P–388 Endometrial extracellular vesicles from recurrent implantation failure patients inhibited embryonic growth and implantation via miR–6131/PAK2 pathway

Abstract

Abstract Study question Could endometrial extracellular vesicles from recurrent implantation failure patients (RIF-EVs) attenuate the growth and implantation potentials of embryos and what are the mechanisms? Summary answer: RIF-EVs inhibited embryonic growth and decreased the trophoblast functions via miR–6131/PAK2 pathway. What is known already Recurrent implantation failure (RIF) is characterized by repeated embryo transfers without pregnancy. To date, the etiology of RIF remains poorly understood. Recent evidence indicated that extracellular vesicles (EVs) secreted by endometrial cells, played a crucial role in the implantation by regulating the development and implantation of embryos. Study design, size, duration Endometrial cells isolated from endometrial tissues of RIF patients (n = 25) and fertile women (n = 16) were cultured and modulated via hormones. Endometrial EVs from RIF patients (RIF-EVs) or fertile women (FER-EVs) were isolated from the conditioned medium. The influence of EVs on embryonic development and implantation was investigated by co-culture models of EVs and 2-cell murine embryos or HTR8/SVneo cells, respectively. High-through put sequencing was performed to identify the miRNA profile in the RIF-EVs. Participants/materials, setting, methods RIF-EVs and FER-EVs were characterized using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. After co-culture with EVs, embryonic blastocyst rate and hatching rate were calculated. Besides, the proliferation, migration, and invasion of EV-treated trophoblast cells were evaluated by CCK–8, wound healing, and transwell invasion assays. miRNA expression profiles were compared between RIF-EVs and FER-EVs, and the regulatory role of significantly upregulated miR–6131 in RIF-EVs was investigated in the trophoblast cells. Main results and the role of chance RIF-EVs and FER-EVs are round bilayer vesicles, ranging mainly at 100 nm and enriched in TSG101, Alix, and CD9. Both RIF-EVs and FER-EVs entered embryonic or trophoblast cytoplasm. The blastocyst rate in the RIF-EV groups was significantly decreased compared to that in the FER-EV groups, at concentrations of 5, 10, and 20 μg/ml. The hatching rate was decreased significantly in embryos treated with 10 or 20 μg/ml RIF-EVs compared to those treated with FER-EVs at the same concentration (p < 0.05). The proliferation, migration, and invasion of trophoblasts were significantly decreased in the RIF-EV group at 20 μg/mL. A total of 11 differently expressed (fold change >2 and p < 0.05) miRNAs were found in the RIF-EVs, and two of them were validated in a larger set of EV samples using RT-PCR. The most significantly different miRNA, 6131, was increased in the RIF-EV-treated HTR8/SVneo cells. The up-regulation of miR–6131 inhibited the growth and invasion of HTR8/SVneo. Bioinformatics coupled with luciferase and western blot assays revealed that PAK2 is a direct target of miR–6131, and the overexpression of PAK2 can rescue the phenotype changes induced by miR–6131 overexpression. Limitations, reasons for caution Our study indicated miRNA in the RIF-EVs dysregulating the growth and function of embryonic cells. However, EVs contained a wide spectrum of bioactive molecules, including proteins, mRNAs, and DNA, which may play an important role in the implantation. Further studies are required to investigate the mechanisms. Wider implications of the findings: This work indicates an important role of EVs from women with RIF in embryonic implantation, potentially providing a novel insight to understand the pathophysiology of RIF. Trial registration number Not applicable