S, V. S. | bioRxiv Science

bioRxiv2019

Listing candidate diagnostic markers and exploring the molecular basis of human spermatogenesis and a type of male infertility (Non-Obstructive Azoospermia), with transcriptomics via the next generation sequencing methods

Studying the molecular basis of Non-Obstructive Azoospermia (NOA), a type of male infertility with failed spermatogenesis at various stages, can also help in exploring molecular basis of human spermatogenesis and possibly pave way to identify new targets for male contraceptive development. Hence, we initiated a functional genomics study by applying RNA-seq. Testicular biopsies collected from donors with Non-Obstructive Azoospermia (NOA), Obstructive Azoospermia (OA), Congenital Bilateral Absence of the Vas Deferens (CBAVD), and Varicocele (VA) conditions. Strong association of 100+ genes with human spermatogenesis and NOA has been detected via NGS-based transcriptomic analysis. In addition, 20 RNA molecules have been short-listed for potential diagnostic applications (non-obstructive azoospermia vs. obstructive azoospermia, varicocele or normal). A hierarchical list of several genes and alternatively spliced mRNAs, transcribed differentially in NOA, is reported - based on a strength of association. Such association with NOA, spermatogenesis or both is a new finding for many genes as revealed by a comparison with a newly prepared comprehensive list of genes having such association with human spermatogenesis/NOA. Many top-ranking genes involved in viral gene expression were up-regulated in testes from NOA-patients, while those associated with an antiviral mechanism were down-regulated. A tangential finding: while most well-established control mRNAs did not qualify, two new ones worked best in RT-qPCR experiments. Needle-aspiration of testicular biopsies, followed by the use of short-listed promising candidate biomarkers (i.e., 16 mRNA & 4 chimeric transcripts) and control mRNAs in RT-qPCR-based diagnostic assays, may help to avoid open surgeries in future.

developmental biology↗

bioRxiv2018

Correlation between the aberrant human testicular germ-cell gene expression and disruption of spermatogenesis leading to male infertility

Spermatogenesis is characterized by sequential gene-expression at precise stages in progression of differentiation of the germ cells. Any alteration in expression of the critical genes is responsible for arrest of spermatogenesis associated with infertility. Inspite of advances the differential gene expression accompanying spermatogenesis, the corresponding regulatory mechanisms and their correlation to human infertility have not been clearly established. This study aims to identify the gene expression pattern of the human testicular germ cells from the patients either with obstructive azoospermia with complete intra-testicular spermatogenesis or non-obstructive azoospermia with spermatogenesis arrested at different stages and correlate the same to infertility. The testicular transcriptomes of 3 OA and 8 NOA patients and pooled testicular RNA (commercial source) were analyzed for their differential gene expression to identify potential regulators of spermatogenesis and the results were further validated in all of the 44 patients clinically diagnosed with azoospermia undergoing sperm retrieval surgery over the study period and 4 control samples included in this study. Analyses of the differential transcriptome led to identification of genes enriched in a specific testicular cell type and subsequently, several regulators of the diploid-double-diploid-haploid transitions in the human spermatogenesis were identified. Perturbations in the expression of these genes were identified as the potential causes of the spermatogenic arrest seen in azoospermia and thus the potential mediators of human male infertility. Another interesting observation was the increased autophagy in the testes of patients with non-obstructive azoospermia. The present study suggests that the regulation of the diploid-double-diploid-haploid transition is multigenic with the tandem alteration of several genes resulting in infertility. In conclusion, this study identified some of the genetic regulators controlling spermatogenesis using comparative transcriptome analyses of testicular tissues from azoospremic individuals and showed how alterations in several genes results in disruption of spermatogenesis and subsequent infertility. This study also provides interesting insights into the gene expression patterns of the Indian population that were not available earlier.

genomics↗