ART影响印记基因Phlda2表达致胎盘发育及功能异常的机制研究

The placental maldevelopment and dysfunction resulted by assisted reproductive technology (ART) is an important cause of adverse pregnancy outcomes and high risk of embryo origins of diseases in later life. However, the related mechanisms that ART resulted abnormal placentation remains to be elucidated. In our previous work, we systematically studied the effects of ART on the placenta development and expression of imprinted genes. We found that ART induced placental overgrowth, placental glycogen and lipids over accumulation, and the expression level of imprinted gene Phlda2 is significantly downregulated. With Phlda2 regulate cell proliferation, glycogenesis and lipidosis through the negative regulation of Akt signal pathway; we proposed the hypothesis that ART down-regulation of Phlda2 expression that lead to excessive activation of the Akt signal, which resulted in placental hypertrophy, excessive accumulation of glycogen and lipids in placenta. In this study, firstly, we will clarify the molecular pathway that abnormal expression of Phlda2 causes placental maldevelopment and dysfunction by using molecular biology and lipidomics technologies. Secondly, to reveal the epigenetic mechanisms that ART induced the aberrant expression of Phlda2 by using BSP and CHIP Technologies. Finally, by activating or inhibiting the Akt pathway, to explore the intervention strategy to save the ART placental function. The completion of this project will reveal new mechanisms of ART induced abnormal placental development and provide theoretical bases for the markers of early warning and intervention strategies.

辅助生殖技术（ART）致胎盘发育和功能异常，是导致新生儿不良妊娠结局及胚胎源性疾病发生风险增加的重要原因。但ART致胎盘异常的机制亟待阐明。本课题组前期系统研究了ART对胎盘发育及印记基因表达的影响，发现ART致妊娠末期小鼠胎盘过度生长、糖原和脂质异常积累，印记基因Phlda2表达水平显著下调。鉴于Phlda2通过负调控Akt信号通路，调控细胞增殖及糖原和脂质合成，我们提出ART致Phlda2表达下调，Akt信号过度激活，致胎盘增生，糖脂异常积累的假说。本课题拟在前期研究基础上，首先，通过分子生物学及脂质组学技术阐明Phlda2表达异常致胎盘发育异常分子通路；其次，通过BSP和CHIP技术揭示ART致Phlda2表达异常的表观遗传机制；最后，通过激活或抑制Akt通路，探索挽救ART胎盘功能的干预策略。项目的完成将进一步揭示ART致胎盘发育异常的新机制，为临床寻找干预策略提供实验及理论依据。

研究表明，辅助生殖技术（Assisted Reproductive Technology，ART）致胎盘发育和功能异常是一种普遍现象。临床研究发现，ART致胎盘重量显著增加。小鼠模型发现，ART致妊娠末期胎盘重量显著增加，胎盘效率降低。ART致胎盘发育和功能异常是导致临床不良妊娠结局发生风险增加的重要原因，但其机制不明。.本项目通过临床数据与小鼠模型发现，ART会影响胎儿的宫内生长轨迹，且导致胎盘重量显著增加、胎盘效率下降。进而研究发现，ART致人和小鼠胎盘PHLDA2表达下调，且致小鼠胎盘磷酸化AKT（p-AKT），p-mTOR和p-Gsk3β的水平显著升高，表明ART致胎盘AKT/mTOR信号过度激活。利用滋养层细胞系HTR-8发现，敲降PHLDA2致滋养层细胞增殖速度显著增加；利用蛋白质组学发现，敲降PHLDA2致滋养层细胞AKT/mTOR信号过度激活；进而利用western-blot验证发现，PHLDA2表达下调致p-AKT，p-mTOR和p-S6K的水平显著升高，表明敲降PHLDA2致滋养层细胞AKT/mTOR信号过度激活。利用临床样本发现，ART可导致胎盘总体DNA甲基化水平显著降低，且调控PHLDA2表达的Long non-coding RNA KCNQ1OT1的表达水平显著升高，表明其可能是ART导致胎盘PHLDA2表达下调的重要原因。.本项目通过临床和动物模型的系统研究，发现ART致胎盘过度生长；首次揭示ART致PHLDA2的表达降低致胎盘AKT/mTOR信号过度激活，是导致胎盘过度生长的重要机制；另外，发现ART致胎盘KCNQ1OT1的表达水平显著升高，可能是导致胎盘PHLDA2表达下调的重要机制。这些发现，为辅助生殖临床导致的胎盘生长和发育异常提供了理论依据，并为今后临床干预和治疗提供了靶点，具有较重要的科学意义及临床价值。