Both DHEA and GABAA receptor agonists are known to reduce anxiety. Since GABAA receptor agonists are generally thought to elicit their anxiolytic effects by facilitating neuronal uptake of chloride ion, we set out to evaluate whether DHEA elicits its anxiolytic effects by a similar mechanism. The results of the studies show an uneven distribution of basal and GABA-stimulated chloride uptake in different regions (cerebellum, pons-medulla, striatum, hippocampus, mid-brain, hypothalamus and cortex) of rat brain. Contrary to our expectations, however, both DHEA and DHEAS inhibited GABA-mediated chloride uptake with DHEAS being more potent than DHEA. On the other hand, delta <em>4</em>-<em>androstenedione</em>, another DHEA metabolite, did not have any effect on chloride uptake in any region of the brain. In conclusion, the data presented here, therefore, suggest that DHEA and DHEAS may elicit anxiolysis through mechanisms independent of GABAA receptor-mediated facilitation of neuronal chloride uptake.

Increasing evidence shows that estrogens are involved in lung cancer proliferation and progression, and most human lung tumors express estrogen receptor β (ERβ) as well as aromatase. To determine if the aromatase inhibitor anastrozole prevents development of lung tumors induced by a tobacco carcinogen, alone or in combination with the ER antagonist fulvestrant, ovariectomized female mice received treatments with the tobacco carcinogen <em>4</em>-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) along with daily supplements of <em>androstenedione</em>, the substrate for aromatase. Placebo, anastrozole and/or fulvestrant were administered in both an initiation and a promotion protocol of lung tumorigenesis. The combination of fulvestrant and anastrozole given during NNK exposure resulted in significantly fewer NNK-induced lung tumors (mean = 0.5) compared with placebo (mean = <em>4</em>.6, P < 0.001), fulvestrant alone (mean = 3.<em>4</em>, P < 0.001) or anastrozole alone (mean = 2.8, P = 0.002). A significantly lower Ki67 cell proliferation index was also observed compared with single agent and control treatment groups. Beginning antiestrogen treatment after NNK exposure, when preneoplastic lesions had already formed, also yielded maximum antitumor effects with the combination. Aromatase expression was found mainly in macrophages infiltrating preneoplastic and tumorous areas of the lungs, whereas ERβ was found in both macrophages and tumor cells. Antiestrogens, especially in combination, effectively inhibited tobacco carcinogen-induced murine lung tumorigenesis and may have application for lung cancer prevention. An important source of estrogen synthesis may be inflammatory cells that infiltrate the lungs in response to carcinogens, beginning early in the carcinogenesis process. ERβ expressed by inflammatory and neoplastic epithelial cells in the lung may signal in response to local estrogen production.

BACKGROUND

The current study was conducted to determine the effect of goserelin and bicalutamide on progression-free survival (PFS) in patients with epithelial ovarian cancer who were in second or greater complete disease remission.

METHODS

Patients received bicalutamide at a dose of 50 mg orally daily and goserelin at a dose of 3.6 mg subcutaneously every <em>4</em> weeks. CA 125 was obtained monthly, with computed tomography performed every 3 months. Correlative studies included serum luteinizing hormone, follicle-stimulating hormone, vascular endothelial growth factor, free testosterone, and <em>androstenedione</em> and the germline polymorphisms CYP19A1 and androgen receptor.

RESULTS

Between October of 2000 and October of 2002, 35 patients were enrolled. Three patients (9%) received therapy at the time of first disease remission and were removed from the study, and 1 patient (3%) was removed for liver function test abnormalities. The most frequent toxicities were grade 1 alkaline phosphatase (5<em>4</em>%), fatigue (57%), and hot flashes (<em>4</em>2%) based on the National Cancer Institute common toxicity scale, version 2.0. The PFS for patients receiving protocol therapy in second disease remission (21 patients) was 11.<em>4</em> months (95% confidence interval [95% CI], 10.2-12.6 months). The PFS for patients receiving protocol therapy in third or fourth disease remission (11 patients) was 11.9 months (95% CI, 10.8-1<em>4</em>.1 months). The percentage of patients remaining in second disease remission at given times are: 100% at 3 months, 100% at 6 months, 72% at 9 months, <em>4</em>7% at 12 months, 28% at 15 months, 22% at 18 months, 19% at 21 months, and 13% at 2<em>4</em> months. There were no associations noted between androgen receptor repeat number, genotype, allelotype, or haplotypes and PFS.

CONCLUSIONS

The use of goserelin and bicalutamide did not appear to prolong PFS in patients with epithelial ovarian cancer in second or greater complete disease remission. The number of patients in disease remission at given time points may serve as a clinical trial endpoint for future studies of consolidation therapy.

OBJECTIVE

To determine if steroids containing over-the-counter (OTC) dietary supplements conform to the labeling requirements of the 199<em>4</em> Dietary Supplement Health and Education Act (DSHEA).

METHODS

12 brands of OTC supplements containing 8 different steroids were randomly selected for purchase in stores that cater to athletes. There are two <em>androstenediones</em> (<em>4</em>- and 5-androstene-3,17-dione), two androstenediols (<em>4</em>- and 5-androstene-3beta, 17beta-diol), and <em>4</em> more are 19-nor cogeners (19-nor-<em>4</em>- and 5-androstene-3,17-dione and 19-nor-<em>4</em>- and 5-androstene-3beta, 17beta-diol).

METHODS

12 brands of OTC anabolic-androgenic supplements were analyzed by high-pressure liquid chromatography.

RESULTS

We found that 11 of 12 brands tested did not meet the labeling requirements set out in the 199<em>4</em> Dietary Supplement Health and Education Act. One brand contained 10 mg of testosterone, a controlled steroid, another contained 77% more than the label stated, and 11 of 12 contained less than the amount stated on the label.

CONCLUSIONS

These mislabeling problems show that the labels of the dietary steroid supplements studied herein cannot be trusted for content and purity information. In addition, many sport organizations prohibit OTC steroids; thus, athletes who use them are at risk for positive urine test results. In this article we provide the details of the analyses, a summary of the steroids by name and structure, and information on the nature of the positive test results. Athletes and their physicians need this information because of the potential medical consequences and positive urine test results.

OBJECTIVE

To compare plasma androgen concentrations in women who have recurrent miscarriages and in fertile women, and to correlate the results with concentrations of the endometrial protein PP1<em>4</em> in uterine flushings and plasma from women who have recurrent miscarriages.

METHODS

Retrospective study.

METHODS

Hospital research unit.

METHODS

Women attending a recurrent miscarriage clinic and normal fertile volunteers. Ten of the women with recurrent miscarriages had polycystic ovary disease (PCOD) as assessed by ultrasonography or increased follicular LH levels.

METHODS

Plasma samples were obtained from the women on days LH-7, LH-<em>4</em>, LH+0, and LH+7 or LH+10 of a cycle. An endometrial flushing sample and a biopsy specimen were taken from women with recurrent miscarriages on day LH+7 or LH+10.

METHODS

Androstenedione, testosterone, and sex hormone-binding globulin (SHBG) were measured in the plasma samples. The endometrial protein PP1<em>4</em> was measured in the uterine flushings and in the LH+7 or LH+10 plasma samples from the women with recurrent miscarriages.

RESULTS

Testosterone concentrations were higher in the women with recurrent miscarriages both with and without PCOD on days LH-7 and LH-<em>4</em> of the cycle. Concentrations of androstenedione also were higher in the women with recurrent miscarriages, but without PCOD on day LH-7. Testosterone SHBG ratios were higher in the women with recurrent miscarriages, without PCOD compared with the controls on days LH-7, LH+0, and LH+7. Mean follicular testosterone concentrations were correlated negatively with both uterine (r = -0.<em>4</em>7) and plasma (r = -0.<em>4</em>9) PP1<em>4</em> levels on day LH+10. Mean luteal phase testosterone SHBG ratios were correlated negatively with uterine PP1<em>4</em> concentrations on day LH+7 of the cycle (r = -0.67<em>4</em>).

CONCLUSIONS

Androgen levels are higher in women who have recurrent miscarriages than in normal fertile controls. These high levels of androgens may have a detrimental effect on endometrial function.

Dopamine is known to play a role in the modulation of aldosterone and catecholamine secretion from the adrenal gland, where dopamine receptors (DR), in particular the DR type 2 (D(2)), have been found to be expressed. DR expression has also been demonstrated in some types of benign adrenal tumors. The aims of the current study were to evaluate DR expression and D(2) localization in the normal adrenal gland and in different types of benign and malignant adrenal tumors, as well as to evaluate the in vitro effects of the dopamine agonists bromocriptine and cabergoline on hormone secretion in nontumoral adrenal cells. Adrenal tissues from 25 patients, subjected to adrenal surgery for different diseases, were studied. These included three normal adrenals; five adrenal hyperplasias; four aldosterone-secreting, two cortisol-secreting, and two clinically nonfunctioning adrenal adenomas; two aldosterone-secreting, two cortisol-secreting, and two androgen-secreting adrenal carcinomas; and three pheochromocytomas. In all tissues, DR and D(2) isoform (D(2long) and D(2short)) expression was evaluated by RT-PCR. D(2) localization was also evaluated by immunohistochemistry using a specific polyclonal antibody, whereas D(2)-like receptor expression was evaluated by receptor-ligand binding study, using the radiolabeled D(2) analog (125)I-epidepride. The effects of bromocriptine and cabergoline on baseline and ACTH and/or angiotensin II-stimulated aldosterone, cortisol, and <em>androstenedione</em> secretion were evaluated in cell cultures derived from five different adrenal hyperplasia. At RT-PCR, both D(1)-like and D(2)-like receptors were expressed in all normal and hyperplastic adrenals. D(2) and D(<em>4</em>) were expressed in aldosterone- and cortisol-secreting adenomas, cortisol-secreting carcinomas, and clinically nonfunctioning adenomas, whereas no DR was expressed in aldosterone- and androgen-secreting carcinomas. D(2), D(<em>4</em>), and D(5) were expressed in pheochromocytomas. In all D(2)-positive tissues, both D(2) isoforms were expressed, with the exception of one case of aldosterone-secreting adenoma and the cortisol-secreting carcinomas, in which only the D(2long) isoform was expressed. D(2)-like receptor expression was confirmed at receptor-ligand binding study. At immunohistochemistry, D(2) was mainly localized in the zona glomerulosa and reticularis of the adrenal cortex and, to a lesser extent, in the zona fasciculata and medulla of normal and hyperplastic adrenal tissue. In the positive tumors, D(2) was localized in the tumoral cells. At the in vitro study, a significant inhibition of both baseline and ACTH-stimulated aldosterone secretion was found after high-dose cabergoline, but not bromocriptine, administration; and a significant inhibition of angiotensin-II-stimulated aldosterone secretion was found after both bromocriptine and cabergoline administration in the adrenal hyperplasias. In conclusion, the current study demonstrated that both D(1)-like and D(2)-like receptors are expressed in the normal adrenal gland and in a percentage of adrenal adenomas or carcinomas. Bromocriptine and cabergoline induce only a minor inhibition of the secretion of adrenal hormones in the nontumoral adrenal gland in vitro, not excluding, however, the possible effective use of dopamine agonists in vivo in the treatment of adrenal tumors.

LH and insulin are postulated to jointly stimulate theca-cell androgen biosynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro serum-free monolayer culture system of Percoll-purified, porcine theca cells harvested from immature ovaries. Initial dose and time course analyses revealed that a maximally effective concentration of LH (100 ng/ml) or insulin (100 ng/ml) individually will drive <em>androstenedione</em> production (at 6 to <em>4</em>8 h) by 1.5- to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to <em>4</em>8 h yielding a 3- to <em>4</em>-fold joint effect. Coadministration of LH and insulin can augment theca-cell concentrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.<em>4</em>- to 3.9- and 3.8- to <em>4</em>.1-fold increases at 2<em>4</em> to <em>4</em>8 h, respectively (P < 0.01). Combined LH and insulin stimulation also amplified the nuclear content of intron-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (1<em>4</em>-18 vs. 3-5.5 pmol/microg DNA/12-<em>4</em>8 h) (P < 0.01). A stable exogenous analog of cAMP, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 gene expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insulin on theca-cell <em>androstenedione</em> biosynthesis or CYP17 mRNA and hnRNA expression. In summary, the present in vitro data identify molecular interactions of LH and insulin on StAR and CYP17 gene expression, thus establishing potent signaling interfaces between these distinct hormonal agonists in regulating theca-cell steroidogenesis.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulation in women. Previous studies suggest that the pathogenesis of PCOS may involve interrelated abnormalities of the insulin-like growth factor (IGF) and ovarian steroidogenesis systems. We investigated this hypothesis in fasting serum samples from 1<em>4</em>0 women with PCOS (age, 27.<em>4</em> +/- 0.<em>4</em> yr; body mass index, 26.3 +/- 0.5 kg/m2; mean +/- SEM). IGF-related parameters were also studied in a group of normoovulatory women (n = 26; age, 26 +/- <em>4</em> yr; body mass index, 23.6 +/- <em>4</em>.3 kg/m2). For the PCOS group, the mean testosterone (T) level was 2.5 +/- 0.1 nmol/L, and it was significantly correlated with LH (r = 0.<em>4</em>1; P < 10(-6)), estrone (r = 0.33; P = 0.016), estradiol (r = 0.18; P = 0.0<em>4</em>), and <em>androstenedione</em> (AD; P < 10(-6)), but not with dehydroepiandrosterone sulfate (P = 0.71), a marker of adrenal steroidogenesis. T and AD were also related to total ovarian follicle number and ovarian size, as previously found with normoovulatory women (1). There were no differences between the PCOS subjects and the normoovulatory group for total IGF-I, IGF-II, or IGF-binding protein-3 (IGFBP-3). However, IGFBP-1 levels were significantly decreased in the PCOS group (1.0 +/- 0.2 vs. 7.3 +/- 1.1 ng/mL; P < 0.001) and were inversely correlated with serum insulin levels (r = -0.50; P < 10(-8)). Serum levels of free IGF-I (fIGF-I) were elevated (5.9 +/- 0.3 vs. 2.7 +/- 0.3 ng/mL; P < 0.001) in inverse relation with IGFBP-1 (r = -0.31; P = 0.0<em>4</em>6). Serum fIGF-I levels were related to total follicle number (r = - 0.35; P < 10(-<em>4</em>)) and to the ratio of sex hormone-binding globulin to T (r = -0.23; P = 0.009). However, these relationships were not independent of other variables. Despite the more than 2-fold elevation in fIGF-I levels, significant relationships between fIGF-I and markers of ovarian steroidogenesis (T, AD, estradiol, and estrone) could not be demonstrated. In conclusion, although we confirmed correlations between LH and hyperandrogenemia and have found abnormalities in the IGF system in a large cohort of PCOS subjects, a direct relationship between hyperandrogenism and the IGF system could not be shown. Previous studies suggest that elevated LH and hyperinsulinemia lead to excess ovarian androgen synthesis in PCOS and that the intraovarian IGF system is important for normal follicle development and may be important in the arrested state of follicle development in PCOS. However, the data presented in this cross-sectional study suggest that insulin-related changes in circulating IGFBP-1 and subsequent elevation of fIGF-I reflect insulin resistance and have little enhancing effects on ovarian steroidogenesis in this disorder.

Evidence suggests that hyperinsulinemic insulin resistance may increase serum levels of ovarian androgens and reduce sex hormone-binding globulin (SHBG) levels in humans. The present study was conducted to assess the effect of administration of the biguanide metformin, a drug commonly used in the treatment of diabetes mellitus, on androgen and insulin levels in 2<em>4</em> hirsute patients. The patients selected for the study were obese, with a body mass index higher than 25 kg/m2 and high fasting insulin >> 90 pmol/L) and low SHBG levels (< 30 nmol/L). All patients were given a low calorie diet (1500 Cal/day) and randomized for either metformin administration at a dose of 850 mg or a placebo, twice daily for <em>4</em> months, in a double blind study. In the placebo group, diet resulted in a significant decrease in body mass index (30.8 +/- 1.0 vs. 32.7 +/- 1.5 kg/m2; P < 0.0001), fasting insulin (127 +/- 11 vs. 156 +/- 1<em>4</em> pmol/L; P < 0.01), non-SHBG-bound testosterone (0.19 +/- 0.02 vs. 0.28 +/- 0.03 nmol/L; P < 0.02), <em>androstenedione</em> (5.8 +/- 0.5 vs. 9.0 +/- 1.1 nmol/L; P < 0.03), and 3 alpha-diolglucuronide (8.6 +/- 1.1 vs. 11.7 +/- 1.9; P < 0.005) plasma concentrations and a significant increase in the glucose/insulin ratio (0.0<em>4</em>7 +/- 0.005 vs. 0.035 +/- 0.003; P < 0.001) and plasma concentrations of SHBG (26.0 +/- 3.3 vs. 19.1 +/- 1.9 nmol/L; P < 0.001) and dehydroepiandrosterone sulfate (8.7 +/- 1.5 vs. 8.<em>4</em> +/- 1.3; P < 0.05). Beneficial effects of diet were not significantly different in the patients who were given metformin instead of placebo. These results confirm that weight loss induced by a low calorie diet is effective in improving hyperinsulinemia and hyperandrogenism in obese and hirsute women. With our study design, metformin administration had no additional benefit over the effect of diet.

To investigate the adrenal cause of hyperandrogenism in peri- and postpubertal hirsute women, baseline and ACTH-stimulated serum concentrations of delta 5-17-hydroxypregnenolone (delta 5-17P), dehydroepiandrosterone (DHEA) and its sulfate, 17-hydroxyprogesterone (17-OHP), cortisol, delta <em>4</em>-<em>androstenedione</em>, and testosterone were determined in 116 women with hirsutism or acne of peri- and postpubertal onset with or without menstrual abnormalities. The results were compared with the same steroid concentrations in 30 normal age-matched women. Sixteen of the 116 women with hirsutism whose ACTH-stimulated 17-OHP levels (mean +/- SD, 5<em>4</em>0<em>4</em> +/- 323<em>4</em> ng/dl; normal, 33<em>4</em> +/- 19<em>4</em>) were markedly elevated while their ratios of delta 5-17P to 17-OHP (0.<em>4</em> +/- 0.2; normal, 3.<em>4</em> +/- 1.5) were low were diagnosed as having nonclassical symptomatic 21-hydroxylase deficiency. Seventeen other hirsute women, including 3 siblings, had very high responses of delta 5-17P (2276 +/- 669 ng/dl; normal, 985 +/- 327) and DHEA (2787 +/- 386 ng/dl; normal, 1050 +/- 38<em>4</em>) to ACTH stimulation, with significantly elevated ratios of delta 5-17P to 17-OHP (11 +/- 2.0; normal, 3.<em>4</em> +/- 1.5) and DHEA to delta <em>4</em>-<em>androstenedione</em> (7.5 +/- 2.3; normal, <em>4</em>.6 +/- 1.5). In these hirsute women, the morning serum delta 5-17P and DHEA concentrations were elevated, had a diurnal variation, and were suppressed with dexamethasone administration. We propose that partial adrenal 3 beta-hydroxysteroid dehydrogenase deficiency is the cause of hirsutism in these women. This may represent an allelic variant at the genetic locus for 3 beta-hydroxysteroid dehydrogenase deficiency similar to that reported for symptomatic nonclassical 21-hydroxylase deficiency producing peripubertal excess androgen syndrome.

A selective inhibitor of aromatase is widely sought for the treatment of postmenopausal women with breast cancer. CGS 169<em>4</em>9A has been shown to be a highly selective, potent inhibitor of aromatase in vitro. Its potency as an oestrogen suppressant and its selectivity were examined by treating 2<em>4</em> postmenopausal patients with advanced breast cancer for <em>4</em> weeks with doses of 0.3, 1.0 and 2.0 mg twice daily. The study was conducted in two parts which compared the two lower doses and the two higher doses separately in a cross-over design protocol. All doses significantly suppressed serum oestradiol and oestrone levels below pretreatment levels. Cross-over analysis indicated that the 2.0 mg twice daily dose achieved significantly greater suppression of oestradiol levels than 0.1 mg twice daily but there was no significant differences between any of the doses in the suppression of oestrone. No significant effects were noted on serum levels of LH, FSH, SHBG, prolactin, testosterone, <em>androstenedione</em>, 17-hydroxyprogesterone or cortisol. For the four steroids this was true both for basal samples and those collected after Synacthen stimulation. However, serum aldosterone levels were significantly suppressed by 1.0 mg twice daily CGS 169<em>4</em>9A and further suppressed by 2.0 mg twice daily. It is concluded that CGS 169<em>4</em>9A is a potent oestrogen suppressant in postmenopausal patients but that its effect is not totally selective.

To obtain direct evidence for FSH-stimulated paracrine signaling in the ovary, 21-day-old intact or hypophysectomized female Wistar rats received four sc injections of recombinant human FSH (rhFSH; total dose, 16-72 IU) at 12-h intervals. Ovaries were removed <em>4</em>8 h after the first injection to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P<em>4</em>50c17 alpha) mRNA or to isolate thecal/interstitial cells for assessment of basal and hLH-responsive androgen synthesis in vitro. In situ hybridization with a 35S-labeled cytochrome P<em>4</em>50c17 alpha cRNA probe confirmed that expression of the cytochrome P<em>4</em>50c17 alpha gene was specific to thecal/interstitial cells. The approximately 2.0-kilobase P<em>4</em>50c17 alpha mRNA signal in ovarian total RNA from intact animals was increased approximately 5-fold by treatment with rhFSH (total dose, 72 IU) or PMSG (15 IU). This effect was shown to be dose dependent, with a approximately 2-fold increase in response to 16 IU (total dose) rhFSH. P<em>4</em>50c17 alpha mRNA levels in isolated granulosa and thecal/interstitial cell total RNA from intact animals were compared to establish which was the principal cellular site of P<em>4</em>50c17 alpha mRNA expression. The P<em>4</em>50c17 alpha mRNA signal was undetectable in control granulosa cells and only barely discernible after treatment with 72 IU (total dose) rhFSH. In contrast, P<em>4</em>50c17 alpha mRNA was abundant in control thecal/interstitial mRNA, and its level was increased <em>4</em>- to 6-fold by treatment with rhFSH. Treatment of hypophysectomized animals with rhFSH did not consistently alter ovarian P<em>4</em>50c17 alpha mRNA levels. During culture for <em>4</em>8 h in serum-free medium, basal androgen (<em>androstenedione</em> plus androsterone) production by thecal/interstitial cells from intact animals was unaffected by treatment with rhFSH in vivo, but hLH-stimulated androgen production by these cells was enhanced approximately 2-fold. Neither basal nor hLH-responsive androgen production by thecal/interstitial cells from hypophysectomized animals was altered by previous treatment with rhFSH in vivo. Treatment of thecal/interstitial cell cultures from both intact and hypophysectomized animals with inhibin (0.1-30 ng/ml), a putative granulosa-derived paracrine factor, did not measurably affect basal androgen synthesis, but potently enhanced LH-responsive androgen synthesis in vitro. Similarly, treatment of thecal/interstitial cell cultures with conditioned medium from FSH-treated granulosa cell cultures significantly enhanced LH-responsive, but not basal, androgen production. We conclude that treatment of pituitary-intact rats with "pure" FSH modulates thecal/interstitial cell androgen synthesis. Granulosa cells, but not thecal cells, possess FSH receptors, and thecal/interstitial cells are the principal ovarian sites of P<em>4</em>50c17 alpha expression.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

CGS 169<em>4</em>9A inhibited the conversion of [<em>4</em>-1<em>4</em>C]<em>androstenedione</em> (A) to [<em>4</em>-1<em>4</em>C]estrone by human placental microsomes in a competitive manner (Ki = 1.6 nM). Aminoglutethimide, also a competitive inhibitor, had a Ki = 0.7 microM in this assay system. The Km for the aromatization of A was 0.11 microM. Using ovarian microsomes from immature rats primed with pregnant mare's serum gonadotrophin and using [<em>4</em>-1<em>4</em>C]testosterone conversion to [<em>4</em>-1<em>4</em>C]estradiol as a measure of aromatase activity, the Km was <em>4</em>2 nM. At a substrate concentration 3-fold the Km, CGS 169<em>4</em>9A was 180 times more potent as an inhibitor than aminoglutethimide, exhibiting half-maximal inhibition at 1.7 nM as compared to 0.3 microM. In vivo CGS 169<em>4</em>9A lowered ovarian estrogen synthesis by gonadotropin-primed, <em>androstenedione</em> treated, immature rats by 90% at a dose of 260 micrograms/kg (PO). A dose of 100 mg/kg of aminoglutethimide was needed to produce this same effect. CGS 169<em>4</em>9A at a dose of <em>4</em> mg/kg (PO) induced uterine atrophy (aromatase inhibition) without inducing adrenal hypertrophy - indicating a lack of inhibition of corticosterone secretion, while aminoglutethimide at <em>4</em>0 mg/kg (PO) induced adrenal hypertrophy without inducing uterine atrophy. CGS 169<em>4</em>9A was neither androgenic nor estrogenic in rats using standard bioassays. The data suggest that CGS 169<em>4</em>9A may serve as a potent and selective agent for modulating estrogen-dependent functions.

OBJECTIVE

Our purpose was to assess the endocrine status of women with polycystic ovaries (PCO) undergoing IVF, and to compare oocyte quality with endocrine markers of the syndrome, in an attempt to define a subpopulation with poor quality oocytes.

METHODS

This was a retrospective study. Patients were first endocrinologically analyzed: serum levels of androgens (T, <em>androstenedione</em>, DHEAS), FSH, and LH as well as glucose and insulin after an oral glucose tolerance test (OGTT) were recorded and are expressed as absolute values and area under the curve (AUC). Subsequently, they were followed over a 2-year period in which patients underwent several attempts of IVF as well as serving as oocyte donors. Patients were divided into three groups: group I (n = <em>4</em>) was women who displayed embryos unable to implant in 15 IVF cycles and 10 ovum donation cycles in which they served as donors; group II (n = 16) was PCO patients in whom IVF (n = 38) and/or oocyte donation cycles (n = <em>4</em>2) resulted in pregnancies; and group III (n = 13) was IVF patients with normal appearance of the ovaries by ultrasound. The endocrine status was compared with the IVF results.

RESULTS

There was no difference among groups in the endocrinological parameters tested, except for the OGTT which identified women in group I as having higher serum glucose and insulin levels than patients in groups II and III. Similarly, the OGTT showed higher serum glucose values in group II compared to group III. Women in group I were also obese. Patients in group III were older than PCO patients and needed more gonadotropins to reach an ovarian response which resulted in a reduced number of oocytes retrieved. Fertilization was also impaired in group I, in which no pregnancy was recorded.

CONCLUSIONS

This study shows that there is a particular subgroup of PCO patients with lower fertilization rates and embryos unable to implant. These patients are obese and nonhyperandrogenic and show derangements of insulin secretion.

Effects of various doses of aminoglutethimide (AG) alone upon adrenal steroidogenesis were studied in normal postmenopausal women, whereas the effects of combined treatment with aminoglutethimide in variable doses together with <em>4</em>0 mg of hydrocortisone were studied in postmenopausal women with advanced mammary cancer and compared to effects of treatment with cortisol alone. Despite the well known inhibitory effect of AG on cortisol biosynthesis, plasma cortisol levels were unaffected by AG in doses of 150-1000 mg/d, probably due to a compensatory increase in ACTH in subjects with an intact pituitary-adrenal axis. The aromatase system appeared to be very sensitive to inhibition by AG, a clearcut inhibition being shown at doses as low as 150 mg/d. Evaluated from the ratio of plasma oestrone (E1) to plasma <em>androstenedione</em> (AN), treatment with AG at a dose of 150 mg/d appeared to reduce the aromatase activity to 33% of the basal value; 250 mg/d resulted in a reduction to 20% and 1 g/d to 5% of basal values. Whereas AG at 150 mg/d did not appear to affect 11 beta-hydroxylase, the latter was clearly inhibited by 250 mg/d and even more so by 1000 mg/d, as indicated by the increase in plasma 11-desoxycortisol and 17-OH progesterone (17-OHP) levels. Due to the increase of the latter, their biosynthetic precursor, AN and to a lesser degree testosterone (TS) levels increased significantly during AG treatment at a dose of 250 or 1000 mg/d. delta 5 steroid levels remained practically unchanged, probably because 11-(as well as the 21-) hydroxylation concerns essentially the delta <em>4</em> pathway. During combined treatment with 500-1000 mg/d of AG and cortisol <em>4</em>0 mg/d, AN and TS were significantly higher than during treatment with cortisol alone, suggesting that cortisol had not completely blocked ACTH secretion. E1 and E2 levels were however lower than during treatment with cortisol alone, a consequence of the inhibition of the aromatase activity. Although at a dose of 500-1000 mg/d AG is a highly effective aromatase inhibitor, oestrogen levels during treatment with AG with or without concomitant administration of cortisol are still significantly different from zero. Therefore if one aims at complete elimination of any oestrogen effect, addition of an antioestrogen to AG treatment may be required.

Hirsutism, acne, alopecia, and oligo-amenorrhea are clinical expressions of hyperandrogenism, one of the most frequent endocrine disorders in women of reproductive age. Women referred to our endocrine clinics for skin symptoms of hyperandrogenism underwent a laboratory workup to evaluate hormone measurements and received antiandrogen therapy. We retrospectively analyzed the outcome of 228 consecutive patients investigated over 6 years.Patients with hirsutism had higher levels of <em>androstenedione</em>, dehydroepiandrosterone sulfate (DHEAS), and salivary testosterone; lower levels of sex hormone-binding globulin (SHBG); and a higher prevalence of oligo-amenorrhea than patients with alopecia, while patients with acne showed intermediate values. Hirsutism score correlated positively with <em>androstenedione</em>, DHEAS, and salivary testosterone, and correlated negatively with SHBG; salivary testosterone showed the highest correlation coefficient. Total testosterone was not significantly different among patients with hirsutism, alopecia, or acne, and did not significantly correlate with hirsutism score. Hirsutism and oligo-amenorrhea were the most sensitive symptoms of hyperandrogenism, and no androgenic parameter alone allowed us to identify all cases of hyperandrogenism.Patients of central European origin sought consultation with milder hirsutism scores than patients of southern European origin. There was, however, no difference in the clinical-biological correlation between these groups, arguing against differences in skin sensitivity to androgens.Polycystic ovary syndrome, defined as hyperandrogenism (hirsutism or elevated androgens) and oligo-amenorrhea, was diagnosed in 63 patients (27.6%), an underestimate compared with other reports that include systematic ovarian ultrasound studies. Neither pelvic ultrasound, used in a limited number of cases, nor the luteinizing hormone/follicle-stimulating hormone ratio helped to distinguish patients with polycystic ovary syndrome from the other diagnostic groups. These included hyperandrogenism (hirsutism or elevated androgens) and eumenorrhea (101 patients; <em>4</em><em>4</em>.3%); normal androgens (acne or alopecia and eumenorrhea) (51 patients; 22.<em>4</em>%); isolated low SHBG (7 patients; 3.1%); nonclassical congenital adrenal hyperplasia (<em>4</em> patients; 1.8% of total, <em>4</em>.9% of patients undergoing cosyntropin stimulation tests); and ovarian tumor (2 patients; 0.9%).Ethinylestradiol and high-dose cyproterone acetate treatment lowered the hirsutism score to 53.5% of baseline at 1 year, and was also effective in treating acne and alopecia. The clinical benefit is ascribed to the peripheral antiandrogenic effect of cyproterone acetate as well as the hormone-suppressive effect of this combination. Salivary testosterone showed the most marked proportional decrease of all the androgens under treatment. Cost-effectiveness and tolerance of ethinylestradiol and high-dose cyproterone acetate compared well with other antiandrogenic drug therapies for hirsutism. The less potent therapy with spironolactone only, a peripheral antiandrogen without hormone-suppressive effect, was effective in treating isolated alopecia in patients with normal androgens.

Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a <em>4</em>6,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H-corticosterone and <em>androstenedione</em> were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T->>A transversion in intron <em>4</em>,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns <em>4</em>, 5 and 6 either with or without the T->>A mutation in intron <em>4</em>. RNase protection assays showed that expression of the vector with normal intron <em>4</em> yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T->>A transversion in intron <em>4</em> was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient.

We have investigated the mechanism by which different natriuretic peptides stimulate steroidogenesis in purified mouse Leydig cells. In addition to atrial natriuretic factor (ANF), we show that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) also stimulate testosterone production in these cells. Testosterone production was increased dramatically to 1<em>4</em>-fold with ANF (EC50 = 0.3 nM) and 15-fold with BNP (EC50 = 0.2 nM); however, the CNP-stimulated level of testosterone production was only 2.5-fold compared with controls. ANF and BNP enhanced the stimulatory effect of LH on testosterone production. The C-ANF(<em>4</em>-23) (a truncated form of ANF) had no effect on testosterone production in these cells. ANF, BNP, and CNP stimulated the production of intermediate precursors of testosterone biosynthesis, which included progesterone, 17 alpha-hydroxy progesterone, <em>androstenedione</em>, pregnenolone, 17 alpha-hydroxy pregnenolone, and dehydroepiandrosterone sulfate. The conversion of pregnenolone and progesterone to testosterone was also significantly enhanced after treatment of Leydig cells with these peptides. All three natriuretic peptides (ANF, BNP, and CNP) stimulated the activity of particulate guanylate cyclase by 8.<em>4</em>-, 8.5-, and <em>4</em>.8-fold and the accumulation of intracellular cGMP by 52-, 58-, and 19-fold, respectively. The cGMP inhibitor LY83583 attenuated both the generation of cGMP as well as testosterone in response to these natriuretic peptides, suggesting the involvement of cGMP as a second messenger. Leydig cells were found to contain high affinity and low capacity binding sites for ANF [dissociation constant (Kd), 2.0 x 10(-10) M; maximum binding capacity (Bmax). 20 fmol/1 x 10(5) cells], BNP (Kd, 2.2 x 10(-10) M; Bmax, 19 fmol/1 x 10(5) cells), and CNP (Kd, 3.1 x 10(-10) M; Bmax, 8.6 fmol/1 x 10(5) cells). The results presented here document that a family of different natriuretic peptides stimulates Leydig cell steroidogenesis in receptor-mediated fashion, beginning at the cholesterol side-chain cleavage enzyme. The data also show that these peptide hormones induce testosterone production in mouse Leydig cells by involving both delta <em>4</em>- and delta 5-pathways of steroidogenesis.

Progression to hormone-refractory growth of prostate cancer has been suggested to be mediated by androgen receptor (AR) gene alterations. We analyzed AR for mutations and amplifications in 21 locally recurrent prostate carcinomas treated with orchiectomy, estrogens, or a combination of orchiectomy and estramustine phosphate using fluorescence in situ hybridization, single-strand conformation polymorphism, and DNA sequence analyses. Amplification was observed in <em>4</em> of 16 (25%) and amino acid changing mutations was observed in 7 of 21 (33%) of the tumors, respectively. Two (50%) tumors with AR amplification also had missense mutation of the gene. Four of five (80%) cancers that were treated with a combination of orchiectomy and estramustine phosphate had a mutation clustered at codons 51<em>4</em> to 533 in the N-terminal domain of AR. In functional studies, these mutations did not render AR more sensitive to testosterone, dihydrotestosterone, <em>androstenedione</em>, or beta-estradiol. Tumors treated by orchiectomy had mutations predominantly in the ligand-binding domain. In summary, we found molecular alterations of AR in more than half of the prostate carcinomas that recurred locally. Some tumors developed both aberrations, possibly enhancing the cancer cell to respond efficiently to low levels of androgens. Furthermore, localization of point mutations in AR seems to be influenced by the type of treatment.

5α-Reductases are crucial enzymes involved in the biosynthesis of dihydrotestosterone, the most potent natural androgen. To date, three types of 5α-reductases, chronologically named types 1, 2 and 3 5α-reductases (SRD5a-1, 2 and 3) have been described. In the present paper, we characterized the activity and compared the mRNA expression levels of SRD5a-3 with those of SRD5a-1 and 2 in various human tissues, and determined its sensitivity to finasteride and dutasteride. We have established HEK-293 cell line that stably expressed SRD5a-3 for studying its activity and the inhibitory effect of finasteride, using [1<em>4</em>C]labeled steroids. mRNA expression levels were quantified using real-time PCR in many male and female human tissues including the prostate, adipose tissue, mammary gland, as well as breast and prostate cancer cell lines. Incubation of HEK-SRD5a-3 cells with [1<em>4</em>C]<em>4</em>-<em>androstenedione</em> and [1<em>4</em>C]testosterone allowed us to show that SRD5a-3 can catalyze very efficiently both substrates <em>4</em>-<em>androstenedione</em> and testosterone into 5α-androstanedione and dihydrotestosterone, respectively. We observed that the affinity of the enzyme for <em>4</em>-<em>androstenedione</em> is higher than for testosterone. The activity of SRD5a-3 and SRD5a-2 are similarly sensitive to finasteride, whereas dutasteride is a much more potent inhibitor of SRD5a-3 than SRD5a-2. Tissue distribution analysis shows that SRD5a-3 mRNA expression levels are higher than those of SRD5a-1 and SRD5a-2 in 20 analyzed tissues. In particular, it is highly expressed in the skin, brain, mammary gland and breast cancer cell lines, thus suggesting that SRD5a-3 could play an important role in the production of androgens in these and other peripheral tissues.

Myo-inositol and D-chiro-inositol are capable of improving the ovarian function and metabolism of polycystic ovary syndrome (PCOS) patients. The aim of this work is to compare the effects of myo-inositol and D-chiro-inositol in PCOS. We enrolled 50 patients, with homogeneous bio-physical features, affected by PCOS and menstrual irregularities, and we randomly divided them into two groups: 25 were treated with <em>4</em> g of myo-inositol/die plus <em>4</em>00 mcg of folic acid/die orally for six months, 25 with 1 g of D-chiro-inositol/die plus <em>4</em>00 mcg of folic acid/die orally for six months. We analyzed in both groups pre-treatment and post-treatment BMI, systolic and diastolic blood pressure, Ferriman-Gallwey score, Cremoncini score, serum LH, LH/FSH ratio, total and free testosterone, dehydroepiandrosterone sulfate (DHEA-S), Δ-<em>4</em>-<em>androstenedione</em>, SHBG, prolactin, glucose/immunoreactive insulin (IRI) ratio, homeostatic model assessment (HOMA) index, and the resumption of regular menstrual cycles. Both the isoforms of inositol were effective in improving ovarian function and metabolism in patients with PCOS, although myo-inositol showed the most marked effect on the metabolic profile, whereas D-chiro-inositol reduced hyperandrogenism better.

The conversion of C19 steroids to estrogens occurs in a number of tissues and is catalyzed by a specific form of cytochrome P<em>4</em>50, namely aromatase cytochrome P<em>4</em>50 (P<em>4</em>50arom). Previously, conversion of radiolabeled <em>androstenedione</em> to estrone has been demonstrated in uterine leiomyomas. By use of reverse transcription followed by polymerase chain reaction amplification of total RNA together with a rat cRNA as an internal standard, we detected and quantified P<em>4</em>50arom transcripts in total RNA isolated from 32 of the 35 leiomyoma (91%) and from 18 of the 2<em>4</em> adjacent myometrial (75%) tissue samples from 26 women. P<em>4</em>50arom transcripts were not detectable in myometrial tissues from disease-free uteri (n = 8). P<em>4</em>50arom transcript levels in leiomyomas were similar to those in adipose tissue (normalized to total RNA) and were 1.5- to 25-fold higher than those in adjacent myometrial tissues. We did not find any correlation between P<em>4</em>50arom transcript levels and leiomyoma size, histopathology, uterine weight, or patient age. In leiomyoma smooth muscle cells in culture (n = <em>4</em>) and tissue explants (n = <em>4</em>), aromatase activity was stimulated by dibutyryl cAMP, and this effect was potentiated by a phorbol ester. These increases in aromatase expression were accompanied by comparable increases in the levels of translatable P<em>4</em>50arom mRNA. Treatment with dexamethasone or platelet-derived growth factor did not stimulate aromatase expression. Consistently higher levels of aromatase activity and P<em>4</em>50arom transcripts were found in the leiomyoma tissues than in smooth muscle cells in culture (2- to 20-fold). Reverse transcription-polymerase chain reaction analysis of untranslated 5'-termini of mRNA species in leiomyomas revealed the use of primarily promoter II (the ovarian-type promoter) for CYP19 gene transcription. Leiomyomas also contain some transcripts with untranslated exon I.<em>4</em> (previously found in adipose stromal cells and skin fibroblasts). Placental-type promoter-specific 5'-ends were not present in leiomyomas. We conclude that aromatase expression in leiomyomas is regulated by the rate of CYP19 gene transcription, which is, in turn, regulated by the use of tissue-specific promoters. These findings are consistent with the hypothesis that localized estrogen biosynthesis may be of pathological significance in the promotion of leiomyoma growth.

BACKGROUND

Pubertal onset is usually defined by breast development in girls and testicular growth in boys. Pubarche is defined as the attainment of pubic hair and is considered as a sign of pubertal transition. Pubarche is preceded by a gradual increase in production of adrenal androgens, DHEA and Δ4-androstenedione (Adione), a process termed adrenarche.

OBJECTIVE

To study the natural course of pubertal transition and the associations with adrenarche, body fat, and linear growth.

METHODS

A longitudinal study of 179 healthy children (89 girls) with higher socioeconomic background examined every 6 months for 5 years. Pubic hair stage, breast stage, genital stage, testicular volume (TV), height, weight, and four skinfolds were measured.

RESULTS

In girls, median age (25th and 75th percentiles) at thelarche (B2+) was 10.1 years (9.3-10.9). In boys, median age at attaining a TV >3 ml was 11.5 years (10.9-12.0). Median age at pubarche (PH2+) was 10.9 years (10.3-11.4) in girls and 11.6 years (10.8-12.4) in boys. Only 6.8% (4/59) of the girls and 24.6% (15/61) of the boys developed pubic hair as the first isolated sign of puberty. Serum DHEAS and Adione increased with age, although the increase in Adione was most pronounced in girls. No associations between early age at thelarche/testicular growth and increased body fat (BMI and sum of four skinfolds) were observed.

CONCLUSIONS

Danish children rarely experience pubarche as the first sign of puberty. No associations between age at pubertal onset and body composition were found. Circulating levels of Adione, but not DHEAS, increased with the onset of puberty, although with large interindividual variability.

Although serum testosterone levels decrease acutely in critically ill patients, estrogen levels rise. We hypothesized that increased rates of aromatization of androgens to estrogens underlie the increase in serum estrogen levels. Eleven men and three women (age <em>4</em>2-69 yr) were prospectively studied before and again after elective coronary artery bypass graft surgery (CABG). Each patient received priming doses of [(1<em>4</em>)C]androgen and [(3)H]estrogen that were immediately followed by peripheral infusions for 210 min. Eight men and three women received <em>androstenedione</em> (A(<em>4</em>))/estrone (E(1)) and three men received testosterone (T)/estradiol (E(2)). Adipose tissue biopsies were obtained in another six men before and after CABG to evaluate levels of P<em>4</em>50 aromatase mRNA. Serum T levels decreased postoperatively in all 17 men (P < 0.001), whereas E(1) levels rose (P = 0.00<em>4</em>), with a trend toward a rise in E(2) (P = 0.23). Peripheral aromatization rates of androgens to estrogens rose markedly in all 1<em>4</em> patients (P < 0.0001). Estrogen clearance rates rose (P < 0.002). Mean serum A(<em>4</em>) levels increased slightly postoperatively (P = 0.0<em>4</em>), although no increase in A(<em>4</em>) production rates (PRs) was observed. T PRs decreased in two of three men, whereas clearance rates increased in all three. Adipose tissue P<em>4</em>50 aromatase mRNA content increased postoperatively (P < 0.001). We conclude that the primary cause of increased estrogen levels in acute illness is increased aromatase P<em>4</em>50 gene expression, resulting in enhanced aromatization of androgens to estrogens, a previously undescribed endocrine response to acute illness. Both increased T clearance and decreased T production contribute to decreased serum T levels. Animal studies suggest that these opposing changes in circulating estrogen and androgen levels may be important to reduce morbidity and mortality in critical illness.