Progesterone synthesis by the human placenta

One of the essential roles of the human placenta is to produce the steroid hormone progesterone, which is required for the maintenance of pregnancy. The rate-determining step of placental progesterone synthesis is the conversion of cholesterol to pregnenolone by cytochrome P450scc (CYP11A1) in placental mitochondria in a reaction requiring electrons delivered via adrenodoxin reductase and adrenodoxin. Pregnenolone is converted to progesterone by type 1 3beta-hydroxysteroid dehydrogenase located in the mitochondrion. Progesterone synthesis by the human placenta displays notable differences from steroid synthesis in the classical steroid producing tissues such as the adrenal cortex and corpus luteum. One important difference is that the placenta lacks short term modulation of steroid synthesis and does not express the steroidogenic acute regulatory (StAR) protein. The most notable difference between the placenta and other steroidogenic tissues is that electron supply to P450scc limits the rate at which cholesterol is converted to pregnenolone in the placenta. The limiting component for electron delivery to P450scc is the concentration of adrenodoxin reductase in the mitochondrial matrix which is insufficient to maintain the adrenodoxin pool in a fully reduced state. Evidence suggests that placental mitochondria have a near-saturating cholesterol concentration for P450scc, likely provided by the StAR-like protein MLN64, and cholesterol translocation to the P450scc is not a major site of regulation of progesterone synthesis. Cyclic AMP stimulates progesterone synthesis by the human placenta but uncertainty remains regarding the key hormones that control cyclic AMP levels. The mechanism of regulation of adrenodoxin reductase levels in the human placenta remains to be studied.     

Expression of messenger ribonucleic acid encoding steroidogenic enzymes in postmenopausal ovaries

To investigate expression of steroidogenic enzymes involved in androgen and estrogen synthesis in the stroma of postmenopausal ovaries.Ovarian stromal tissue samples were obtained at hysterectomy from postmenopausal women who had hysterectomy and oophorectomy. Tissues were frozen immediately in liquid nitrogen and kept frozen until RNA was extracted. Total RNA was examined by Northern blot analysis using (32)P-labeled cDNA probes encoding human P450 side chain cleavage (P450(SSC)), P450(17alpha), and cytochrome P450 aromatase (P450(arom)). Concentrations of mRNA encoding cytochrome P450(SSC), cytochrome P450 17alpha-hydroxylase (CYP17), and P450(arom) were determined in the ovarian stroma.We detected P450(SSC) mRNA in all postmenopausal ovaries studied. P450(SSC) mRNA was increased threefold in the ovarian stroma of postmenopausal women with endometrial cancer and endometrial hyperplasia. CYP17 mRNA was detected in the ovarian stroma of all women with endometrial cancer. P450(arom) mRNA was not detected in the ovaries studied.Postmenopausal ovaries possess enzymes for initiation of steroidogenesis. Enzymes essential for androgen synthesis were expressed in the ovarian stroma of postmenopausal women with endometrial cancer or hyperplasia.     

Congenital Imperforate Hymen with Hydrocolpos and Hydronephrosis associated with Severe Hydramnios and Increase of Maternal Ovarian Steroidogenic Enzymes

Study ObjectiveTo study clinical features of patient presented with severe hydramnios, associated with hydronephrosis, that was antenatally diagnosed and has been successfully treated immediately after birth. At a molecular level, we investigated the gene expression of key steroidogenic enzymes from the maternal ovary.DesignUltrasound scan, MRI, semi-quantitative RT-PCRSettingThe patient was admitted to the University Hospital, University of Crete, Medical School, Greece, where all clinical data has been obtained. Gene expression studies took place at Biosciences, Brunel University, UK.ResultsSemi-quantitative RT-PCR analyses revealed that there is upregulation of key steroidogenic genes in the maternal ovary, including steroidogenic acute regulatory protein, and the cytochrome P450 heme-containing proteins CYP11A, CYP17 and CYP19. From a clinical perspective, the prenatal ultrasound scan and MRI findings showed a multicystic pelvic mass, bilateral hydronephrosis and prior to delivery severe polyhydramnios.ConclusionThis clinical case is the only one that we have found in the current literature where congenital imperforate hymen accompanied with hematocolpos is associated with renal obstruction in combination with polyhydramnios and increase in maternal steroidogenic enzymes.     

Effect of ketoconazole on steroidogenic human granulosa-luteal cells in culture

Ketoconazole (KCZ), a widely used antifungal drug, has been reported in humans to inhibit adrenal and testicular steroidogenesis by interfering with the cytochrome P-450-dependent enzymes. The purpose of this study was to investigate the drug effect on steroidogenic human granulosa-luteal cells, obtained by follicular aspiration from mature follicles of gonadotropin-treated women. Cells were cultured in long-term monolayers, and the steroid production was assayed by radioimmunoassay. A profound inhibition of ovarian cell secretion of progesterone (P), testosterone (T) and estradiol was found. At a low concentration (5 micrograms/ml), KCZ failed to inhibit the conversion of pregnenolone to P, mediated by the non-cytochrome 3 beta-hydroxysteroid dehydrogenase-isomerase enzyme (3 beta-HSDH). At a similar concentration, P secretion by human chorionic gonadotropin (hCG; 100 mIU/ml) -treated cells was decreased by 68% (P less than 0.001) and therefore, an inhibitory effect of KCZ on the cholesterol side-chain cleavage enzyme (P-450SCC) was assumed. A similar marked inhibitory effect (81%) (P less than 0.001) on T secretion was observed for hCG-stimulated cells given pregnenolone as substrate. The P-450 aromatase was profoundly inhibited (86%) (P less than 0.001) in a reversible manner, by a similar concentration (5 micrograms/ml) of KCZ. These findings suggest that KCZ has the capability to suppress human ovarian steroidogenesis similarly as in testis and adrenal.     

Paraquat inhibits progesterone synthesis in human placental mitochondria

IntroductionHuman placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis.MethodsPlacentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography.ResultsPQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin.DiscussionWe conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.     

Disorders of androgen biosynthesis

Disorders of androgen biosynthesis are a relatively rare cause of sexual ambiguity in 46,XY genetic males, but genetic disorders characterized by excessive androgen synthesis are a common cause of virilization in 46,XX genetic females. Understanding of these disorders is relatively simple if one is familiar with the components of the various steroidogenic pathways. The biosynthesis of androgens requires the steroidogenic acute regulatory protein (StAR) and the steroidogenic enzymes P450scc, P450c17, 3betaHSDII, 17betaHSDIII, and 5alpha-reductase. Deficiencies have been described in all of these, leading to male pseudohermaphroditism. Other steroidogenic enzymes not involved in androgen biosynthesis, such as P450c21, P450c11beta, and P450aro, must also be considered, as mutations in these can result in overproduction of androgens, resulting in female pseudohermaphroditism.     

Metformin directly inhibits androgen production in human thecal cells

OBJECTIVE: To examine the direct effect of metformin on thecal cell androgen production. SETTING: Basic science research laboratory, University of Texas Southwestern, Dallas, Texas. INTERVENTION(S): Human ovarian theca-like tumor cells were treated with various concentrations of metformin in the presence and absence of forskolin for 48 hours. MAIN OUTCOME MEASURE(S): Media were collected, and radioimmunoassay (RIA) for progesterone, 17 alpha-hydroxyprogesterone (17OHP), androstenedione, and testosterone was performed. The effect of metformin on the expression of various enzymes involved in theca cell steroidogenesis was examined. RESULT(S): Metformin (50 microM and 200 microM) significantly inhibited androstenedione production from both forskolin-stimulated and unstimulated theca cells. Testosterone production was also significantly inhibited in forskolin-treated cells in the presence of 200 microM of metformin-treated compared with forskolin-only-treated cells. Western blot analysis revealed that metformin significantly inhibited the expression of steroidogenic acute regulatory (StAR) protein and 17 alpha-hydroxylase (CYP17) expression in cells stimulated with forskolin compared with forskolin treatment alone. There was no significant change in either 3beta-hydroxysteroid dehydrogenase (3 beta HSD) or cholesterol side-chain cleavage (CYP11A1) protein expression. Northern analysis revealed a significant decrease in the expression of CYP17 mRNA in forskolin-stimulated cells treated with metformin (200 microM) compared with forskolin-only-treated cells, however, there was no significant change in steroidogenic acute regulatory protein mRNA expression.CONCLUSION(S): Our results suggest that metformin may have a direct effect on thecal cells' androgen production.     

A trypsin-sensitive protein is required for utilization of exogenous cholesterol for pregnenolone synthesis by placental mitochondria

The utilization of cholesterol for steroid hormone synthesis by human placental mitochondria is poorly understood. The human placenta does not express the steroidogenic acute regulator protein, which is critical for cholesterol delivery to the cholesterol side chain cleavage system in adrenal and gonadal mitochondria. We explored the mechanism underlying cholesterol transport in human placental mitochondria by measuring its transformation into pregnenolone. Mitochondria of syncytiotrophoblast from human term placenta were isolated by centrifugation through a sucrose gradient. The synthesis of pregnenolone in the presence of exogenous cholesterol was increased two-fold in syncytiotrophoblast mitochondria. Treatment of mitochondria with trypsin prevented the increase in the synthesis of pregnenolone in the presence of exogenous cholesterol. However, when 22-OH cholesterol, a substrate that readily crosses membranes, was added, the trypsin-treated mitochondria synthesized increased amounts of pregnenolone. The trypsin-treated mitochondria were intact, since oxygen consumption, succinate dehydrogenase and the adenine nucleotide translocase activities were not significantly different from in untreated mitochondria. However, activity of NADH cytochrome c oxidoreductase, an outer mitochondrial membrane enzyme, was reduced in the trypsin-treated mitochondria, reflecting the selective degradation of proteins. In addition, SDS-PAGE analysis revealed the loss of a prominent 34 kDa band which proved to be a novel porin-like protein that binds to cholesterol. These results support our previous assumption that human placental mitochondria employ a novel protein(s)-mediated the mechanism to take up cholesterol for steroidogenesis.     

Importance of CYP1A1 polymorphism and its transcriptional regulation in ovarian and endometrial cancer

Human cytochrome P450 1A 1 is one of the most important enzymes participating in human carcinogenesis because it metabolites several procarcinogens to active carcinogenic metabolites. Additionally enzymes of CYP450 family play an important role in estrogenes catabolization (17-beta-estradiol and estron) to intermediate products (2-, 4-hydroxyestradiol and 2-, 4-hydroxyestrone) including CYP1A1 that catalyses hydroxylation to 2-hydroxyestrogens in the endometrium. Derivates of these compounds (4-hydroxyestrogens) are carcinogenic and could induce DNA damage leading to tumour transformation. The presence of CYP1A1 enzyme in genital tract tissues could induce chemical carcinogenesis initiating cancer development. Recent studies also confirmed the role of CYP1A1 in the development of ovarian and endometrial cancer in humans. The presence of mutated CYP1A1 polymorphic variants influencing the CYP1A 1 activity could be responsible for different interindividual susceptibility to genital cancers in women.     

Ovarian enzyme activities in women with polycystic ovary syndrome

Studies using primary ovarian tissue and cultured human theca and granulosa cells have shown that steroidogenic enzyme activities are up-regulated in theca cells in polycystic ovary syndrome (PCOS). Although granulosa cells in arrested follicles in PCOS fail to express significant amounts of aromatase, there is an overexpression of 5alpha-reductase activity and premature expression of cholesterol side-chain cleavage cytochrome P450.     

Association of endometriosis risk and genetic polymorphisms involving biosynthesis of sex steroids and their receptors: an updating meta-analysis

The objective of our study is to assess the association of endometriosis risk and genetic polymorphisms involving biosynthesis of sex steroids and their receptors. A systematic search of three databases was conducted. Twenty-seven studies on the association of the cytochrome P450 subfamily 17 (CYP17), estrogen receptor gene (ER), progesterone receptor gene (PR), 17-beta-hydroxysteroid dehydrogenase type 1 gene (HSD17B1), and cytochrome P450 subfamily 19 (CYP19) polymorphisms with endometriosis risk were identified. When all groups were pooled, we found an association between HSD17B1 (A variant allele vs. G wild allele: odds ratio (OR)=1.42, 95% confidence interval (CI)=1.10-1.84, P=0.007) and PR (P2 variant allele vs. P1 wild allele, OR=1.43, 95% CI=0.99-2.08, P=0.058) polymorphisms and endometriosis risk, while failing to detect links with CYP17, ER, and CYP19 polymorphisms examined. In the subgroup analysis, a significant association of CYP17 and ERα-PvuII polymorphisms with endometriosis was found neither in a Caucasian population nor in an Asian population. The findings of our study suggest that HSD17B1 and PR polymorphisms are associated with an increased risk of endometriosis. Further investigation into the association between CYP17, ER, PR, HSD17B1, and CYP19 polymorphisms and endometriosis risk is warranted and should include larger sample sizes.     

Congenital adrenal hyperplasia: 11beta-hydroxylase deficiency

The most potent corticosteroids are 11beta-hydroxylated compounds. In humans, two cytochrome P450 isoenzymes with 11beta-hydroxylase activity, catalyzing the biosynthesis of cortisol and aldosterone, are present in the adrenal cortex. CYP11B1, the gene encoding 11beta-hydroxylase (P450c11), is expressed in high levels in the zona fasciculata and is regulated by adrenocorticotropic hormone (ACTH). CYP11B2, the gene encoding aldosterone synthase (P450c11Aldo), is expressed in the zona glomerulosa under primary control of the renin-angiotensin system. The substrate for P450c11 is 11-deoxycortisol. Mutations in CYP11B1 cause congenital adrenal hyperplasia (CAH) due to 11beta-hydroxylase deficiency. This disorder is characterized by androgen excess and hypertension and is autosomal recessively inherited. Classical and nonclassical forms of 11beta-hydroxylase deficiency can be distinguished. Studies in heterozygotes for classical 11beta-hydroxylase deficiency show inconsistent results with no or only mild hormonal abnormalities (elevated plasma levels of 11-deoxycortisol after ACTH stimulation). Molecular genetic studies of the CYP11B1 gene in 11beta-hydroxylase deficiency have led to the identification of several mutations. Transfection experiments showed loss of enzyme activity in vitro. Molecular genetic studies have practical importance for the prenatal diagnosis of virilizing CAH forms.     

Ovarian and adrenal contributions to peripheral steroid levels in postmenopausal women.

Serum levels of cortisol (F), pregnenolone (delta5-P), 17-hydroxypregnenolone (17-delta5-P), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), progesterone (P), 17-hydroxyprogesterone (17-P), androstenedione (A), testosterone (T), dihydrotestosterone (DHT), androst-5-ene-3beta, 17beta-diol (delta5-diol), estrone (E1), and estradiol-17beta (E2) were measured in 10 postmenopausal and 5 premenopausal women before (control) and after 7 days of Dexamethasone (post-dex). Control and post-dex levels of delta5-P, P, DHT, T, and F were not different (P greater than 0.05) in premenopausal versus postmenopausal women, while the control levels of 17-P, DHEA, DHEA-S, A, delta5-diol, and E2 were lower in the postmenopausal women (P less than 0.05). Assuming post-dex levels equal ovarian contribution, the ovarian contribution of 17-delta5-P, 17-P, DHEA, delta5-diol, A, E1, and E2 and the adrenal contribution (control-post-dex) to DHEA, DHEA-S, 17-P, A, and delta5-diol was significantly lower (P less than 0.05) in postmenopausal subjects.     

Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice

BackgroundFormaldehyde, a ubiquitous environmental pollutant, is used extensively and has been proved to impair male reproduction in mammals. However, no trials have explored whether formaldehyde affects sexual function.AimTo evaluate the effect of long-term formaldehyde exposure on sexual behavior and to investigate the potential mechanism.MethodsForty C57BL/6 male mice were randomly allocated to four equally sized groups. Mice were exposed to formaldehyde at a dose of 0 (control), 0.5, 5.0, or 10.0 mg/m³ by inhalation for 60 days.OutcomesSexual behavior, body and reproductive organ weights, testosterone concentration in serum and testicular tissue, expression of steroidogenic enzymes, quality of sperm, and testicular structure were measured.ResultsFormaldehyde inhibited sexual behavior and decreased reproductive organ weights in mice. Serum testosterone levels and intratesticular testosterone concentrations were decreased in the formaldehyde-treated groups. Expression levels of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase (3β-HSD), also were decreased in the testes of mice exposed to formaldehyde. Moreover, the structure of seminiferous tubules was destroyed and sperm quality decreased after formaldehyde exposure. In addition, the results indicated that the effects of formaldehyde were dose dependent.Clinical ImplicationsEfforts should be undertaken to decrease impairment of sexual function caused by formaldehyde exposure.Strengths and LimitationsThe relatively small sample might have affected the outcomes. Further experiments are needed to study the mechanism of action of formaldehyde.ConclusionExposure to formaldehyde gas inhibited sexual behavior, caused reproductive organ atrophy, and impaired spermatogenesis in male mice, which might have been induced by suppressed expression of steroidogenic enzymes in Leydig cells and decreased testosterone synthesis.Zang Z-J, Fang Y-Q, Ji S-Y, et al. Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice. J Sex Med 2017;14:1297–1306.     

Molecular analysis of WNT4 gene in four adolescent girls with mullerian duct abnormality and hyperandrogenism (atypical Mayer-Rokitansky-Küster-Hauser syndrome)

In a collaborative study, we investigated four 46,XX adolescent girls with Mayer-Rokitansky-Küster-Hauser syndrome and hyperandrogenism. Molecular analysis of the WNT4 gene permitted us to identify a new mutation (p.A233T). Functional studies revealed partial repression of steroidogenic enzymes (normal repression of HSD3B2) contrasting with the abnormal reexpression of CYP17A1 enzyme in the OVCAR3 cell line. This fourth new WNT4 mutation confirms that this signaling molecule is involved in mullerian development and androgen biosynthesis repression in the ovary. Interestingly, this mutant partially lacks the capability to repress ovarian steroidogenic enzymes, with abnormal expression of 17α- hydroxylase.     

Peripheral steroid levels in a patient with congenital absence of the uterus.

In a patient with congenital absence of the uterus, daily measurements of progesterone (P), 17-hydroxyprogesterone (17-P), pregnenolone (delta5P), 17-hydroxypregnenolone (17-delta5P), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), testosterone (T), dihydrotestosterone (DHT), androstenedione (A), estrone (E1), estradiol-17beta (E2), and cortisol (F) were performed over a period of 48 consecutive days. Serum P, 17-P, and E2 determinations revealed normal, cyclical ovarian activity consisting of two luteal phases and one follicular phase. Serum E1, delta5P, and DHEA-S levels were normal throughout the study, whereas DHEA and DHT levels were persistently elevated. Serum levels of 17-delta5P and A showed peak levels above normal. Serum T and F concentrations were normal during the initial part of the study, but some elevation was seen during the second luteal phase. In the patient studied, absence of the uterus did not seem to interfere with cyclic ovarian activity. The role of the uterus in the metabolism of androgens remains subject to further investigation.     

Dysregulation of cytochrome P450c 17 alpha as the cause of polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) appears to be due to a previously unrecognized type of steroidogenic abnormality, one in which hyperandrogenism arises from a regulatory abnormality (dysregulation) rather than from enzyme deficiency. It appears that PCOS typically arises from masculinized regulation of the androgen-forming enzyme (cytochrome P450c17 alpha) within ovarian thecal cells. This may arise by either excessive stimulation by luteinizing hormone (LH) or by escape from desensitization to LH. We review evidence which is compatible with the concept that the latter situation may result from an intrinsic intraovarian flaw in the paracrine feedback mechanism by which thecal androgen biosynthesis is inhibited and that coexistent adrenal 17-ketosteroid hyper-responsiveness to corticotropin (ACTH) may be due to a similar type of dysregulation of adrenocortical P450c17 alpha.