1993; 8(9): 1525-39. Sakkas D, Jaquenoud N, Leppens G, Campana A. Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on Vero cells: a randomized study. Fertil Steril 1994; 61(3): 521-5. Turner K, Lenton EA. The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro. Hum Reprod 1996; 11(9): 1966-74. Ben-Chetrit A, Jurisicova A, Casper RF. Coculture with ovarian cancer cell enhances human blastocyst formation in vitro. Fertil Steril 1996; 65(3): 664-6. Hu Y, Maxson W, Hoffman D, et al. Co-culture with assisted hatching of human embryos using Buffalo rat liver cells. Hum Reprod 1998; 13(1): 165-8. Wetzels AM, Bastiaans BA, Hendriks JC, et al. The effects of coculture with human fibroblasts on human embryo development in vitro and implantation. Hum Reprod 1998; 13(5): 1325-30. Kattal N, Cohen J, Barmat LI. Role of coculture in human in vitro fertilization: a meta-analysis. Fertil Steril 2008; 90(4): 1069-76. Weichselbaum A, Paltieli Y, Philosoph R, et al. Improved development of very-poor-quality human preembryos by coculture with human fallopian ampullary cells. J Assist Reprod Genet 2002; 19(1): 7-13. Eyheremendy V, Raffo FG, Papayannis M, Barnes J, Granados C, Blaquier J. Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure. Fertil Steril 2010; 93(3): 769-73 Desai N, Abdelhafez F, Bedaiwy MA, Goldfarb J. Live births in poor prognosis IVF patients using a novel non-contact human endometrial co-culture system. Reprod Biomed Online 2008; 16(6): 869-74. Richter KS. The importance of growth factors for preimplantation embryo development and in-vitro culture. Curr Opin Obstet Gynecol 2008; 20(3): 292-304. d'Estaing SG, Lornage J, Hadj S, Boulieu D, Salle B, Guérin JF. Comparison of two blastocyst culture systems: coculture on Vero cells and sequential media. Fertil Steril 2001; 76(5): 1032-5. 214 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] Griffin DK. The incidence, origin, and etiology of aneuploidy. Int Rev Cytol 1996; 167: 263-96. Maroulis GB, Koutlaki N. Preimplantation genetic diagnosis. Ann N Y Acad Sci 2006; 1092: 279-84. Farfalli VI, Magli MC, Ferraretti AP, Gianaroli L.Role of aneuploidy on embryo implantation. Gynecol Obstet Invest 2007; 64(3): 161-5. Fragouli E, Katz-Jaffe M, Alfarawati S, et al. Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure. Fertil Steril 2009. [Epub ahead of print] Raziel A, Friedler S, Schachter M, Kasterstein E, Strassburger D, Ron-El R. Increased frequency of female partner chromosomal abnormalities in patients with high-order implantation failure after in vitro fertilization. Fertil Steril 2002; 78(3): 515-9. Gianaroli L, Magli MC, Munné S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod 1997; 12(8): 1762-7. Pehlivan T, Rubio C, Rodrigo L, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 2003; 6(2): 232-7. ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001). Hum Reprod 2002; 17(1): 233-46. Gianaroli L, Magli MC, Ferraretti AP, Munné S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72(5): 837-44. Goossens V, Harton G, Moutou C, Traeger-Synodinos J, Van Rij M, Harper JC. ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009; 24(8): 1786-810. Platteau P, Staessen C, Michiels A, Van Steirteghem A, Liebaers I, Devroey P. Which patients with recurrent implantation failure after IVF benefit from PGD for aneuploidy screening? Reprod Biomed Online 2006; 12(3): 334-9. Mantzouratou A, Mania A, Fragouli E, et al. Variable aneuploidy mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening. Hum Reprod 2007; 22(7): p. 1844-53. Voullaire L, Wilton L, McBain J, Callaghan T, Williamson R. Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure. Mol Hum Reprod 2002; 8(11): 1035-41. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988; 332(6163): 459-61. Wells D, Bermudez MG, Steuerwald N, et al. Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development. Hum Reprod 2005; 20(5): 1339-48. Voullaire L, Collins V, Callaghan T, McBain J, Williamson R, Wilton L. High incidence of complex chromosome abnormality in cleavage embryos from patients with repeated implantation failure. Fertil Steril 2007; 87(5): 1053-8. Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation and treatment of repeated implantation failure following IVF-ET. Hum Reprod 2006; 21(12): 3036-43. Munné S, Magli C, Bahçe M, et al. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn 1998;18(13): 1459-66. Kahraman S, Bahçe M, Samli H, et al. Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure. Hum Reprod 2000; 15(9): 2003-7. Munné S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003; 7(1): 91-7. Arefi S, Soltanghoraei H, Sadeghi MR, Tabaei AS, Zeraati H, Zarnani H. Findings on hysteroscopy in patients with in vitro fertilization by intra cytoplasmic single sperm injection and embryo transfer failures. Saudi Med J 2008; 29(8): 1201-2. Lorusso F, Ceci O, Bettocchi S, et al. Office hysteroscopy in an in vitro fertilization program. Gynecol Endocrinol 2008; 24(8): 465-9. Ly et al. [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] Oliveira FG, Abdelmassih VG, Diamond MP, et al. Uterine cavity findings and hysteroscopic interventions in patients undergoing in vitro fertilization-embryo transfer who repeatedly cannot conceive. Fertil Steril 2003; 80(6): 1371-5. Makrakis E, Hassiakos D, Stathis D, Vaxevanoglou T, Orfanoudaki E, Pantos K. Hysteroscopy in women with implantation failures after in vitro fertilization: findings and effect on subsequent pregnancy rates. J Minim Invasive Gynecol 2009; 16(2): 181-7. Madani T, Ghaffari F, Kiani K, Hosseini F. Hysteroscopic polypectomy without cycle cancellation in IVF cycles. Reprod Biomed Online 2009; 18(3):412-5. Küçük T, Safali M. "Chromohysteroscopy" for evaluation of endometrium in recurrent in vitro fertilization failure. J Assist Reprod Genet 2008; 25(2-3): 79-82. Boolell M, Allen MJ, Ballard SA, et al. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res 1996; 8(2): 47-52. Goodman C, Jeyendran RS, Coulam CB. Coulam, P53 tumor suppressor factor, plasminogen activator inhibitor, and vascular endothelial growth factor gene polymorphisms and recurrent implantation failure. Fertil Steril 2009; 92(2): 494-8. Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. Hum Reprod 2000; 15(4): 806-9. Sher G, Fisch JD. Effect of vaginal sildenafil on the outcome of in vitro fertilization (IVF) after multiple IVF failures attributed to poor endometrial development. Fertil Steril 2002; 78(5): 1073-6. Check JH, Graziano V. Multiple confounders--measured with error? Fertil Steril 2003; 79(5): 1256-7; author reply 1257-8. Zinger M, Liu JH, Thomas MA. Successful use of vaginal sildenafil citrate in two infertility patients with Asherman's syndrome. J Womens Health (Larchmt) 2006; 15(4): 442-4. Paulus WE, Strehler E, Zhang M, Jelinkova L, El-Danasouri I, Sterzik K. Benefit of vaginal sildenafil citrate in assisted reproduction therapy. Fertil Steril 2002; 77(4): 846-7. Quenby S, Bates M, Doig T, et al. Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod 1999; 14(9): 2386-91. Gilman-Sachs A, DuChateau BK, Aslakson CJ, et al. Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions. Am J Reprod Immunol 1999; 41(1): 99-105. Aoki K, Kajiura S, Matsumoto Y, et al. Preconceptional naturalkiller-cell activity as a predictor of miscarriage. Lancet 1995; 345(8961): 1340-2. Strandell A, Lindhard A, Waldenström U, Thorburn J. Nitric oxide inhibits development of embryos and implantation in mice. Mol Hum Reprod 1998; 4(5): 503-7. Battaglia C, Ciotti P, Notarangelo L, Fratto R, Facchinetti F, de Aloysio D. Embryonic production of nitric oxide and its role in implantation: a pilot study. J Assist Reprod Genet 2003; 20(11): 44954. Strandell A, Waldenström U, Nilsson L, Hamberger L. Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum Reprod 1994; 9(5): 861-3. Bontis JN, Dinas KD. Management of hydrosalpinx: reconstructive surgery or IVF? Ann N Y Acad Sci 2000; 900: 260-71. Strandell A, Lindhard A, Waldenström U, Thorburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a randomized controlled trial. Hum Reprod 2001; 16(11): 2403-10. Mansour R, Aboulghar M, Serour GI. Controversies in the surgical management of hydrosalpinx. Curr Opin Obstet Gynecol 2000; 12(4): 297-301. Granot I, Dekel N, Segal I, Fieldust S, Shoham Z, Barash A. Is hydrosalpinx fluid cytotoxic? Hum Reprod 1998; 13(6): 1620-4. Sharara FI. The role of hydrosalpinx in IVF: simply mechanical? Hum Reprod 1999; 14(3): 577-8. Seli E, Kayisli UA, Cakmak H, et al. Removal of hydrosalpinges increases endometrial leukaemia inhibitory factor (LIF) expression at the time of the implantation window. Hum Reprod 2005; 20(11): 3012-7. Bildirici I, Bukulmez O, Ensari A, Yarali H, Gurgan T. A prospective evaluation of the effect of salpingectomy on endometrial re- Evidence-Based Management of Infertile Couples [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] ceptivity in cases of women with communicating hydrosalpinges. Hum Reprod 2001; 16(11): 2422-6. Meyer WR, Lessey BA, Castelbaum AJ.Hydrosalpinx and altered uterine receptivity. Fertil Steril 1997;68(5): 944-5. Grifo JA, Jeremias J, Ledger WJ, Witkin SS.Interferon-gamma in the diagnosis and pathogenesis of pelvic inflammatory disease. Am J Obstet Gynecol 1989; 160(1): 26-31. Ben-Rafael Z, Orvieto R. Cytokines--involvement in reproduction. Fertil Steril 1992; 58(6): 1093-9. Jones RB, Mammel JB, Shepard MK, Fisher RR. Recovery of Chlamydia trachomatis from the endometrium of women at risk for chlamydial infection. Am J Obstet Gynecol 1986; 155(1): 35-9. Lass A. What effect does hydrosalpinx have on assisted reproduction? What is the preferred treatment for hydrosalpines? The ovary's perspective. Hum Reprod 1999; 14(7): 1674-7. Dechaud H, Anahory T, Aligier N, Arnal F, Humeau H, Hedon B. Salpingectomy for repeated embryo nonimplantation after in vitro fertilization in patients with severe tubal factor infertility. J Assist Reprod Genet 2000; 17(4): 200-6. Johnson NP, Mak W, Sowter MC. Surgical treatment for tubal disease in women due to undergo in vitro fertilisation. Cochrane Database Syst Rev 2004; (3): CD002125. Johnson NP, Mak W, Sowter MC. Laparoscopic salpingectomy for women with hydrosalpinges enhances the success of IVF: a Cochrane review. Hum Reprod 2002; 17(3): 543-8. Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive Technology. Fertil Steril 2008; 90(5 Suppl): S30-44. Klein J, Sauer MV.Oocyte donation. Best Pract Res Clin Obstet Gynaecol 2002; 16(3): 277-91. Sauer MV, Kavic SM.Oocyte and embryo donation 2006: reviewing two decades of innovation and controversy. Reprod Biomed Online 2006; 12(2): 153-62. Lee RM, Silver RM. Recurrent pregnancy loss: summary and clinical recommendations. Semin Reprod Med 2000; 18(4): 433-40. Lee RM, Silver RM. The efficacy and safety of human oocyte vitrification. Semin Reprod Med 2009; 27(6): 450-5. ASRM Practice Committee response to Rybak and Lieman: elective self-donation of oocytes. Fertil Steril 2009; 92(5): 1513-4. Rienzi L, Romano S, Albricci L, et al. Embryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod 2010; 25(1): 66-73. Azem F, Yaron Y, Amit A, et al. Transfer of six or more embryos improves success rates in patients with repeated in vitro fertilization failures. Fertil Steril 1995; 63(5): 1043-6. Hu Y, Maxson WS, Hoffman DI, et al. Maximizing pregnancy rates and limiting higher-order multiple conceptions by determining the optimal number of embryos to transfer based on quality. Fertil Steril 1998; 69(4): 650-7. Stern JE, Cedars MI, Jain T, et al. Assisted reproductive technology practice patterns and the impact of embryo transfer guidelines in the United States. Fertil Steril 2007; 88(2): 275-82. Campbell B, Erickson L. The number of embryos to transfer: current practices in Minnesota. Minn Med 2009; 92(4): 40-1. Guidelines on number of embryos transferred. Fertil Steril 2009; 92(5): 1518-9. Guidelines for the number of embryos to transfer following in vitro fertilization No. 182, September 2006. Int J Gynaecol Obstet 2008; 102(2): 203-16. Pandian Z, Bhattacharya S, Ozturk O, Serour G, Templeton A. Number of embryos for transfer following in-vitro fertilisation or intra-cytoplasmic sperm injection. Cochrane Database Syst Rev 2009(2): CD003416. Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001; 76(5): 863-70. Levran D, Farhi J, Nahum H, Royburt M, Glezerman M, Weissman A. Prospective evaluation of blastocyst stage transfer vs. zygote intrafallopian tube transfer in patients with repeated implantation failure. Fertil Steril 2002; 77(5): 971-7. Levran D, Mashiach S, Dor J, Levron J, Farhi J. Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated failure of implantation. Fertil Steril 1998; 69(1): 26-30. Urman B, Yakin K, Balaban B. Recurrent implantation failure in assisted reproduction: how to counsel and manage. B. Treatment Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] 215 options that have not been proven to benefit the couple. Reprod Biomed Online 2005; 11(3): 382-91. Jansen RP. Endocrine response in the fallopian tube. Endocr Rev 1984; 5(4): 525-51. Devroey P, Staessen C, Camus M, De Grauwe E, Wisanto A, Van Steirteghem AC.Zygote intrafallopian transfer as a successful treatment for unexplained infertility. Fertil Steril 1989; 52(2): 2469. Hammitt DG, Syrop CH, Hahn SJ, Walker DL, Butkowski CR, Donovan JF.Comparison of concurrent pregnancy rates for in-vitro fertilization--embryo transfer, pronuclear stage embryo transfer and gamete intra-fallopian transfer. Hum Reprod 1990; 5(8): 947-54. Asch RH.Uterine versus tubal embryo transfer in the human. Comparative analysis of implantation, pregnancy, and live-birth rates. Ann N Y Acad Sci 1991; 626: 461-6. Farhi J, Weissman A, Nahum H, Levran D.Zygote intrafallopian transfer in patients with tubal factor infertility after repeated failure of implantation with in vitro fertilization-embryo transfer. Fertil Steril 2000; 74(2): 390-3. 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(2): 286-93. Aslan D, Elizur SE, Levron J, et al. Comparison of zygote intrafallopian tube transfer and transcervical uterine embryo transfer in patients with repeated implantation failure. Eur J Obstet Gynecol Reprod Biol 2005; 122(2): 191-4. Weissman A, Eldar I, Ravhon A, et al. Timing intra-Fallopian transfer procedures. Reprod Biomed Online 2007; 15(4): 445-50. Weissman A, F.J., Levran D. Zygote intrafallopian transfer. Reproductive Techniques: Laboratory and Clinical Perspectives, 2nd ed. W.A. Gardner DK, Howies CM, Shoham Z, Eds. London Taylor and Francis 2004; pp. 735-48. Ubaldi F, Rienzi L, Ferrero S, et al. Natural in vitro fertilization cycles. Ann N Y Acad Sci 2004; 1034: 245-51. Morgia F, Sbracia M, Schimberni M, et al. A controlled trial of natural cycle versus microdose gonadotropin-releasing hormone analog flare cycles in poor responders undergoing in vitro fertilization. Fertil Steril 2004; 81(6): 1542-7. Bassil S, Godin PA, Donnez J. Outcome of in-vitro fertilization through natural cycles in poor responders. Hum Reprod 1999; 14(5): 1262-5. Schimberni M, Morgia F, Colabianchi J, et al. Natural-cycle in vitro fertilization in poor responder patients: a survey of 500 consecutive cycles. Fertil Steril 2009; 92(4): 1297-301. Pelinck MJ, Hoek A, Simons AH, Heineman MJ. Efficacy of natural cycle IVF: a review of the literature. Hum Reprod Update 2002; 8(2): 129-39. Paria BC, Lim H, Das SK, Reese J, Dey SK. Molecular signaling in uterine receptivity for implantation. Semin Cell Dev Biol 2000; 11(2): 67-76. Paria BC, Lim H, Das SK, Reese J, Dey SK. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999; 340(23): 1796-9. Martín J, Domínguez F, Avila S, et al. Human endometrial receptivity: gene regulation. J Reprod Immunol 2002; 55(1-2): 131-9. Davis GE, Senger DR. Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res 2005; 97(11): 1093-107. Bischof P, Campana A. Molecular mediators of implantation. Baillieres Best Pract Res Clin Obstet Gynaecol 2000; 14(5): 80114. Basir GS, O WS, Ng EH, Ho PC. Morphometric analysis of periimplantation endometrium in patients having excessively high oestradiol concentrations after ovarian stimulation. Hum Reprod 2001; 16(3): 435-40. Devroey P, Bourgain C, Macklon NS, Fauser BC. Reproductive biology and IVF: ovarian stimulation and endometrial receptivity. Trends Endocrinol Metab 2004; 15(2): 84-90. Tavaniotou A, Albano C, Smitz J, Devroey P. Impact of ovarian stimulation on corpus luteum function and embryonic implantation. J Reprod Immunol 2002; 55(1-2): 123-30. Basille C, Fay S, Hesters L, Frydman N, Frydman R. In vitro fertilization (IVF): why doing it in unstimulated cycles?. Gynecol Obstet Fertil 2007; 35(9): 877-80. Lédée-Bataille N, Dubanchet S, Kadoch J, Castelo-Branco A, Frydman R, Chaouat G. Controlled natural in vitro fertilization 216 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] may be an alternative for patients with repeated unexplained implantation failure and a high uterine natural killer cell count. Fertil Steril 2004; 82(1): 234-6. Croy BA, Ashkar AA, Minhas K, Greenwood JD.Can murine uterine natural killer cells give insights into the pathogenesis of preeclampsia? J Soc Gynecol Investig 2000; 7(1): 12-20. Horcajadas JA, Riesewijk A, Polman J, et al. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Hum Reprod 2005; 11(3): 195-205. Haouzi D, Mahmoud K, Fourar M, et al. Identification of new biomarkers of human endometrial receptivity in the natural cycle. Hum Reprod 2009; 24(1): 198-205. Di Simone N, Meroni PL, de Papa N, et al. Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness by binding directly and through adhered beta2-glycoprotein I. Arthritis Rheum 2000; 43(1): 140-50. Kutteh WH.Antiphospholipid antibodies and reproduction. J Reprod Immunol 1997; 35(2): 151-71. Girardi G, Yarilin D, Thurman JM, Holers VM, Salmon JE.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction. J Exp Med 2006; 203(9): 2165-75. el-Roeiy A, Gleicher N, Friberg J, Confino E, Dudkiewicz A. Correlation between peripheral blood and follicular fluid autoantibodies and impact on in vitro fertilization. Obstet Gynecol 1987; 70(2): 163-70. Buckingham KL, Stone PR, Smith JF, Chamley LW. Antiphospholipid antibodies in serum and follicular fluid--is there a correlation with IVF implantation failure? Hum Reprod 2006; 21(3): 72834. Matsubayashi H, Sugi T, Arai T, et al. IgG-antiphospholipid antibodies in follicular fluid of IVF-ET patients are related to low fertilization rate of their oocytes. Am J Reprod Immunol 2006; 55(5): 341-8. Choudhury SR, Knapp LA.Human reproductive failure I: immunological factors. Hum Reprod Update 2001; 7(2): 113-34. Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335(8705): 1544-7. Meroni PL, Gerosa M, Raschi E, Scurati S, Grossi C, Borghi MO.Updating on the pathogenic mechanisms 5 of the antiphospholipid antibodies-associated pregnancy loss. Clin Rev Allergy Immunol 2008; 34(3): 332-7. Stern C, Chamley L, Hale L, Kloss M, Speirs A, Baker HW. Antibodies to beta2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of a prevalence study. Fertil Steril 1998; 70(5): 938-44. Birkenfeld A, Mukaida T, Minichiello L, Jackson M, Kase NG, Yemini M. Incidence of autoimmune antibodies in failed embryo transfer cycles. Am J Reprod Immunol 1994; 31(2-3): 65-8. Gleicher N, Liu HC, Dudkiewicz A, et al. Autoantibody profiles and immunoglobulin levels as predictors of in vitro fertilization success. Am J Obstet Gynecol 1994; 170(4): 1145-9. Coulam CB, Kaider BD, Kaider AS, Janowicz P, Roussev RG. Antiphospholipid antibodies associated with implantation failure after IVF/ET. J Assist Reprod Genet 1997; 14(10): 6038. Denis AL, Guido M, Adler RD, Bergh PA, Brenner C, Scott RT Jr. Antiphospholipid antibodies and pregnancy rates and outcome in in vitro fertilization patients. Fertil Steril 1997; 67(6): 1084-90. Geva E, Yaron Y, Lessing JB, et al. Circulating autoimmune antibodies may be responsible for implantation failure in in vitro fertilization. Fertil Steril 1994; 62(4): 802-6. Kowalik A, Vichnin M, Liu HC, Branch W, Berkeley AS. Midfollicular anticardiolipin and antiphosphatidylserine antibody titers do not correlate with in vitro fertilization outcome. Fertil Steril 1997; 68(2): 298-304. Hill JA, Choi BC. Maternal immunological aspects of pregnancy success and failure. J Reprod Fertil Suppl 2000; 55: 91-7. Hornstein MD, Davis OK, Massey JB, Paulson RJ, Collins JA. Antiphospholipid antibodies and in vitro fertilization success: a meta-analysis. Fertil Steril 2000; 73(2): 330-3. Gleicher N, Vidali A, Karande V. The immunological "Wars of the Roses": disagreements amongst reproductive immunologists. Hum Reprod 2002; 17(3): 539-42. Ly et al. [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] [181] [182] Matsubayashi H, Arai T, Izumi S, Sugi T, McIntyre JA, Makino T. Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures. Fertil Steril 2001; 76(4): 694-9. Eldar-Geva T, Wood C, Lolatgis N, et al. Cumulative pregnancy and live birth rates in women with antiphospholipid antibodies undergoing assisted reproduction. Hum Reprod 1999; 14(6): 1461-6. Practice Committee of American Society for Reproductive Medicine. Anti-phospholipid antibodies do not affect IVF success. Fertil Steril 2008; 90(5 Suppl): S172-3. Ermel LD, Marshburn PB, Kutteh WH. Interaction of heparin with antiphospholipid antibodies (APA) from the sera of women with recurrent pregnancy loss (RPL). Am J Reprod Immunol 1995; 33(1): 14-20. Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone. Am J Obstet Gynecol 1996; 174(5): 1584-9. Kutteh WH, Yetman DL, Chantilis SJ, Crain J. Effect of antiphospholipid antibodies in women undergoing in-vitro fertilization: role of heparin and aspirin. Hum Reprod 1997; 12(6): 1171-5. Stern C, Chamley L, Norris H, Hale L, Baker HW. A randomized, double-blind, placebo-controlled trial of heparin and aspirin for women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies. Fertil Steril 2003; 80(2): 376-83. Empson M, Lassere M, Craig J, Scott J. Prevention of recurrent miscarriage for women with antiphospholipid antibody or lupus anticoagulant. Cochrane Database Syst Rev 2005; (2): CD002859. Vaquero E, Lazzarin N, Valensise Het, et al. Pregnancy outcome in recurrent spontaneous abortion associated with antiphospholipid antibodies: a comparative study of intravenous immunoglobulin versus prednisone plus low-dose aspirin. Am J Reprod Immunol 2001; 45(3): 174-9. Triolo G, Ferrante A, Ciccia F, et al. Randomized study of subcutaneous low molecular weight heparin plus aspirin versus intravenous immunoglobulin in the treatment of recurrent fetal loss associated with antiphospholipid antibodies. Arthritis Rheum 2003; 48(3): 728-31. Dendrinos S, Sakkas E, Makrakis E. Low-molecular-weight heparin versus intravenous immunoglobulin for recurrent abortion associated with antiphospholipid antibody syndrome. Int J Gynaecol Obstet 2009; 104(3): 223-5. Clark AL, Branch DW, Silver RM, Harris EN, Pierangeli S, Spinnato JA.Pregnancy complicated by the antiphospholipid syndrome: outcomes with intravenous immunoglobulin therapy. Obstet Gynecol 1999; 93(3): 437-41. Wong RC, Favaloro EJ. Clinical features, diagnosis, and management of the antiphospholipid syndrome. Semin Thromb Hemost 2008; 34(3): 295-304. Takeshita T. Diagnosis and treatment of recurrent miscarriage associated with immunologic disorders: Is paternal lymphocyte immunization a relic of the past? J Nippon Med Sch 2004; 71(5): 308-13. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 1993; 14(7): 353-6. Lédée-Bataille N, Bonnet-Chea K, Hosny G, Dubanchet S, Frydman R, Chaouat G. Role of the endometrial tripod interleukin18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization-embryo transfer failure. Fertil Steril 2005; 83(3): 598-605. Quenby S, Farquharson R. Uterine natural killer cells, implantation failure and recurrent miscarriage. Reprod Biomed Online 2006; 13(1): 24-8. Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J Immunol 1996; 156(10): 4027-34. Ntrivalas EI, Kwak-Kim JY, Gilman-Sachs A, et al. Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology. Hum Reprod 2001; 16(5): 855-61. Kwak JY, Beaman KD, Gilman-Sachs A, Ruiz JE, Schewitz D, Beer AE. Up-regulated expression of CD56+, CD56+/CD16+, and CD19+ cells in peripheral blood lymphocytes in pregnant women Evidence-Based Management of Infertile Couples [183] [184] [185] [186] [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] with recurrent pregnancy losses. Am J Reprod Immunol 1995; 34(2): 93-9. Yamada H, Kato EH, Kobashi G, et al. High NK cell activity in early pregnancy correlates with subsequent abortion with normal chromosomes in women with recurrent abortion. Am J Reprod Immunol 2001; 46(2): 132-6. Yamamoto T, Takahashi Y, Kase N, Mori H. Role of decidual natural killer (NK) cells in patients with missed abortion: differences between cases with normal and abnormal chromosome. Clin Exp Immunol 1999; 116(3): 449-52. Quack KC, Vassiliadou N, Pudney J, Anderson DJ, Hill JA. Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion. Hum Reprod 2001; 16(5): 949-55. Lédée-Bataille N, Laprée-Delage G, Taupin JL, Dubanchet S, Frydman R, Chaouat G. Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is highly predictive of embryo implantation. Hum Reprod 2002; 17(1):213-8. Kwak-Kim JY, Chung-Bang HS, Ng SC, et al. Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF. Hum Reprod 2003; 18(4): 767-73. Ng SC, Gilman-Sachs A, Thaker P, Beaman KD, Beer AE, KwakKim J. Expression of intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion, implantation failures after IVF/ET or normal pregnancy. Am J Reprod Immunol 2002; 48(2): 77-86. Lim KJ, Odukoya OA, Ajjan RA, Li TC, Weetman AP, Cooke ID. The role of T-helper cytokines in human reproduction. Fertil Steril 2000; 73(1): 136-42. Hill JA, Polgar K, Anderson DJ. T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA 1995; 273(24): 1933-6. Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med 1998; 4(9): 1020-4. Bates MD, Quenby S, Takakuwa K, Johnson PM, Vince GS. Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss? Hum Reprod 2002; 17(9): 2439-44. Kruse C, Varming K, Christiansen OB. Prospective, serial investigations of in-vitro lymphocyte cytokine production, CD62L expression and proliferative response to microbial antigens in women with recurrent miscarriage. Hum Reprod 2003; 18(11): 2465-72. Borzychowski AM, Croy BA, Chan WL, Redman CW, Sargent IL. Changes in systemic type 1 and type 2 immunity in normal pregnancy and pre-eclampsia may be mediated by natural killer cells. Eur J Immunol 2005; 35(10): 3054-63. Chaouat G, Ledée-Bataille N, Dubanchet S, Zourbas S, Sandra O, Martal J. TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm. Int Arch Allergy Immunol 2004; 134(2): 93-119. Steck T, Giess R, Suetterlin MW, et al. Leukaemia inhibitory factor (LIF) gene mutations in women with unexplained infertility and recurrent failure of implantation after IVF and embryo transfer. Eur J Obstet Gynecol Reprod Biol 2004; 112(1): 69-73. Jack DL, Read RC, Tenner AJ, Frosch M, Turner MW, Klein NJ. Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B. J Infect Dis 2001; 184(9): 1152-62. Kilpatrick DC, Bevan BH, Liston WA. Association between mannan binding protein deficiency and recurrent miscarriage. Hum Reprod 1995; 10(9): 2501-5. Dahl M, Tybjaerg-Hansen A, Schnohr P, Nordestgaard BG. Morbidity and mortality in persons with mannose-binding lectin deficiency. The Osterbro study. Ugeskr Laeger 2004; 166(50): 4606-9. Kruse C, Rosgaard A, Steffensen R, Varming K, Jensenius JC, Christiansen OB. Low serum level of mannan-binding lectin is a determinant for pregnancy outcome in women with recurrent spontaneous abortion. Am J Obstet Gynecol 2002; 187(5): 1313-20. Shetty S, Ghosh K. Anti-phospholipid antibodies and other immunological causes of recurrent foetal loss--a review of literature of various therapeutic protocols. Am J Reprod Immunol 2009; 62(1): 9-24. Morikawa M, Yamada H, Kato EH, et al. Massive intravenous immunoglobulin treatment in women with four or more recurrent Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [203] [204] [205] [206] [207] [208] [209] [210] [211] [212] [213] [214] [215] [216] [217] [218] [219] [220] [221] [222] [223] 217 spontaneous abortions of unexplained etiology: down-regulation of NK cell activity and subsets. Am J Reprod Immunol 2001; 46(6): 399-404. Clark DA, Chaouat G. Loss of surface CD200 on stored allogeneic leukocytes may impair anti-abortive effect in vivo. Am J Reprod Immunol 2005; 53(1): 13-20. Gorczynski RM. Thymocyte/splenocyte-derived CD4+CD25+Treg stimulated by anti-CD200R2 derived dendritic cells suppress mixed leukocyte cultures and skin graft rejection. Transplantation 2006; 81(7): 1027-34. Wright GJ, Cherwinski H, Foster-Cuevas M, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 2003; 171(6): 3034-46. Daya S, Gunby J, Porter F, Scott J, Clark DA. Critical analysis of intravenous immunoglobulin therapy for recurrent miscarriage. Hum Reprod Update 1999; 5(5): 475-82. Stephenson MD, Dreher K, Houlihan E, Wu V. Prevention of unexplained recurrent spontaneous abortion using intravenous immunoglobulin: a prospective, randomized, double-blinded, placebocontrolled trial. Am J Reprod Immunol 1998; 39(2): 82-8. Coulam CB, Krysa L, Stern JJ, Bustillo M.Intravenous immunoglobulin for treatment of recurrent pregnancy loss. Am J Reprod Immunol 1995; 34(6): 333-7. Christiansen OB, Pedersen B, Rosgaard A, Husth M.A randomized, double-blind, placebo-controlled trial of intravenous immunoglobulin in the prevention of recurrent miscarriage: evidence for a therapeutic effect in women with secondary recurrent miscarriage. Hum Reprod 2002; 17(3): 809-16. Hutton B, Sharma R, Fergusson D, et al. Use of intravenous immunoglobulin for treatment of recurrent miscarriage: a systematic review. BJOG 2007; 114(2): 134-42. Clark DA, Coulam CB, Stricker RB. Is intravenous immunoglobulins (IVIG) efficacious in early pregnancy failure? A critical review and meta-analysis for patients who fail in vitro fertilization and embryo transfer (IVF). J Assist Reprod Genet 2006, 23(1): 1-13. Carp HJ. Intravenous immunoglobulin: effect on infertility and recurrent pregnancy loss. Isr Med Assoc J 2007; 9(12): 877-80. van den Heuvel MJ, Peralta CG, Hatta K, Han VK, Clark DA. Decline in number of elevated blood CD3(+) CD56(+) NKT cells in response to intravenous immunoglobulin treatment correlates with successful pregnancy. Am J Reprod Immunol 2007; 58(5): 447-59. Winger EE, Reed JL, Ashoush S, Ahuja S, El-Toukhy T, Taranissi M. Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF. Am J Reprod Immunol 2009; 61(2): 113-20. Porter TF, LaCoursiere Y, Scott JR. Immunotherapy for recurrent miscarriage. Cochrane Database Syst Rev 2006; (2): CD000112. Pandey MK, Rani R, Agrawal S. An update in recurrent spontaneous abortion. Arch Gynecol Obstet 2005; 272(2): 95-108. McIntyre JA, Coulam CB, Faulk WP. Recurrent spontaneous abortion. Am J Reprod Immunol 1989; 21(3-4): 100-4. Tamura M, Takakuwa K, Arakawa M, Yasuda M, Kazama Y, Tanaka K. Relationship between MLR blocking antibodies and the outcome of the third pregnancy in patients with two consecutive spontaneous abortions. J Perinat Med 1998; 26(1): 49-53. Daya S, Gunby J. The effectiveness of allogeneic leukocyte immunization in unexplained primary recurrent spontaneous abortion. Recurrent Miscarriage Immunotherapy Trialists Group. Am J Reprod Immunol 1994; 32(4): 294-302. Pandey MK, Thakur S, Agrawal S. Lymphocyte immunotherapy and its probable mechanism in the maintenance of pregnancy in women with recurrent spontaneous abortion. Arch Gynecol Obstet 2004; 269(3): 161-72. Gatenby PA, Cameron K, Simes RJ, et al. Treatment of recurrent spontaneous abortion by immunization with paternal lymphocytes: results of a controlled trial. Am J Reprod Immunol 1993; 29(2): 8894. Mowbray JF, Gibbings C, Liddell H, et al. Controlled trial of treatment of recurrent spontaneous abortion by immunisation with paternal cells. Lancet 1985; 1(8435): 941-3. Worldwide collaborative observational study and meta-analysis on allogenic leukocyte immunotherapy for recurrent spontaneous abortion. Recurrent Miscarriage Immunotherapy Trialists Group. Am J Reprod Immunol 1994; 32(2): 55-72. 218 Current Women’s Health Reviews, 2010, Vol. 6, No. 3 [224] [225] [226] Ly et al. Kling C, Steinmann J, Westphal E, Magez J,Kabelitz D. Experience with allogeneic leukocyte immunization for implantation failure in the in-vitro fertilization program. Am J Reprod Immunol 2002; 48: 147. Ober C, Karrison T, Odem RR, et al. Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial. Lancet 1999; 354(9176): 365-9. Clark DA, Chaouat G. Immunomodulation by CD200 as a basis for prevention of cytokine-dependent fetal loss syndrome. Am J Reprod Immunol 2003; 49: 350-1. Received: January 17, 2010 [227] [228] [229] Research, C.f.B.E.a., Lymphocyte Immune Therapy. 2001. Kling C, Steinmann J, Westphal E, Magez J, Kabelitz D. Adverse effects of intradermal allogeneic lymphocyte immunotherapy: acute reactions and role of autoimmunity. Hum Reprod 2006; 21(2): 42935. Pearlman SA, Meek RS, Cowchock FS, Smith JB, McFarland J, Aster RH. Neonatal alloimmune thrombocytopenia after maternal immunization with paternal mononuclear cells: successful treatment with intravenous gamma globulin. Am J Perinatol 992; 9(56): 448-51. Revised: March 29, 2010 Accepted: June 15, 2010