P-098 The proteome of extracellular vesicle cargo from human seminal plasma
Authors: Governini L , Landi C , Haxhiu A , Zarovni N , Ponchia R , Morgante G , Piomboni P , Luddi A 
Published: July 2024

Abstract

Study question
Could Extracellular Vesicles be employed as a new tool to characterize the male fertility potential?

Summary answer
Extracellular Vesicles cargo is involved in mechanisms strictly linked to reproductive processes.

What is known already
Extracellular vesicle contain a plethora of bioactive molecules, such as lipids, DNA and RNA, mainly proteins, which are delivered to target cells to elicit a specific response. This multivarious EV functionality is accomplished by the transfer of their content to the sperm cell, followed by either downregulating or upregulating a series of proteins expressed throughout different sites of sperm cell structure. Despite various studies exploring the proteomic composition of these vesicles, our comprehension of their specific influence on sperm maturation, motility, and the attainment of fertilizing capacity is currently restricted.

Study design, size, duration
Semen samples were collected from subjects undergoing semen analysis at the UOSA PMA, Siena University Hospital, Italy. All participants were of Caucasian origin and signed a written informed consent to be enrolled in this study. The median age of the patients was 36.7 years (range: 20-45 years); the BMI ranged between 18 and 25 (normal weight). Patients were divided on three groups based on seminal parameters (WHO, 2021) into: Normozoospermic (NORMO); OligoAsthenoTeratozoospermic (OAT); Azoospermic (AZO).

Participants/materials, setting, methods
EVs from human semen samples were isolated following an in-house optimized protocol using a chemical precipitation reagent ev-GAG®. Samples were subjected to Gel-free and gel-based proteomic analysis. Comprehensive analysis of the vesicles involved acquiring a detailed proteomic profile through the use of 2-D electrophoresis and mass spectrometry. Subsequent inquiries included functional Omics analysis, utilizing bioinformatic tools like Metacore and STRING. We have combined and compared results from several proteomic approaches, both gel-free and gel-based ones.

Main results and the role of chance
A total of 1133 proteins were identified in NORMO EV samples from the Gel-free approach by LC-MS/MS. These proteins are involved in numerous functions important for reproduction activity. GO enrichment analysis identified involvement of pathways such as cytoskeleton regulation, cell adhesion, cell cycle, development and neurophysiology, proteostasis mechanisms, immune response, and inflammation, reproduction as progesterone signalling and spermatogenesis, protein translation, and signal transduction. 2DE analysis extrapolated a total of 80 differentially abundant spots among the three conditions. Biological Process analysis report that highly abundant proteins in NORMO EVs were significantly involved in chaperone-mediated protein complex assembly, autophagy and in protein folding, in negative regulation of intrinsic apoptotic signaling pathway, in positive regulation of protein- containing complex assembly, positive regulation of tau-protein kinase activity, in regulation of apoptotic processes, in protein stabilization and in regulation of protein ubiquitination. Biological processes related to the three group of proteins suggested that in OAT and even more in AZO EVs, the altered molecular cargo is involved in mechanism strictly linked to reproductive processes, protein folding, inflammation, immune responses, proteolysis, signal transduction. All these mechanisms, in healthy conditions, cooperate for a successful pregnancy.

Limitations, reasons for caution
To enhance characterization, it is essential to conduct a confirmatory investigation focused on the distinct group of different EVs such as epididymosomes and prostasomes, aiming to offer more precise insight.

Wider implications of the findings
EVs released along the male reproductive tract, specifically in seminal fluid, have been shown to play a role in sperm maturation. They have the potential to serve as an attractive diagnostic and therapeutic tool in the realm of human reproduction, particularly for issues related to male fertility and infertility.

Trial registration number
NA