Endocrine Abstracts

Background: Differences of sex development (DSD) in 46,XY individuals include conditions that impair androgen action, such as complete androgen insensitivity syndrome (CAIS) and 5α-reductase type 2 deficiency (SRD5A2 deficiency). While CAIS affects androgen receptor signaling, SRD5A2 deficiency impairs the conversion of testosterone to dihydrotestosterone (DHT). We hypothesized that these conditions exhibit distinct intracrine androgen steroid patterns in genital skin fibroblasts compared with 46,XY controls. We aim to map intracrine androgen biosynthesis pathways in these conditins and link functional steroid profiles to underlying genetic mutations.

Methods: We analysed GSFs from molecular proven CAIS and SRD5A2 individuals and GSFs from 46, XY controls. GSFs were cultured in DMEM-based medium at 37°C, 5% CO2. Cells were seeded in 6-well plates (6×10⁴ cells/2 ml/well) and incubated with testosterone, DHEA, and 17-hydoxyprogesterone (17OHP) at three concentrations (1, 10 and 100 nM). Supernatants were analyzed by LC-MS/MS.

Results: CAIS cell lines showed reduced DHT production compared to controls (9.4 ± 14.05 nmol/l vs. 26.47 ± 25.09 nmol/l at 100 nM testosterone). In contrast, androstenedione (A4) levels were elevated in CAIS cells (55.26 ± 50.35 nmol/l vs. 0.63 ± 0.49 nmol/l), suggesting altered downstream androgen metabolism and possible back-conversion. Mutation-specific differences were observed, with exon 7 AR mutations showing the highest A4 accumulation. Similarly, SRD5A2-deficient cells demonstrated markedly reduced DHT production (5.39 ± 1.78 nmol/l vs. 26.94 ± 25.09 nmol/l in controls at 100 nM testosterone) and increased A4 levels, indicating enhanced back-conversion of testosterone to A4.

Conclusions: Our findings demonstrate that both CAIS and SRD5A2 deficiency are associated with markedly reduced DHT production but differ in their underlying metabolic patterns. In CAIS, defective AR function is accompanied by altered downstream androgen metabolism and increased accumulation of precursor metabolites such as androstenedione. In SRD5A2-deficient cells, reduced enzymatic conversion of testosterone to DHT leads to pronounced back-conversion and pathway shifts consistent with the molecular defect. These results support our hypothesis that molecularly defined XY-DSD subtypes display distinct intracrine androgen signatures in genital target tissue.