Abstract
To characterize surface molecules of the extracellular vesicles (EVs) present in seminal plasma (exosomes, EXOs; and microvesicles, MVs) by high-resolution flow cytometry. The study was performed between March 2019 to January 2021 in IVI Madrid and UNEX. All the samples included (n=65) were obtained from semen donors (18-35 yo, and with high quality seminal samples) of IVI Sperm Bank. The exclusion criteria were men with infectious or chronic diseases or men undergoing drug treatment. After performing a conventional semen analysis for each semen sample, seminal samples were ultra centrifuged in order to obtain EVs enriched seminal plasma. To perform the flow cytometric analysis, the samples were first stained with fluorescent antibodies against the following surface molecules: CD81, CD63, CD9, CD40, CD44, CD49d, CD73, CD284 and CD29. After staining protocol, samples were analyzed by flow cytometry (Cytoflex, Beckman Coulter). The percentage and the mean fluorescence signal (MFI) were analyzed of each molecule in EVs populations. Statistical differences between EXOs and MVs were analyzed using t-test two tailed. Differences were considered statistically significant when p-value < 0.05. Flow cytometric analyses demonstrated the presence of both MVs and EXOs populations, being MVs population larger than the EXOs population (p < 0.001). With respect to the surface molecules, the proportion of EVs expressing CD81, CD63, CD40, CD282 and CD29 was higher in EXOs (p < 0.001 in all cases) than in MVs. The MFI of CD63, CD73, CD282, CD284 and CD29 was higher in MVs (p < 0.001, p < 0.01, p < 0.01, p < 0.05, p < 0.05, respectively) than in EXOs. This study demonstrates the presence of both EXOs and MVs in human seminal plasma, but with differences in their proportion, being MVs more abundant in all semen samples collected. The differences found in the expression profiles of tetraspanins (CD81, CD9 and CD63) and CD40, CD73, CD282, CD284 and CD29 between EXOs and MVs suggest the presence of a large heterogeneity in the EVs populations, reflecting possible differences in cargo and function of the different EVs.
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