Joint analysis of m6A and mRNA expression profiles in the testes of idiopathic nonobstructive azoospermia patients

Abstract

Background: Growing evidence has indicated that epigenetic factors might be associated with the pathophysiology of idiopathic nonobstructive azoospermia (iNOA). As the most common RNA modification, N6-methyladenosine (m⁶A) methylation has recently attracted more attention in the regulation of spermatogenesis; however, its role in the mechanisms of iNOA is still unclear.

Objective: To determine the differential expression of mRNA and m⁶A methylation status in the testes of iNOA patients.

Methods: Testes tissues from diagnosed iNOA and controlled obstructive azoospermia (OA) patients were collected and grouped according to the histological examinations. Total RNA was isolated and quantified by an m⁶A RNA Methylation Quantification Kit. The expression level of mRNAs was detected by qRT-PCR analysis. Differentially expressed m⁶A genes were analyzed using the human ArrayStar m⁶A epitranscriptomic microarray, and bioinformatics analyses were applied.

Results: A total of 36 iNOA and 8 controlled patients were included. The global expression of m⁶A in the iNOA group was significantly decreased. A dosage relationship was observed between the m⁶A decline and the degree of impaired spermatogenesis, with the successive process of normal spermatogeneis, hypospermatogenesis (HP), maturation arrest (MA), and Sertoli cell-only syndrome (SO). Four down-expressed genes (BDNF, TMEM38B, RPL3L, and C22orf42) displayed significantly lower expression of m⁶A methylation. Additionally, they also showed a gradually down-expressed tendency in the three groups (OA, HP, SO/MA groups). Moreover, m⁶A reader EIF3A was approved to have differential expression through microarrays analysis, which was consistent with the result from the qRT-PCR test.

Conclusions: The m⁶A expression was gradually downregulated in the testes tissue from iNOA patients in accordance with the degree of spermatogenic dysfunction. The determined differential expression of mRNA and m⁶A methylation status may represent potentially novel molecular targets for the mechanism study of iNOA in the epigenetic level, which could benefit the understanding of the pathophysiology of iNOA.

Keywords: azoospermia; m6A; male infertility; methylation; spermatogenesis.