Y-chromosome microdeletions and recurrent pregnancy loss

Transcription

1 RECURRENT PREGNANCY LOSS Y-chromosome microdeletions and recurrent pregnancy loss Sheri Dewan, M.S., a Elizabeth E. Puscheck, M.D., b Carolyn B. Coulam, M.D., c Alexander J. Wilcox, B.S., d and Rajasingam S. Jeyendran, D.V.M., Ph.D. a a Andrology Laboratory Services Inc., Chicago, Illinois; b Department of Obstetrics and Gynecology, Wayne State University Medical School, Detroit, Michigan; c Sher Institute of Recurrent Pregnancy Loss, Chicago, Illinois; and d Northwestern University Graduate School, Department of Biochemistry, Molecular Biology and Cellular Biology, Evanston, Illinois Objective: To determine the prevalence of Y-chromosome microdeletions in recurrent pregnancy loss (RPL) couples as compared with couples with male factor infertility and fertile couples. Design: Controlled clinical study. Setting: Andrology laboratory and RPL clinic. Patient(s): Seventeen men from RPL couples, 18 men from couples with a live birth and no history of miscarriages, and 10 men from couples with male factor infertility. Intervention(s): Buccal smears for Y-chromosome microdeletion testing. Main Outcome Measure(s): The DNA was tested for microdeletions in the proximal AZFc region by polymerase chain reaction (PCR). Result(s): Fourteen of the 17 men (82%) tested had microdeletions in one or more of the four segments studied. Two of the 10 male factor infertility patients (20%) had microdeletions in 2 different segments. None of the 18 fertile men had any microdeletions in the 4 segments of the proximal AZFc region studied. Conclusion(s): The prevalence of the Y-chromosome microdeletions in the proximal AZFc region was much higher in men from RPL couples than from fertile or infertile couples. Although these patients are from a tertiary referral center that may skew the population and findings, one may consider Y-chromosome microdeletion testing particularly of the AZFc region in the evaluation of RPL couples when all other tests fail to reveal the etiology. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Y-chromosome, microdeletions, recurrent pregnancy loss, miscarriages, infertility, male factor infertility, habitual abortions Received August 23, 2004; revised and accepted August 5, Presented on October 22, 2001 at the Annual Meeting of the American Society for Reproductive Medicine in Orlando, Florida (October 21 25, 2001). Reprint requests: Elizabeth Puscheck, M.D., 3750 Woodward Ave, Suite 200D, Detroit, Michigan (FAX: ; epuschec@ med.wayne.edu). Miscarriages are very common, affecting about 15% of all known pregnancies (1), and others report a much higher incidence if those pregnancies that are lost before the first missed period are included. A subset of this population will have three or more consecutive miscarriages and that defines recurrent pregnancy loss (RPL) also known as recurrent abortion or recurrent miscarriages. Recurrent pregnancy loss is devastating to couples who desire children. The etiologies of RPL vary considerably; they range from genetic abnormalities of the conceptus or one or both of the parents, to hormonal, structural (uterine or cervical), immunologic, or hematological abnormalities, and to chronic illness, especially if it is uncontrolled (i.e., diabetes) (2 5). Unfortunately, only about 30% of these RPL have identifiable etiologies (6). Thus, treatments are only marginally successful. The majority of the testing for RPL assesses the woman. The male, though important for conception, has been less thoroughly investigated than the female in the RPL evaluation. Semen analysis is not generally a part of the initial assessment for RPL because it is for an infertility evaluation. Yet, sperm integrity is required for sperm egg interactions, fertilization, and early embryonic development (7 9). Sperm quality is associated with the embryo s ability to reach the blastocyst stage and progress to implantation. Paternally expressed genes modulate the proliferation and invasiveness of trophoblast cells and later placental proliferation (10 12). What is more interesting is that the men of RPL couples have higher sperm chromosome abnormalities and poor sperm membrane integrity (13, 14). To date, men with very low sperm counts ( 1 million/ml), identified through the infertility office, have a higher incidence of chromosomal abnormalities (0.5% 2.8%) or Y-chromosome microdeletions (up to 17%). Y-chromosome microdeletions were ini /06/$32.00 Fertility and Sterility Vol. 85, No. 2, February 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 441

2 tially reported in 1995 as DAZ (deletion in azoospermia) or AZF (azoospermia factor) (15). The three regions of the AZF gene where pathology-associated microdeletions occur are simply labeled a, b, and c. Patients with AZFa microdeletions often present with congenital oligozoospermia or partial spermatogenic arrest, whereas patients with AZFb and AZFc usually present with azoospermia or oligozoospermia. However, microdeletions in the overlapping area between the latter two regions may present with range of sperm counts (azoospermia to normal sperm count). Consequently, this region of AZF was evaluated in RPL patients. Our preliminary study (Nani et al., Proceedings of the American Society for Reproductive Medicine Meeting, Orlando, Florida, October 20 25, 2001; O-32, S13) utilized the Promega Kit Version 1.1 (Promega, Madison, WI) to screen for 18 different microdeletions in the AZFa d regions of the Y chromosome. The AZFd region is now recognized as the proximal portion of the AZFc region. In this preliminary study, seven men from couples with RPL were evaluated. Some sequence tagged sites (STS) in the proximal AZFc region of the Y chromosome were microdeleted in RPL patients. This preliminary study only evaluated three sequences tagged sites within the AZFc region that were developed mainly for evaluating azoospermic and oligospermic men. This current investigation expanded these initial studies by evaluating a larger number of sequence tagged sites in the proximal portion of the AZFc region, to determine whether there existed an association between microdeletions in this region and RPL. In addition, we added two other groups for comparison: fertile couples in whom we expected few or no microdeletions and couples with male infertility in whom we expected microdeletions. MATERIALS AND METHODS Recurrent pregnancy loss couples, male factor infertile couples, and fertile controls were recruited from a tertiary referral center. Informed consent was obtained. Both male and female partners of couples experiencing RPL were evaluated as follows. Chromosome analyses were performed on both partners in the couples. All women with RPL were examined by ultrasonography or hysterosalpingogram for detection of anatomic abnormalities of the genital tract, had blood drawn for testing for immunologic and thrombophilic risk factors, and had buccal swabs taken for evaluation of inherited thrombophilic gene mutations. Immunologic risk factors included anticoagulant, reproductive immunophenotype, natural killer cell activation assay, and embryotoxicity assay. Those with a negative RPL work-up were considered candidates for the study. Couples with male infertility were identified as men who had very low sperm concentrations (i.e., 1 million sperm/ ml) and possibly other semen parameter abnormalities on semen analyses or azoospermia, but no other significant findings in their medical history or examination. Seventeen men from couples with a history of 3 or more consecutive miscarriages, 18 men from couples with at least 1 live birth and no miscarriages, and 10 men with diagnosed male factor infertility were recruited; they had buccal swabs taken for analysis of the Y-chromosome AZFc region for microdeletions. Women were recruited as negative controls because they do not have a Y chromosome and thus no Y-chromosome AZF regions either. Buccal swabs were taken from these women and processed similarly. The DNA was extracted from the buccal swab samples using ethanol precipitation and standard methods (16 18). Polymerase chain reaction (PCR) was performed on the DNA with the Promega PCR Core System reagents per the manufacturer s instructions (Promega, Madison, WI). Amplification conditions were as follows: 2 minutes of the initial denaturation at 94 C, followed by 40 cycles of 2 minutes at 94 C; 30 seconds at 57 C; and 1 minute at 72 C, followed by final extension at 72 C for 5 minutes. The PCR products were analyzed on a 3% agarose gel with ethidium bromide staining, using a molecular ruler ( bp; Bio-Rad Laboratories, Hercules, CA). These experiments were performed at least twice. Negative and positive controls were run concurrently. Eight STSs from the human male genome, which were located in the proximal AZFc region of the Y chromosome, were selected from previous reports (19 21). Four of these STSs (DYF84S1, DYF83S1, DYF51S1, DYF87S1) revealed deletions in the fertile population, and were eliminated from the remainder of the study. The remaining four STSs (DYS262, DYS220, DYF8551, DYF8651) were studied. Statistical Analysis The results were evaluated with Pearson s chi-square and Fisher s exact test; P.05 was considered statistically significant. RESULTS The mean age of female partners experiencing RPL was 34.7 years old, and the number of previous pregnancy losses ranged from The results of the analysis are detailed in the Table 1. Fourteen men from 17 RPL couples (82%) had microdeletions in 1 or more of the 4 sequence tagged sites tested, whereas none of the fertile patients (0%) had any microdeletions for the STSs tested. Eleven of these 17 (65%) men from couples with RPL had 3 or more microdeletions in the same STS of the human Y chromosome. The detailed results revealed that 1 RPL test patient had microdeletions in all 4 STSs tested, 10 had microdeletions in 3 STSs, 2 had microdeletions in 2 STSs, 1 had a microdeletion in 1 STS, and 3 had no microdeletions. Two of the 10 men from couples with male factor infertility (20%) tested had separate single microdeletions, with 1 sequence tagged site each (not overlapping). The total number of mutations for the 17 couples with RPL was 39 as compared with 2 from the Dewan et al. Y-chromosome and recurrent miscarriages Vol. 85, No. 2, February 2006

3 TABLE 1 Incidence of individual Y-chromosome microdeletions for the STSs in the proximal AZFc region tested for various groups of men. Subjects No. of men DYF85S1 DYF86S1 DYS220 DYS262 RPL a Infertile b Fertile c a Men of recurrent pregnancy loss couples. b Men of couples with diagnosed male factor infertility. c Men of fertile couples with at least one child and no miscarriages. Dewan. Y-chromosome and recurrent miscarriages. Fertil Steril couples with male factor infertility. None of the DNA samples tested for the STSs of the proximal AZFc region of the Y chromosome from the women subjects had mutations detected, as expected because women do not have a Y chromosome. Statistical results are listed in Table 2. For statistical ease, groups were compared by the assessment of those with one TABLE 2 Total number of men with one or more Y-chromosome microdeletions among the various groups. RPL a Infertile b Fertile c Microdeletions No microdeletions Total P value d for each compared with the fertile group P value overall e.001 a Men of recurrent pregnancy loss couples. b Men of couple with diagnosed male factor infertility. c Men of fertile couples with at least one child and no miscarriages. d P values are regarding each group compared with the fertile group individually using the Fisher s exact test: RPL with microdeletion mutations vs. fertility with microdeletion mutations was statistically significant at P.001, but male factor infertility with mutations vs. fertility with mutations was not significant (P.119). In addition, RPL with mutations vs. male factor infertility with mutations was also statistically significant at P.003. e P value overall: Pearson chi-square test comparing all three groups gives a Z value of with 2 degrees of freedom, and it is statistically significant (P.001). Dewan. Y-chromosome and recurrent miscarriages. Fertil Steril or more microdeletions. The results of comparing all three groups were statistically significant (P.001). When the groups (labeled RPL, Male Infertility, and Fertility) were individually compared with each other (RPL vs. Fertility, RPL vs. Male Infertility, Male Infertility vs. Fertility), the differences for the RPL group were significant when compared with either the Fertility group (P.001) or the Male Infertility group (P.003). No statistically significant difference was, however, observed between the Male Infertility and the Fertility groups (P.119). A power analysis regarding the Male Infertility group with microdeletion mutations vs. the Fertility group with microdeletion mutations was performed with an alpha of To detect a 20% difference, at least 40 subjects would be needed in each arm (Male Infertility and Fertility) to confirm whether this last test was meaningful. Therefore, the sample size to compare results from male factor infertile and fertile couples regarding these Y-chromosome microdeletions is too small. DISCUSSION Our results indicate that a significant proportion of our RPL couples had microdeletions in the proximal portion of the AZFc region of the Y chromosome. Specifically, 82.4% of RPL couples had at least one microdeletion in this region of the human Y-chromosome, and 65% had three or more microdeletions in this area. Men from infertile couples had about 20% microdeletions (2 of 10) in this same region, and neither was overlapping with each other. None of the fertile men had microdeletions in this region. As expected, the negative control (i.e., DNA extracted from the buccal swabs of women) revealed deletions for all the STSs tested, indicating that the primers used to amplify the STSs were specific to the Y chromosome only. Microdeletions were found in all four STSs of the proximal AZFc region of the Y chromosome studied in the RPL population. The STSs labeled DYF85S1 and DYF86S1 yielded the most deletions; 13 of the 17 RPL patients had Fertility and Sterility 443

4 microdeletions in these 2 STSs also. It is not uncommon to find one abnormality in any given laboratory panel testing, but in this study, 11 of the 17 patients had 3 or more microdeletions. This cannot be attributed simply to laboratory panel screening, and statistical analysis demonstrated that the difference in frequency between the RPL and fertile populations is significant at the P.001 level. This finding suggests an additive effect of several microdeletions contributing to a more severe phenotype. Further, the microdeletions detected in these 4 sequence tagged sites among the infertile men (2 of 10) are markedly less than those detected among men from RPL couples (14 of 17). Additionally, no cumulative effects could be detected among the infertile men because both positive men had only single microdeletions, each which were located in different STSs of the proximal AZFc region. Although the frequency of the Y-chromosome microdeletions detected by these four STSs was significantly less among men in the male factor infertility group compared with men from RPL couples, the frequency of microdeletions was not statistically significantly different when men with male factor infertility were compared with fertile men (see Table 2, P.119). Despite the lack of significance between the male factor infertile men and fertile men, this finding of about 20% Y-chromosome microdeletions in male factor infertility is within the expected range previously reported of up to 15% in severely oligospermic or azoospermic men, given this small sample size. Alternatively, this negative result comparing fertile and men with male factor infertility could be the consequence of small sample size. A power analysis revealed that at least 40 subjects would be needed in each group (fertile and infertile) to detect a difference of 20% and to confirm significance with an alpha of 0.05 and a beta of 80%. Another alternative explanation and possible mechanism for RPL is that these Y-chromosome microdeletions are polymorphisms. This region of the Y-chromosome is known to have a number of palindromic areas that may make it prone to polymorphisms. Such polymorphisms may make it more likely for a crossing over event with the X-chromosome to yield a genetic abnormality leading to RPL. Such polymorphism testing may be useful in diagnosing an at risk population even if the findings do not yield a treatment. Furthermore, these patients are purposely selected from a tertiary referral center, which receives a disproportionate number of RPL patients. It is likely that these patients may have failed evaluations and treatments elsewhere before the referral; thus, this population set may skew the outcome. Consequently, these results may be different than those found in the general population or the RPL patients presenting to the general practitioner. Finally, this data still suggests a potential connection between RPL and microdeletions in the proximal portion of the AZFc region of the Y-chromosome that have not been noted before. The much higher prevalence of Y-chromosome microdeletions found in the group of RPL couples than in either the fertile or male factor infertile groups supports the explanation that the proximal AZFc region of the Y chromosome may play an important role in embryo competency or in maintaining gestation instead of in embryo generation. One may consider Y-chromosome microdeletion testing at these loci in the RPL population when no other explanation for RPL is apparent. 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