Joint analysis of m(6)A and mRNA expression profiles in the testes of idiopathic nonobstructive azoospermia patients

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 (m(6)A) 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 m(6)A 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 m(6)A RNA Methylation Quantification Kit. The expression level of mRNAs was detected by qRT-PCR analysis. Differentially expressed m(6)A genes were analyzed using the human ArrayStar m(6)A epitranscriptomic microarray, and bioinformatics analyses were applied. RESULTS: A total of 36 iNOA and 8 controlled patients were included. The global expression of m(6)A in the iNOA group was significantly decreased. A dosage relationship was observed between the m(6)A 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 m(6)A methylation. Additionally, they also showed a gradually down-expressed tendency in the three groups (OA, HP, SO/MA groups). Moreover, m(6)A reader EIF3A was approved to have differential expression through microarrays analysis, which was consistent with the result from the qRT-PCR test. CONCLUSIONS: The m(6)A 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 m(6)A 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.