人卵泡不同发育时期卵泡液游离DNA甲基化修饰及其功能研究

Cell-free DNA(cfDNA) has been confirmed existing in human follicular fluid, and the concentration of cfDNA were significant difference during the folliculogenesis stages, however, the characteristics and the methylation of follicular fluid cfDNA during folliculogenesis remains unclear. Our investigation first noticed the followings: 1. the size of the cfDNA fragmentation distribution was times of nucleosome length. 2. The absolute global methylation of follicular fluid cfDNA was low in preovulatory follicular, which is significant lower than global methylation of follicular fluid cfDNA in 13-17mm follicular. 3. The methylated CpG sites were mainly located in functional regulation regions and introns of genes. It is highly essential to evaluate the global methylation status in other stages of human follicular. Hence, we hypothesized that the global and single-base DNA methylation dynamic erase and reconstruction during the folliculogenesis. In order to prove this hypothesis, we will recruit the patients in assisted reproductive technology and collect the follicular fluid from different development stages. Antibody based global DNA methylation detection method; whole genome DNA methylation sequencing and bioinformatics analysis were used to investigate the global and single base DNA methylation and function during human follicular development stages. Our study will provide new insight into epigenetic mechanism of human follicular development.

人卵泡液中存在游离DNA，卵泡发育不同时期其含量差异显著，但其在卵泡发育不同时期的特性、甲基化修饰与功能尚不清楚。我们前期研究结果提示：排卵前卵泡内卵泡液游离DNA片段长度呈核小体长度倍数分布，其整体甲基化低于大卵泡内卵泡液游离DNA整体甲基化；全基组甲基化测序提示排卵前卵泡内卵泡液游离DNA甲基化位点主要位于功能调控与内含子区域，但其功能及在卵泡发育其他时期有待进一步探讨。因此，我们提出假说：人卵泡发育过程中，卵泡液游离DNA 整体甲基化与其全基因组甲基化呈动态变化。为验证这一假说，我们收集辅助生殖过程中卵泡不同发育时期的卵泡液，采用荧光定量、全基因组甲基化测序与生物信息学分析等手段，从整体和单碱基水平探讨卵泡液游离DNA在卵泡发育不同时期甲基化情况，解析卵泡不同发育时期卵泡液游离DNA甲基化擦除与重建及功能。本研究将从卵泡液游离DNA这个新视角为研究人卵泡发育表观遗传机制提供新思路。

项目承担人主要针对胚胎发育染色质开放性和染色体易位子代传递阻断问题进行研究。针对人类胚胎早期发育染色质重编程这一科学问题，揭示了人类早期发育过程中染色质重编程的变化规律，首次在人类胚胎ZGA前发现大量染色质开放区域，阐明人类早期胚胎染色质开放规律依赖基因组激活的机制，发现染色质调控规律在人和小鼠胚胎发育过程中同时存在保守性和物种特异性。提出染色质海湾(chromatin harbor)新理论：这种早期胚胎基因组激活前特有的开放染色质区域可能作为一种特殊的染色质海湾，可以暂时储存转录因子。一旦基因座激活，这些位点被关闭，转录因子可被释放至启动子区参与基因组激活。平衡易位是最常见的染色体结构异常，人群中的发生率为1‰到2‰。平衡易位携带者面临较高的反复流产、生育畸形儿的风险。传统方法只能筛选整倍体胚胎进行移植，仍有50%的概率将染色体平衡易位遗传给后代，导致后代依然有面临反复流产、生育畸形胎儿的风险。针对阻断染色体平衡易位向子代传递这一辅助生殖医学领域的难题，申请人联合亿康基因团队，研发了等位基因映射识别胚胎染色体易位（MaReCs）技术，克服了传统方法无法识别胚胎的染色体平衡易位携带状态，无法阻断平衡易位向子代传递的难题，改写了此类患者生育结局。