High Levels of Aberrent DNA Methylation at Loci Associated with Reduced Male Fertility Are Correlated with Increased DNA Fragementation

In recent years, the use of sperm DNA fragmentation as a potential diagnostic for male infertility has been the focus of many studies. More recently, there has been growing interest in the role of DNA methylation in sperm function and fertility. It has been shown that disrupted sperm epigenetic profiles correlate with reduced rates of conception and poor embryo quality in IVF patients. Aberrant sperm epigenetic profiles are also associated with abnormal routine semen parameters. Despite the potential for both DNA fragmentation rates and methylation profiles to bolster understanding of male infertility on the DNA level, the relationship between the two is still not well understood. Here we aim to study the association between sperm DNA methylation disruptions and DNA fragmentation index (DFI) throughout the genome in men from infertile couples. Using array technology, we profiled DNA methylation levels at more than 480,000 sperm CpG sites in 39 patients. Samples were collected from two major fertility clinics in the United States and Canada, and were derived from couples seeking infertility treatment. Data was normalized to correct for potential batch effects. Failed probes and those within 2 base pairs of known SNPs were removed from analysis. Differentially methylated regions (DMRs) were detected using the DMRcate1 package in R with a p-value cut-off of 0.05 for statistical significance after Bonferroni correction. Fisher's exact test was used to determine the significance of enrichment for imprinted genes. DNA fragmentation index is calculated using a SCSA test. Using control ranges previously established by Episona Inc., we identified CpGs with methylation levels that deviated from normal. We filtered these loci to include only those which show a significant association with fertility status using data previously collected by Episona Inc. The number of abnormalities in these loci was positively correlated with the DFI of the sample (spearman's rho=0.316, p-value=.047). This association was most apparent in samples with high DFI. In samples with low DFI, we still observed epigenetic abnormalities despite normal DNA fragmentation. In addition, we probed for specific genomic loci showing strong association between methylation level and the DFI of the sample. We identified 1,053 such regions, overlapping 843 genes. These loci were highly enriched for imprinted genes (odds ratio = 5, p-value < 1x10-5), an enrichment that increases with the threshold for significance. The high number of significant DMRs detected in our results suggests a strong association between sperm DFI and epigenetic profile. Our results showed an enrichment of these locations for imprinted genes, which are assumed to have an important role in reproduction, specifically early embryo development. Our results suggest that in addition to abnormal semen parameters, sperm epigenetic profile shows a close correlation with DFI.