STUDY QUESTION What is the transcriptomic response of human blastocysts following internalization of extracellular vesicles (EVs) secreted by the human endometrium? SUMMARY ANSWER EVs secreted by the maternal endometrium induce a transcriptomic response in human embryos that modulates molecular mechanisms related to embryo development and implantation. WHAT IS KNOWN ALREADY EVs mediate intercellular communication by transporting various molecules, and endometrial EVs have been postulated to be involved in the molecular regulation of embryo implantation. Our previous studies showed that endometrial EVs carry miRNAs and proteins associated with implantation events that can be taken up by human blastocysts; however, no studies have yet investigated the transcriptomic response of human embryos to this EV uptake, which is crucial to demonstrate the functional significance of this communication system. STUDY DESIGN, SIZE, DURATION A prospective descriptive study was performed. Primary human endometrial epithelial cells (pHEECs), derived from endometrial biopsies collected from fertile oocyte donors (n = 20), were cultured in vitro to isolate secreted EVs. Following EV characterization, Day 5 human blastocysts (n = 24) were cultured in the presence or absence of the EVs for 24 h and evaluated by RNA-sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blot, nanoparticle tracking analysis, and transmission electron microscopy. Human blastocysts were devitrified, divided into two groups (n = 12/group), and cultured in vitro for 24 h with or without previously isolated EVs. RNA-sequencing analysis was performed, and DESeq2 was used to identify differentially expressed genes (DEGs) (FDR < 0.05). QIAGEN Ingenuity Pathway Analysis was used to perform the functional enrichment analysis and integration with our recently published data from the pHEECs’ EV-miRNA cargo. MAIN RESULTS AND THE ROLE OF CHANCE Characterization confirmed the isolation of EVs from pHEECs’ conditioned culture media. Among the 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. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS 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. It provides potential biomarkers that could become useful diagnostic targets for predicting implantation success. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139), and Instituto de Salud Carlos III and cofounded by the European Social Fund (ESF) “Investing in your future” through the Miguel Servet Program (CP20/00120 [H.F.]; CP19/00149 [I.C.]). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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子宫内膜细胞外囊泡调节与人类囊胚胚胎发育和植入相关的过程


研究问题 人子宫内膜分泌的细胞外囊泡 （EV） 内化后，人囊胚的转录组反应是什么？摘要答案 母体子宫内膜分泌的 EV 在人类胚胎中诱导转录组反应，调节与胚胎发育和植入相关的分子机制。已知的 EV 通过运输各种分子来介导细胞间通讯，并且子宫内膜 EV 已被假定参与胚胎植入的分子调控。我们之前的研究表明，子宫内膜 EV 携带与植入事件相关的 miRNA 和蛋白质，这些 mi RNA 和蛋白质可以被人类囊胚吸收;然而，目前还没有研究调查人类胚胎对这种 EV 摄取的转录组反应，这对于证明这种通信系统的功能意义至关重要。研究设计、规模、持续时间 进行了一项前瞻性描述性研究。原代人子宫内膜上皮细胞 （pHEECs） 来源于从可育卵母细胞供体 （n = 20） 收集的子宫内膜活检，在体外培养以分离分泌的 EV。在 EV 表征之后，在存在或不存在 EV 的情况下培养第 5 天人囊胚 （n = 24） 24 小时，并通过 RNA 测序进行评估。参与者/材料、设置、方法 使用超速离心从条件培养基中分离 EV，并使用 WESTERN BLOT、纳米颗粒跟踪分析和透射电子显微镜进行表征。将人囊胚分卵，分成两组 （n = 12/组），并在体外培养 24 小时，有或没有先前分离的 EV。 进行 RNA 测序分析，并使用 DESeq2 鉴定差异表达基因 （DEGs） （FDR < 0.05）。使用 QIAGEN Ingenuity Pathway Analysis 进行功能富集分析，并与我们最近发表的 pHEECs EV-miRNA 货物数据进行整合。主要结果和机会的作用 表征证实了 EV 从 pHEECs 的条件培养基中分离出来。在与 EV 共培养的囊胚中的 DEGs 中，我们发现 519 个显著上调，395 个显著下调。这些 DEGs 在与胚胎发育、细胞侵袭和迁移、细胞周期、细胞组织和组装、基因表达和细胞活力相关的上调功能中显著富集;以及与细胞死亡和 DNA 片段化相关的功能下调。此外，受子宫内膜 EV 内化调节的细胞内信号通路以前与早期胚胎发育和植入潜力有关，因为它们在多能性、细胞稳态、早期胚胎发生和植入相关过程中的作用。最后，整合来自 EV 的 miRNA 货物的数据，我们发现子宫内膜 EV 携带的 miRNA 靶向人囊胚中近 80% 的 DEGs。局限性，谨慎的原因 这是一项体外研究，其中子宫内膜细胞培养的条件无法模拟宫内环境。研究结果的更广泛意义 本研究为人类子宫内膜分泌的 EV 的功能相关性，特别是 EV-miRNA 调节对早期胚胎发生和胚胎植入过程中人类囊胚整体转录组行为的作用提供了新的见解。 它提供了潜在的生物标志物，可以成为预测植入成功的有用诊断靶标。研究资金/利益争夺 本研究由西班牙教育部通过授予 MS-B 的 FPU 提供支持。（FPU18/03735），巴伦西亚政府通过 VALi+d 计划授予 M.C.C.-G。（ACIF/2019/139） 和 Instituto de Salud Carlos III 共同创立，由欧洲社会基金 （ESF） 通过 Miguel Servet 计划 （CP20/00120 [H.F.];CP19/00149 [I.C.]）。作者没有需要披露的利益冲突。试验注册号 N/A。