Key Findings

• Widespread use, limited data — 100% of gender dysphoria patients in this pediatric biorepository were on puberty blockers, yet long-term effects on testicular development remain poorly understood.
• Physical atrophy observed — Histology revealed mild-to-severe seminiferous tubule atrophy in PB-treated children, with some patients showing fully atrophied glands and microlithiasis.
• Developmental block at stem cell stage — Single-cell analysis showed >90% of germ cells in PB-treated juveniles were arrested at the spermatogonial stage, failing to progress toward meiosis.
• Machine learning flags "prepubertal" profile — Models trained on normal developmental data classified PB-treated patients as prepubertal across all cell types, suggesting incomplete or absent maturation of the testicular niche.
• Reversibility questioned — The combination of gland atrophy, abnormal cell proportions, and persistently immature gene expression signatures raises concerns about whether complete reproductive recovery is guaranteed after discontinuing puberty blockers.

Abstract

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as 'puberty blocker' (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete 'reversibility' and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

Summary

This study from Mayo Clinic researchers presents the largest clinically annotated juvenile testicular biorepository to date, examining how puberty blockers (GnRH analogs and other hormone suppressants) affect testicular cell development and reproductive fitness in children with gender dysphoria. Using histological analysis of 400+ tissue sections and single-cell RNA sequencing of >130,000 cells from 25 patients (including a novel 52-month PB-treated case), the authors found that chronic puberty blocker exposure is associated with sex gland atrophy, developmental blocks at the spermatogonial stem cell stage, and transcriptomic profiles that appear prepubertal even after extended treatment. Machine learning models trained on cell-type proportions and gene expression patterns classified PB-treated patients as prepubertal, raising concerns about whether the effects on spermatogonial stem cells are fully reversible. The study also discovered rare post-meiotic cells (spermatid-like) in newborns and prepubertal children, challenging the assumption that meiosis only begins at puberty.

Conclusion

This study from Mayo Clinic researchers presents the largest clinically annotated juvenile testicular biorepository to date, examining how puberty blockers (GnRH analogs and other hormone suppressants) affect testicular cell development and reproductive fitness in children with gender dysphoria. Using histological analysis of 400+ tissue sections and single-cell RNA sequencing of >130,000 cells from 25 patients (including a novel 52-month PB-treated case), the authors found that chronic puberty blocker exposure is associated with sex gland atrophy, developmental blocks at the spermatogonial stem cell stage, and transcriptomic profiles that appear prepubertal even after extended treatment. Machine learning models trained on cell-type proportions and gene expression patterns classified PB-treated patients as prepubertal, raising concerns about whether the effects on spermatogonial stem cells are fully reversible. The study also discovered rare post-meiotic cells (spermatid-like) in newborns and prepubertal children, challenging the assumption that meiosis only begins at puberty.