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Numerous factors involved in the precise regulation of the spermatogenesis, and any disruptions or alterations in these regulatory mechanisms can lead to spermatogenesis arrest, which may result in male infertility. Among these factors, genetic influences play essential role in regulating the process. This study aimed to identify genes that are differentially expressed in relation to spermatogenesis arrest. Testicular biopsy samples were collected from 22 non-obstructive azoospermic patients diagnosed with spermatogenesis arrest (cases) and nine obstructive azoospermic patients (controls). RNA sequencing (RNA-seq) was performed on five samples from the 22 non-obstructive azoospermic patients and compared to previously published transcriptomic data from obstructive azoospermic patients, which served as the control group. Differential expression analysis of the RNA-seq data identified 1,915 differentially expressed genes, comprising 337 upregulated and 1,578 downregulated genes. Among these, several key candidate genes were identified for further analysis, including the upregulation of <jats:italic>FOS, FOSB, RGS1, and CXCL8<\/jats:italic>, as well as the downregulation of <jats:italic>TNP2, SPRR2C, LINC02314, and C16orf78<\/jats:italic>. RT-qPCR validation confirmed the RNA-seq findings for these genes in the tested samples. Subsequently, RT-qPCR was performed on the remaining 17 non-obstructive (n\u2009=\u200917) and obstructive azoospermic samples (n\u2009=\u20099) collected in this study. The results from these additional samples were consistent with the RNA-seq data, further supporting the findings. Using gene ontology (GO) analysis and published literature, we linked these genes with spermatogenesis arrest, identifying promising targets that could serve as potential biomarkers for this condition in the future.<\/jats:p>","DOI":"10.1371\/journal.pone.0332025","type":"journal-article","created":{"date-parts":[[2025,9,12]],"date-time":"2025-09-12T17:27:38Z","timestamp":1757698058000},"page":"e0332025","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Gene expression alterations in testicular biopsies from males with spermatogenesis arrest identified by transcriptome analysis"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2415-324X","authenticated-orcid":true,"given":"Osamah","family":"Batiha","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3093-2143","authenticated-orcid":true,"given":"Esra\u2019a","family":"Al-Zoubi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rowida","family":"Almomani","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammad A.","family":"Al Smadi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sura","family":"Alrawabdeh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Omar","family":"Alshokaibi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5463-4979","authenticated-orcid":true,"given":"Hussam","family":"Abu-Farsakh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1751-7570","authenticated-orcid":true,"given":"Abedalrhman","family":"Alkhateeb","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masood","family":"Abu-Halima","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,9,12]]},"reference":[{"key":"pone.0332025.ref001","unstructured":"WHO. 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[corrected]","volume":"66","author":"SC Esteves","year":"2011","journal-title":"Clinics (Sao Paulo)"},{"key":"pone.0332025.ref023","doi-asserted-by":"crossref","first-page":"99","DOI":"10.6061\/clinics\/2013(Sup01)11","article-title":"An update on sperm retrieval techniques for azoospermic males","author":"SC Esteves","year":"2013","journal-title":"Clinics (Sao Paulo)"},{"issue":"1","key":"pone.0332025.ref024","doi-asserted-by":"crossref","first-page":"21518","DOI":"10.1038\/s41598-022-26011-6","article-title":"Transcriptomic differences between fibrotic and non-fibrotic testicular tissue reveal possible key players in Klinefelter syndrome-related testicular fibrosis","volume":"12","author":"M Willems","year":"2022","journal-title":"Sci Rep"},{"key":"pone.0332025.ref025","doi-asserted-by":"crossref","DOI":"10.1007\/978-1-4419-9863-7_1215","volume-title":"Benjamini\u2013Hochberg Method","author":"W Haynes","year":"2013"},{"key":"pone.0332025.ref026","article-title":"Using clusterProfiler to characterize multiomics data","author":"S Xu","year":"2024","journal-title":"Nat Protoc"},{"issue":"4","key":"pone.0332025.ref027","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1002\/mrd.10105","article-title":"Protamine 1: protamine 2 stoichiometry in the sperm of eutherian mammals","volume":"61","author":"M Corzett","year":"2002","journal-title":"Mol Reprod Dev"},{"key":"pone.0332025.ref028","doi-asserted-by":"crossref","DOI":"10.1016\/S0079-6603(08)60839-9","article-title":"Vertebrute protamine genes and the histone-to protamine replacement reaction","volume":"40","author":"RGHD Oliva","year":"1991","journal-title":"Progress in Nucleic Acid Research and Molecular Biology"},{"issue":"1","key":"pone.0332025.ref029","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1006\/dbio.1993.1173","article-title":"Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II","volume":"158","author":"SM McPherson","year":"1993","journal-title":"Dev Biol"},{"key":"pone.0332025.ref030","doi-asserted-by":"crossref","DOI":"10.1210\/en.2010-0133","article-title":"Cannabinoid Receptor 1 Influences Chromatin Remodeling in Mouse Spermatids by Affecting Content of Transition Protein 2 mRNA and Histone Displacement","volume":"151","author":"T Chioccarelli","year":"2010","journal-title":"Endocrinology"},{"key":"pone.0332025.ref031","article-title":"Quantification of sperm specific mRNA transcripts (PRM1, PRM2, and TNP2) in teratozoospermia and normozoospermia: new correlations between mRNA content and morphology of sperm","author":"HE Elhm Savadi-Shiraz","year":"2014","journal-title":"Molecular Reproduction & Development"},{"issue":"6","key":"pone.0332025.ref032","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1093\/molehr\/7.6.513","article-title":"Teratozoospermia in mice lacking the transition protein 2 (Tnp2)","volume":"7","author":"IM Adham","year":"2001","journal-title":"Mol Hum 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Anbazhagan","year":"2020","journal-title":"Front Cell Dev Biol"},{"issue":"20","key":"pone.0332025.ref036","doi-asserted-by":"crossref","first-page":"7796","DOI":"10.3390\/ijms21207796","article-title":"The Role of Zinc in Male Fertility","volume":"21","author":"D Allouche-Fitoussi","year":"2020","journal-title":"Int J Mol Sci"},{"key":"pone.0332025.ref037","article-title":"Effects of dietary zinc depletion on seminal volume and zinc loss, serum testosterone concentrations, and sperm morphology in young men","volume":"56","author":"D Curtiss","year":"1992","journal-title":"American Society for Clinical Nutrition"},{"issue":"5","key":"pone.0332025.ref038","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1111\/j.1365-2605.1982.tb00279.x","article-title":"Removal of zinc from subcellular regions of human spermatozoa by EDTA treatment studied by X-ray microanalysis","volume":"5","author":"GM Roomans","year":"1982","journal-title":"Int J 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Amjad","year":"2021","journal-title":"Andrologia"},{"key":"pone.0332025.ref046","doi-asserted-by":"crossref","first-page":"1330161","DOI":"10.3389\/frph.2024.1330161","article-title":"MAPKs signaling is obligatory for male reproductive function in a development-specific manner","volume":"6","author":"L Kumar","year":"2024","journal-title":"Front Reprod Health"},{"issue":"12","key":"pone.0332025.ref047","first-page":"1515","article-title":"Expression of the proto-oncogene c-fos and the immunolocalization of c-fos, phosphorylated c-fos and estrogen receptor beta in the human testis","volume":"24","author":"FC Ara\u00fajo","year":"2009","journal-title":"Histol Histopathol"},{"key":"pone.0332025.ref048","doi-asserted-by":"crossref","first-page":"988047","DOI":"10.3389\/fgene.2022.988047","article-title":"Integrative analyses of potential biomarkers and pathways for non-obstructive azoospermia","volume":"13","author":"Y Zhong","year":"2022","journal-title":"Front 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