多囊卵巢综合征对卵子和胚胎DNA甲基化修饰的影响及机制研究

Polycystic ovary syndrome (PCOS) is an endocrine disorder, which is characterized by hyperandrogenism, oligovulation or anovulation and insulin resistance. It is one of the key factors resulting in reproductive dysfunction. The dysfunction is related to the genetics, but cannot be completely explained by genetics. Epigenetics is the study of mitotically or meiotically heritable changes in gene expression without altering nucleotide sequence. Therefore, we infer that epigenetic changes may play a key role for the fertility decline of reproductive-age females with PCOS. In this study, we will investigate the effects of PCOS on global DNA methylation levels of granulosa cells using Med-CHIP. Meanwhile, we will research whether the mitochondrial functions in granulosa cells are affected by PCOS and the possible mechanisms underlying them by qRT-PCR and confocal microscopy. The communication between cells also will be checked by testing the expression of proteins including in gap junctions. Furthermore, we will study whether global DNA methylation level of oocytes and early embryos is influenced by PCOS using RRBS, as well as the demethylation process of 5mC and 5mhC at pronucleus stage. This study may reveal the possible mechanisms underlying abnormal development of oocytes and embryos caused by PCOS in reproductive-age women, which would provide theoretical foundation for the pathogenesis of PCOS.

多囊卵巢综合征(PCOS)是一种以高雄激素血症、排卵障碍、胰岛素抵抗为主要特征的内分泌紊乱性疾病，是导致育龄女性生育力下降的重要原因之一。虽然PCOS导致的生育力下降与遗传学有关，但不能完全由遗传学解释。表观遗传学是在不改变核苷酸序列的前提下通过有丝分裂或减数分裂将性状遗传给后代的遗传方式。因此我们推测PCOS可能引起卵子和胚胎表观遗传修饰改变，进而影响生育力。本研究采用Med-CHIP等方法研究PCOS患者颗粒细胞基因组DNA甲基化水平的改变。同时通过qRT-PCR技术及显微共聚焦技术分析PCOS对颗粒细胞线粒体功能和细胞间缝隙连接的影响，并进一步用RRBS等方法研究PCOS对卵子和早期胚胎基因组DNA甲基化水平的影响。我们也将研究PCOS对原核期胚胎5mC和5hmC变化的影响。该研究将揭示PCOS对卵子和胚胎DNA甲基化修饰的影响及可能机制，为PCOS的发病机理研究提供新的理论依据。

多囊卵巢综合征(PCOS)是一种以高雄激素血症、排卵障碍、胰岛素抵抗为主要特征的内分泌紊乱性疾病，是导致育龄女性生育力下降的重要原因之一。虽然PCOS导致的生育力下降与遗传学有关，但不能完全由遗传学解释。表观遗传学是在不改变核苷酸序列的前提下通过有丝分裂或减数分裂将性状遗传给后代的遗传方式。因此本项目旨在PCOS对卵子和胚胎DNA甲基化的影响及机制研究。我们研究发现PCOS组颗粒细胞YAP1启动子区域甲基化水平明显低于对照组，且在12个位点(443，431，403，371，331，120，49，5，1，9，15，22)的甲基化水平显著低于对照组(P<0.05)。PCOS组患者颗粒细胞YAP1的mRNA和蛋白表达水平也显著高于对照组，体外培养添加睾酮颗粒细胞YAP1的甲基化水平以浓度依赖式下降。我们随后对人MII期卵母细胞进行单细胞重亚硫酸盐测序，两组卵母细胞基因组DNA甲基化水平无显著差异。我们遂构建糖尿病小鼠模型以模拟PCOS的糖代谢异常，从该角度研究PCOS内分泌异常对颗粒细胞及卵子DNA甲基化水平的影响，我们分别对两组卵子和颗粒细胞进行全基因组甲基化测序并进行GO注释和分析发现差异性甲基化的基因与生长发育相关，且卵子差异性甲基化的相关基因与透明带相关，并且糖尿病组的卵子的透明带比对照组薄，受精率下降，囊胚的线粒体copy数的增加。研究表明PCOS导致的高雄状态可能会引起颗粒细胞YAP1启动子区域DNA甲基化水平的改变，但PCOS不一定引起人卵基因组甲基化水平的显著改变，而PCOS引起的糖代谢异常可导致颗粒细胞和卵母细胞DNA基因组甲基化水平的变化，囊胚线粒体copy数增加进而导致卵子胚胎质量的下降影响女性生育力。