P-054 Significant differences of micro RNA expression pattern in extracellular vesicles between fertile and infertile individuals: new markers of male reproductive status?
Authors: Sánchez L , Alvarez-Barrientos A , García-Velasco J A , Requena A , Rubio S , Pacheco Castro A 
Published: June 2023

Abstract

Study question
Is the miRNAs cargo of the extracellular vesicles (EVs) present in seminal plasma different between healthy semen donors and infertile patients?

Summary answer
There are significant differences in the miRNAs expression patterns in the load of seminal EVs between infertile patients and healthy semen donors

What is known already
Extracellular vesicles (EVs) present in seminal plasma contain a wide range of different proteins, lipids and nucleic acids, such as microRNA. These microRNA can execute their action by regulating gene expression of target cells triggering an important role in different reproductive processes. It is well stablished in different pathologies that the EVs cargo is representative for the functional status of the producer cell. In this sense, the alteration in microRNAs expression patterns in seminal plasma has been associated with spermatogenic impairments, subfertility and azoospermia, indicating that microRNAs in seminal EVs could work as biomarkers of the male fertility status

Study design, size, duration
This prospective study, performed between March 2021 to December 2022, included 30 semen samples from infertile males seeking infertility treatment (study group), and 7 semen samples from healthy semen donors (control group) from our donor semen bank. All the semen samples from patients showed abnormal seminal parameters (oligo- and/or asthenozoospermia). Men under drug treatment and/or with infectious or chronic diseases were excluded from the study

Participants/materials, setting, methods
After conventional semen analysis, EVs were isolated from seminal plasma by ultracentrifugation. To confirm EVs isolation, an aliquot of each sample was used for nanoparticle tracking analysis (NTA). Then, microRNAs from EVs were extracted and miRNAseq was performed. Raw data were standardized using counts per millions and ANOVA was performed to assess differences in the miRNAs expression between groups.Target genes were annotated using miRTarBase and functional enrichment analysis was performed by MIENTURNET and GeneCards

Main results and the role of chance
The presence of EVs after ultracentrifugation of seminal plasma was confirmed by NTA, showing a range size within 40-140 nm. miRNAseq generated reads for 914 annotated miRNAs and significant differences in the expression of 17 of them were found between patient and control samples. These miRNAs target a total of 1310 genes and functional enrichment analysis indicated that they participate in several processes related to cell differentiation, cell proliferation, immunomodulation, cellular adhesion and apoptosis. Specifically, patient group showed significant higher expression of some members of the hsa-let-7 family (p &lt; 0.001 for let-7i-5p, let-7f-5p, let-7c-5p and let-7a-5p; p &lt; 0.05 for let-7b-5p), which are implicated in adaptative immune system activation and differentiation by gene modulation. Results also found significant (p &lt; 0.01) higher expression in patient group of hsa-miR-92a-3p, hsa-miR-200c-3p and hsa-miR-888-5p, which target genes are related to apoptotic processes. Finally, patient group showed significant (p &lt; 0.001) lower expression of hsa-miR-320b and miR-320a-3p, which interact with transcription factor sequences (DLX5, MYC) implicated in cell proliferation and differentiation; and also lower expression in hsa-miR-423-5p (p &lt; 0.01) and miR-423-3p (p &lt; 0.05) that interact with the sequence of transcription factors as p21, a regulator of cell cycle progression at G1, and with RNA-binding proteins involved in ribosome assembly.

Limitations, reasons for caution
This study included males with dissimilar seminal disorders (astheno, oligo, and teratozoospermia). Therefore, further studies are needed lowering the heterogeneity of the study group. At present, no data about reproductive outcome is available

Wider implications of the findings
This work suggests that the significant differences found in the expression pattern in microRNAs in seminal EVs between infertile and fertile men may be related with gene modulation in the reproductive processes and could be used as novel biomarkers of male fertility status.

Trial registration number
1904-MAD-045-AP