高通量测序解析CTNND1调控人类囊胚植入子宫内膜的分子机制研究

Assisted reproductive technology (ART) is one of the most important treatments for infertility. However, the implantation rate after blastocyst transfer is only about 50%. Little is known about the molecular mechanisms in the implantation process especially at the implantation “trigger” sites. In our previous work, we established the in vitro embryo implantation model with human embryos and endometrial cells, and observed that the time window of implantation takes place at 7.5-8.5d. After examining the gene expression profiles in trophoblast (TE) from the pre- and post-implanted human embryos (7.5d) by single cell RNA sequencing (scRNA-seq), we supposed the key hub gene CTNND1 (catenin delta 1) which could regulate blastocyst implantation and guarantee the potential differentiation of TE. In this proposal, we like to further utilize scRNA-seq technology and in vitro implantation model to analyze the temporal (6.5, 7.5, 8.5d) and spatial (polar TE and mural TE) gene regulatory networks by scRNA-seq. We will characterize the key gene CTNND1 Knock Out in human trophoblast stem cells (hTSCs) created by CRISPR/Cas9 gene editing tool. In vitro and/or in vivo levels, we investigate CTNND1 gene’s function and relative molecular epigenetic mechanisms by RNA-seq and DNA methylation sequencing. Then, we use scRNA-seq to establish clinical single blastocyst transfer-PGS system. By tracking its clinical pregnancy outcome data, we will discuss the feasibility of CTNND1 as a potential molecular marker for screening better transplantable blastocyst. Our proposal is to provide scientific theoretical support and basis of clinical practice to greatly improve the success rate of single blastocyst transfer and to decrease maternal and fetal complications in multiple pregnancies.

试管助孕中约50%的囊胚种植失败，且囊胚植入相关时间节点及界面的分子机制尚不够明确。我们前期借助人囊胚体外植入模型，观察到第7.5-8.5天为囊胚植入发生的窗口期；利用单细胞转录组测序(scRNA-seq)比较第7.5天植入和非植入囊胚滋养外胚层细胞(TE)基因表达谱，明确了调控囊胚植入和保障TE分化潜能的关键枢纽基因CTNND1(catenin delta 1)。本项目将继续采用我们现有成熟的scRNA-seq和人囊胚体外植入模型，精细解析囊胚植入“时间窗”及空间的基因表达规律，以及参与囊胚植入和保障TE分化潜能的分子调控网络；借助CRISPR/Cas9敲除人滋养层干细胞CTNND1、采用RNA-seq和DNA甲基化测序研究其功能和表观遗传学分子机制；建立临床单囊胚移植TE-PGS系统，追踪临床结局，并探讨利用CTNND1筛选优质单囊胚移植的可行性，提高移植成功率、降低多胎母婴并发症。

辅助生殖技术中囊胚的成功植入与多种因素相关，而胚胎植入能力、子宫内膜容受性最为关键。目前人类胚胎着床具体机制尚缺乏有效的系统性研究，临床治疗中对胚胎种植失败尚缺乏有效的干预手段。.为找寻及探究调控人类囊胚植入子宫内膜关键基因及其分子机制，本项目建立了人类囊胚-子宫内膜体外共培养模型，利用该模型收集了植入窗口期胚胎和子宫内膜的样本并进行单细胞转录组测序分析，通过生物信息学分析以及自主开发的歧化分析算法，我们发现了两个从胞体滋养层细胞CTB向合胞体滋养细胞STB及绒毛膜外滋养细胞EVT分化的过渡态细胞亚群，进一步鉴定出调控植入期胚胎滋养层分化为STB细胞的关键基因TBX3并进行了功能验证。同时我们发现CTNND1在随着胚胎的植入，其表达量逐渐升高，在滋养细胞出现显著分化的Day8天表达最高，提示CTNND1对滋养细胞分化的调控作用。鉴于人类胚胎的来源限制，我们通过合作研究分离了来源于人胎盘和人囊胚的滋养干细胞TSC细胞系，并自行优化建立了由primed iPS细胞以及naïve H9细胞诱导的TSC细胞系。通过定向诱导TSC分化成EVT和STB，我们发现CTNND1的敲低会显著影响人滋养层干细胞的增殖及分化。进一步通过在临床上收集移植胚胎活检的滋养细胞进行单细胞测序，结合临床妊娠结局，进一步发现CTNND1在未着床胚胎中降低表达，这些数据证明了CTNND1对滋养细胞分化和胚胎植入的关键调控作用。在此基础上，进一步分析了植入期子宫内膜细胞的基因表达变化特征。通过对胚胎植入前后子宫内膜细胞的单细胞分析发现，植入后内膜细胞的血管新生相关基因显著升高，而与滋养细胞的信号交互分析发现整合素和成纤维细胞生长因子通路在着床过程中扮演重要角色。.此外围绕胚胎植入的核心问题，探索了不同辅助生殖技术，包括冷冻、光照、线粒体替代疗法等对胚胎发育的影响机制，鉴定出了miR-148b-3p、Mrpl11基因、细胞松弛素B等多个影响胚胎发育的关键因子。同时为进一步提高临床移植率，探索了利用间充质干细胞卵巢早衰的效果，并在PCOS患者卵泡液中筛选得到了影响胚胎发育的外泌体蛋白和miRNA，可作为预测胚胎质量的潜在生物标志物。.本项目的研究成果为理解胚胎植入过程和提高单囊胚移植成功率提供了有力的参考依据。