Institute of Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Clinics of Münster, Germany.
Andrology. 2013 Sep;1(5):731-40. doi: 10.1111/j.2047-2927.2013.00118.x.
Recent studies have shown associations of aberrant DNA methylation in spermatozoa with idiopathic infertility. The analysis of DNA methylation of specific genes could therefore serve as a valuable diagnostic marker in clinical andrology. For this purpose, rapid and reliable detection methods, reference values and the temporal stability of spermatozoal DNA methylation need to be established and demonstrated. In this prospective study, swim-up purified semen samples from 212 consecutive patients (single samples), 31 normozoospermic volunteers (single samples) and 10 normozoospermic volunteers (four samples at days 1, 3, 42 and 45 plus a fifth sample after 180-951 days) were collected. Spermatozoal DNA was isolated, bisulphite converted and DNA methylation was analysed by pyrosequencing. DNA methylation of the maternally imprinted gene MEST was measured in samples of 212 patients and 31 normozoospermic volunteers and the temporal stability of eight different genes and two repetitive elements was examined in consecutive samples of 10 normozoospermic volunteers. MEST DNA methylation was significantly associated with oligozoospermia, decreased bi-testicular volume and increased FSH levels. A reference range for spermatozoal MEST DNA methylation (0-15%) was established using the 95th percentile of DNA methylation in normozoospermic volunteers. Using this reference range, around 23% of our patient cohort displayed an aberrant MEST DNA methylation. This epigenetic aberration was found to be significantly associated with bi-testicular volume, sperm concentration and total sperm count. DNA methylation in normozoospermic volunteers was stable over a time period of up to 951 days in contrast to classical semen parameters. Our data show that MEST DNA methylation fulfils the prerequisites to be used as routine parameter and support its use during andrological workup if a prognostic value can be shown in future.
最近的研究表明，精子中异常的 DNA 甲基化与特发性不育有关。因此，特定基因的 DNA 甲基化分析可以作为临床男科有价值的诊断标志物。为此，需要建立并证明快速、可靠的检测方法、参考值以及精子 DNA 甲基化的时间稳定性。在这项前瞻性研究中，采集了 212 名连续患者（单样本）、31 名正常精子志愿者（单样本）和 10 名正常精子志愿者（4 天、3 天、42 天和 45 天的 4 个样本，以及 180-951 天后的第 5 个样本）的泳动纯化精液样本。分离精子 DNA，亚硫酸氢盐转化，焦磷酸测序分析 DNA 甲基化。在 212 名患者和 31 名正常精子志愿者的样本中测量了母系印迹基因 MEST 的 DNA 甲基化，在 10 名正常精子志愿者的连续样本中检查了 8 个不同基因和 2 个重复元件的时间稳定性。MEST DNA 甲基化与少精子症、双侧睾丸体积减少和 FSH 水平升高显著相关。使用正常精子志愿者 DNA 甲基化的第 95 百分位数建立了精子 MEST DNA 甲基化（0-15%）的参考范围。使用这个参考范围，我们的患者队列中约有 23%显示出异常的 MEST DNA 甲基化。这种表观遗传异常与双侧睾丸体积、精子浓度和总精子计数显著相关。与经典精液参数相比，正常精子志愿者的 DNA 甲基化在长达 951 天的时间内保持稳定。我们的数据表明，MEST DNA 甲基化满足作为常规参数使用的前提条件，如果在未来能够显示出预测价值，支持在男科检查中使用。
