Abstract
BackgroundSuccessful 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.MethodsIshikawa 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.ResultsUltracentrifugation 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.ConclusionsEV 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.
Highlights
Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of extracellular vesicles (EVs) in the required cross-talk cell-to-cell has been recently established
To elucidate the molecular mechanisms involved in endometrial receptivity and implantation success, we used the most efficient EV isolation method to isolate EVs secreted by primary human endometrial epithelial cells (pHEECs) and analyzed their protein content via proteomic approaches
Size distribution and concentration of EVs isolated with different methods To confirm that all three methodologies isolate EVs from the culture medium of endometrial epithelial cells, an nanoparticle tracking analysis (NTA), which shows the size distribution and concentration of nanoparticles, was performed in EVs isolated from the extracellular medium of Ishikawa cells
Summary
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 transfer‐ ring their contents and enhancing their adhesive and invasive capacity. The probability of pregnancy in one menstrual cycle is only 30% [3] due to different factors, among them the short period of time (4–5 days) in which the endometrium is receptive This “window of implantation” depends on an adequate transformation regulated by estrogen and progesterone [1, 4]. Embryo implantation and pregnancy likely involve a successful crosstalk between the embryo and the maternal endometrium; the molecular mechanisms involved in this process are not well understood
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