Alternative (backdoor) androgen production and masculinization in the human fetus

Abstract

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

Abstract

Fetal human masculinisation depends on testosterone production by the testes and an alternative “backdoor” androgen. This study shows that this androgen is likely to be androsterone, which is sexually dimorphic in the fetus but does not come from the testes; instead, synthesis probably depends on placental substrates.

Author summary

The human penis starts to develop before birth from a structure called the genital tubercle. This process is dependent on the secretion of testosterone from the fetal testes and subsequent conversion of testosterone into dihydrotestosterone (DHT) by enzymes in the genital tubercle. Recently, an alternative "backdoor" route to the formation of DHT, which does not require testosterone, has also been shown to be essential for normal development of the human penis. In this study we provide evidence indicating that androsterone is the major backdoor androgen involved in human masculinization and that it is produced in nongonadal tissues. Steroid hormone levels were measured in the plasma of second trimester human fetuses, and testosterone and androsterone were the only androgens with higher levels in males than in females. Analysis of tissue steroid levels showed that plasma androsterone did not primarily originate from the testes but, instead, was probably formed in other tissues via metabolism of placental progesterone. These data indicate, therefore, that masculinization of the human fetus depends on steroid hormone secretion from both the testes and the placenta, and would explain why placental dysfunction is associated with disorders of sex development.