Endocrine Abstracts

Background: Despite the growing identification of genetic variants that cause Differences of Sex Development (DSD), it is still unclear how these mutations impair testicular cellular organization and cell-cell communication at the tissue level. This knowledge gap is critical: clinical management decisions have historically relied on limited understanding of gonadal tissue biology. One prevailing hypothesis suggests that DSD gonads retain features of fetal development, resulting in developmental arrest or delay in tissue differentiation.

Methods: To elucidate the molecular and spatial etiology of DSD, we applied Visium HD spatial transcriptomics at near-single-cell resolution to compare an adult testis from an individual with a MYRF mutation to a healthy adult control.

Results: Spatial clustering revealed a striking paradox: despite the complete absence of germ cells, which comprise multiple developmental stages in healthy testis, the MYRF-mutant sample exhibited greater cellular fragmentation (16 spatial clusters) compared to the healthy control (10 clusters). In healthy adult testis, clusters represented expected cellular diversity, including germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells. In contrast, the MYRF-mutant testis displayed fragmented spatial organization that could suggest pronounced Sertoli cell heterogeneity, indicating retention of transitional or immature cell states rather than terminal differentiation. The proportional distribution of cells per cluster shifted from balanced diversity in healthy tissue to highly skewed distribution in MYRF tissue, with several clusters showing restricted spatial domains suggestive of incomplete differentiation. These spatial patterns support the hypothesis of retained fetal characteristics: rather than progressing to stable, mature cell types, the DSD tissue maintains molecular signatures of developmental intermediates.

Conclusions: Our study establishes a framework for spatially mapping gonadal dysgenesis and reveals unexpected cellular heterogeneity that may reflect differentiation arrest, altered proliferative states, or retention of fetal characteristics. To distinguish between these hypotheses, we are integrating publicly available single-cell atlases spanning human fetal testis maturation, which will enable molecular comparison of MYRF-mutant clusters to normal developmental trajectories. This foundational spatial atlas contributes to closing the knowledge gap in DSD gonad biology and provides molecular evidence that may inform re-evaluation of clinical decision-making based on tissue developmental status rather than assumed risk alone.