冷冻对父源印记基因H19DNA甲基化影响的长时程效应研究  

Long term effect of cryopreservation on DNA methylation status of paternal imprinted gene H19

作　　者：梁新新[1] 李博[1] 马夜肥[1] 李建波[1] 李超波[1] 张永琦[1] 闵保华[1] 王晓红[1] 

机构地区：[1]第四军医大学唐都医院妇产科生殖医学中心,西安710038

出　　处：《中国男科学杂志》2013年第3期3-9,共7页Chinese Journal of Andrology

摘　　要：目的检测冷冻及冷冻时间长短对精子的父源印记基因H19印记控制区域（ICR）的DNA甲基化程度的影响.方法以15例健康男性精液为研究对象,将每份精液样品除留少许作为对照外,其余分为两部分（每部分又等分为若干份）,一部分连同精浆一起进行程序化冷冻,另一部分则去除精浆后冷冻,并分别于冷冻后1个月、3个月和6个月时,通过亚硫酸氢盐测序PCR的方法分析H19ICR的DNA甲基化状态.结果连同精浆一起冷冻的精子样品,冷冻1个月、3个月和6个月后,H19ICR的 DNA甲基化丢失率分别为（13.62±2.06）%、（14.88±1.96）%和（15.89±2.26）%,与对照相比（12.74±2.85）%差异无统计学意义（P〉0.05）;去除精浆后冷冻的精子样品,冷冻1个月、3个月后,H19ICR的 DNA甲基化丢失率分别为（15.24±1.91）%、（13.49±2.63）%,与对照相比差异无统计学意义（P〉0.05）,而冷冻6个月后,H19ICR的 DNA甲基化丢失率高达（30.92±3.08）%,差异极显著（P〈0.01）.结论程序化冷冻对精子H19DNA甲基化的影响具有长时程的累积效应,本研究为少精子症患者或者无精子症患者通过经附睾精子抽吸术（PESA）取精后的精子冷冻保存的安全期限提供了一定的参考依据.Objective To detect the effects of frozen and freezing timeperiod on DNA methylation status of paternal imprinting genes H19 imprint control region. Methods Semen samples of 15 cases of healthymale were collected as theresearch object. each semen sample was divided into two parts （one part was further subdivided into in addition to leave a little outside a control （Divide the two parts into sections）. Along with the seminal plasma part undergoing programmed freezing, the other part is to remove seminal plasma freezing. Respectively after 1 month 3 months and 6 months freezing, through the bisulphite sequencing PCR method analysises DNA methylation status of H19ICR. Results For frozen sperm samples with seminal plasma, H19ICR DNA methylation loss rate were （13.62±2.06）%, （14.88±1.96）%, （15.89±2.26）%after frozen 1 month, 3 months and 6 months respectively. No significant differences were found compared with that of control （12.74 ±2.85）%（P〉0.05）. For frozen sperm samples without seminal plasma, H19ICR DNA methylation loss rate were （15.24 ±1.91）%, （13.49±2.63）%at1 month, 3 months after frozen, respectively. No significant differences were found compared with that of the control （P〉0.05）. But at 6 months after frozen , H19ICR DNA methylation losss rate was （30.92 ±3.08）%, it is a＆nbsp;significant difference compared with that of the control（P〈0.01）. Conclusion Programmed freezing had long range accumulation effects on sperm DNA methylation. The data in the study provideda safety period referenece of sperm cryopreservation for semen collection of less refined disease patients or aspermia disease patients through.

关 键 词：后成说 遗传 DNA甲基化 基因组印迹 精液保存 

分 类 号：R394.113[医药卫生—医学遗传学] R321.1[医药卫生—基础医学]