Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

Objective: To study the uptake by human embryos of extracellular vesicles secreted by the maternal endometrium, and to investigate their miRNA cargo, in order to describe their role in implantation and early embryo development. Design: Prospective descriptive study. Subjects: Healthy women oocyte donors with confirmed fertility and day 5 human blastocysts. Intervention: Endometrial biopsies were collected from healthy oocyte donors undergoing transvaginal ultrasound-guided cyst aspiration for oocyte retrieval. Main Outcome Measures: Extracellular vesicle were isolated from culture media of primary human endometrial epithelial cells by ultracentrifugation. Concentration and size were analyzed by nanoparticle tracking analysis, their morphology visualized by transmission electron microscopy and extracellular vesicle protein markers expression was determined by western blotting. Vesicles were fluorescently labelled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of extracellular vesicles was performed using miRNAseq, target genes of the most expressed miRNAs were annotated and functional enrichment analysis was performed. Results: Extracellular vesicle characterization revealed a size within 100-300 nm, and expression of extracellular vesicle protein markers HSP70, TSG101, CD9 and CD81. Fluorescent microscopy showed an efficient extracellular vesicle internalization by human blastocysts within 1-2h, being the fluorescent signal stronger in the hatched area of the embryo. miRNAseq described 149 annotated miRNAs and top 37 most expressed miRNAs targeted 6,592 genes. Functional enrichment analysis of these targeted genes indicated that they participate in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization or gene expression. Among miRNAs contained in these EVs, hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p and hsa-let-7a-5p were highly implicated in all these biological processes. Conclusion: Data suggest that extracellular vesicles secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Knowledge of the communication system between human endometrium and embryo via miRNA cargo of these vesicles could describe new biomarkers of implantation success and embryo competence. Overall design: Descriptive analysis of miRNA content of extracellular vesicles secreted by primary human endometrial epithelial cells (n = 3 pools of cells). Samples were collected 36 hours after the LH surge on the day of oocyte retrieval from healthy women, oocyte donors (18–35 years old with a body mass index of <30 kg/m2)

研究目标：探究人类胚胎对母体子宫内膜分泌的细胞外囊泡（extracellular vesicles）的摄取情况，并解析其miRNA载荷，以阐明其在着床与早期胚胎发育中的作用。
研究设计：前瞻性描述性研究。
研究对象：经确认生育能力正常的健康卵母细胞捐赠者，以及第5天人类囊胚。
干预措施：从接受经阴道超声引导下囊肿抽吸取卵术的健康卵母细胞捐赠者处采集子宫内膜活检样本。
主要结局指标：采用超速离心法从原代人子宫内膜上皮细胞的培养基中分离细胞外囊泡；通过纳米颗粒追踪分析（nanoparticle tracking analysis）测定其浓度与粒径，借助透射电子显微镜（transmission electron microscopy）观察其形态，采用蛋白质印迹法（western blotting）检测细胞外囊泡蛋白标志物的表达水平；使用Bodipy-TR神经酰胺对囊泡进行荧光标记，通过共聚焦显微镜分析人类囊胚对其的摄取情况；采用miRNA测序（miRNAseq）分析细胞外囊泡的miRNA载荷，对高表达miRNA的靶基因进行注释，并开展功能富集分析。
研究结果：细胞外囊泡表征结果显示其粒径介于100~300 nm，且表达细胞外囊泡蛋白标志物HSP70、TSG101、CD9与CD81。荧光显微镜观察表明，人类囊胚可在1~2小时内高效内化细胞外囊泡，且胚胎孵出区域的荧光信号更强。miRNA测序共鉴定出149个已注释的miRNA，前37个高表达miRNA的靶基因共计6592个。对这些靶基因的功能富集分析显示，它们参与多种与胚胎发育、氧代谢、细胞周期、细胞分化、凋亡、代谢、细胞结构组织或基因表达相关的生物学过程。在这类细胞外囊泡所含的miRNA中，hsa-miR-92a-3p、hsa-let-7b-5p、hsa-miR-30a-5p、hsa-miR-24-3p、hsa-miR-21-5p及hsa-let-7a-5p在上述所有生物学过程中均发挥关键调控作用。
研究结论：本研究数据表明，人子宫内膜上皮细胞分泌的细胞外囊泡可被人类囊胚内化，并通过转运miRNA调控与着床事件及早期胚胎发育相关的生物学过程。阐明人类子宫内膜与胚胎通过此类囊泡的miRNA载荷实现的通讯系统，可为着床成功率及胚胎发育潜能的新型生物标志物研发提供理论支撑。
总体研究设计：对原代人子宫内膜上皮细胞（n=3个细胞混合池）分泌的细胞外囊泡的miRNA含量开展描述性分析。样本采集自健康卵母细胞捐赠者（年龄18~35岁，体质量指数<30 kg/m²），于取卵当日LH峰出现后36小时采集。