17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17β-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17β-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17β-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF.

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A hallmark of severe congenital adrenal hyperplasia due to 21-hydroxylase deficiency is pre- and postnatal virilization. The most characteristic biochemical abnormality is the elevation of 17α-hydroxyprogesterone, which is metabolized to the most potent androgen receptor agonist dihydrotestosterone. 17α-Hydroxyprogesterone can be metabolized to dihydrotestosterone via 4-androstenedione through the classical Δ⁴-pathway or via 17α-hydroxypregnenolone and dehydroepiandrosterone through the classical Δ⁵-pathway, as well as through an alternative route, called the 'backdoor pathway', that bypasses dehydroepiandrosterone, 4-androstenedione, and testosterone as intermediates. This review article will summarize recent advances in the understanding of the activities of androgen synthesis pathways in patients with 21-hydroxylase deficiency obtained by urinary steroid metabolomics based on gas chromatography-mass spectrometry. Compared with healthy controls, the relative activities of the backdoor and Δ⁴-pathways increase in patients with congenital adrenal hyperplasia during neonatal age and infancy, whereas the activity of the Δ⁵-pathway remains unchanged. Thereafter, the activity of the Δ⁵-pathway dominates, whereas a decreasing 5α-reductase activity leads to a diminished role of the backdoor pathway for androgenic steroid production. Beside the backdoor pathway, the Δ⁴-pathway seems to be responsible for increased androgen generation in patients with 21-hydroxylase deficiency before the onset of adrenarche, whereas the Δ⁵-pathway might contribute to the increased androgen formation in those patients only after the onset of adrenarche.