O-137 Mapping microRNA profiling and dysregulated pathways in non-obstructive azoospermia cases with maturation arrest: A systematic review and in-silico analysis of the affected pathways
Authors: Zikopoulos A , Grigoriadis S , Karagkouni D , Maziotis E , Tomara P , Kotsifaki A , Agapitou K , Pappas A , Hatzigeorgiou A , Pantos K , Sfakianoudis K , Simopoulou M 
Published: June 2023

Abstract

Study question
Could microRNA profiling be a useful tool towards better understanding the pathophysiological mechanisms leading to non-obstructive azoospermia (NOA) due to maturation arrest?

Summary answer
MicroRNA profiling may be an efficient method towards unveiling maturation arrest pathophysiology as microRNAs constitute master posttranscriptional regulators of cell-cycle control during spermatogenesis.

What is known already
Non-obstructive azoospermia constitutes the most severe form of male factor infertility, affecting approximately 10% of infertile men. According to the histological subtype, NOA is classified into three main categories namely hypospermatogenesis, maturation arrest, and Sertoli-cell only syndrome. Maturation arrest is one of the most complex forms of NOA characterized by failure of spermatogenesis. Despite advances, the pathophysiology of maturation arrest remains a conundrum rendering management complex. Recent studies indicate that alterations on microRNA expression patterns could lead to maturation arrest. Mapping microRNA profiling and highlighting dysregulated pathways of maturation arrest cases may lead the way forward.

Study design, size, duration
A systematic review was performed in PubMed/Medline and Embase up to April 2022. Only full-length original studies in humans were included. Strict inclusion-exclusion criteria were applied aiming to isolate studies comparing microRNA profiling between maturation arrest cases versus normal fertile men, and/or men with obstructive azoospermia (OA). Following study selection, data on altered microRNA expression patterns were analyzed to underline differences between the abovementioned groups. Subsequently, in-silico analysis was performed to compare affected gene pathways.

Participants/materials, setting, methods
The studied population consisted of maturation arrest cases. Control groups consisted of men with normal spermatogenesis and/or men with OA. Predicted microRNA–target pairs were retrieved from microT-CDS, while a 0.8 cutoff threshold was applied. The GTEx repository was used to identify microRNA-targeted genes in the testis. Annotations derived from Ensembl and miRbase. Gene-set enrichment analysis was performed employing the KEGG-database. Fisher’s exact test was performed in R package limma, setting a 0.01 p-value threshold.

Main results and the role of chance
Ten studies reported altered microRNA expression patterns in maturation arrest versus normal spermatogenesis cases and six studies in maturation arrest versus OA cases. Considering the maturation arrest-normal spermatogenesis arm, analysis revealed that eight microRNAs, which were upregulated in maturation arrest cases, namely hsa-miR-449a, hsa-miR-370-3p, hsa-miR-10b-3p, hsa-miR-539-5p, hsa-miR-22-5p, hsa-miR-605-5p, hsa-miR-491-3p and hsa-miR-302d-3p, affected 106 statistically significant gene-targets in the testis. The three most significantly affected pathways included the “microRNAs in cancer” pathway (55 affected genes, p-value &lt;0.001), the “EGFR tyrosine kinase inhibitor resistance pathway” (29 affected genes, p-value = 0.007), and the “Hedgehog signaling pathway” (22 affected genes, p-value=0.008). Regarding the second arm of maturation arrest-OA, functional analysis indicated that four microRNAs, presenting to be downregulated in maturation arrest cases, namely hsa-miR-181a-5p, hsa-miR-449a, hsa-miR-182-5p and hsa-miR-138-5p, affected 421 statistically significant gene-targets in testis. The three most significantly affected pathways included the “pathways in cancer” pathway (86 affected genes, p-value =0.009), the “microRNAs in cancer” pathway (48 affected genes, p-value &lt; 0.001), and the “ cGMP-PKG signaling” pathway (36 affected genes, p-value=0.008). In summary, microRNAs affect the expression of master genes regulating cell-cycle and maturation processes during spermatogenesis, leading to maturation arrest as well as to other associated pathologies, including cancer.

Limitations, reasons for caution
The limited number of the included studies, as well as the small size population characterizing the great majority of them, constitute the main limitations of this study. Another reason for caution is the great heterogeneity observed among the studies regarding the molecular methods employed for microRNA profiling.

Wider implications of the findings
Data presented herein indicate that microRNA functional analysis may be a significant tool towards better understanding the pathophysiological basis of spermatogenesis maturation arrest, indicating future personalized diagnostic and therapeutic targets. Moreover, microRNA analysis may also constitute an efficient method of evaluating predisposition of NOA patients to other pathologies including cancer.

Trial registration number
Not applicable