Abstract
Background: Small extracellular vesicles (sEVs) mediate intercellular communication by transporting microRNAs and proteins. Endometrial sEVs have been implicated in embryo implantation due to their content of implantation-related molecules; however, their precise functions and underlying mechanisms remain unclear. This study aimed to identify proteins in endometrial sEVs that contribute to embryo implantation and to clarify their functional significance. Methods: Uterine fluid (UF) samples were collected from five women in each of the ovulatory and implantation phases. UF-derived sEVs (UF-sEVs) were isolated using a combination of size-exclusion chromatography and ultrafiltration, and their proteomic profiles were compared between phases. In parallel, hormone-treated Ishikawa cells-a human endometrial adenocarcinoma cell line-were used to model the proliferative and implantation phases in vitro, and sEVs derived from these cells (Ishikawa-sEVs) were similarly analysed. Galectin-3, a protein upregulated in both UF-sEVs and Ishikawa-sEVs during the implantation phase and known to be involved in cytotrophoblast (CT) cell fusion, was selected for functional validation. CT cell fusion assays were performed using Ishikawa-sEVs from galectin-3 knockdown (Gal3-KD) and non-target knockdown (NT-KD) cells. Results: We identified 2,041 and 1,386 proteins in UF-sEVs from the ovulatory and implantation phases, respectively. Comparative analysis revealed 318 differentially expressed proteins (140 upregulated and 178 downregulated) during the implantation phase, including proteins related to immune response, cell adhesion, and migration. In Ishikawa-sEVs, we identified 913 and 915 proteins in the proliferative and implantation phases, respectively, with 68 differentially expressed proteins (44 upregulated and 24 downregulated). Implantation-phase Ishikawa-sEVs significantly promoted CT cell fusion compared with proliferative-phase sEVs. Moreover, Gal3-KD in Ishikawa cells significantly reduced the fusion-promoting effect of the sEVs, suggesting that galectin-3-enriched endometrial sEVs may facilitate CT fusion and subsequent differentiation into syncytiotrophoblast (ST) during implantation. Conclusions: Our findings demonstrate that endometrial sEVs enriched in galectin-3 during the implantation phase promote CT differentiation into ST, underscoring a novel role for sEV-mediated protein delivery in regulating early placental development and suggesting potential diagnostic and therapeutic applications in reproductive medicine.