Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

Abstract

Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape

“…Comparative proteome analysis of EP-sEVs ( versus E-sEVs) revealed molecular players of epithelial cell migration (BMPR2, DDR1, IGSF8, MST1R), embryo development (COPS3, CUL3, NOTCH1, PLCG1, ADAM10), cell-cell adhesion (SDCBP, EPCAM, NOTCH1), nitric oxide biosynthesis (e.g., DDAH2, AKT1 and SPR) and cell invasion (CSTB, DDR1, RAB25, ST14, TXN) ( Supplementary Figure S3 ; Supplementary Table S2 ). Importantly, EP-sEVs are enriched in key players shown to remodel endometrium to promote receptivity, or embryo to promote implantation processes ( Illera et al, 2000 ; Haslinger et al, 2013 ; Zhao et al, 2018 ; Segura-Benitez et al, 2022 ); enriched in regulators of endometrial receptivity (CTNNB1, EGFR, EPCAM, NOTCH1 and DDRI), as well as promote implantation processes (BMPR2, BTF3, COPS3, PLCG1, RAB14, ADAM10, CUL3, ITGAV and AKT1) ( Table 1 ). This further supports the successful hormonal priming of endometrial epithelial cells, the distinct molecular composition of sEVs, and highlights the pro-invasive function of EP-primed endometrial cell sEVs.…”

“…While such components including ITGAV are enriched in EP-sEVs ( Table 1 ); suggests that sEVs may transfer integrins to modulate target cell surface expression ( Park et al, 2019 )—whether such targets (on sEVs or following their transfer to target cells) implicate trophectoderm cell interaction [e.g., ITGAV and B3 ( Altei et al, 2020 )], delivery/cell uptake ( Chen and Brigstock, 2016 ; Li et al, 2021 ) or tissue-specific homing ( Park et al, 2019 ) remain to be investigated. Moreover, ITGAV forms part of the integrin αVβ3 receptor expressed maternally and on embryo to mediate successful implantation ( Illera et al, 2000 ; Segura-Benitez et al, 2022 ). In addition to these invasion-associated surfaceome components, SERPINE1 is also expressed in extravillous trophoblasts (EVTs)—a highly invasive trophoblast lineage ( Bilban et al, 2010 ).…”

Exosomes: New regulators of reproductive development

“…Endometrial reactivity and readiness for embryo implantation also result from the participation of exosomes in the communication process originating from endometrium and embryo [ 79 , 80 ]. One study reported that the presence of uterine fluid-derived exosomes during the preimplantation stage contains a higher abundance of proteins involved in cell apoptosis, while the uterine fluid-derived exosomes in the implantation stage had a higher abundance of proteins involved in cell adhesion [ 81 ]. Moreover, the highest number of exosomes are observed in the uterine fluid during the luteal phase of the menstrual cycle [ 82 , 83 ].…”

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

“…Importantly, from women associated with a pregnancy outcome, transcripts were identified associated with implantation success (i.e., PMS2P5 , ANKRD18A , GLIS2‐AS1 , NPTN‐IT1 , and CERC7 ). Further, Rai and colleagues described that purified sEVs from UF of fertile women during the receptive phase were enriched in receptivity protein markers, including annexins (ANXA2/5/6), enzymes (DPP4, TGM2), and CAMs (ITGA1/2/6/B1, ICAM1) [173]. Such findings were further supported by a meta‐analysis of nine receptivity studies reporting 28 markers as co‐identified in sEVs from UF (including ANXA2/4, SPP1, DPP4, GPX3).…”