从子宫内膜蜕膜化及胚胎早期发育重要相关基因表达的甲基化调控探索叶酸缺乏所致胚胎发育异常的分子机制

Folate deficiency has been reported for the association with many birth defects. But the molecular mechanisms involved in it remain poorly understood. Folate, one of the B vitamins, provides the one-carbon units required for nucleic acid biosynthesis and methylation. It plays a key role in epigenetic regulation of gene expression. Folate deficiency, either by insufficient nutritional intake or dysregulated metabolism, will result in an impaired DNA synthesis and repair, an abnormal genomic methylation of DNA and other molecules..Embryo implantation, uterine decidualization, placentation, and embryo development are all crucial for successful pregnancy. In our last National Natural Science Funds project(No.30973195), we previously found that folate, at the deficiency status, was not able to influence embryo implantation, and the methylation and expression of moleculars essential for uterine receptivity was not altered either. Successful implantation does not mean to successful pregnancy. As the results shown in the our previous research, the pregnant outcome in folate deficiency was unsuccessful . The birth rate of fetal was significantly decreased and the mortality rate of pregnant mouse was obviously increased at the status of folate deficiency. As we known, normal uterine decidualization, normal placentation, normal embryo are all much crucial for pregnancy after implantation. It has been confirmed genes related to placentation could be regulated by methylation and maternal folate level. However, it is not known whether the methylation and expression of genes participated in uterine decidualization and embryo development could be affected by folate level, potentially resulting in the failure of embryo development. .To explore whether folate deficiency affected the epigenetic regulation of genes related to decidualization and embryo development, and their influence on pregnancy, in this study we will investigate the methylation and expression of genes related to uterine decidualization and embryo development using a folate-deficient pregnant mouse model, and we will utilize methylation specific oligonucleotide (MSO), methylation-specific PCR (MSP), bisulfite genomic sequencing (BSP) and a series of molecular biology techniques to investigate the genes methylation and expression during decidualization and embryo development. By determining the effect of folate deficiency on DNA methylation, this study help elucidate the mechanism of folate deficiency resulting in birth defects from uterine decidualization and embryo development in two aspects.

叶酸缺乏是胚胎发育异常的多因素病因之一。课题组前期研究发现在叶酸缺乏的情况下，孕鼠胚胎能够正常着床，但妊娠结局不利。子宫内膜蜕膜化、胎盘正常形成以及胚胎早期自身正常发育是着床以后妊娠建立和维持的重要生理环节，胎盘形成中各基因DNA甲基化模式可受到母体叶酸水平的影响。子宫内膜蜕膜化进程以及胚胎早期自身的发育过程中相关基因DNA甲基化是否也会受到叶酸水平的影响，最终造成胚胎发育异常，目前尚不清楚，值得关注。本项目通过构建叶酸缺乏孕鼠模型，采用甲基化特异性寡核苷酸微阵列分析技术、甲基化特异性PCR和亚硫酸氢钠测序等一系列分子生物学技术对子宫内膜蜕膜化进程以及胚胎早期发育重要相关基因DNA甲基化及其表达进行系统深入研究，旨在阐明低叶酸水平可能通过DNA甲基化修饰调控子宫内膜蜕膜化进程以及胚胎早期发育重要相关基因的表达从而导致胚胎异常发育的分子机制，为出生缺陷的防治探寻新的线索。

本研究着眼于胚胎着床后子宫内膜蜕膜化及后续的胚胎发育是否受到低叶酸水平的影响，从母体与胚胎两个方面研究叶酸缺乏导致不良妊娠结局的作用机制。.在第一部分研究中，我们发现叶酸缺乏组孕鼠所形成的蜕膜鼓包减小，蜕膜化标志基因表达异常，证实低叶酸水平会抑制小鼠子宫内膜蜕膜化进程；进一步研究发现叶酸缺乏孕鼠子宫内膜凋亡的阳性细胞数明显减少，细胞凋亡相关基因异常表达，伴随血管密度明显降低，血管生成相关基因异常表达，表明叶酸缺乏会影响基质细胞的凋亡和血管生成；利用RRBS技术对两组小鼠蜕膜化时期子宫内膜组织的全基因组甲基化模式进行分析，发现两组小鼠孕6到8天子宫内膜组织基因组Promoter和CGI区不同类型C碱基（CG、CHG和CHH）的分布和甲基化水平存在差异，部分蜕膜化相关基因甲基化水平升高的同时表达下降，提示低叶酸水平会改变基因组的甲基化模式，影响关键基因的表达抑制子宫内膜蜕膜化进程。.在第二部分研究中，发现叶酸缺乏状态下的小鼠产仔数减少，叶酸缺乏组小鼠自D10开始出现子宫出血，D12、D13胚胎吸收明显，提示叶酸缺乏严重损害小鼠胚胎发育；利用RRBS对两组小鼠胚胎组织的基因组甲基化模式进行分析，发现两组小鼠孕9到11天胚胎组织基因组Promoter和CGI区不同类型C碱基的分布和甲基化水平存在差异，部分发育相关基因甲基化水平升高的同时表达下降，提示低叶酸水平会影响关键基因的表达损害胚胎发育；此外，叶酸缺乏组小鼠胚胎组织中Vangl基因的表达和功能显著被抑制，提示叶酸缺乏可能通过抑制PCP信号通路影响胚胎发育。.以上结果证实了母体叶酸摄入不足会影响孕早期子宫内微环境、损害胚胎发育，导致出生缺陷。其作用机制与基因组甲基化模式的改变以及胚胎发育过程中PCP信号通路的抑制有关。本研究以“营养环境→表观遗传修饰→早期子宫内膜微环境和胚胎发育”的视角，从母体与胚胎两方面系统探讨了叶酸缺乏所致胚胎发育异常的分子机制，为出生缺陷的研究和预防提供了新的实验依据。