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目的:探讨超促排卵(controlled ovarian hyperstimulation,COH)和未成熟卵母细胞体外培养(in vitro maturation,IVM)是否引起印迹基因甲基化异常。方法:采用单细胞低熔点琼脂糖包埋亚硫酸氢钠处理、半巢式PCR扩增及克隆测序的方法,检测人单个卵子印迹基因H19和PEG1的甲基化状态。结果:共检测分析了94个(58个来自COH,36个来自IVM)处于不同成熟阶段的卵子中H19基因和PEG1(MEST)基因的甲基化状态,分别有6个COH卵子和2个IVM卵子出现H19或PEG1的1~2个CpG位点甲基化异常。绝大多数COH卵子的H19(91.4%,53/58)和PEG1(91.7%,33/36)的所有检测CpG位点甲基化正常,H19和PEG1所有CpG位点的甲基化率分别为0.8%(8/1044)和99.4%(859/864)。结论:经超促排卵和体外成熟培养卵子的印迹基因H19和PEG1甲基化状态是正常的,1~2个CpG位点甲基化异常的卵子是否导致胚胎的基因印迹异常需进一步研究。
Objective: To investigate whether abnormal ovarian hyperstimulation (COH) and in vitro maturation (IVM) induced methylation of imprinted genes. Methods: Methylation status of human single-marker gene H19 and PEG1 was detected by single cell low melting point agarose embedded sodium bisulfite treatment, semi-nested PCR amplification and cloning sequencing. RESULTS: The methylation status of the H19 and PEG1 (MEST) genes in 94 (58 COH, 36 from IVM) eggs at different stages of maturation were examined and analyzed, with 6 COH eggs and 2 IVM Egg methylation at 1 or 2 CpG sites in H19 or PEG1 is abnormal. Methylation of all CpG sites detected in most of the COH eggs at H19 (91.4%, 53/58) and PEG1 (91.7%, 33/36) was normal. The methylation rates at all CpG sites of H19 and PEG1 were 0.8% (8/1044) and 99.4% (859/864). CONCLUSION: The methylation status of H19 and PEG1 genes imprinted by overexpression and in vitro maturation is normal. Whether oocytes methylated by one or two CpG sites lead to abnormal gene imprinting of embryos needs to be further studied.