Endometrial extracellular vesicles regulate processes related to embryo development and implantation in human blastocysts.. Endometrial extracellular vesicles regulate processes related to embryo development and implantation in human blastocysts.

Extracellular vesicles (EVs) mediate intercellular communication by transporting various molecules, and endometrial EVs have been postulated to be involved in the molecular regulation of embryo implantation. The objective of this study was to investigate the transcriptomic response of human blastocysts following internalization of endometrial EVs in vitro. Primary human endometrial epithelial cells, derived from endometrial biopsies collected from fertile oocyte donors, were cultured in vitro to isolate secreted EVs. Following EV characterization, day 5 human blastocysts were cultured in the presence or absence of the EVs for 24 hours and evaluated by RNA-sequencing. Among the differentially expressed genes (DEGs) in blastocysts co-cultured with EVs, we found 519 were significantly upregulated and 395 were significantly downregulated. These DEGs were significantly enriched in upregulated functions related to embryonic development, cellular invasion and migration, cell cycle, cellular organization and assembly, gene expression and cell viability; and downregulated functions related to cell death and DNA fragmentation. Further, the intracellular signaling pathways regulated by the internalization of endometrial EVs were previously related to early embryo development and implantation potential, for their role in pluripotency, cellular homeostasis, early embryogenesis and implantation-related processes. Finally, integrating data from miRNA cargo of EVs, we found that the miRNAs carried by endometrial EVs targeted nearly 80% of the DEGs in human blastocysts. This study provides novel insights into the functional relevance of EVs secreted by the human endometrium, and particularly the role of EV-miRNA regulation on global transcriptome behavior of human blastocysts during early embryogenesis and embryo implantation. Overall design: To investigate endometrium-embryo crosstalk during implantation, this study uses next-generation sequencing and compares the differential gene expression profiles of day 5 human embryos, cultured with and without extracellular vesicles secreted by primary human endometrial epithelial cells in a secretory-phase hormonal environment.

细胞外囊泡（Extracellular vesicles, EVs）通过转运多种分子介导细胞间通讯，而子宫内膜来源的细胞外囊泡被认为参与胚胎着床的分子调控过程。本研究旨在探究体外内化子宫内膜EVs后，人类囊胚的转录组应答反应。
本研究从可生育卵母细胞供者的子宫内膜活检组织中分离原代人子宫内膜上皮细胞，体外培养以获取分泌的细胞外囊泡。完成EVs的表征鉴定后，将第5天人类囊胚在有/无EVs的培养体系中培养24小时，随后通过RNA测序（RNA-sequencing）进行分析。
在与EVs共培养的囊胚差异表达基因（differentially expressed genes, DEGs）中，我们发现519个基因显著上调，395个基因显著下调。这些差异表达基因显著富集于与胚胎发育、细胞侵袭与迁移、细胞周期、细胞组织与组装、基因表达及细胞存活相关的上调功能通路；而下调功能则与细胞死亡及DNA片段化相关。
进一步研究发现，子宫内膜EVs内化所调控的细胞内信号通路此前已被证实与早期胚胎发育及着床潜能相关，其涉及多能性维持、细胞稳态、早期胚胎发生及着床相关过程。此外，结合EVs的microRNA（miRNA）载荷，我们发现子宫内膜EVs所携带的miRNA靶向了人类囊胚中近80%的差异表达基因。
本研究为人类子宫内膜分泌的细胞外囊泡的功能相关性提供了新的见解，特别是EVs携带的miRNA对早期胚胎发生及胚胎着床过程中人类囊胚整体转录组行为的调控作用。
整体实验设计：为探究着床过程中子宫内膜与胚胎的交叉对话，本研究采用下一代测序技术，对比分析了在分泌期激素环境下，分别添加/不添加原代人子宫内膜上皮细胞分泌的细胞外囊泡的培养体系中，第5天人类胚胎的基因表达差异谱。