Correlation between sperm DNA fragmentation and methylation in male partners of couples with idiopathic recurrent pregnancy loss.

With ∼50% recurrent pregnancy loss cases being termed idiopathic (iRPL), understanding of contribution of male factors to iRPL is still lacking. Higher prevalence of sperm DNA fragmentation index (DFI) and lower sperm 5-methylcytosine (5-mC) levels have been previously reported in male partners of iRPL couples and shed light on importance of the male gamete in maintenance of a successful pregnancy. The present study aimed to determine the serum sex steroid hormone levels, sperm DFI and 5-mC and correlation between them in male partners of fertile and iRPL couples. Further, correlation between sperm DFI and 5-mC with semen parameters and paternal age in both groups were determined. 36 male partners of fertile couples and 45 male partners of women experiencing iRPL were enrolled for this study and semen and blood samples were collected. Serum testosterone and estradiol levels were measured by ELISA; sperm DFI and global 5-mC were determined by TUNEL assay and ELISA respectively. Significantly higher serum testosterone levels were noted in the iRPL group (p = 0.028). Incidence of sperm DNA fragmentation was found to be higher in the iRPL study group but with no significance difference. No significant differences in sperm 5-mC values were noted. Upon correlation analysis within both groups, strong significant negative correlation of sperm DFI % and 5-mC % was observed in the control group (p < 0.001) but not the iRPL group (p = 0.249). Hence, we infer that with lower 5-mC levels in sperm genome, there is a higher incidence of sperm DFI in fertile men. However, this trend is not noted in men of iRPL group which could possibly be due to other underlying epigenetic alterations in genomic regions probably unsusceptible to fragmentation. On the other hand, no significant correlations of semen parameters, testosterone, estradiol and paternal age with sperm DFI and 5-mC were noted in both groups.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss.

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Whole genome bisulfite sequencing of sperm reveals differentially methylated regions in male partners of idiopathic recurrent pregnancy loss cases.

OBJECTIVE: To study the genome wide alterations in sperm DNA methylation in male partners of idiopathic recurrent pregnancy loss (iRPL) cases and note regions as potential diagnostic markers. DESIGN: Case-control study and methylome analysis of human sperm. SETTING: Obstetrics and Gynaecology clinics. PATIENT(S): Control group consists of apparently healthy fertile men having fathered a child within the last 2 years (n = 39); and case group consists of male partners of iRPL cases having ≥2 consecutive 1st trimester pregnancy losses (n = 47). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm DNA samples of controls and cases were selected for whole genome bisulfite sequencing analysis based on the previously set thresholds of global methylation levels and methylation levels of imprinted genes (KvDMR and ZAC). Whole genome bisulfite sequencing of selected sperm genomic DNA was performed to identify differentially methylated CpG sites of iRPL cases compared with fertile controls. Pathway analysis of all the differentially methylated genes was done by Database for Annotation, Visualization, and Integrated Discovery annotation tool and Kyoto Encyclopedia of Genes and Genomes tool. Differentially methylated CpGs within genes relevant to embryo and placenta development were selected to further validate their methylation levels in study population by pyrosequencing. RESULT(S): A total of 9497 differentially methylated CpGs with highest enrichment in intronic regions were obtained. In addition, 5352 differentially methylated regions and 2087 differentially methylated genes were noted. Signaling pathways involved in development were enriched on pathway analysis. Select CpGs within genes PPARG, KCNQ1, SETD2, and MAP3K4 showed distinct hypomethylated subpopulations within iRPL study population. CONCLUSION(S): Our study highlights the altered methylation landscape of iRPL sperm, and their possible implications in pathways of embryo and placental development. The CpG sites that are hypomethylated specifically in sperm of iRPL subpopulation can be further assessed as predictive biomarkers.

Maternal cypermethrin exposure during perinatal period dysregulates gonadal steroidogenesis, gametogenesis and sperm epigenome in F1 rat offspring.

Evidences suggest that maternal exposure to endocrine disruptor insecticide cypermethrin (CYP) affects the reproductive functions of offspring. However, its molecular effects are not well researched. This study elucidates the effects of CYP on gonadal steroidogenesis, gametogenesis and sperm epigenome of perinatally exposed F1 rat offspring. CYP (1, 10, 25 mg/kg bw/day) and Diethylestilbestrol (10 µg/kg bw/day; positive control) were gavaged once daily to pregnant dams from gestational day 6 to postnatal day 21 and their effects were assessed in F1 adults. Dysregulated steroidogenesis was observed in F1 testis; expression of Star, Cyp11a1 and Cyp19a1 were upregulated and Hsd3b1, Cyp17a1, Hsd17b3 downregulated. Expression of spermatogenesis cell-type markers Pcna, Plzf, Sohlh2 were upregulated and Ccna1, Sycp1, Ccnb1, Acrv, Amh downregulated. Upon epigenetic investigations of F1 spermatozoa; global hypermethylation, H19 DMR hypomethylation and increased expression of testicular Dnmts were observed. In F1 ovaries, expression of all studied steroidogenesis genes and oogenesis markers such as Amh, Gdf9, Ccnb1 and Pcna were altered. Amh was downregulated and Gdf9 was found to be upregulated. Overall, perinatal CYP exposure hampers molecular control of steroidogenesis and gametogenesis processes along with sperm epigenome in CYP exposed F1 offspring.

Cypermethrin exposure during perinatal period affects fetal development and impairs reproductive functions of F1 female rats.

Cypermethrin (CYP) is a ubiquitously present synthetic pyrethroid insecticide. It has endocrine disrupting activities which may adversely affect reproductive development and functions of offspring if exposed during critical developmental period. The present study was undertaken to delineate the effects of CYP exposure in pregnant female rats during perinatal period on the sexual maturation, hormonal regulation, reproductive development and fertility of F1 female offspring and its molecular mechanism of action. Pregnant rats (F0) were gavaged daily with 0, 1, 10, 25 mg/kg bw/day CYP and 10 µg/kg bw/day Diethylstilbestrol (DES; positive control) from gestation day (GD) 6 to postnatal day (PND) 21. The reproductive development and function parameters were evaluated at PND 45 and 75. Reduced body weight, delayed vaginal opening, and disrupted estrous cyclicity were observed at 25 mg/kg CYP dose. CYP exposure significantly affected the reproductive organ development and their functions at all doses. Significant alterations in ovarian and uterine histology such as luteinization, reduction of primordial follicular reserves, presence of multi-oocyte follicles and thin degenerative luminal and glandular uterine epithelium were observed at adulthood. Altered circulatory steroid hormone levels and expression of ovarian and uterine steroid hormone receptors were observed at PND 75 in the F1 female offspring. Expression of HOXA10 and α-SMA which are important for uterine integrity and functions, were found to be altered at PND 75. Increased pre-implantation loss (PIL%), post-implantation loss (POL%), and reduced litter size in F1 females when cohabitated with unexposed fertile male rats were observed. Overall, perinatal exposure of pregnant rats to CYP led to significant long lasting effects on the reproductive functions of F1 female offspring. The adverse effects were passed on to F2 generation via female germ line and posed developmental anomalies. The present finding necessitates additional molecular studies to understand its trans-generational mechanism of action via female germline.