{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T06:22:31Z","timestamp":1791872551455,"version":"4.3.4"},"reference-count":47,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,2,26]],"date-time":"2021-02-26T00:00:00Z","timestamp":1614297600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100013853","name":"Shenzhen Hospital of Southern Medical University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100013853","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Genet."],"abstract":"<jats:p>Non-obstructive azoospermia (NOA) is one of the most important causes of male infertility. It is mainly characterized by the absence of sperm in semen repeatedly or the number of sperm is small and not fully developed. At present, its pathogenesis remains largely unknown. The goal of this study is to identify hub genes that might affect biomarkers related to spermatogenesis. Using the clinically significant transcriptome and single-cell sequencing data sets on the Gene Expression Omnibus (GEO) database, we identified candidate hub genes related to spermatogenesis. Based on them, we performed Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analyses, protein-protein interaction (PPI) network analysis, principal component analysis (PCA), cell cluster analysis, and pseudo-chronological analysis. We identified a total of 430 differentially expressed genes, of which three have not been reported related to spermatogenesis (C22orf23, TSACC, and TTC25), and the expression of these three hub genes was different in each type of sperm cells. The results of the pseudo-chronological analysis of the three hub genes indicated that TTC25 was in a low expression state during the whole process of sperm development, while the expression of C22orf23 had two fluctuations in the differentiating spermatogonia and late primary spermatocyte stages, and TSACC showed an upward trend from the spermatogonial stem cell stage to the spermatogenesis stage. Our research found that the three hub genes were different in the trajectory of sperm development, indicating that they might play important roles in different sperm cells. This result is of great significance for revealing the pathogenic mechanism of NOA and further research.<\/jats:p>","DOI":"10.3389\/fgene.2021.608629","type":"journal-article","created":{"date-parts":[[2021,2,26]],"date-time":"2021-02-26T07:26:58Z","timestamp":1614324418000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["The Novel Key Genes of Non-obstructive Azoospermia Affect Spermatogenesis: Transcriptomic Analysis Based on RNA-Seq and scRNA-Seq Data"],"prefix":"10.3389","volume":"12","author":[{"given":"Haihong","family":"He","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fan","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wang","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keyan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuang","family":"Lou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiaomin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siping","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinhua","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingwang","family":"Jia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiwen","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,2,26]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"633","DOI":"10.1136\/jmedgenet-2017-104514","article-title":"Mutation in TDRD9 causes non-obstructive azoospermia in infertile men","volume":"54","author":"Arafat","year":"2017","journal-title":"J. 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Reprod."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"1972","DOI":"10.1093\/humrep\/deaa163","article-title":"Prediction model for clinical pregnancy for ICSI after surgical sperm retrieval in different types of azoospermia","volume":"35","author":"Song","year":"2020","journal-title":"Hum. Reprod."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"4250","DOI":"10.1128\/MCB.25.10.4250-4261.2005","article-title":"Identification and characterization of SSTK, a serine\/threonine protein kinase essential for male fertility","volume":"25","author":"Spiridonov","year":"2005","journal-title":"Mol. Cell. Biol."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"234","DOI":"10.1038\/aja.2009.80","article-title":"c.822+126T>G\/C: a novel triallelic polymorphism of the TSSK6 gene associated with spermatogenic impairment in a Chinese population","volume":"12","author":"Su","year":"2010","journal-title":"Asian J. Androl."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"151","DOI":"10.3389\/fcell.2019.00151","article-title":"CCDC42 localizes to manchette, HTCA and tail and interacts with ODF1 and ODF2 in the formation of the male germ cell cytoskeleton","volume":"7","author":"Tapia Contreras","year":"2019","journal-title":"Front. Cell Dev. Biol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"R209","DOI":"10.1530\/REP-18-0480","article-title":"The contribution of epididymosomes to the sperm small RNA profile","volume":"157","author":"Trigg","year":"2019","journal-title":"Reproduction"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1080\/14712598.2018.1380622","article-title":"Biological therapy for non-obstructive azoospermia","volume":"18","author":"Vij","year":"2018","journal-title":"Expert Opin. Biol. 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Genet."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"1199","DOI":"10.1002\/mrd.23236","article-title":"Functional role of GKAP1 in the regulation of male germ cell spontaneous apoptosis and sperm number","volume":"86","author":"Wang","year":"","journal-title":"Mol. Reprod. Dev."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1007\/s11596-015-1417-2","article-title":"Testis specific serine\/threonine protein kinase 4 (TSSK4) leads to cell apoptosis relying on its kinase activity","volume":"35","author":"Wang","year":"2015","journal-title":"J. Huazhong Univ. Sci. Technolog. Med. Sci."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"e28218","DOI":"10.4161\/spmg.28218","article-title":"Review of azoospermia","volume":"4","author":"Wosnitzer","year":"2014","journal-title":"Spermatogenesis"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"3584","DOI":"10.1073\/pnas.1817018116","article-title":"Motile cilia of the male reproductive system require miR-34\/miR-449 for development and function to generate luminal turbulence","volume":"116","author":"Yuan","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1661","DOI":"10.1002\/jat.4026","article-title":"Histone demethylase JHDM2A regulates H3K9 dimethylation in response to arsenic-induced DNA damage and repair in normal human liver cells","volume":"40","author":"Zhang","year":"2020","journal-title":"J. Appl. 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