体外胚胎出生性别比例失衡的分子机制研究

Previous studies have proven that in vitro fertilization (IVF) could lead to a skewed sex-ratio of IVF embryos or offspring. However, the underlying mechanisms are still unclear. Based on our preliminary work, we found that there is a biased female lethality during per-implantation period after the IVF embryos were transferred into recipients, which lead to a skewed sex-ratio at birth. Together with the high-throughput data of transcriptome and methylome, we hypothesize that an impaired X chromosome inactivation (XCI) of female IVF embryos may be involved in the skewed sex-ratio. Moreover, we have proven that the expression of Xist, the key regulator of XCI, was consistently depressed in IVF embryos during pre-implantation period. Therefore, in this project, we will firstly conduct a systematic and dynamic analysis of XCI status, as well as developmental profiling of IVF embryos. In addition, through the functional overexpression and knock down experiments, we aim to prove that the inhibited Xist expression is the key factor for the impaired XCI of IVF embryos. Furthermore, we will explore the mechanisms responsible for the inhibited Xist expression in IVF embryos, with focus on the Rnf12, the upstream inactivator of Xist, and the methylation status of Xist promoter. Finally, using some epigenetic modifying drugs, or physical and chemical factors, we will try to improve the XCI status of IVF embryos and revise the skewed sex-ratio of IVF offspring.

家畜和人类相关研究已证实体外受精（IVF）胚胎及后代存在性别失衡现象。然而，这一现象的内在机制目前尚不清楚。我们发现移植后IVF胚胎在附植前后会出现较高比例的雌性死亡现象，并导致出生后代性别失衡。胚胎转录组和甲基化组的高通量数据进一步提示，雌性IVF胚胎X染色体失活不足可能导致了出生性别比例失衡，并已经证实X染色体失活的核心调控基因Xist在附植前的IVF胚胎中持续低表达。针对这一科学假设，本项目将对IVF胚胎的X失活状态及发育表型进行系统的动态分析，并通过过表达和敲降等功能实验证实Xist低表达是导致IVF胚胎X失活不足的关键因素。项目还将针对Xist上游激活因子Rnf12和Xist promoter的甲基化状态，探索IVF胚胎Xist低表达的调控机制。此外，将利用表观修饰药物，或通过调整IVF胚胎发育环境，优化现有的体外发育体系，改善胚胎失活状态，进而校正IVF胚胎及后代性别比例。

体外受精技术（IVF）广泛应用于人类不育症治疗，部分发达国家IVF出生后代已占新生人口1-5%。同时，IVF还是快速繁育优良种畜最有最具潜力的技术之一。然而，越来越多的动物实验和临床证据表明，IVF会引发一系列健康问题，包括流产、早产、低初生重，及出生后患病风险增加等。其中，性别比例失衡，作为重要生殖指标，意味着严重的胚胎发育障碍，受到广泛的关注。然而，其中的机制却长期无法回答。本课题揭示了IVF导致出生性别失衡的内在机制。利用小鼠作为模型，我们发现IVF引发的性别失衡是由于围植入期雌性胚胎偏向性发育异常导致的。进一步，我们证实了雌性特有的表观修饰事件-X染色体印记失活（iXCI）不充分是导致IVF后代性别失衡的主要原因，并发现启动iXCI的关键通路Rnf12-Xist受到抑制，引发iXCI不充分。同时，利用兔子和牛胚胎，发现IVF引发XCI不足的现象在物种间普遍存在。通过筛选具有表观修饰活性的小分子物质，发现维生素A的代谢产物，视黄酸（RA），可以上调Rnf12/Xist表达，进而改善IVF雌性胚胎iXCI状态，减少雌性胚胎死亡，最终IVF性别比例恢复至自然状态。这项工作，首次为表观错误引发IVF胚胎发育异常的机制，提供了功能学证据，并率先提出通过优化IVF体系，校正表观错误，解决IVF安全性问题的新思路。