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 to 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 (adrenals, 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.

Clinical outcomes and characteristics of P30L mutations in congenital adrenal hyperplasia due to 21-hydroxylase deficiency

“…The presence of unidentified rare mutations modulating the phenotype cannot be excluded; however, it is unlikely due to the frequent occurrence of SV form with P30L mutation in studies with meticulous genotyping. Some other factors modifying phenotype have also been suggested such as the CAG repeats of the androgen receptor and other genes encoding proteins other than cytochrome P450 type II enzyme with a 21-hydroxylase activity, as well as alternative pathway of androgen biosynthesis causing fetal virilization in females [ 58 – 60 ].…”

Reply to Flück et al.: Alternative androgen pathway biosynthesis drives fetal female virilization in P450 oxidoreductase deficiency

“…Mutations in the electron donor enzyme POR invariably disrupt CYP17A1 and CYP21A2 activities ( 3 ), but have variable effects on CYP19A1 aromatase ( 4 ). We observed wild-type–equivalent CYP19A1 activity for the POR A287P mutant, within the linear range of the enzymatic reaction ( 2 ). When assessing full enzyme kinetics, Flück and coworkers ( 4 ) observed that POR A287P mildly impairs CYP19A1 activity, although catalytic efficiency was similar to wild-type POR.…”

“…Newborn girls with P450 oxidoreductase (POR) deficiency regularly present with virilized external genitalia despite low circulating androgens ( 1 ). In PNAS, we ( 2 ) explain this conundrum by enhanced prenatal activity of an alternative androgen pathway ( Fig. 1 ) while classic androgen biosynthesis is disrupted.…”

“…Flück et al ( 5 ) argue that mild impairment of placental aromatase activity may explain maternal virilization in POR deficiency. Our paper in PNAS ( 2 ) is primarily about fetal female virilization in POR deficiency. However, no maternal virilization was observed in three of four pregnancies with children homozygous for POR A287P ( 6 ).…”

“…1 ), and therefore nonaromatizable. In POR deficiency, classic androgen biosynthesis is disrupted, resulting in low circulating androgens ( 2 , 7 – 9 ). Thus, with little substrate provided via the classic pathway, impaired aromatase activity will not result in clinically relevant androgen accumulation and consequently is unlikely to contribute to prenatal fetal female virilization in POR deficiency.…”