Overexpression of hsa-miR-30a-5p and non-obstructive azoospermia: A case-control study

Abstract Background Some previous human and animal studies have supported the idea that KDM3A down-regulation might be the main cause of male infertility, especially in non-obstructive azoospermia (NOA). The regulatory role of micro-RNAs (miRNA) has been investigated in the development of male infertility. Objective The expression level of hsa-miR-30a-5p in azoospermia was evaluated to reveal its possible association with the etiology of male infertility. Materials and Methods In this case-control study, 30 men with azoospermia (19 of whom had NOA) were selected as the case individuals, and 11 men with obstructive azoospermia (OA) were selected as control individuals. The best miRNA with the strongest ability to target the KDM3A gene was detected via comprehensive bioinformatics analysis. Reverse transcriptase quantitative polymerase chain reaction was used to assess the expression level of hsa-miR-30a-5p. After analyzing the data, the expression level of hsa-miR-30a-5p wascompared between men with NOA and men with OA. Results The findings supported the idea that hsa-miR-30a-5p is the miRNA with the best ability to target the KDM3A transcript. The expression analysis of hsa-miR-30a-5p indicated a significant overexpression (p = 0.04) in men with NOA compared to in men with OA. Conclusion Hsa-miR-30a-5p was overexpressed in men with NOA compared to in control individuals. Hsa-miR-30a-5p could target the KDM3A transcript and may suppress its expression.


Introduction
Infertility is defined by the World Health Organization as an inability to conceive after at least 1 yr of regular unprotected intercourse (1). It is estimated that 15% of all couples in the world may experience infertility. Half of these failures are thought to have roots in male factors (2). Azoospermia is considered to be one of the main causes of male infertility, described as the lack of sperm in ejaculation. Azoospermia can result in infertility if an obstruction occurs in the seminiferous tubules; however, non-obstructive azoospermia (NOA) can develop independently of physical defects (3). Therefore, in people suffering from obstructive azoospermia (OA), the process of spermatogenesis is normal, but individuals with NOA show abnormal testicular spermatogenesis (4).  (8,9). The pioneer study (conducted in 2009) introduced mir-122a as a regulatory molecule of the Tnp2 gene (10). Other studies have highlighted the significance of miRNAs in spermatogenesis and the development of the masculine gametes (11)(12)(13).
Aberrant expression of KDM3A was reported previously, but the role of miRNAs controller was not discussed (5). To answer this question, hsa-miR-30a-5p expression in men with OA and NOA was investigated in this study.

Sample size
Based on the prevalence of azoospermia in the population (1%) and previous studies (14,15) the size of the sample needed was determined to be 30. The sample size was calculated by the formula: The current research was performed in 2020

Statistical analysis
To calculate and compare the mean Cq of hsa-miR-30a-5p between men with OA vs. NO, t test were calculated using GraphPad Prism 7.05 (USA) software. P-values < 0.05 were considered significant.

Bioinformatics exploration
Having explored various databases, we screened the existing information and hsa-miR-30a-5p was chosen. It was observed that hsa-miR-30a-5p showed the highest frequency among different databases with the highest score for the potential to impact KDM3A transcription.

RNA assessment
Agarose gel electrophoresis showed a 28S rRNA band intensity 2 times greater than

Discussion
In the current study, 30 testicular tissues of idiopathic azoospermia from 19 individuals with NOA and 11 individuals with OA (control) were explored. RT-qPCR data analyses indicated that the expression of hsa-miR-30a-5p was 11.3 times higher in men with NOA compared to OA-control individuals (p = 0.04).
In contrast, another study showed the downregulation of hsa-miR-30a-5p in men with NOA. The inconsistency might be ascribed to the control individuals as they used men with testicular carcinomas as their control group (18). In accordance with our results, hsa-miR-30a-5p was previously identified by microarray data analysis as a suggested biomarker in asthenozoospermia (19).

Conclusion
According to the current study, hsa-miR-30a-5p is overexpressed in NOA patients compared to OA patients, and hsa-miR-30a-5p could potentially target the KDM3A transcript and hinder it from being translated. As a result, the downstream genes TNP1, PRM1, and PRM2 remain silent. All of these factors combine to induce male infertility in men with NOA.