人卵胞浆内单精子显微注射对囊胚印迹基因H19甲基化的影响

Study on the effect of human intracytoplasmic sperm microinjection on the blastocyst imprinted gene methylation of the H19

目的探讨人卵胞浆内单精子显微注射技术(ICSI)侵入性操作对囊胚期胚胎印迹基因H19差异性甲基化区(DMR)Cp G岛甲基化的影响。方法对行人类辅助生殖技术(ART)助孕患者自愿放弃并签署科研捐献知情同意书后的胚胎,依据胚胎来源分成常规IVF组、ICSI组、早期补救ICSI(R-ICSI)三组,继续培养至囊胚阶段;并采用重亚硫酸盐DNA测序法(BSP)对所获得的囊胚行H19这个经典的且对环境敏感的印迹基因差异性甲基化区(DMR)Cp G岛甲基化程度进行检测,并统计三组的甲基化程度差异。结果 BSP法单囊胚扩增成功率为2.4%,ICSI组和R-ICSI组囊胚H19基因甲基化阳性率为27.3%和25.5%,低于常规IVF组49.5%的甲基化阳性率,差异有统计学意义(P<0.05);但ICSI组和R-ICSI组两组间无统计学差异(P>0.05);结论 ICSI技术侵入性操作可造成囊胚期胚胎印迹基因H19差异性甲基化区(DMR)Cp G岛甲基化程度的降低,造成甲基化的丢失和甲基化模式的异常,其甲基化模式的异常对胎儿发育及子代影响需要进一步研究。

Objective：To investigate the effect of the human intracytoplasmic sperm injection techniques（ICSI）invasive procedure on blastocyst stage embryos imprinted gene H19 differential methylation region（DMR）Cp G island methylation. Methods：Patients who Carryed human assisted reproductive technology（ART）,respect fertility patient′s willingness fully,informed them abandon embryos scientific research donated and signed informed consent,Continue to cultured to the blastocyst stage;On the basis of the blastocyst sources,samples divided into conventional IVF embryo group,ICSI group,early remedial ICSI（R-ICSI）three groups,to continue to foster or After thawing the culture to the blastocyst stage;and the use of DNA with bisulfite sequencing method（BSP）to the blastocyst this classic H19 gene obtained and are sensitive to environmental changes imprinted genes differentially methylated region（DMR）Cp G island methylation of detection,and statistical differences methylation three groups. Results：of ICSI group and R-ICSI group blastocysts H19 gene methylation positive rate of 27.3% and 25.5%,lower than the 49.5% positive rate of methylation of conventional IVF group,the difference was statistically significant（P〈0.05）;but there is no significant difference between the group R-ICSI and the ICSI group（P〉0.05）. Conclusion：Invasive procedure of ICSI technology can cause a blastocyst stage embryos H19 imprinted gene methylation differences（DMR）Cp G island methylation,causing loss of methylation and methylation patterns of abnormalities,abnormal methylation,patterns and their impact on fetal development requires further study.