Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study

Early pregnancy loss is defined as a miscarriage occurring before 12 weeks of gestation. The most common cause of these losses is embryo aneuploidy. Risk factors include uterine anomalies, metabolic and hormonal disorders, infection, chronic endometritis, autoimmune anomalies, thrombophilia and parental chromosomal translocations [1]. However, around 50% of miscarriages remain unexplained. Recurrent pregnancy loss (RPL) is defined as three or more consecutive early pregnancy losses in the first trimester of gestation. Studies on male factors involved in these pregnancy losses are recent, and several factors have been found, notably related to semen characteristics. The total number of motile spermatozoa has also been found to be significantly decreased in RPL compared with fertile men [2, 3] and morphological sperm alterations are more numerous [4–11].

It is noteworthy that sperm DNA fragmentation is involved in the late paternal effect on human embryo development in cases of repeated failures of assisted reproduction techniques (ART), with a presumed effect on implantation failure [12], post-implantation embryo development and increased pregnancy loss [13]. Gil-Villa et al.. found an increased DNA fragmentation index (DFI) in 9 out of 17 patients with RPL after spontaneous pregnancy [14]. Kumar et al. found that DFI values differed significantly between RPL cases and controls [15]. Using the TUNEL technique, Brahem et al. found that men in the RPL group had significantly higher values of DNA fragmentation than the control group [6]. Furthermore, Zidi-Jrah et al.. showed similar results since 45% of men in the RPL group had an abnormal TUNEL value [7].

Abnormal sperm aneuploidy has been investigated in a few studies as a possible cause of RPL. In men whose partners experienced RPL, Bernardini et al found significantly higher values for chromosome 1 and 8 disomy and total aneuploidy [3], as did Collodel et al. The latter authors also found increased diploidy, disomy and total aneuploidy for chromosomes 18, X and Y [5]. Two studies on chromosomes 18, 13, 21 X and Y found an increased aneuploidy rate in the case group compared with normospermic men [16], with the general population and with fertile men [11]. A recent study which scanned all the chromosomes in sperm from 11 male partners of couples experiencing RPL found increased aneuploidy for chromosomes 16, 2, 1, 21, 4 and 6 [17]. Lastly, Zidi-Jrah et al. observed that all men in the RPL group (n = 22) presented a significant increase in sex chromosome disomy and nullisomy as well as a significantly higher rate of diploidy compared with the control group (n = 20) [7].

In the light of the few studies on sperm fragmentation and RPL and of the sparse research focusing on sperm aneuploidy and RPL, our prospective case-control study focused on male implication in RPL by studying all three parameters (sperm DNA fragmentation, sperm chromatin condensation status and sperm aneuploidy) in 33 men whose partners experienced unexplained RPL (cases) and by comparing results with those of 27 men of proven fertility (control group). We also sought risk factors with an extensive questionnaire and thorough andrological examination.

To our knowledge, our study is the only one that includes an extensive andrological examination and a questionnaire about family history, living environment, lifestyle and exposure. Our prospective case-control study, including 33 cases of men from couples experiencing URPL and 27 controls of proven fertility, analyzed semen characteristics, sperm morphology, sperm DNA status and sperm aneuploidy. To date, only one other study has performed the same type of analyses, but in a smaller population [7]. All other research has focused either on sperm characteristics, morphology and DNA status [2, 4, 6, 8–10, 13–15, 21–26], on sperm characteristics, morphology and aneuploidy [3, 5, 11, 17], or on DNA status and aneuploidy [16].

Our results highlight a significant difference in family history of infertility and miscarriage in couples experiencing URPL. This could suggest the implication of a potential genetic factor. However, patient bias is also a possibility, as the cases are probably more aware of issues related to miscarriages and infertility than the control group and have a better knowledge of family infertility and miscarriage history. Furthermore, the questionnaire revealed a significant difference in the living environment between controls and cases. Controls predominantly lived in large urban areas, whereas cases more frequently lived in small urban areas. People living in the country may be more prone to exposure to various factors such as pesticides that could have an impact on pregnancy loss, as it has been shown that exposure to some types of pesticides was associated with early abortions [27]. However, this cannot be confirmed as we can also argue that the controls were enrolled in three large urban maternity departments where the population is mainly composed of people living in the city or close suburbs, while the cases more frequently came from the whole region, including close suburbs and more rural areas.

We found that TMSC was significantly lower in men from the URPL group than in men from the control group. This finding is in agreement with previously published results [2, 3, 6–9], with the exception of two studies [5, 28]. Battacharya et al. suggested that motility alteration was related to damaged plasma membrane which can affect the sperm’s DNA integrity to an extent where although the oocyte can be fertilized, the embryo later fails to grow correctly, leading to a miscarriage [2]. In the same field, Gil-Villa et al found increased levels of oxidant components in the semen of men in an URPL group, and these possibly affect the cellular components necessary for correct embryo development and sperm parameters such as motility [9]. Kazerooni et al found that abnormal sperm parameters in men in a URPL group were correlated with protamine deficiency, which is known to affect normal embryo development [8]. These three studies seem to link, though indirectly, impaired motility to impaired embryo development and miscarriage. In conclusion, we do not know if impaired motility is directly involved in miscarriage, but it could reflect sperm dysfunction in these men that could lead to URPL.

We observed a significant increase in chromosome 18 disomy, hyperhaploidy and total aneuploidy in the men from the URPL group compared with the fertile controls. This confirms previously published data that show a trend to increased chromosome 18 disomy in men in an URPL group [3]. Other studies on URPL and the search for chromosomal abnormalities have found increased incidence of sperm aneuploidy [7, 11, 16, 17]. Collodel et al. found increased diploidy, disomy and total aneuploidy in 50% of their cases when comparing the median and 75th percentile with their controls [5]. In our study, we found that sex chromosome sperm nondisjunction (XY18) and 18 disomy values were above the 90th percentiles of the control group in 79% and 49% of cases, respectively. These data all seem to indicate that increased anomalies in chromosome numbers have an impact on URPL, possibly involving a male factor, as has already been suggested in pregnancy losses after intracytoplasmic sperm injection (ICSI) [25, 29] as well as in implantation failure [25, 29]. Regarding DNA fragmentation and URPL, there seems to be a discrepancy in published results: some show a tendency to an increased fragmentation in cases of URPL [2, 4, 6–8, 10, 13–15, 21–24, 28] whilst others report no differences between cases and controls [9]. In our study we found no significant difference in DNA fragmentation between cases and controls, using either SCSA or TUNEL.

One limitation of our study is the small number of chromosomes studied by FISH (X, Y, 18). However, an interchromosomal effect has been reported using array-comparative genomic hybridization (CGH) [30, 31] and it has been suggested that the results obtained by studying one set of chromosomes could be extended to others [32]. While this study is one of the largest concerning the number of participants, further extensive population studies are required to investigate whether environmental factors may be associated with URPL.

To conclude, the increased aneuploidy rate in spermatozoa could explain URPL. In ICSI, selection of euploid spermatozoa could possibly improve the chances of these couples of successfully carrying a pregnancy to term. However, as yet no such technique is available.

Preimplantation genetic screening (PGS) could also be useful in managing URPL, especially as our results indicate that spermatozoa in these patients show an increased aneuploidy that could lead to an aneuploid embryo. Screening genetically normal embryos could improve chances of successful implantation and pregnancy. But PGS in cases of URPL is controversial. One study showed that in cases of increased sperm aneuploidy PGS seemed to prevent miscarriages and increased the chances of a successful pregnancy [33], whereas another more recent study showed that PGS did not improve the chances of a successful pregnancy compared with expectant management [34]. However, in this latter study, sperm aneuploidy status was not investigated. In view of our results and those of Rubio et al., study of sperm chromosome status in men whose partners experience URPL must be continued. PGS may be a useful option in the management of URPL when sperm are found to be aneuploid, but further studies on the results of PGS in this context should be done to confirm this hypothesis.