少精症患者H19DNA甲基化丢失导致胚胎发育潜能下降及子代健康风险增加的分子机制

The rate of abortion and imprinting defect diseases of infertile male offspring is increased significantly followed by introcytoplasmic single sperm injection technology, but the molecular mechanism is still unknown, which is the key point in the reproductive health field. Our previous study firstly demonstrated that the loss of DNA methylation of H19-imprinting control region(ICR) was only occurred in oligozoospermia patients, and raised a hypothesis that H19 gene affected fetus development through their own placenta by regulating impinted genes network. It was suggested that we might avoid the health risks by screen out sperm with normal imprinted status of H19-ICR. Therefore, we conducted infertile mice model of H19-ICR imprinting loss. On the basis of the mice model, we would combine the epigenetics, embryology and molecular biology study methods, take trophoblast of blastula and placenta of different developmental stage as materials, to observe the expression changes and remodeling process of H19 and plancenta blood supply and nutrient transfer-related imprinted genes, approach the role played in embryo development regulation. Furtherly,we will investigate the process of remove and re-establishment of imprinted genes at E12.5 of mice fetus.Finally try to screen out normal imprinted sperm, reveal a new mechanism of inheritance of imprinting defected sperm and provide a new clinical treatment for oligozoospermia patients, which have important academic and practical significance.

男性不育患者行卵胞浆内单精子注射助孕后胚胎发育潜能下降及子代健康风险增加的分子机制不明，成为生殖健康领域的研究热点。我们的前期研究表明，单纯少精症患者精子H19印记控制区域DNA甲基化丢失严重，提出H19通过调控胎盘印记基因网络影响胎儿发育的假说，提示，筛选出H19印记正常的精子有可能降低甚至规避男性不育患者后代的健康风险。为此，我们成功构建了H19印记丢失的不育小鼠模型，采用表观遗传学、胚胎学和分子生物学相结合的技术路线，以囊胚滋养层细胞、不同发育节点胎盘为研究材料，观察H19以及与胎盘血供、营养代谢相关印记基因的表达变化和重塑过程，探讨其在胚胎发育调控中的作用，并进一步研究孕12.5天胎鼠生殖细胞中印记基因的擦除和重建过程，揭示精子印记基因DNA甲基化缺陷遗传性的新机制，最后尝试无创筛选印记正常的精子，为少精症患者临床辅助生殖治疗的安全性提供新的方法，具有重要的理论和实际意义。

男性不育患者行卵胞浆内单精子注射助孕后胚胎发育潜能下降及子代健康风险增加的分子机制不明，成为生殖健康领域的研究热点。前期研究表明，单纯少精症患者精子H19印记控制区域DNA甲基化丢失严重，据此，我们提出H19通过调控胎盘印记基因网络影响胎儿发育的假说。. 首先，本项目通过亚磷酸氢钠测序PCR技术（BSP），首次研究发现精子H19-DMR区域DNA甲基化丢失仅发生于少精症患者中，深入研究发现H19-DMR区域DNA甲基化丢失主要发生于H19-DMR区域的CTCF结合位点6处，且甲基化丢失程度与精子密度显著负相关，而与精子活力无关，并且识别出2×1000000/ml的精子浓度为风险阈值；其次，本项目通过研究精子形态与胚胎发育及临床结局的相关性，发现单一畸形精子症患者行ICSI助孕后胚胎正常受精率、卵裂率、优胚率、临床妊娠率并无显著差异，但是胎儿流产率显著升高；最后，通过构建ART小鼠模型，采用表观遗传学、胚胎学和分子生物学相结合的技术路线，以不同发育节点胎盘为研究材料，首次发现ART操作致小鼠植入前胚胎H19印记丢失，并确定印记丢失的开始时间为胚胎8-细胞期，同时发现ART操作不仅影响小鼠胎儿宫内生长轨迹，也影响小鼠胎盘宫内生长轨迹，致妊娠中期胎盘重量减小，ART操作致妊娠末期胎盘过度生长且功能紊乱，并且致小鼠胎盘H19印记水平发生动态变化。. 综上，本研究发现H19印记控制区域DNA甲基化丢失可能通过影响胎盘的结构和功能导致胚胎发育潜能下降，具有重要的理论和实际意义。