Evolving Insights into the Relationship Between miR-23a/b-3p and Target Genes in Subfertile Patients

Spermatogenesis is a complex, highly regulated process by which spermatogonial stem cells (SSCs) replicate and develop into spermatozoa(1). A series of specialized differentiation events in the seminiferous tubules facilitates the spatial and temporal progression into functional sperm. Consequently, aberrations in these testicular regulatory mechanisms and gene expression patterns can contribute to male infertility. Our understanding of the genetic underpinnings of male infertility continues to evolve as new biomarkers for the condition are discovered.

MicroRNAs (miRNA) are a class of small, non-coding RNAs that are involved in transcriptional and post-transcriptional gene regulation. Profiling has revealed that miRNAs are expressed in a phase specific manner within both somatic and germ cells in the testicle(1). Moreover, androgens regulate spermatogenesis in part through miRNA-based mechanisms. Imperfect sequence complementarity between a miRNA and its target mRNA enables a single miRNA transcript to modulate potentially hundreds of genes, and a single gene can be also be modulated by multiple miRNAs. The complete landscape of these regulatory interactions in the setting of spermatogenesis is incompletely understood.

The authors of the present study have undertaken several prior investigations into the relationship between the miRNA-23 family and male infertility. In 2013, Abu-Halima et al. investigated whether miRNAs are differentially expressed in sperm samples from normozoospermic versus asthenozoospermic and oligoasthenozoospermic men(2). Microarray analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR) revealed significant differences in microRNA profiles between men with normal and impaired spermatogenesis. The authors discovered five novel miRNAs - miR-429, miR-1973, miR-1274a, miR-4286 and miR-34b - that were newly implicated in spermatogenesis. The authors also confirmed the deregulated expression of several previously identified miRNA transcripts including miR-23 family members. Ultimately, the identification of these miRNA transcripts added to the growing body of evidence that miRNA profiling could provide diagnostic information for male infertility. Despite this insight, the question remained as to whether these miRNAs would also be dysregulated directly within the testis.

In 2014, Abu-Halima et al. undertook miRNA profiling of formalin-fixed testicular biopsy samples from azoospermic men undergoing intracytoplasmic sperm injection (ICSI)(3). Patient samples were classified into Sertoli Cell only, mixed atrophy, germ cell arrest or normal seminiferous tubules groups. Microarray analysis combined with RT-qPCR validation identified various differential expression between these histopathological groups of a number of miRNA clusters, including members of the miR-23 family. Correlating expression of these miRNA clusters with testicular histology further underscored the regulatory role that these miRNAs could play in male reproduction. In a subsequent study, Abu-Halima et al. investigated whether miRNA expression profiles also vary in extracellular microvesicles obtained from the seminal plasma of oligoasthenozoospermic men(4). The authors once again identified that miRNA-23 expression remained dysregulated within the seminal plasma, which motivated the authors to elucidate the potential regulatory function of the miRNA-23 family on spermatogenesis-specific genes.

In the present study, the authors present the results of an investigation into the role of differential expression of miR-23a/b-3p and its target genes in patients with subfertility(5). This study examined gene and protein expression in semen samples and formalin-fixed testicular biopsies from men presenting to an infertility clinic. RT-qPCR validation revealed that miR-23a/b-3p was more abundant in men with impaired spermatogenesis. In silico prediction and dual luciferase assays revealed that potential links exist between overexpression of miR-23a/b-3p and the coincident suppression of four expressed genes - PFKFB4, HMMR, SPATA6, and TEX15. Additionally, mutations introduced into miR-23a/b-3p binding sites within the 3’ untranslated regions (3’-UTR) of their mRNAs diminished the suppression of gene translation by of these miRNAs, suggesting that the transcript is involved with post-transcriptional regulation. Lastly, correlation analysis highlighted that semen parameters including sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p expression and positively correlated with lower expression of the identified target genes.

Abu-Halima et al. should be applauded for their work towards elucidating the function of miRNAs during spermatogenesis. Their iterative progress illustrates how the authors have systematically identified and refined our understanding of this miRNA family. However, whether these results will be applicable to animal models and whether they will ultimately translate into a bona fide regulatory pathway involved in male fertility remains to be determined. Future work should aim to more effectively recapitulate the in vivo molecular environment of the testis in order to provide a more complete picture of miRNA-driven regulation in the setting of male infertility. Employing fresh testicular biopsies may also help broaden these conclusions by facilitating discovery of other miRNAs and their targets. Furthermore, refinement of this study’s experimental system will help deepen our understanding of the extent of binding between the miR-23 family and the 3’-UTRs of PFKFB4, HMMR, SPATA6, and TEX15.