O-255 microRNA-23a/b-3p regulate expression levels of testis-specific transcripts in men with impaired fecundity
Authors: Becker L S , Al Smadi M A , Koch H , Meese E , Abu-Halima M 
Published: June 2023

Abstract

Study question
To investigate whether microRNA-23a/b-3p targets spermatogenesis-related transcripts and whether this targeting impacts their expression contents in patients with subfertility.

Summary answer
The higher expression of microRNA-23a/b-3p and lower expression of 11 tested target genes are associated with men subfertility

What is known already
Spermatogenesis is a complex and highly regulated process, many genes are involved, the expression levels of which are strongly or partially coordinated by microRNAs (miRNAs). MiRNAs are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Transcriptome analysis shows that hundreds of genes are expressed exclusively or predominantly in male germ cells including CEP41, G2E3, GOLGA6B, GOLGA6C, LMLN, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 genes, which play a crucial role during spermatogenesis and/or sperm function. However, the expression regulation of these genes is still unclear.

Study design, size, duration
Reverse transcription-quantitative PCR (RT-qPCR), dual luciferase assay, Western blot, and bioinformatics analysis were used to validate the lower expression of 11 target genes as a result of the known higher expression of microRNA-23a/b-3p in men with subfertility. A total of 82 men were included for RT-qPCR, consisting of 41 oligoasthenozoospermic subfertile men who attended the IVF center for infertility treatment and 41 age-matched normozoospermic volunteers who served as controls.

Participants/materials, setting, methods
In silico prediction and dual-luciferase assays were performed to evaluate the potential links between the higher expression of microRNA-23a/b-3p and the lower expression of 11 genes. Total RNA, including miRNA, was isolated from the sperm of oligoasthenozoospermic (n = 41) and normozoospermic men (n = 41). RT-qPCR was used to detect the expression levels of 11 target genes. Correlation analyses between the mRNA expression levels and basic semen parameters were carried out.

Main results and the role of chance
The expression levels of microRNA-23a/b-3p were significantly up-regulated and 11 genes were significantly down-regulated in oligoasthenozoospermic men compared with age-matched normozoospermic men as determined by RT-qPCR. Using dual-luciferase assays, 9 genes including CEP41, G2E3, GOLGA6C, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 were identified as direct targets of miR-23a-3p and 4 genes including GOLGA6C, PAPOLB, SOX6, and ZNF695 were identified as direct targets of miR-23b-3p. Mutations in the miR-23a/b-3p binding site within the 3ˊuntranslated regions (3ˊUTRs) of the 9 target genes, which target either miR-23a-3p and/or miR-23b-3p, resulted in abrogated responsiveness to microRNA-23a/b-3p and confirmed that CEP41, GOLGA6C, NOL4, PCDHA9, and SOX6 as direct targets for miR-23a-3p and NOL4, SOX6 and PCDHA9 as direct targets for miR-23b-3p. Correlation analysis highlighted sperm count, motility, and morphology were positively correlated with the lower expression level of these validated genes.

Limitations, reasons for caution
Despite the correlation between the higher expression of microRNA-23a/b-3p and the lower expression of the validated genes, further validation by Western blotting in human sperm and testicular tissues is needed.

Wider implications of the findings
Findings suggest that the higher expression of microRNA-23a/b-3p or the lower expression of validated target genes are associated with male subfertility, probably through influencing the basic semen parameters. This study lay the groundwork for future studies focused on investigating therapies for male infertility.

Trial registration number
Hedwig-Stalter foundation (2016)