Exploring the underlying gene expression profiles of differences of sex development phenotypes through transcriptome analysis

Differences of sex development (DSD) comprise a heterogeneous group of conditions involving atypical chromosomal, gonadal, or anatomical sex. While genetic causes have been extensively studied, transcriptional programs underlying human gonadal differentiation remain poorly understood. In this study, we performed transcriptome profiling of gonadal tissue from 11 individuals with DSD and compared them to developmental stage-matched control samples from public datasets. Dimensionality reduction using sex-differentiation genes revealed that DSD samples do not align with typical male or female transcriptomes. Instead, they occupy an intermediate transcriptional space, reflecting varying degrees of masculinization or feminization, independent of chromosomal sex. Key sex-determining genes showed different expression in DSD samples, with reduced ovarian markers and dysregulated testicular factors. In 46,XY DSD individuals, testis-specific pathways, especially those related to spermatogenesis, cell proliferation, and metabolism, were downregulated. In contrast, 46,XX DSD samples exhibited a mixed expression profile, activating both testicular and ovarian gene programs, aligning with their variable histological features. These results suggest that gonadal identity in DSD is defined by a transcriptional range rather than a binary model. This is the first study to characterize gene expression directly in gonadal tissue from DSD patients, providing new insights into the molecular complexity of human sex development.