Endocrine Abstracts

JOINT1597

Introduction: Klinefelter Syndrome (KS) is a genetic condition characterized by an extra X chromosome in males, resulting in a 47,XXY karyotype. This leads to health issues, including hypogonadism, increased risk of metabolic disorders and altered body composition - characterized by higher fat mass and lower muscle mass compared to 46,XY males. KS males routinely receive testosterone replacement therapy (TRT) to lessen these complications, with documented benefits for metabolism and body composition. However, the cellular-level effects of TRT on skeletal muscle in KS remain unclear.

Methods: This study utilized single-nucleus RNA sequencing (snRNAseq) to investigate muscle tissue changes associated with TRT in KS. Skeletal muscle biopsies were obtained from untreated KS males (n = 4) before initiating TRT, and after one year of treatment. Age-matched 46,XY males (n = 4) was served as controls. Muscle samples were processed to isolate nuclei, followed by fluorescence-activated nuclei sorting (FANS), and subsequently subjected to snRNAseq using the 10X Genomics platform. The obtained snRNAseq data was integrated and clustered using the Seurat (v5) workflow. Comparative analyses of untreated KS, TRT-treated KS, and 46,XY control muscle samples were performed, enabling detailed insights into cell-specific transcriptional changes – including differential expression analysis (MAST) and cell-cell communication patterns (MultiNicheNet).

Results: TRT increased testosterone levels to normal ranges in KS males while reducing FSH/lH levels, though not to normal levels. A total of 81,768 nuclei were sequenced, revealing 11 major cell populations: type I, type IIa, and type IIx myonuclei, neuromuscular junctions, fibro-adipogenic progenitors, endothelial cells, muscle stem cells, T-cells, and macrophages. Differential gene expression (DEG) analysis revealed DEGs across all cell types, with 630 DEGs associated with TRT and 287 DEGs associated with KS. Key processes affected included differentiation, proliferation, growth, vascular remodeling, and steroid/androgen signaling pathways. To contextualize these transcriptional changes, cell-to-cell communication analysis was conducted to assess how these changes affected communication pathways. TRT was found to enhance vascular and extracellular matrix remodeling and muscle stem cell activation, while untreated KS muscle relied on maintenance and repair pathways, potentially favoring fibrosis over active regeneration.

Conclusion: This study provides insights into the cellular effects of TRT on skeletal muscle in KS. The findings suggest that TRT promotes a more regenerative and angiogenic muscle state, enhancing structural adaption and tissue remodeling. By identifying key transcriptional and intercellular signaling changes, these results contribute to understanding TRT’s therapeutic impact and may aid in optimizing treatment strategies for KS individuals.