Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo 33006, Spain.
Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo 33006, Spain Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid 28049, Spain.
Hum Reprod. 2015 May;30(5):1014-28. doi: 10.1093/humrep/dev053. Epub 2015 Mar 9.
Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility?
DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls.
Aberrant DNA methylation of sperm has been associated with human male infertility in patients demonstrating either deficiencies in the process of spermatogenesis or low semen quality.
STUDY DESIGN, SIZE, DURATION: Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Illumina Infinium HD Human Methylation 450K arrays were used to identify genomic regions showing differences in sperm DNA methylation patterns between five fertile and seven infertile individuals. Additionally, global DNA methylation of sperm was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek) in 14 samples, and DNA methylation at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4) measured by bisulfite pyrosequencing in 44 sperm samples. A sperm-specific DNA methylation pattern was obtained by comparing the sperm methylomes with the DNA methylomes of differentiated somatic cells using data obtained from methylation arrays (Illumina 450 K) of blood, neural and glial cells deposited in public databases.
In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls.
LIMITATIONS, REASONS FOR CAUTION: Our results are descriptive and further studies would be needed to elucidate the functional effects of aberrant DNA methylation on male fertility.
Overall, our data suggest that aberrant sperm DNA methylation might contribute to fertility impairment in couples with unexplained infertility and they provide a promising basis for future research.
STUDY FUNDING/COMPETING INTERESTS: This work has been financially supported by Fundación Cientifica de la AECC (to R.G.U.); IUOPA (to G.F.B.); FICYT (to E.G.T.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F); the Plan Nacional de I+D+I 2008-2011/2013-2016/FEDER (PI11/01728 to AF.F., PI12/01080 to M.F.F. and PI12/00361 to S.L.); the PN de I+D+I 2008-20011 and the Generalitat de Catalunya (2009SGR01490). A.F.F. is sponsored by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (CP11/00131). S.L. is sponsored by the Researchers Stabilization Program from the Spanish National Health System (CES09/020). The IUOPA is supported by the Obra Social Cajastur, Spain.
男性不育夫妇中无法解释的不孕原因是否与精子中的 DNA 甲基化改变有关？
与生育能力正常的对照组相比，不孕个体的精子中 DNA 甲基化模式不仅在特定基因座发生改变，而且在 Alu Yb8 重复序列中也发生改变。
在表现出生精过程缺陷或精液质量低的患者中，精子的异常 DNA 甲基化与男性不育有关。
研究设计、规模、持续时间：病例对照前瞻性研究。本研究比较了 17 名正常生育能力的男性和 29 名正常生育能力的不孕患者的 46 个精子样本。
参与者/材料、设置、方法：使用 Illumina Infinium HD 人类甲基化 450K 阵列来鉴定精子 DNA 甲基化模式在五个生育能力正常和七个生育能力异常个体之间存在差异的基因组区域。此外，使用 Epigentek 的 Methylamp Global DNA Methylation Quantification Ultra 试剂盒测量了 14 个样本中的精子全基因组 DNA 甲基化，并用 bisulfite pyrosequencing 测量了 44 个精子样本中的几个重复序列（LINE-1、Alu Yb8、NBL2、D4Z4）的 DNA 甲基化。通过将从公共数据库中存储的血液、神经和神经胶质细胞的甲基化阵列（Illumina 450 K）获得的数据与分化体细胞的 DNA 甲基化进行比较，获得了精子特异性的 DNA 甲基化模式。
在这项研究中，我们首次进行了一项全基因组研究，以确定不明原因不孕个体精子 DNA 甲基化的改变，这些改变可能导致他们的生物学生育能力与生育能力正常的个体不同。我们已经确定了 2752 个表现出异常 DNA 甲基化模式的 CpG 位点，更重要的是，这些差异甲基化的 CpG 位点与在体细胞中特异性甲基化的精子 CpG 位点显著相关。我们还发现，DNA 低甲基化与对应于在体细胞中富含抑制性组蛋白标记 H3K9me3 的区域之间，以及 DNA 高甲基化与富含 H3K4me1 和 CTCF 的区域之间存在统计学上显著的关联（P < 0.001），这表明不孕男性精子中染色质结构与异常 DNA 甲基化之间的关系可能与基因座有关。最后，我们还表明，精子中的 DNA 甲基化模式不仅在特定基因座，而且在几个重复序列（LINE-1、Alu Yb8、NBL2、D4Z4）中都低于体细胞。有趣的是，不孕患者的精子样本在 Alu Yb8 重复序列中的 DNA 甲基化水平明显低于对照组。
局限性、谨慎的原因：我们的结果是描述性的，需要进一步的研究来阐明异常 DNA 甲基化对男性生育力的功能影响。
总的来说，我们的数据表明，精子 DNA 甲基化的异常可能导致不明原因不孕夫妇的生育能力受损，并为未来的研究提供了有希望的基础。
研究资金/利益冲突：这项工作得到了 Fundación Cientifica de la AECC（R.G.U.）；IUOPA（G.F.B.）；FICYT（E.G.T.）；西班牙国家研究委员会（CSIC；200820I172 至 M.F.F.）；Ramón Areces 基金会（M.F.F.）；国家研究、发展和创新计划 2008-2011/2013-2016/FEDER（PI11/01728 至 AF.F.，PI12/01080 至 M.F.F.，PI12/00361 至 S.L.）；2008-2011 年的国家研究、发展和创新计划以及加泰罗尼亚政府（2009SGR01490）。AF.F. 得到了 ISCIII-Subdirección General de Evaluación y Fomento de la Investigación（CP11/00131）的资助。S.L. 得到了西班牙国家卫生系统的研究人员稳定计划的资助。IUOPA 得到了 Obra Social Cajastur 的支持，西班牙。
