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. 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.

A peer-reviewed open access journal published by the National Institute of Environmental Health Sciences

Context: Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data. Objective: The objective of the study was to analyze second-trimester human testicular morphology and function. Design: This was an observational study of second-trimester testis development. Setting: The study was conducted at the Universities of Glasgow and Aberdeen. Patients/Participants: Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11–19 wk gestation). Main Outcome Measure(s): Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined. Results: Sertoli cell and germ cell number increased exponentially throughout the second trimester. Leydig cell number initially increased exponentially but slowed toward 19 wk. Transcripts encoding Sertoli (KITL, FGF9, SOX9, FSHR, WT1) and germ (CKIT, TFAP2C) cell-specific products increased per testis through the second trimester, but expression per cell was static apart from TFAP2C, which declined. Leydig cell transcripts (HSD17B3, CYP11A1, PTC1, CYP17, LHR, INSL3) also remained static per cell. Testicular expression of adrenal transcripts MC2R, CYP11B1, and CYP21 was detectable but unchanged. Expression of other transcripts known or postulated to be involved in testicular development (GATA4, GATA6, CXORF6, WNT2B, WNT4, WNT5A) increased significantly per testis during the second trimester. Conclusions: The second trimester is essential for the establishment of Sertoli and germ cell numbers. Sertoli and Leydig cells are active throughout the period, but there is no evidence of changing transcript levels.