辅助生育技术对胚胎组蛋白修饰及其跨代遗传的影响和机制研究

Over the last four decades assisted reproductive technologies (ART) have been used increasingly around the world. Consequently more than seven million infants have been born after ART treatments. However, emerging evidence suggests that ART babies have more adverse short-term and long-term health outcomes than naturally conceived individuals. Altered epigenetic modifications due to ART manipulations may be the underlying mechanism. Our preliminary data suggests that two components of ART, ovarian hyperstimulation and in vitro culture, impair developmental potential of mouse embryos, and alter the gene expression as well as histone modifications, both before and after implantation. This study aims to examine the effects of different ART treatment protocols on transcriptome and histone modifications of pre-implanted and post-implanted embryos and F1 and F2 offspring. The focus will be on the effects of two specific ART procedures on histone modifications, gene expression and its transgenerational inheritance. Such an analysis may uncover the underlying mechanism by which changes to histone modifications contribute to impaired embryo development and abnormal transgenerational inheritance. Also the study will aim to confirm if mild ovarian stimulation or the addition of melatonin to culture media will reduce the impact of ART on histone modifications and health risks of offspring. In essence this study aims to provide the theoretical basis for optimizing ART procedures, hence reducing health risk and transgenerational inheritance for ART offspring, thereby playing an important role in improving population health outcomes.

近40年来辅助生殖技术（ART）在全世界的应用持续增加，至今有超过七百万婴儿经ART治疗后出生，但研究表明ART子代的近期和远期的健康风险增加，而ART操作导致的表观遗传修饰改变可能是主要的发病机制。我们前期的研究发现ART治疗的大剂量促排卵及体外培养都影响小鼠胚胎的发育潜能，并影响胚胎着床前后的基因表达和组蛋白修饰状态。本项目将研究不同ART治疗方案对小鼠植入前后胚胎、F1及F2子代转录组和组蛋白修饰的影响，探讨ART两个环节对胚胎组蛋白修饰模式、基因表达及跨代遗传的不同影响；通过对异常组蛋白修饰及相关基因的研究阐明组蛋白修饰改变引起ART子代发育异常及跨代遗传的机制；明确微刺激或培养液中添加褪黑素是否能减轻ART对组蛋白修饰的影响并降低子代的风险。本研究将为进一步改善ART技术操作提供理论基础，将有助于降低ART子代的健康风险，并阻断健康风险的跨代遗传，对提高人口素质有十分重要的意义。

研究表明辅助生殖技术（ART）子代的近期和远期的健康风险增加，而体外受精（IVF）操作导致的组蛋白修饰改变可能是主要的发病机制之一。本项目主要研究了不同ART治疗对植入前后胚胎发育、胚胎基因表达谱及组蛋白修饰的影响，并明确不同ART治疗通过表观修饰调控子代糖代谢的分子机制。.我们发现IVF胚胎在植入前阶段发育质量下降，胚外组织的发育受到了更加显著的影响，植入后这种胚外组织的发育缺陷进一步扩大，表现为胚胎植入失败、胎盘效率降低、胎盘结构紊乱等。转录组分析显示IVF胚胎的胚外外胚层（ExE）中存在上胚层（Epi）特异性基因与特异性转录因子的异常表达现象。.IVF胚胎的ExE表现出与正常胚胎Epi相似的组蛋白修饰状态，并且这种异常的聚类是由于H3K4me3修饰的异常占位导致的。此外，在IVF胚胎ExE的异常高表达基因的转录起始位点与启动子区域存在异位H3K4me3修饰。.本课题在前期研究基础上进一步研究了Fbxl7基因的上游调控机制，结果表明表达Fbxl7受到上游转录因子Foxa2的调控。在小鼠上进行活体注射Fbxl7过表达和敲降AAV病毒的体内功能验证，对照组和mIVF组敲降Fbxl7后糖耐量异常；IVF组过表达Fbxl7后糖耐量改善。这些结果验证了IVF通过FBXL7泛素化调控RHOA蛋白，进而影响小鼠IVF子代的糖代谢表型。.本项目明确了IVF植入前后胚胎的组蛋白H3K4me3与H3K27me3修饰的变化，阐明了组蛋白H3K4me3修饰改变引起IVF子代发育异常的机制；验证了IVF上调肝脏Fbxl7影响子代糖代谢的分子机制，而在培养液中添加褪黑素通过回调肝脏Fbxl7基因表达，进而逆转了小鼠IVF子代的糖代谢异常表型。这些结果为进一步改善ART技术操作提供理论基础，将有助于提高辅助生殖成功率，降低ART子代的健康风险。