冷冻技术对精子印记基因DNA甲基化影响的研究

Effect of cryopreservation on DNA methylation status of imprinted genes in human sperm

目的检测常规精液冷冻技术中冷冻保护剂、冷冻过程及冷冻时间长短对精子父源印记基因H19及母源印记基因MEST印记控制区域(imprinting control region,ICR)的DNA甲基化的影响程度。方法以10例符合精子库捐精条件的正式志愿者为研究对象,将每份精液样品平均分为4组:A组为新鲜精液,作为对照;B组加入同体积冷冻保护剂,不冷冻;C组加同体积冷冻保护剂,冷冻2d;D组加同等体积冷冻保护剂,冷冻两个月。通过亚硫酸氢盐克隆测序法分析H19及MEST ICR的DNA甲基化状态。结果四组(A、B、C、D组)H19基因ICR区的DNA甲基化率以克隆数计算时分别为58.70%、53.85%、49.46%和45.74%,以CpG岛数计算时分别为97.57%、97.33%、97.13%和96.56%,两者都有降低趋势,但组间差异均无统计学意义(P>0.05);四组MEST基因ICR区甲基化率以克隆数计算时分别为41.10%、45.33%、47.30%和50.68%,以CpG岛数计算甲基化率时分别为1.77%、1.74%、2.00%和2.26%,有升高趋势,但差异亦无统计学意义(P>0.05)。结论常规精液冷冻复苏技术对精子父源印记基因H19及母源印记基因MEST的ICR区甲基化程度未见明显影响。

Objective：To assess the effect of cryoprotectant,frozen process and cryopreservation duration on DNA methylation status of paternal imprinted gene H19 and maternal imprinted gene MEST imprinting control region（ICR）in human sperm.Methods：The semen samples from 10 qualified donors were collected in Beijing Human Sperm Bank.The samples were divided into four equal aliquots：（A）untreated,（B）diluted in cryoprotectant,（C）diluted in cryoprotectant and cryopreserved for 2days,and（D）diluted in cryoprotectant and cryopreserved for 2months.The DNA methylation degree of H19 and MEST ICR was determined by bisulfite sequencing PCR（BSP）method.Results： H19 ICR DNA methylation degree analyzed by clone numbers was 58.70%,53.85%,49.46% and 45.74%,while analyzed by CpG island numbers,it was 97.57%,97.33%,97.13% and96.56%in group A,B,C and D,respectively.The results from both analytical methods showed a decreasetrend,but no significant difference was found among the four groups（P 〉0.05）.MEST ICR DNA methylation degree analyzed by clone numbers in the four groups was 41.10%,45.33%,47.30% and50.68%,while analyzed by CpG island numbers,it was 1.77%,1.74%,2.00%and 2.26%in group A,B,C and D,respectively.There was also no significant difference among the four groups（P〉0.05）despite a increasing trend.Conclusions：The lack of significant difference in the DNA methylation degree of H19 and MEST ICR in our study indicates that DNA methylation of human sperm is not affected by cryopreservation and the cryopreservation technology.