【摘要】 目的:比较研究常规慢速冷冻和玻璃化冷冻后的人类早期胚胎印记基因H19的DNA甲基化状态,初步了解低温冷冻对胚胎基因印记的影响。方法:收集胚胎移植术后正常受精的低质量的第3天胚胎152个,进行冷冻、复苏,其中常规慢速冷冻胚胎78个,玻璃化冷冻胚胎74个,另收集70个新鲜未冷冻胚胎作为对照;记录3种类型单个胚胎Bisulfite-PCR扩增个数,采用结合重亚硫酸盐限制性酶分析法检测胚胎印记基因H19的DNA甲基化状态。结果:慢速冷冻胚胎、玻璃化冷冻胚胎及新鲜未冷冻胚胎的单个胚胎Bisulfite-PCR扩增分别成功40、53、58个;慢速冷冻胚胎、玻璃化冷冻胚胎及新鲜未冷冻胚胎H19基因的平均甲基化程度分别为(44.8±6.4)%、(47.5±5.6)%及(46.7±6.0)%,异常甲基化率分别为10.0%(4/40)、13.2%(7/53)及3.4%(2/58);3组胚胎的甲基化程度和异常甲基化率比较,差异无统计学意义(P>0.05)。结论:低温冷冻不增加人类胚胎甲基化异常的发生率。更多还原

【Abstract】 Objective: To investigate the DNA methylation status of human early embryonic imprinting H19 gene after slow-freezing or vitrification freezing,and preliminarily explore the effect of low temperature freezing on embryonic imprinting gene. Methods: 152 Day3-embryos( 2PN,bad quality)with normal fertilization after embryo transplantation were collected and underwent freezing and resuscitation,of which 78 embryos were conducted by slow-freezing,74 embryos by vitrification freezing. Besides,70 fresh non-frozen embryos were collected as controls. The number of Bisulfite-PCR amplification of a single embryo was recorded in the three types. The Combined Bisulfite Restriction Analysis( COBRA) was used to determine H19 methylation status after embryos thawing. Results: The number of Bisulfite-PCR amplification of a single embryo in the three groups was 40( slow-freezing),53( vitrification freezing) and 58( fresh non-frozen),respectively. The average degree of H19 methylation in the three groups was( 44. 8 ± 6. 4) %( slow-freezing),( 47. 5 ± 5. 6) %( vitrification freezing) and46. 7% ± 6. 0%( fresh non-frozen),respectively. The rate of abnormal methylation was 10. 0%( slow-freezing),13. 2%( vitrification freezing) and 3. 4%( fresh non-frozen),respectively. There was no statistical difference of H19 methylation degree and rate of abnormal methylation between three groups. Conclusion: The freezing technique may not increase the incidence of abnormal methylation in human embryos.更多还原