不同类型早期稽留流产中的甲基化异常探讨

Aberrant DNA Methylation in Different Types of Missed Abortion

目的:探讨不同类型早期稽留流产(停经6~9^(+6)周)中的甲基化异常情况。方法:选择早期稽留流产绒毛61例,根据B超检查的胚胎发育情况分为空囊型(18例)、有胚芽无胎心型(30例)、有胎心后停育型(13例)3组。采用RT-PCR法及Western-blot检测10-11易位蛋白(TET)(包括TET1、TET2、TET3)、5-mC、5-hmC在3组中的表达。对所有流产绒毛行G显带染色体分析,并比较染色体正常组和异常组间TET、5-mC、5-hmC的表达。结果:1流产绒毛组织中的TET1、TET2、TET3 mRNA的表达和蛋白的表达在3组间及组间两两比较,差异均无统计学意义(P>0.05)。25-mC含量在空囊型中明显降低,且与其他两组比较,差异有统计学意义(P<0.05),而5-hmC在绒毛组织中的表达量整体偏低,但3组间比较,差异无统计学意义(P>0.05)。3染色体正常组与异常组间TET1、TET2、TET3、5-mC、5-hmC表达的比较,差异均无统计学意义(P>0.05)。结论:甲基化水平低下可能阻碍了形成胚囊后的胚胎进一步发育,甲基化的改变影响了绒毛组织的发育,但可能与染色体的异常无直接关系。

Objective：According to whether an embryo or a foetal heart was present,we evaluated the expres- sion of the ten-eleven translocation （TET） family, 5-methylcytosine （ 5-mC ） and 5-hydroxymethylcytosine （ 5-hmC ） in villi of the missed abortion. Methods： Embryonic villi were collected from the missed abortion patients at gesta- tion ages of 6 -9 ^＋8 weeks. The 61 villi were divided into three groups based on the findings of ultrasonic examina- tion,namely GS - gestational sac only group（n = 18）, EM - having an embryo group（n =30） and FH-having a foetal heart rate group（ n = 13 ）. TET family mRNAs were analyzed using quantitative polymerase chain reaction （qPCR） ,and TET proteins were examined using Western-blot. The MethylFlash^TM Kit was used to quantify the absolute amount of 5-mC and 5-hmC. Results： No statistically significant difference was seen at both gene tran- scription and translation levels of TET1 ,TET2 and TET3 expression among these groups （GS, EM, and FH） （ P 〉 0.05）. Besides,our results showed that the expression of 5-mC in the GS group was decreased in comparison to EM and FH groups （ P 〈 0. 05 ） but no significant difference was seen in 5-hmC expression level among these three groups. Further subgroup analysis was performed based on the chromosome abnormalities in each group, the results showed that no significant difference was seen in the expression of TET1 、TET2、TET3、5-mC、5-hmC proteins between normal and abnormal chromosome group （ P 〉 0.05）. Conclusions： Insufficient maintenance DNA methylation would influence embryonic development. DNA methylation has long-term effects on the develop- ment of the villi. Perhaps,these changes have no relation with chromosomal changes.