The Effects And Mechanisms Of Endometrial Extracellular Vesicles From Women With Recurrent Implantation Failure On Embryonic Growth And Implantation

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The effects of endometrial extracellular vesicles from women with recurrent implantation failure on embryonic developmentObjective:To investigate whether endometrial extracellular vesicles(EVs)from recurrent implantation failure(RIF)patients attenuate the growth and development of embryos.Methods:Endometrial tissues obtained from RIF patients and fertile women were used for endometrial cell isolation.Endometrial cells were cultured and modulated in vitro via hormones to mimic the receptive phase.Conditioned medium was collected for EV isolation.EVs secreted by endometrial cells of RIF patients(RIF-EVs)or fertile women(FER-EVs)were characterized using western blotting,transmission electron microscopy,and nanoparticle tracking analysis.EVs from the two groups were co-cultured with 2-cell murine embryos.Fluorescence labeled EVs were used to visualize internalization by embryos.Following co-culture,blastocyst rate and hatching rate were calculated.Blastocysts were stained with diamidino-2-phenylindole(DAPI)to visualize the nuclei of blastomeres,and the total cell number of blastocysts were counted.Results:RIF-EVs and FER-EVs are bilayered vesicles,of approximately 100 nm and enriched with TSG101,Alix,and CD9.EVs were internalized within 12 h.The blastocyst rate in the RIF-EV groups was significantly decreased compared to that in the FER-EVs groups,at 5,10,and 20 ?g/mL.The hatching rate and total cell number of blastocysts were also decreased significantly in the RIF-EVs groups compared to that in the FER-EVs groups at 10 and 20 ?g/mL.Conclusion:Endometrial EVs from RIF patients attenuate the development of embryos.Part ? The effects of endometrial extracellular vesicles from women with recurrent implantation failure on trophoblastsObjective:This study aims to investigate whether endometrial extracellular vesicles(EVs)from recurrent implantation failure(RIF)patients inhibit the proliferation,invasive capacity,and migration of HTR8/SVneo cells.Methods:Endometrial cells isolated from the endometria of recurrent implantation failure patients and fertile women were cultured and modulated by hormones,and the conditioned medium was used for EV isolation.Fluorescence-labeled EVs were used to visualize internalization by HTR8/SVneo cells.EVs secreted by the endometrial cells of RIF patients(RIF-EVs)or fertile women(FER-EVs)were co-cultured with HTR8/SVneo cells.After co-culture,the proliferation,invasion,and migration of HTR8/SVneo cells were determined using CCK-8 assays,Transwell invasion assays,and wound healing assays,respectively.Results:RIF-EVs and FER-EVs were internalized by HTR8/SVneo cells within 2 h.The proliferation rate of trophoblast cells was significantly decreased in the RIF-EVs group relative to the FER-EVs group at 20 ?g/mL and 40 ?g/mL.The invasion and migration capacity of trophoblast cells were significantly decreased in the RIF-EVs group relative to the FER-EVs group at 20 ?g/mL.Conclusion:Endometrial EVs from RIF patients inhibited the functions of trophoblasts by decreasing their proliferation,migration,and invasive capacity.Such dysregulations induced by RIF-EVs may provide novel insights for better understanding the pathogenesis of implantation failure.Part ? miRNA expression in the endometrial extracellular vesicles of women with recurrent implantation failureObjective:To investigate whether extracellular vesicles(EVs)secreted by RIF patients'endometria have a different miRNA expression profile compare to endometrial EVs of fertile women.Methods:Endometrial tissues from fifteen RIF patients and nine fertile women were collected and digested to cells for culture.Endometrial cells were modulated by estrogen and progesterone to mimic the secretory phase,and the conditioned medium was collected for EV isolation.Three pairs of EV samples from two groups were used for miRNA sequencing.Each of the left samples was divided into two parts.One part was used for miRNA validation using quantitative reverse transcription polymerase chain reaction(qRT-PCR).The other part was used to co-culture with HTR8/SVneo cells.After co-culture,the expression of miRNAs in the HTR8/SVneo cells were detected using qRT-PCR.Results:A total of 11 miRNAs were differently expressed in the RIF-EVs.Target genes of the differently expressed miRNAs were predicted,and the functional analysis was performed.Besides,we proved that miR-6131,miR-1246,miR-1307-3p,and miR-218-5p were significanty up-regulated in the RIF-EVs using qRT-PCR.Moreover,miR-6131,miR-1307-3p,and miR-218-5p were up-regulated in the HTR8/SVneo cells after co-culture with RIF-EVs.Conclusion:Our study confirmed an altered miRNA expression profile in the RIF-EVs.Moreover,the differently expressed miRNAs could be transferred to trophoblast cells via EVs,which might enhance our understanding of the pathogenesis of RIF.Part ? miR-6131 inhibits the proliferation,migration and invasion of HTR8/SVneo cells by targeting PAK2.Objective:To investigate whether miR-6131 inhibits the growth and function of HTR8/SVneo cells and the detailed mechanisms.Methods:HTR8/SVneo cells were transfected with miR-6131 mimics or scramble,and the proliferation,migration,and invasive capacity of transfected cells were evaluated using CCK-8 assays,wound healing assays,and Transwell invasion assays,respectively.The target gene(PAK2)of miR-6131 was predicted,and the regulatory role of miR-6131 on PAK2 was investigated using quantitative reverse transcription polymerase chain reaction(qRT-PCR),western blotting,and luciferase reporter gene assays.Moreover,HTR8/SVneo cells were transfected with plasmids to up-regulated PAK2 expression,and whether overexpressed PAK2 could reverse the effects of miR-6131 on the HTR8/SVneo cells was investigated.Results:The up-regulation of miR-6131 significantly decreased the proliferation,migration and invasion of HTR8/SVneo cells.miR-6131 directly down-regulated the expression of PAK2 by binding a specific sequence of its 3'-untranslated region(3'UTR).Furthermore,PAK2 overexpression partly reversed the effects of miR-6131 on the growth,invasion and migration of HTR8/SVneo cells.Conclusion:miR-6131 inhibits the proliferation,migration and invasion of HTR8/SVneo cells by targeting PAK2.