Gamete Immunobiology Department, Indian Council of Medical Research-National Institute for Research in Reproductive and Child Health, Mumbai, India.
Biostatistics Department, Indian Council of Medical Research-National Institute for Research in Reproductive and Child Health, Mumbai, India.
Epigenetics. 2023 Dec;18(1):2252244. doi: 10.1080/15592294.2023.2252244.
Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.
先前，我们表明，反复妊娠丢失（RPL）夫妇的男性伴侣的精子中 DNA 甲基化缺陷可能是一个促成的父系因素。在本研究中，我们旨在确定选定印迹基因的甲基化水平是否可用作诊断标记，以识别这些情况下的表观遗传异常精子样本。使用多元逻辑回归，将先前发现存在差异甲基化的选定印迹基因的精子甲基化水平组合成概率评分（0-1 之间）。使用接收器操作特征分析研究了这些基因的不同组合，并在 38 个对照和 45 个 RPL 精子样本的独立队列中通过实验验证了这些阈值值。在所研究的不同组合中，包含 IGF2-H19 DMR、IG-DMR、ZAC、KvDMR 和 PEG3 的五个印迹基因的组合（AUC=0.88），阈值为 0.61，特异性为 90.41%，灵敏度为 70%。验证研究的结果表明，97%的对照样本的概率评分低于此阈值，而 40%的 RPL 样本高于此阈值，后验概率为 97.8%。因此，这种组合可以正确分类对照样本，并可能识别 RPL 夫妇中男性伴侣的表观遗传异常精子样本。我们提出，这些印迹基因的组合 DNA 甲基化水平可用作诊断工具，以识别具有表观遗传缺陷的精子样本，这些缺陷可能导致 RPL 的病理生理学，并可以对夫妇进行适当的咨询。
