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Study Question: What is the molecular landscape underlying the functional decline of human testicular ageing?, Summary Answer: The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing., What Is Known Already: Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing., Study Design, Size, Duration: Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors., Participants/materials, Setting, Methods: scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat., Main Results and the Role of Chance: The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing., Large Scale Data: The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349., Limitations, Reasons for Caution: Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future., Wider Implications of the Findings: These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism., Study Funding/competing Interest(s): This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)