P-089 Characterization of Epididymosomes and Prostasomes in Seminal Plasma of Infertile Males
Authors: Governini L , Haxhiu A , Landi C , Ponchia R , Morgante G , Piomboni P , Luddi A 
Published: June 2022

Abstract

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

Summary answer
Extracellular vesicles (EVs) content represents a new approach to the discovery of novel molecular mechanisms involved in sperm cell maturation and acquisition of fertilizing ability.

What is known already
Cells throughout the reproductive tract release EVs that are sorted by the endosomal compartment. These vesicles attract great attention because they contain lipids, proteins, nucleic acids that have been shown to be transferred to maturing sperm cells. EVs in seminal plasma, such as epididymosomes and prostasomes, contribute about 3% of the total associated seminal plasma proteins and are recognized as being responsible for sperm maturation, positively influencing actual fertilization. Several studies have documented the proteomic profile of these vesicles, but little is known about their specific contribution to sperm maturation, motility and acquisition of fertilizing capacity.

Study design, size, duration
48 Caucasian males underwent semen evaluation at the IVF Unit, Siena University Hospital, from October 2020 to November 2021. According to seminal parameters (WHO,2010), they were divided into Normozoospermic (N; n = 18); OligoAsthenoTeratozoospermic (OAT; n = 15); Azoospermic (A; n = 15). We excluded patients with an intake of spermiotoxic drugs, smoking, alcohol, or drugs abuse. The mean age was 33 years; the BMI ranged between 18-25. All participants signed a written informed consent, approved by the ethics committee.

Participants/materials, setting, methods
EVs from seminal plasma were isolated by an in-house modified EvGAG®-polymer precipitation-based protocol and characterized for their size and morphology by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively. Full proteomic profiling of these vesicles was obtained by 2-D electrophoresis and mass spectrometry, and further investigations by functional Omics analysis by bioinformatic tools (as. Metacore, STRING). Gene expression was evaluated by digital PCR (ddPCR).

Main results and the role of chance
The present work highlights the impact of EVs on male fertility due to their ability to transport selected molecules to maturing sperm cells, thus providing a specific proteomic and transcriptomic profile based on their fertilizing capacity. Proteomic profiling of OAT and A vs N samples provided crucial information regarding differential protein expression, thus obtaining information on the correlation between the encapsulated content of EVs and sperm parameters.
The ddPCR analysis of specific genes encoding for proteins expressed differently among patients confirmed that EVs may not only be potential biomarkers for male infertility, but at the same time contribute as an important regulator of specific pathways concerning sperm cell modification through the male reproductive tract. Genes involved in sperm-egg binding and zona pellucida interactions were overexpressed in N compared to OAT and A. Congruent results were obtained for genes involved in sperm cell metabolism. Meanwhile, regarding the acquisition of motility, some genes were found overexpressed in the OAT groups compared to N, indicating that some proteins delivered by EVs have a negative impact on sperm cell motility and function.

Limitations, reasons for caution
This study represents a preliminary experiment. A confirmatory study on an enlarged number of samples is needed to provide more statistical strength by reducing the effect of inter-sample variability.

Wider implications of the findings
Seminal extracellular vesicles secreted along the male reproductive tract have been demonstrated to be involved in the process of sperm maturation and could represent a new appealing therapeutic and diagnostic tool in the field of human reproduction for male fertility/infertility.

Trial registration number
Not applicable