Several studies have reported an association between male infertility and aberrant sperm DNA methylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfite sequencing, we have not found any evidence for such an association, but have demonstrated that somatic DNA contamination and genetic variation confound methylation studies in sperm of severely oligozoospermic men. To find out whether testicular germ cells (TGCs) of such patients might carry aberrant DNA methylation, we compared the TGC methylomes of four men with cryptozoospermia (CZ) and four men with obstructive azoospermia, who had normal spermatogenesis and served as controls (CTR). There was no difference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 differentially methylated regions (DMRs), 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10–16). The DMRs were enriched for distal regulatory elements (p = 1.0 × 10–6) and associated with 132 genes, 61 of which are differentially expressed at various stages of spermatogenesis. Almost all of the 67 DMRs associated with the 61 genes (94%) are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As judged by single-cell RNA sequencing, 13 DMR-associated genes, which are mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter is hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, eight of which downregulated in CZ, germ cell-specific enhancers may be affected. We found that impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome. We hypothesize that the described DNA methylation changes may reflect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm.

中文翻译：

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来自隐精子症男性的睾丸生殖细胞的全基因组甲基化分析指向反复发生的和功能相关的 DNA 甲基化变化

一些研究报告了男性不育与异常精子 DNA 甲基化模式之间的关联，特别是在印记基因中。在最近一项基于全甲基化组和深度亚硫酸氢盐测序的调查中，我们没有发现任何证据表明这种关联，但已经证明体细胞 DNA 污染和遗传变异混淆了严重少精症男性精子中的甲基化研究。为了查明此类患者的睾丸生殖细胞 (TGC) 是否可能携带异常 DNA 甲基化，我们比较了四名隐精症患者 (CZ) 和四名精子发生正常并作为对照 (CTR) 的梗阻性无精子症患者的 TGC 甲基化组. 在全基因组水平或 CZ 和 CTR 样本之间的印迹区域，DNA 甲基化没有差异。然而，使用严格的过滤器来识别组特异性甲基化差异，我们检测到 271 个差异甲基化区域 (DMR)，其中 238 个在 CZ 中被高甲基化（二项式检验，p < 2.2 × 10-16）。DMRs 富含远端调控元件 (p = 1.0 × 10–6) 并与 132 个基因相关，其中 61 个基因在精子发生的不同阶段差异表达。几乎所有与 61 个基因相关的 67 个 DMR (94%) 在 CZ 中都是高甲基化的 (63/67, p = 1.107 × 10–14)。通过单细胞 RNA 测序判断，主要在减数分裂和精子发生过程中表达的 13 个 DMR 相关基因在 CZ 患者中表现出显着不同的表达模式。在这些基因中的四个中，启动子在 CZ 男性中高度甲基化，这与这些患者中较低的表达水平相关。在其他九个基因中，其中八个在 CZ 中下调，生殖细胞特异性增强子可能受到影响。我们发现精子发生受损与基因组功能相关区域的睾丸生殖细胞 DNA 甲基化变化有关。我们假设所描述的 DNA 甲基化变化可能反映或促成精子发生的早产，因此不会出现在成熟的活动精子中。