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

Abstract

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).

Small RNAs, spermatogenesis, and male infertility: a decade of retrospect

“…The piRNA expression studies on exosomes isolated from seminal plasma of asthenozoospermic patients reported downregulated piRNAs (piR-1207, piR-2107, piR-5937 and piR-5939) as the molecular biomarkers for male infertility [ 147 ]. Another study on seminal plasma of asthenozoospemic and normal healthy males identified 114 upregulated and 169 downregulated piRNAs, The top 10 upregulated piRNAs included piR-26,591, piR-32,694, piR-18,725, piR-16,782, piR-25,491, piR-5952, piR-18,611, piR-18,586, piR-15,811, piR-17,725 and top 10 downregulated piRNAs included piR-15,551, piR-29,529, piR-25,106, piR-2645, piR-9170, piR-2912, piR-23,203, piR-6148, piR-32,713, piR-29,155 [ 148 ].…”

“…22 piRNAs were downregulated (<-1.5) in sperm sample of asthenozoospermic men when compared to normozoospermic men. He et al [ 148 ] 3 asthenozoospermic and 3 fertile controls Seminal plasma Small RNA sequencing Top 10 upregulated piRNAs: piR-26,591, piR-32,694, piR-18,725, piR-16,782, piR-25,491, piR-5952, piR-18,611, piR-18,586, piR-15,811, piR-17,725; top 10 downregulated piRNAs piR-15,551, piR-29,529, piR-25,106, piR-2645, piR-9170, piR-2912, piR-23,203, piR-6148, piR-32,713, piR-29,155. Cui et al [ 149 ] 186 infertile patients and 40 fertile controls Sperm qRT-PCR piR-31,704 and piR-39,888 were decreased in infertile group compared to control men.…”

“…Sharma et al [ 156 ] showed that there is a change in sperm RNA dynamics from piRNAs to tRNA fragments as spermatozoa transits from testis to the epididymis [ 151 ]. The tRNA derived small RNAs are transported to spermatozoa via vesicles called epididymosomes, which are released from the epididymal epithelium during maturation [ 148 , 149 , 151 ]. All the above studies suggest that tRNA fragments could have key role in fertilization and embryonic development.…”

The Sperm Small RNA Transcriptome: Implications beyond Reproductive Disorder

“…In men, microarray analysis revealed more than one hundred differentially expressed piRNAs in the sperm samples of asthenozoospermic patients compared to the normozoospermia cohort [ 58 ]. Among the significantly upregulated piRNAs, piR-26951 was predicted to target transcripts encoding flagella-associated protein 45 (CFAP45) and bromodomain-containing protein 2 (BRD2), both found to be differentially expressed in the sperm of patients with asthenospermia [ 59 , 60 ].…”