Alternative pathway androgen biosynthesis and human fetal female virilization

Abstract

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted towards testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenal, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.SIGNIFICANCEIn the classic androgen biosynthesis pathway, testosterone is converted to 5α-dihydrotestosterone, a step crucially required for normal male genital virilization. Congenital adrenal hyperplasia (CAH) due to P450 oxidoreductase deficiency (PORD) is an inborn disorder that disrupts classic androgen biosynthesis. However, some affected girls present with severe genital virilization at birth. We hypothesized that this is explained by a prenatally active, alternative biosynthesis pathway to 5α-dihydrotestosterone. We show that adrenals and genital skin cooperate to produce androgens via the alternative pathway during the major period of human sexual differentiation and that neonates with PORD still produce alternative pathway androgens during the first weeks of life. This indicates that alternative pathway androgen biosynthesis drives prenatal virilization in CAH due to PORD.

The underlying cause of the simple virilizing phenotype in patients with 21-hydroxylase deficiency harboring P31L variant

“…may be involved in fetal virilization in females, through which the 17-OHP accumulated in the 21-OHD may be Normal reference interval:17-OHP: 0.31-2.17(M);0.10-0.80ng/ml(F);T:1.75-7.81ng/ml(M), 0.10-0.75ng/ml(F);ACTH: 0-46pg/ml, F:4-22.3ug/dl. converted to the androgen and thus aggravate female virilization (37).…”

Multi-steroid profiling by uPLC-MS/MS with post-column infusion of ammonium fluoride

“…Additionally, while other assays focus mainly on adrenal steroids [28,33,[37][38][39] or androgen panels [14,40], our assay comprehensively covers multiple steroid classes (Figure 1). In addition to classic androgens, such as A4 and T, our assay covers the alternative pathway of DHT biosynthesis, which contributes to androgen excess in CAH [41,42], and the 11oxygenated androgens, which have only recently been shown to have major relevance for the diagnosis and mechanistic understanding of androgen excess conditions [6,[43][44][45]].…”

Isolated 17, 20 Lyase Deficiency Secondary to a Novel CYB5A Variant: Comparison of Steroid Metabolomic Findings with Published Cases Provides Diagnostic Guidelines and Greater Insight into Its Biological Role

“…Several authors, most recently Reisch et al [18], have presented steroid metabolomic data that are interpreted to support the existence in man of a “backdoor pathway” of androgen synthesis [18], with the potent androgen dihydrotestosterone as the final product (Fig. 1).…”