ESPE Abstracts

Background: Previous descriptions of the steroidogenic capacity of human foetal adrenal glands in an ex vivo culture system focused on the classic androgen pathway and the alternative DHT pathway. However, no information is available on the abundance and role of the adrenal-derived 11-oxygenated androgens during foetal development, and how their levels correlate to classic androgens.

Aim: To characterise the steroidogenic capacity of androgen production by classic and 11-oxygenated androgen pathways in human foetal adrenal cultures.

Methods: Human foetal adrenal glands (n = 62 [25 female and 27 male]; gestational week 8-22) were isolated and cultured ex vivo as 1mm³ intact tissue fragments in a hanging drop culture approach over 14 days. In samples from 13 foetuses, the culture medium was supplemented with ACTH (1 nM) to stimulate adrenal steroidogenesis. The culture medium was replaced every 48 hours and harvested for pooled analysis over the culture period. Steroid analysis was performed using a liquid chromatography-tandem mass spectrometry profiling method, allowing for the detection of 22 steroids, including 14 androgens and precursors from the classic, alternative, and 11-oxygenated androgen pathways. Steroid concentrations are presented as the median of all samples.

Results: 11-hydroxyandrostenedione (11A4) was the most abundant androgen detected in foetal adrenal cultures (median: 1,587 nmol/L; range: 583-4,000), exceeding androstenedione (A4) from the classic pathway by 52-fold and increasing even further after ACTH stimulation. 11-ketoandrostenedione (11KA4) was higher compared to 11-ketotestosterone (11KT) or 11-hydroxytestosterone (11OHT) (11KA4: 60.9 nmol/L; 11KT: 2.97 nmol/L; 11OHT: 4.69 nmol/l), of which only 11KT responded to ACTH stimulation. The classic androgen pathway was dominated by delta-5 steroid production including pregnenolone (31.3 nmol/L), 17-hydroxypregnenolone (191.5 nmol/L) and dehydroepiandrosterone (38.3 nmol/L), all of which responded to ACTH stimulation, whilst A4 concentrations were comparatively lower (16.6 nmol/L). Testosterone and dihydrotestosterone were detectable but too low for quantification. In the alternative pathway, only androstanediol was detected at low levels (3.0 nmol/L), whereas 5a-androstanedione and androsterone were mostly below the lower limit of detection. Thus, androgen production was already robust in early first-trimester samples, persisted through the second trimester, and did not differ significantly between males and females.

Discussion: 11-oxygenated androgens are abundantly produced in foetal adrenal glands from 8 weeks gestation, exceeding the amounts of steroids generated in classic and alternative androgen pathways. This is in accordance with previous findings indicating that foetal adrenals express CYP11B1, the ‘entry enzyme’ for 11-oxygenated androgen pathway and cortisol biosynthesis.