Analysis of the expression of four microRNAs in spermatozoa and their role in male fertility

Introduction: MicroRNAs (miRNAs) are 22-24. nt molecules involved in the gene expression regulation of many biological processes. Several authors have recently identified altered miRNA expression profiles in cases of male idiopathic infertility, suggesting its fundamental role in fertility regulation. Objective: To characterize the expression of four miRNAs in sperm ribonucleic acid from fertile and infertile men. Material and methods: Four ejaculated samples from fertile donors and four samples from idiopathic infertile patients were obtained. Total RNA was isolated using the . TRIzol method followed by a DNase treatment. To confirm proper ribonucleic acid purification, a polymerase chain reaction for the Protamine-1 gene (PRM-1) was performed. Afterwards, real-time polymerase chain reaction with specific primers for the miRNAs hsa-miR-23a, hsa-miR-744, hsa-let-7f and hsa-miR-1, together with the normalization control Mamm-U6, were performed. Differences in expression were quantified by the Fold-Change statistic and 95% confidence intervals. Results: For each individual, an average of 2.6×10-5ng ribonucleic acid/spermatozoa was obtained, with a purity of 1.72 (±0.05) and no traces of deoxyribonucleic acid. TaqMan analysis confirmed the presence of hsa-miR-23a, hsa-miR-744 y hsa-let-7f in all the samples. The expression of hsa-let-7f was significantly lower in three of the four samples from men with idiopathic infertility. Conclusion: Human spermatozoa contain the miRNAs hsa-miR-23a, hsa-miR-744 and hsa-let-7f. Hsa-let-7f expression was significantly reduced in three out of four infertile patients, suggesting the involvement of alterations in miRNA expression as the underlying cause of some cases of male infertility. © 2012 Sociedad Española de Andrología.