The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

The serum steroid signature of PCOS hints at the involvement of novel pathways for excess androgen biosynthesis.

CONTEXT: Polycystic ovary syndrome (PCOS) is defined by androgen excess and ovarian dysfunction in the absence of a specific physiological diagnosis. The best clinical marker of androgen excess is hirsutism, while the best biochemical parameter is still a matter of debate. Current consensus guidelines recommend, among other hormones, serum free testosterone as an important serum parameter to measure androgen excess. Recently, however, novel active androgens and androgen metabolic pathways have been discovered. OBJECTIVE: To assess the contribution of novel androgens and related steroid biosynthetic pathways to the serum steroid pool in PCOS women in comparison to healthy controls. DESIGN: This is a case control study, wherein PCOS was diagnosed according to the AE-PCOS 2009 criteria. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. SETTING: Yeditepe University and associated clinics in Istanbul, Turkey, together with Bern University Hospital Inselspital, Bern, Switzerland. PARTICIPANTS: 42 PCOS women and 42 matched, healthy control women. MAIN OUTCOME MEASURES: Assessment of 34 steroids compartmentalized in four androgen related pathways: the classic androgen pathway, the backdoor pathway, the C11-oxy backdoor pathway, and the C11-oxy (11ß-hydroxyandrostenedione) pathway. RESULTS: Metabolites of all four pathways were identified in healthy and PCOS women. Highest concentrations were found for progesterone in controls and androstenedione in PCOS. Lowest levels were found for 11-ketotestosterone in controls compared to PCOS, and for 20α-hydroxyprogesterone in PCOS compared to controls. PCOS also had higher serum testosterone levels compared to the controls. PCOS women had overall higher levels of steroid metabolites of all four androgen pathways compared to healthy controls. CONCLUSIONS: Novel alternative pathways contribute to the androgen production in healthy and PCOS women. Hyperandrogenism in PCOS is characterized by an overall increase of serum androgens in the classic, backdoor and C11-oxy pathways. While monogenetic disorders of steroid biosynthesis can be recognized by a specific pattern in the steroid profile, no diagnostic pattern or classifier was found in the serum for PCOS.

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17ß-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel.

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.

Classic and backdoor pathways of androgen biosynthesis in human sexual development.

Both genes and hormones regulate human sexual development. Although ovarian hormones are not essential for female external genitalia development, male sexual development requires the action of testicular testosterone and dihydrotestosterone (DHT). DHT is the most active endogenous androgen formed by the conversion of testosterone in genital skin. This synthesis route from cholesterol to DHT is called the conventional classic pathway. Recent investigations have reported an alternative ("backdoor") route for DHT formation that bypasses fetal testicular testosterone. This alternative route plays a crucial role in human hyperandrogenic disorders like congenital adrenal hyperplasia caused by P450c21 deficiency, polycystic ovary syndrome, and P450 oxidoreductase deficiency. In addition, mutations in AKR1C2 and AKR1C4, genes encoding 3α-reductases, have been implicated in disorders of sexual development, indicating that both the classic and backdoor routes are required for normal human male sexual development. More recently, androsterone was found to be the primary androgen of the human backdoor route. Androsterone and steroidal substrates specific to the backdoor route are predominantly found in the placenta, liver, and adrenal glands rather than in the testes. These findings are essential to understanding human sexual development.

Classic and current concepts in adrenal steroidogenesis: a reappraisal.

Adrenal steroid biosynthesis and its related pathology are constant evolving disciplines. In this paper, we review classic and current concepts of adrenal steroidogenesis, plus control mechanisms of steroid pathways, distribution of unique enzymes and cofactors, and major steroid families. We highlight the presence of a "mineralocorticoid (MC) pathway of zona fasciculata (ZF)", where most circulating corticosterone and deoxycorticosterone (DOC) originate together with 18OHDOC, under ACTH control, a claim based on functional studies in normal subjects and in patients with 11ß-, and 17α-hydroxylase deficiencies. We emphasize key differences between CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) and the onset of a hybrid enzyme - CYP11B1/CYP11B2 -, responsible for aldosterone formation in ZF under ACTH control, in "type I familial hyperaldosteronism" (dexamethasone suppressible). In "apparent MC excess syndrome", peripheral conversion of cortisol to cortisone is impaired by lack of 11ß-hydroxysteroid dehydrogenase type 2, permitting free cortisol access to MC receptors resulting in severe hypertension. We discuss two novel conditions involving the synthesis of adrenal androgens: the "backdoor pathway", through which dihydrotestosterone is formed directly from androsterone, being relevant for the fetoplacental setting and sexual differentiation of male fetuses, and the rediscovery of C19 11-oxygenated steroids (11-hydroxyandrostenedione and 11-ketotestosterone), active androgens and important markers of virilization in 21-hydroxylase deficiency and polycystic ovaries syndrome. Finally, we underline two enzyme cofactor deficiencies: cytochrome P450 oxidoreductase which partially affects 21- and 17α-hydroxylation, producing a combined clinical/hormonal picture and causing typical skeletal malformations (Antley-Bixler syndrome), and PAPSS2, coupled to SULT2A1, that promotes sulfation of DHEA to DHEAS, preventing active androgens to accumulate. Its deficiency results in reduced DHEAS and elevated DHEA and androgens with virilization. Future and necessary studies will shed light on remaining issues and questions on adrenal steroidogenesis.

Classic and current concepts in adrenal steroidogenesis: a reappraisal

ABSTRACT Adrenal steroid biosynthesis and its related pathology are constant evolving disciplines. In this paper, we review classic and current concepts of adrenal steroidogenesis, plus control mechanisms of steroid pathways, distribution of unique enzymes and cofactors, and major steroid families. We highlight the presence of a "mineralocorticoid (MC) pathway of zona fasciculata (ZF)", where most circulating corticosterone and deoxycorticosterone (DOC) originate together with 18OHDOC, under ACTH control, a claim based on functional studies in normal subjects and in patients with 11β-, and 17α-hydroxylase deficiencies. We emphasize key differences between CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) and the onset of a hybrid enzyme - CYP11B1/CYP11B2 -, responsible for aldosterone formation in ZF under ACTH control, in "type I familial hyperaldosteronism" (dexamethasone suppressible). In "apparent MC excess syndrome", peripheral conversion of cortisol to cortisone is impaired by lack of 11β-hydroxysteroid dehydrogenase type 2, permitting free cortisol access to MC receptors resulting in severe hypertension. We discuss two novel conditions involving the synthesis of adrenal androgens: the "backdoor pathway", through which dihydrotestosterone is formed directly from androsterone, being relevant for the fetoplacental setting and sexual differentiation of male fetuses, and the rediscovery of C19 11-oxygenated steroids (11-hydroxyandrostenedione and 11-ketotestosterone), active androgens and important markers of virilization in 21-hydroxylase deficiency and polycystic ovaries syndrome. Finally, we underline two enzyme cofactor deficiencies: cytochrome P450 oxidoreductase which partially affects 21- and 17α-hydroxylation, producing a combined clinical/hormonal picture and causing typical skeletal malformations (Antley-Bixler syndrome), and PAPSS2, coupled to SULT2A1, that promotes sulfation of DHEA to DHEAS, preventing active androgens to accumulate. Its deficiency results in reduced DHEAS and elevated DHEA and androgens with virilization. Future and necessary studies will shed light on remaining issues and questions on adrenal steroidogenesis.

Back where it belongs: 11ß-hydroxyandrostenedione compels the re-assessment of C11-oxy androgens in steroidogenesis.

Adrenal steroidogenesis has, for decades, been depicted as three biosynthesis pathways -the mineralocorticoid, glucocorticoid and androgen pathways with aldosterone, cortisol and androstenedione as the respective end products. 11ß-hydroxyandrostenedione was not included as an adrenal steroid despite the adrenal output of this steroid being twice that of androstenedione. While it is the end of the line for aldosterone and cortisol, as it is in these forms that they exhibit their most potent receptor activities prior to inactivation and conjugation, 11ß-hydroxyandrostenedione is another matter entirely. The steroid, which is weakly androgenic, has its own designated pathway yielding 11-ketoandrostenedione, 11ß-hydroxytestosterone and the potent androgens, 11-ketotestosterone and 11-ketodihydrotestosterone, primarily in the periphery. Over the last decade, these C11-oxy C19 steroids have once again come to the fore with the rising number of studies contradicting the generally accepted notion that testosterone and it's 5α-reduced product, dihydrotestosterone, are the principal potent androgens in humans. These C11-oxy androgens have been shown to contribute to the androgen milieu in adrenal disorders associated with androgen excess and in androgen dependant disease progression. In this review, we will highlight these overlooked C11-oxy C19 steroids as well as the C11-oxy C21 steroids and their contribution to congenital adrenal hyperplasia, polycystic ovarian syndrome and prostate cancer. The focus is on new findings over the past decade which are slowly but surely reshaping our current outlook on human sex steroid biology.

Steroidogenesis in Peripheral and Transition Zones of Human Prostate Cancer Tissue.

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms.

Androgens and the masculinization programming window: human-rodent differences.

Human male reproductive disorders are common and may have a fetal origin - the testicular dysgenesis syndrome (TDS) hypothesis. In rats, experimentally induced TDS disorders result from disruption of fetal androgen production/action specifically in the masculinization programming window (MPW). MPW androgen action also programs longer anogenital distance (AGD) in male versus female rats; shorter male AGD is correlated with risk and severity of induced TDS disorders. AGD thus provides a lifelong, calibrated readout of MPW androgen exposure and predicts likelihood of reproductive dysfunction. Pregnant rat exposure to environmental chemicals, notably certain phthalates (e.g. diethyl hexl phthalate, DEHP; dibutyl phthalate, DBP), pesticides or paracetamol, can reduce fetal testis testosterone and AGD and induce TDS disorders, provided exposure includes the MPW. In humans, AGD is longer in males than females and the presumptive MPW is 8-14 weeks' gestation. Some, but not all, epidemiological studies of maternal DEHP (or pesticides) exposure reported shorter AGD in sons, but this occurred at DEHP exposure levels several thousand-fold lower than are effective in rats. In fetal human testis culture/xenografts, DEHP/DBP do not reduce testosterone production, whereas therapeutic paracetamol exposure does. In humans, androgen production in the MPW is controlled differently (human chorionic gonadotrophin-driven) than in rats (paracrine controlled), and other organs (placenta, liver, adrenals) contribute to MPW androgens, essential for normal masculinization, via the 'backdoor pathway'. Consequently, early placental dysfunction, which is affected by maternal lifestyle and diet, and maternal painkiller use, may be more important than environmental chemical exposures in the origin of TDS in humans.

Molecular genetic analysis of AKR1C2-4 and HSD17B6 genes in subjects 46,XY with hypospadias.

BACKGROUND: The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into 5α-dihydrotestosterone (DHT). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→DHT) mediated by SRD5A2 and 2) a "backdoor" pathway in which DHT is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate. OBJECTIVE: We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias. DESIGN AND PATIENTS: The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies. RESULTS: Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes. DISCUSSION: Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically 5α-DHT. We selected four candidate genes involved in the "backdoor" pathway for the formation of 5α-DHT. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" 5α-DHT pathway during human sexual development, specifically, the differentiation of male external genitalia. CONCLUSION: These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization.

Characterization of Precursor-Dependent Steroidogenesis in Human Prostate Cancer Models.

Castration-resistant prostate tumors acquire the independent capacity to generate androgens by upregulating steroidogenic enzymes or using steroid precursors produced by the adrenal glands for continued growth and sustainability. The formation of steroids was measured by liquid chromatography-mass spectrometry in LNCaP and 22Rv1 prostate cancer cells, and in human prostate tissues, following incubation with steroid precursors (22-OH-cholesterol, pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone). Pregnenolone, progesterone, 17-OH-pregnenolone, and 17-OH-progesterone increased C21 steroid (5-pregnan-3,20-dione, 5-pregnan-3,17-diol-20-one, 5-pregnan-3-ol-20-one) formation in the backdoor pathway, and demonstrated a trend of stimulating dihydroepiandrosterone or its precursors in the backdoor pathway in LNCaP and 22Rv1 cells. The precursors differentially affected steroidogenic enzyme messenger RNA (mRNA) expressions in the cell lines. The steroidogenesis following incubation of human prostate tissue with 17-OH-pregnenolone and progesterone produced trends similar to those observed in cell lines. Interestingly, the formation of C21 steroids from classical pathway was not stimulated but backdoor pathway steroids (e.g., 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one) were elevated following incubations with prostate tissues. Overall, C21 steroids were predominantly formed in the classical as well as backdoor pathways, and steroid precursors induced a diversion of steroidogenesis to the backdoor pathway in both cell lines and human prostate tissue, and influenced adaptive steroidogenesis to form C21 steroids.

Longitudinal serum and urine steroid metabolite profiling in a 46,XY infant with prenatally identified POR deficiency.

Although POR deficiency (PORD) is assumed to be accompanied by excessive placental androgen accumulation and enhanced adrenal and testicular androgen production via the backdoor pathway as well as compromised testicular androgen production via the frontdoor pathway, there is no direct evidence for the flux of excessive placental androgens into the fetal circulation and for the production of dihydrotestosterone (DHT) via the backdoor pathway. We examined longitudinal serum and urine steroid metabolite profiles in a 46,XY infant with PORD who was prenatally identified because of the progressive fetal masculinization and maternal virilization from the mid-gestation and the presence of fetal radio-humeral synostosis and was confirmed to have compound heterozygous mutations of POR (p.Q201X and p.R457H). The results showed (1) markedly and inappropriately elevated serum androstenedione and testosterone (T) values at birth, (2) a markedly increased serum DHT value with a normal DHT/T ratio at birth, (3) transient elevation of serum T and DHT values accompanied by a normal DHT/T ratio and concomitant elevations of intermediate steroid metabolites on both the frontdoor and backdoor pathways at 30 days of age, and (4) persistent PORD-compatible urine steroid profiles. Although the data obtained from a single infantile patient are too premature to be generalized, they imply: (1) the transfer of excessive placental androgens into the fetal as well as the maternal circulations from the mid-gestation, (2) lack of a clinically discernible amount of DHT production via the adrenal backdoor pathway around birth, and (3) the activation of both the frontdoor and backdoor pathways in the testis around the mini-puberty, with no production of a clinically discernible amount of DHT via the testicular backdoor pathway.

Androgen production in pediatric adrenocortical tumors may occur via both the classic and/or the alternative backdoor pathway.

Children with adrenocortical tumors (ACTs) often present with virilization due to high tumoral androgen production, with dihydrotestosterone (DHT) as most potent androgen. Recent work revealed two pathways for DHT biosynthesis, the classic and the backdoor pathway. Usage of alternate routes for DHT production has been reported in castration-resistant prostate cancer, CAH and PCOS. To assess whether the backdoor pathway may contribute to the virilization of pediatric ACTs, we investigated seven children suffering from androgen producing tumors using steroid profiling and immunohistochemical expression studies. All cases produced large amounts of androgens of the classic and/or backdoor pathway. Variable expression of steroid enzymes was observed in carcinomas and adenomas. We found no discriminative pattern. This suggests that enhanced androgen production in pediatric ACTs is the result of deregulated steroidogenesis through multiple steroid pathways. Thus future treatments of ACTs targeting androgen overproduction should consider these novel steroid production pathways.

Genes and proteins of the alternative steroid backdoor pathway for dihydrotestosterone synthesis are expressed in the human ovary and seem enhanced in the polycystic ovary syndrome.

Recently, dihydrotestosterone biosynthesis through the backdoor pathway has been implicated for the human testis in addition to the classic pathway for testosterone (T) synthesis. In the human ovary, androgen precursors are crucial for estrogen synthesis and hyperandrogenism in pathologies such as the polycystic ovary syndrome is partially due to ovarian overproduction. However, a role for the backdoor pathway is only established for the testis and the adrenal, but not for the human ovary. To investigate whether the backdoor pathway exists in normal and PCOS ovaries, we performed specific gene and protein expression studies on ovarian tissues. We found aldo-keto reductases (AKR1C1-1C4), 5α-reductases (SRD5A1/2) and retinol dehydrogenase (RoDH) expressed in the human ovary, indicating that the ovary might produce dihydrotestosterone via the backdoor pathway. Immunohistochemical studies showed specific localization of these proteins to the theca cells. PCOS ovaries show enhanced expression, what may account for the hyperandrogenism.

Steroidogenic pathways involved in androgen biosynthesis in eumenorrheic women and patients with polycystic ovary syndrome.

The conventional Δ5 and Δ4 steroidogenic pathways mediate androgen production in females. While multiple non-conventional pathways to dihydrotestosterone (DHT) have recently been postulated in humans, the functional significance of these pathways remains to be elucidated. The aim of this study was to clarify the origin of androgens in healthy women and in patients with polycystic ovary syndrome (PCOS), a multifactorial disorder characterized by androgen overproduction. We measured 13 steroids in blood samples of 31 eumenorrheic females and 28 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay. We found that 17-hydroxy (17-OH) progesterone (17-OHP), androstenedione (Δ4A), testosterone, androstanedione, androsterone, and androstanediol levels were higher in the patient group than in the eumenorrheic group, while levels of other steroids were comparable between the two groups. In the eumenorrheic group, DHT levels were correlated with testosterone, androstanedione, and androstanediol. Quantitative correlations were also observed among 17-OH allopregnanolone, androsterone, androstanediol, and DHT, and among Δ4A, androstanedione, androsterone, and androstanediol. In the patient group, DHT levels were correlated with testosterone levels, but not with androstanedione or androstanediol levels. Δ4A and testosterone paralleled 17-OHP. Androstanedione, androsterone, androstanediol, and 17-OH allopregnanolone were quantitatively correlated. In both groups, multivariable linear regression analyses suggested relationships between androsterone and androstanedione, as well as between androsterone and 17-OH allopregnanolone. These results indicate that multiple androgen biosynthesis pathways are operating in eumenorrheic females and PCOS patients. In PCOS patients, excessive androgens are produced primarily via the conventional pathways, while two alternative pathways; i.e., an androstanedione-mediated pathway and a so-called backdoor pathway, likely serve as sources of a weak androgen and potential precursors of DHT.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies.

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Regulation of local steroidogenesis in the brain and in prostate cancer: lessons learned from interdisciplinary collaboration.

Sex steroids play critical roles in the regulation of the brain and many other organs. Traditionally, researchers have focused on sex steroid signaling that involves travel from the gonads via the circulation to intracellular receptors in target tissues. This classic concept has been challenged, however, by the growing number of cases in which steroids are synthesized locally and act locally within diverse tissues. For example, the brain and prostate carcinoma were previously considered targets of gonadal sex steroids, but under certain circumstances, these tissues can upregulate their steroidogenic potential, particularly when circulating sex steroid concentrations are low. We review some of the similarities and differences between local sex steroid synthesis in the brain and prostate cancer. We also share five lessons that we have learned during the course of our interdisciplinary collaboration, which brought together neuroendocrinologists and cancer biologists. These lessons have important implications for future research in both fields.

Roles for the backdoor pathway of androgen metabolism in prostate cancer response to castration and drug treatment.

Almost all men who present with advanced prostate cancer (CaP) and many men who fail potentially curative therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. The goal of this review is to describe the evolution of adrenal androgen blockade, how new androgen measurement methods have furthered understanding of androgen metabolism, and how further understanding of the backdoor pathway of androgen metabolism may lead to interventions that extend survival even more.

Assessment of steroidogenesis and steroidogenic enzyme functions.

There is some confusion in the literature about steroidogenesis in endocrine glands and steroidogenesis in peripheral intracrine tissues. The objective of the present review is to bring some clarifications and better understanding about steroidogenesis in these two types of tissues. Concerns about substrate specificity, kinetic constants and place of enzymes in the pathway have been discussed. The role of 17α-hydroxylase/17-20 lyase (CYP17A1) in the production of dehydroepiandrosterone and back-door pathways of dihydrotestosterone biosynthesis is also analyzed. This article is part of a Special Issue entitled "Synthesis and biological testing of steroid derivatives as inhibitors".