Genetic alterations in azoospermia patients may reveal potential biomarkers for male infertility: A bioinformatic study

Abstract

Background/Aim: Azoospermia is defined as the absence of sperm in semen and is one of the most common causes of male infertility, with a prevalence of 10-15% in infertile men. Conventional methods for semen analysis do not provide a clear understanding of the etiology of azoospermia. Although testicular biopsy may exclude obstructive cases, non-obstructive azoospermia (NOA) treatment is limited due to a limited understanding of the underlying molecular mechanisms. Analysis of genetic alterations in azoospermia patients compared to the fertile population may be a valuable tool for determining diagnostic biomarkers for male infertility. This study aims to use bioinformatic tools to determine the top candidates in certain pathways altered in azoospermia. Methods: Expression data (GSE108886) of the differential testicular transcriptome in patients with NOA was selected from the Gene Expression Omnibus (GEO) database. Testicular RNA was harvested from azoospermia patients (n=11) and healthy controls (n=1, pooled sample). The differentially expressed genes (DEGs) were examined using GEO2R software. Biological pathways were identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG). Construction of the protein network and detection of hub genes were conducted in the STRING database. Data validation was performed via ELISA assay for the FOXO3 gene in obstructive and NOA patients. Significance was set at P-value Results: In NOA patients, 2115 genes were upregulated, and 1753 genes were downregulated compared to the control group. Ninety-one genes involved in spermatogenesis were downregulated. KEGG analysis revealed that the glucagon signaling, AMPK signaling, insulin and estrogen signaling, and oocyte meiosis pathways were upregulated, while the regulation of actin cytoskeleton, MAPK signaling pathway, focal adhesion, and chemical carcinogenesis – reactive oxygen species pathways were downregulated. Downstream genes with the highest score were PSMA4, PSMA6, PSMC1, PSME4, and UBA52, which are responsible for the ubiquitin-dependent protein degradation. The top hub genes with increasing expression were RPS18, RPS2, and RPS4X Conclusion: Although hub genes selected within the altering pathways may serve as a diagnostic tool for NOA, further validation of the presented data is necessary, as protein-protein interactions may not reflect alterations in gene expression in vivo. '+st+' Katz DJ, Teloken P, Shoshany O. Male infertility-the other side of the equation. Aust Fam Phys. 2017;46(9):641-6. Ghieh F, Mitchell V, Mandon-Pepin B, Vialard F. Genetic defects in human azoospermia. Bas Clin Androl. 2019;29(1):1-16. DOI: https://doi.org/10.1186/s12610-019-0086-6 Dong M, Li H, Zhang X, Tan J. Weighted correlation gene network analysis reveals new potential mechanisms and biomarkers in non-obstructive azoospermia. Frontiers in Genetics. 2021;12:617133. DOI: https://doi.org/10.3389/fgene.2021.617133 Malcher A, Rozwadowska N, Stokowy T, Kolanowski T, Jedrzejczak P, Zietkowiak W, et al. Potential biomarkers of non-obstructive azoospermia identified in microarray gene expression analysis. Fertil Steril. 2013;100(6):1686-94. e7. DOI: https://doi.org/10.1016/j.fertnstert.2013.07.1999 Peña VN, Kohn TP, Herati AS. Genetic mutations contributing to non-obstructive azoospermia. Best Prac Rest CL EN. 2020;34(6):101479. DOI: https://doi.org/10.1016/j.beem.2020.101479 Dodé C, Hardelin J-P. Kallmann syndrome. Eur J Hum Genet. 2009;17(2):139-46. DOI: https://doi.org/10.1038/ejhg.2008.206 Batista RL, Costa EMF, Rodrigues AdS, Gomes NL, Faria Jr JA, Nishi MY, et al. Androgen insensitivity syndrome: a review. Arch Endocrin Metab. 2018;62:227-35. DOI: https://doi.org/10.20945/2359-3997000000031 Yatsenko AN, Georgiadis AP, Röpke A, Berman AJ, Jaffe T, Olszewska M, et al. X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men. New Engl J Med. 2015;372(22):2097-107. DOI: https://doi.org/10.1056/NEJMoa1406192 Boroujeni PB, Sabbaghian M, Totonchi M, Sodeifi N, Sarkardeh H, Samadian A, et al. Expression analysis of genes encoding TEX11, TEX12, TEX14 and TEX15 in testis tissues of men with non-obstructive azoospermia. JBRA Assist Reprod. 2018;22(3):185. DOI: https://doi.org/10.5935/1518-0557.20180030 Massart A, Lissens W, Tournaye H, Stouffs K. Genetic causes of spermatogenic failure. Asian J Androl. 2012;14(1):40. DOI: https://doi.org/10.1038/aja.2011.67 Marchetti F, Wyrobek AJ. Mechanisms and consequences of paternally‐transmitted chromosomal abnormalities. Birth Defects Res C. 2005;75(2):112-29. DOI: https://doi.org/10.1002/bdrc.20040 Adler I-D. Comparison of the duration of spermatogenesis between male rodents and humans. Mutat Res-Fund Mol M. 1996;352(1-2):169-72. DOI: https://doi.org/10.1016/0027-5107(95)00223-5 Cannarella R, Condorelli RA, Mongioì LM, La Vignera S, Calogero AE. Molecular biology of spermatogenesis: novel targets of apparently idiopathic male infertility. Int J Mol Sci 2020;21(5):1728. DOI: https://doi.org/10.3390/ijms21051728 Xia W, Mruk DD, Lee WM, Cheng CY. Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model. The J Endoc. 2007;192(3):563. DOI: https://doi.org/10.1677/JOE-06-0158 Zheng H, Zhou X, Li D-k, Yang F, Pan H, Li T, et al. Genome-wide alteration in DNA hydroxymethylation in the sperm from bisphenol A-exposed men. Plos One. 2017;12(6):e0178535. DOI: https://doi.org/10.1371/journal.pone.0178535 Hadziselimovic F, Hadziselimovic NO, Demougin P, Krey G, Oakeley E. Piwi-pathway alteration induces LINE-1 transposon derepression and infertility development in cryptorchidism. Sex Dev. 2015;9(2):98-104. DOI: https://doi.org/10.1159/000375351 Yanaka N, Kobayashi K, Wakimoto K, Yamada E, Imahie H, Imai Y, et al. Insertional mutation of the murine...