Endocrine Abstracts

JOINT879

Introduction: Differences of sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. Disorders of steroid biosynthesis are often directly or indirectly involved. Steroid hormones are typically measured in the bloodstream, while locally produced intracrine steroids in peripheral tissues remain underexplored. 5-α-Reductase type 2(SRD5A2) converts testosterone to dihydrotestosterone (DHT), a potent androgen critical for male genital development in the fetal period and puberty. Deficiency of this enzyme, caused by mutations in the SRD5A2 gene, leads to an autosomal recessive DSD with varying degrees of undervirilization in individuals with a 46, XY karyotype. This study focuses on the analysis of the intracrine steroidome associated with SRD5A2 deficiency in cultured genital skin fibroblasts (GSF) to improve the understanding of peripheral androgen biosynthesis.

Method and Cell culture: GSFs from molecular proven SRD5A2-deficient persons (labioscrotal folds) and controls (scrotum) 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 after 24 h pre-incubation (before hormone incubation) or after additional 72 h of hormone incubation.

•ARD515: homozygous p.Arg227Gln.

•ARD249: homozygous Gly196Ser.

•GS451: homozygous p.Ile199Asn.

Results: DHT production was significantly reduced in SRD5A2-deficient cells compared to control cell lines confirming the underlying molecular defect. At 100 nM testosterone, the mean DHT production in SRD5A2-deficient cells was 5.54 nM ± 2.69 (n = 3), while in control cells it was 46.65 nM ± 30.58 (n = 3). Additionally, there was a notable back-conversion to androstenedione in the SRD5A2-deficient cells, with levels of 8.84 nM ± 2.79 (n = 3), compared to 0.15 nM ± 0.22 (n = 3) in control cells. Similarly, the metabolism of DHEA and 17OHP showed distinct differences between the SRD5A2-deficient and controls.

Conclusion: A limited but detectable conversion of testosterone to DHT occurs in SRD5A2-deficient cells, suggesting that residual enzymatic activity may be involved. The observed back-conversion of testosterone to androstenedione highlights the complexity of steroidogenesis in SRD5A2 deficiency. To fully explain this back-reaction, Further androgen biosynthesis pathways should be included in steroid measurements to understand androgen biochemistry in these cells better.