Abstract
BackgroundIn the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown.ResultsUsing deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 patients diagnosed as oligoasthenoteratozoospermic, oligoteratozoospermic or oligozoospermic, which are termed OATs throughout the manuscript, for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated oligozoospermic (AMO; n = 7) and normally methylated oligozoospermic (NMO; n = 86). Whole-genome bisulfite sequencing (WGBS) of five NC and five AMO samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMO sample. To investigate whether this finding reflected epigenetic germline mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell-specific DNA methylation. We found that > 2000 germ cell-specific genes are promoter-methylated in blood and that AMO samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMO samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype.ConclusionsOur results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.
Highlights
In the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown
Apart from that, a number of studies have shown that male infertility itself is associated with aberrant DNA methylation profiles, of imprinted genes [3,4,5,6], suggesting that assisted reproductive technology (ART) may facilitate the transmission of imprinting errors in sperm cells to the generation
We subdivided the patients into normally methylated normal controls (NC, n = 40), abnormally methylated oligozoospermic (AMO, n = 7) and normally methylated oligozoospermic men (NMO, n = 86) (Fig. 1c, Additional file 2: Fig. S2)
Summary
In the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown. Apart from that, a number of studies have shown that male infertility itself is associated with aberrant DNA methylation profiles, of imprinted genes [3,4,5,6], suggesting that ART may facilitate the transmission of imprinting errors in sperm cells to the generation. This latter aspect is still, notably, a matter of much debate [7]. Incorrect erasure or re-establishment of methylation patterns in individual PGCs could lead to a population of spermatogonia giving rise, via clonal divisions, to a subpopulation of sperm with aberrant methylation profiles
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