O-250 Strong differences in surface molecule expression profile in both microvesicles and exosomes between patient and semen donor groups: new fertility biomarker?
Authors: Sánchez L , Álvarez-Barrientos A , García-Velasco J.A , Soraya R , Pacheco Castro A 
Published: June 2022

Abstract

Study question
Are the extracellular vesicles (exosomes and microvesicles) presented in seminal plasma a potential novel biomarkers of male infertility?

Summary answer
There are significant differences in surface molecules (CD9, CD29, CD40, CD49d, CD73, CD282 and CD284) expressed in extracellular vesicles of patient and control groups

What is known already
Male infertility has been progressively increasing in recent decades. Therefore, it is essential to find new tools that help us to diagnose the male factor. One of the potential markers could be the study of the extracellular vesicles (EV), mainly exosomes (EXO) and microvesicles (MV), present in the seminal plasma. Recent works have shown that these extracellular vesicles would have an important role in the modulation of the maternal immune system, as well as in regulating the sperm activation. Therefore, the study of the extracellular vesicles can be a useful model to increase the diagnosis of the infertile male

Study design, size, duration
This prospective study, performed between March 2019 to January 2022, included 44 semen samples from healthy semen donors (control group) and 44 semen samples from infertile males seeking infertility treatment with their partners (study group). Men under drug treatment and/or with infectious or chronic diseases were excluded from the study

Participants/materials, setting, methods
After conventional semen analysis, semen samples were ultra-centrifuged to obtain EVs enriched plasma seminal. The samples were then stained with fluorescent antibodies against the following surface molecules: CD81, CD63, CD9, CD40, CD44, CD49d, CD73, CD284 and CD29. After incubation, samples were analyzed by flow cytometry (Cytoflex cytometer) The percentage and mean expression of each molecule in EVs populations (EXOs and MVs) were determined. Statistical analysis was performed by t-Student test; significance established under 0.05

Main results and the role of chance
Conventional semen analysis showed significant differences between control and patient group in sperm concentration (77.2 · 106 vs 40.2 · 106 cells / ml, respectively, p &lt; 0.01), percentage of progressive sperm (60 vs 40.3 %, respectively, p &lt; 0.01) and total progressive sperm cells (170 · 106 vs 69 · 106 spermatozoa, respectively, p &lt; 0.01). Flow cytometry analysis demonstrated that the MVs population is larger than the EXOs population in both control and patient group. With respect to the surface molecule expression in EXOs population, patient group showed significant higher expression of CD9 (31.18 vs 20.1 % control group, p &lt; 0.01), CD40 (8.94 vs 3.84 % control group, p &lt; 0.001), CD49d (69.07 vs 46.73 %, control group, p &lt; 0.001), CD73 (68.17 vs 51.41 % control group, p &lt; 0.001) and CD284 (61.48 vs 53.45 % control group, p &lt; 0.01), and significant lower expression of CD29 (19.49 vs 29.54 % control group, p &lt; 0.01) molecules compared to control group. When MVs populations were analyzed, significant differences between patient and control group were found in the expression of CD63 (13.01 vs 1.65 %, respectively p &lt; 0.001), CD40 (9.23 vs 1.34 %, respectively p &lt; 0.001), CD282 (17 vs 10.22 %, respectively p &lt; 0.01) and CD29 (17.11 vs 7.8 %, respectively p &lt; 0.001) molecules

Limitations, reasons for caution
This study enrolled male partners of couples seeking infertility treatment with dissimilar disorders and seminal diagnosis (astheno, oligo, and teratozoospermia). Therefore, further studies are needed lowering the heterogeneity of the study group. No data about reproductive outcome of these patients are available at this moment

Wider implications of the findings
These results suggest that the differences found in the expression profiles between men with proven fertility and infertile men may be related to molecular mechanisms implicated in sperm function, and/or female immune system modulation and, for that reason, may be used as a new potential marker of male fertility

Trial registration number
1904-MAD-045-AP