冷冻胚胎印记基因H19的DNA甲基化状态研究

Research of DNA Methylation Status of Frozen Embryonic Imprinting H19 Gene

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。

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.