BACKGROUND 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. RESULTS Using 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. CONCLUSIONS Our 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.

中文翻译：

---

在精子发生严重受损的患者中，精子表观基因组不会表现出复发性的突变。

背景技术在过去的15年中，许多研究已经描述了少精症男性精子中印迹基因（例如MEST和H19）的异常DNA甲基化，但是异常甲基化模式的发生率和基因组范围仍然未知。结果我们使用亚硫酸氢盐深层测序（DBS），从40名正常精子和93名被诊断为少精，少精或少精子的患者中筛选出精子样本，在整个手稿中H19和MEST甲基化被称为OAT。基于此筛选，我们定义了三个患者组：正常对照（NC），异常甲基化的少精子症（AMO； n = 7）和正常甲基化的少精子症（NMO； n = 86）。五个NC和五个AMO样品的全基因组亚硫酸氢盐测序（WGBS）显示了每个AMO样品中所有50个印迹控制区域的异常甲基化水平。为了调查这一发现是否反映了表观遗传的种系镶嵌或残留的体细胞DNA的存在，我们进行了全基因组的生殖细胞特异性DNA甲基化清单。我们发现> 2000个生殖细胞特异性基因在血液中被甲基化，而AMO样品在这些基因上的甲基化水平异常，与体细胞DNA的存在一致。在五个NC和六个NMO样品之间的比较显示了19个差异甲基化区域（DMR），没有一个可以在40名男性的独立队列中得到验证。先前的研究报道了不育患者H19的CTCF结合区域6中单个CpG位点的表位突变发生率更高。然而，该位点的DBS分析显示DNA甲基化水平与基因型（rs2071094）之间存在关联，而与生育力表型无关。结论我们的结果表明，在不育男性精子中体细胞DNA污染和遗传变异混淆了甲基化研究。尽管我们不能排除在非复发性CpG部位存在精子甲基化稍有异常的罕见患者，但可能会高估了不育男性精制后的精子中异常甲基化的患病率，这为接受辅助生殖的患者提供了保证。揭示了DNA甲基化水平与基因型（rs2071094）之间的关联，但与生育表型无关。结论我们的结果表明，在不育男性精子中体细胞DNA污染和遗传变异混淆了甲基化研究。尽管我们不能排除在非复发性CpG部位存在精子甲基化稍有异常的罕见患者，但可能会高估了不育男性精制后精制精子中异常甲基化的患病率，这为接受辅助生殖的患者提供了保证。揭示了DNA甲基化水平与基因型（rs2071094）之间的关联，但与生育力表型无关。结论我们的结果表明，在不育男性精子中体细胞DNA污染和遗传变异混淆了甲基化研究。尽管我们不能排除在非复发性CpG部位存在精子甲基化稍有异常的罕见患者，但可能会高估了不育男性精制后精制精子中异常甲基化的患病率，这为接受辅助生殖的患者提供了保证。