L26/P-026 Single-cell profiling reveals aberrant germ-cell expression as predictor of TESE failure and identifies targets for regenerative interventions in NOA testes

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Abstract Study question Can comprehensive single-cell characterisation of germ cells identify molecular predictors of testicular sperm extraction (TESE) outcome and therapeutic targets in patients with non-obstructive azoospermia (NOA)? Summary answer Late-stage germ-cell maturation defects distinguish TESE outcomes, while spermatogonial heterogeneity highlights germ-cell niche restoration as therapeutic strategy. What is known already TESE success varies markedly across NOA histological subtypes, yet molecular predictors distinguishing successful from failed sperm retrieval remain elusive. Previous single-cell studies have characterised germ-cell populations in healthy testes but were limited by small sample sizes and insufficient representation of NOA phenotypes. Spermatogonial stem cell (SSC) heterogeneity and stage-specific markers predictive of sperm retrieval success have not been comprehensively mapped. Emerging evidence suggests growth factor-based therapies may support germ-cell viability and differentiation in vitro; however, their clinical translation requires an improved understanding which germ-cell states are the finest targets to regenerative interventions in NOA patients. Study design, size, duration Single-cell RNA sequencing (scRNA-seq) was performed on testicular biopsies from fourteen NOA patients stratified according to TESE outcome. All samples were collected and cryopreserved as single-cell suspensions at the time of testicular biopsy for assisted reproductive technologies. Germ-cell proportions and transcriptional profiles were compared between patient with successful (TESE+) and unsuccessful (TESE-) sperm retrieval to identify specific cell clusters and differentially expressed genes in each condition and germ-cell stages. Participants/materials, setting, methods Patients were comprehensively classified with embryologist support into TESE + (n = 3) or TESE- (n = 11). scRNA-seq libraries were generated using 10x Genomics Next GEM technology, followed by stringent quality control filtering. Over 20,000 germ-cell transcriptomes were retained and annotated using automated and manual approaches based on established markers such as UTF1, MAGEA4, HORMAD1 or PRM1. Trajectory inference and differential expression analyses were performed using Scanpy. Main results and the role of chance We initially defined 20 subclusters encompassing all germ-cell types. High-resolution single-cell profiling revealed previously unappreciated SSC heterogeneity, resolving four transcriptionally distinct populations arranged along a differentiation continuum. A primitive SSC subset characterised by high expression of GFRA1, FGFR2, and ICA1L was positioned at the root of the spermatogenic lineage by trajectory analysis, likely representing an early stem-cell pool. Additionally, late-stage germ cells exhibited TESE outcome-associated molecular signatures. Elongated spermatids from TESE- samples showed significantly increased mitochondrial gene expression, whereas TESE+ samples displayed marked upregulation of genes involved in flagellar assembly and essential structural components for sperm maturation, including SAXO1 and HOOK1. Spermatozoa from TESE+ patients showed elevated expression of chromatin remodelling genes like protamines (PRM1, PRM2) and genes encoding sperm-specific proteins (SPEM2, TSSK6, SMCP), consistent with complete spermiogenesis. Collectively, these findings suggest that TESE failure reflects late-stage transcriptional defects rather than germ-cell depletion alone, highlighting specific molecular checkpoints as potential therapeutic targets. Limitations, reasons for caution The sample size is limited and requires validation in larger and independent cohorts. Functional validation of identified SCC subsets and late-stage markers is necessary to confirm their biological relevance before clinical prior to translation. Wider implications of the findings This comprehensive germ-cell characterisation identifies early undifferentiated SSC populations in NOA testes. Growth factor-based therapies, such as platelet-rich plasma (PRP), which show promise in supporting SSC viability and differentiation, warrant further clinical investigation. Late-stage molecular markers may enable prediction of TESE outcomes and inform associations with fertilisation and implantation success. Trial registration number No

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