Seminal plasma piRNA array analysis and identification of possible biomarker piRNAs for the diagnosis of asthenozoospermia

Asthenozoospermia (AZS) is characterized by reduced sperm motility and its pathogenesis remains poorly understood. Piwi-interacting RNAs (piRNAs) have been indicated to serve important roles in spermatogenesis. However, little is known about the correlation of piRNA expression with AZS. In the present study, small RNA sequencing (small RNA-seq) was performed on sperm samples from AZS patients and fertile controls. Reverse transcription-quantitative (RT-q) PCR was used to validate the small RNA-seq results. Bioinformatics analyses were performed to predict the functions of differentially expressed piRNAs (DEpiRNAs). Logistic regression models were constructed and receiver operating characteristic curve (ROC) analysis was used to evaluate their diagnostic performance. A total of 114 upregulated and 169 downregulated piRNAs were detected in AZS patients. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the DEpiRNAs were mainly associated with transcription, signal transduction, cell differentiation, metal ion binding and focal adhesion. These results were verified by RT-qPCR analysis of eight selected piRNAs. The PCR results were consistent with the sequencing results in patients with AZS compared with controls in the first cohort. The expression of piR-hsa-32694, piR-hsa-26591, piR-hsa-18725 and piR-hsa-18586 was significantly upregulated in patients with AZS. The diagnostic power of the four piRNAs was further analyzed using ROC analysis; piR-hsa-26591 exhibited an area under the ROC curve (AUC) of 0.913 (95% CI: 0.795-0.994). Logistic regression modelling and subsequent ROC analysis indicated that the combination of the 4 piRNAs achieved good diagnostic efficacy (AUC: 0.935).