施晓鋆,陈士岭,郑海燕,王乐乐,吴雅琴
SHI Xiaoyun,CHEN Shiling,ZHENG Haiyan,WANG Lele,WU Yaqin

• 1:贵阳市第二人民医院妇产科
• 2:南方医科大学南方医院妇产科生殖中心

摘要(Abstract)：

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.

关键词(KeyWords)： 胚胎研究;胚胎移植;甲基化;生殖技术,辅助;印记基因,H19;慢速冷冻;玻璃化冷冻
embryo research;embryo transplantation;methylation;reproductive technology,auxiliary;imprinted genes,H19;slow-freezing;vitrification freezing

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81460237)

作者(Author): 施晓鋆,陈士岭,郑海燕,王乐乐,吴雅琴
SHI Xiaoyun,CHEN Shiling,ZHENG Haiyan,WANG Lele,WU Yaqin

DOI: 10.19367/j.cnki.1000-2707.2016.05.007

参考文献(References)：

• [1]Hiura H,Okae H,Chiba H,et al.Imprinting methylation errors in ART[J].Reprod Med Biol,2014(4):193-202.
• [2]Halliday J,Oke K,Breheny S,et al.Beckwith-Wiedemann syndrome and IVF:a case-control study[J].Am J Hum Genet,2004(3):526-528.
• [3]Ishida M,Moore GE.The role of imprinted genes in humans[J].Mol Aspects Med,2013(4):826-840.
• [4]Choufani S,Shuman C,Weksberg R.Molecular findings in Beckwith-Wiedemann syndrome.[J].Am J Med Genet C Semin Med Genet,2013(2):131-140.
• [5]施晓鋆,洪焱,黄绘,等.不同载体和冷冻保护剂用于人胚胎玻璃化冷冻的初步研究.[J].中国计划生育学杂志,2009(9):546-549.
• [6]Chen SL,Shi XY,Zheng HY,et al.Aberrant DNA methylation of imprinted H19 gene in human preimplantation embryos.[J].Fertil Steril,2010(6):2356-2358.
• [7]Wang Z,Xu L,He F.Embryo vitrification affects the methylation of the H19/Igf2 differentially methylated domain and the expression of H19 and Igf2[J].Fertil Steril,2010(8):2729-2733.
• [8]Shi XY,Chen SL,Zheng HY,et al.Abnormal DNA Methylation of Imprinted Loci in Human Preimplantation Embryos[J].Reprod Sci,2014(8):978-983.
• [9]Petrussa L,Van de Velde H,De Rycke M.Dynamic regulation of DNA methyltransferases in human oocytes and preimplantation embryos after assisted reproductive technologies[J].Mol Hum Reprod,2014(9):861-874.
• [10]Denomme MM,Mann MR.Maternal control of genomic imprint maintenance[J].Reprod Biomed Online,2013(6):629-636.

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