超促排卵和体外成熟人卵子中印迹基因H19和PEG1的甲基化状态

Methylation Status of the Imprinted Genes of H19 and PEG1 in Human Superovulated and in vitro Maturation Cultured Oocytes

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 evaluate whether controlled ovarian hyperstimulation （COH） and in vitro maturation of oocyte （IVM） result in aberrant methylation of H19 and PEG1 in human oocytes. Methods： Single human superovulated oocyte or oocyte following in vitro maturation was embedded into low melting point （LMP） agarose, followed by bisulphate treatment and polymerase chain reaction （PCR） amplification of the H19 and PEG1 genes. The PCR products were then subjected to TA cloning and sequencing to determine the methylation status. Results： Fifty-eight and 36 oocytes at various stages from COH and IVM, respectively, were analyzed. Six oocytes from COH and 3 from IVM had abnormal methylation in 1-2 CpG sites of H19 and PEG1. The normal methylation of all CpG sites occurred in 91.4% （53/58） and 91.7% （33/36） oocytes for H19 and PEG1. The rates of methylated CpG sites were 0.8% （8/1 044） and 99.4% （859/864） for H19 and PEG1, respectively. Conclusion： Most oocytes from COH and IVM showed normal methylation of H19 or PEG1. It warrants further study whether the aberrant methylation in 1-2 CpG sites causes abnormal imprinting of the embryos.