Despite significant improvement in the treatment outcome of hormone responsive postmenopausal breast cancer, some patients eventually acquire resistance to aromatase inhibitors (AIs). Using our MCF-7Ca xenograft model, we observed that although AIs such as anastrozole initially inhibit tumor growth effectively, tumors eventually began to grow. Our previous data show that anastrozole-resistant tumors upregulate growth factor receptor pathways as they adapt to grow in the low estrogen environment. Therefore, in the current study, we investigated the effect of inhibiting the growth factor receptor pathways with a MEK-1/2 inhibitor selumetinib (AZD62<em>4</em><em>4</em>, ARRY-1<em>4</em>2866). We treated the mice with anastrozole-resistant tumors with selumetinib alone or in combination with anastrozole. MCF-7Ca cells were inoculated sc into ovariectomized athymic nude mice supplemented throughout the experiment with <em>androstenedione</em> (100 μg/day), the substrate for aromatase conversion to estrogen. Once the tumors reached a measurable size (~300 mm(3)), the mice were treated with anastrozole (200 μg/day), supplemented with <em>androstenedione</em> (Δ(<em>4</em>)A). The tumors in the anastrozole group doubled in volume after 6 weeks, at which time the animals were regrouped to receive the following treatments: (i) anastrozole, (ii) anastrozole withdrawal (Δ(<em>4</em>)A alone), (iii) selumetinib (25 mg/kg/d, bid, po), and (iv) selumetinib + anastrozole, (n = 10 mice/group). The treatments were given for 6 weeks (till week 12) and then the mice were euthanized, the tumors were collected and analyzed. The tumors of mice treated with selumetinib + anastrozole had significantly lower growth rates than those treated with single agents (p = 0.008). Western blot analysis of the tumors showed that treatment with anastrozole resulted in upregulation of proteins in the growth factor receptor cascade such as p-mTOR, pAkt, pMEK, and pMAPK. This was accompanied by downregulation of ERα protein, consistent with previous findings. The treatment of mice with selumetinib resulted in downregulation of activated MAPK, along with p-mTOR, which likely resulted in upregulation of ERα. Our results suggest that inhibition of the growth factor receptor pathway with selumetinib can reverse anastrozole resistance.

BACKGROUND

Ovaries surgically removed for fertility preservation served as a source of follicle fluid from human small antral follicles.

OBJECTIVE

The objective of the study was to measure intrafollicular concentrations of anti-Müllerian Hormone (AMH), inhibin-B, progesterone, <em>androstenedione</em>, testosterone, estradiol, and IGF binding protein-<em>4</em>.

METHODS

The study was conducted at a university hospital.

METHODS

Patients included <em>4</em>3 women having one ovary removed prior to receiving gonadotoxic treatment due to malignant disease.

METHODS

Fluid from 100 follicles (diameter of 3-9 mm) were included.

METHODS

Intrafollicular concentrations of the measured hormones, their possible intercorrelation, and correlation with age were measured.

RESULTS

Concentrations of AMH were unrelated to follicular fluid concentrations of <em>androstenedione</em> and testosterone. There was a significant negative correlation between estradiol, inhibin-B, progesterone, and AMH. In four age groups spanning 11-37 yr, levels of AMH, estradiol, <em>androstenedione</em>, testosterone and inhibin-B remained constant, whereas progesterone showed significant variations. IGF binding protein-<em>4</em> was unrelated to any other measured hormone.

CONCLUSIONS

This study was unable to confirm a stimulatory effect of androgens on AMH secretion but did enforce a close intimate correlation between AMH and estradiol expressions in the developing human follicle. The insignificant variation of the AMH concentration with age, even in prepubertal girls, suggests that AMH expression is unrelated to menstrual cycle FSH cyclicity.

Cyclic hamsters hypophysectomized at estrus (Day 1 of the cycle) and injected with 5 micrograms follicle-stimulating hormone (FSH) on Day 1 and 20 micrograms luteinizing hormone (LH) in polyvinylpyrrolidone (PVP) from Days 1-<em>4</em> ovulated 15.3 ova, in response to 30 IU human chorionic gonadotropin (hCG) administered at 1500 h on Day <em>4</em> (Kim and Greenwald, 198<em>4</em>). When 1 mg progesterone (P<em>4</em>) was administered daily from Days 1-<em>4</em> concurrent with the above regimen, ovulation increased to 38 ova, a clearcut superovulatory response. However, daily injection of 1, 10, or 100 micrograms P<em>4</em> plus FSH and LH reduced the number of antral follicles present on the afternoon of Day <em>4</em> to 3-<em>4</em> per ovary, compared to 9 per ovary after FSH-LH alone, and the ovulation rate was drastically reduced with most animals being anovulatory. Substituting 1 mg 17 alpha-hydroxyprogesterone or estradiol cyclopentylpropionate for P<em>4</em> on Days 1-<em>4</em> did not alter the number of antral follicles on Day <em>4</em> from FSH-LH alone, whereas 1 mg <em>androstenedione</em> or 1 mg testosterone cyclopentylpropionate reduced the number of antral follicles to 3 or less. Hence, the stimulatory effects of 1 mg P<em>4</em> are not attributable to its conversion to other P<em>4</em> derivatives. After the concurrent injection of 1 mg P<em>4</em> and FSH-LH, on the afternoon of Day 3, an average of only 1.8 large preantral follicles was present per ovary. By the morning of Day <em>4</em>, however, the ovary contained 1<em>4</em> large preantral and early antral follicles in addition to 8 large antral follicles. Injection of hCG at this time resulted in the ovulation of 1<em>4</em>.5 ova.(ABSTRACT TRUNCATED AT 250 WORDS)

Sebum excretion has been shown to demonstrate a circadian rhythm using a gravimetric method (cigarette paper). With the newly introduced method of Sebutape, we confirmed this periodicity and showed that the elevation in sebum excretion is correlated with an increase in the number of secreting follicles. We found, furthermore, that the number of secreting follicles on the forehead showed a distinct and statistically significant circadian rhythmicity, in contrast to those of the chest, which remained almost constant. The quantification in plasma levels of cortisol, melatonin, delta-<em>4</em>-<em>androstenedione</em>, dehydroepiandrosterone sulphate, and free testosterone showed no correlation with sebum excretion at either site. These observations suggest that local factors are involved in control of sebum secretion.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17β-HSD type 3 (17β-HSD3) catalyses the reduction of the weakly androgenic <em>androstenedione</em> (adione) to testosterone, suggesting that specific inhibitors of 17β-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17β-HSD3 inhibitor with an IC(50) of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17β-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17β-HSD3 inhibitor, STX1383 (SCH-<em>4</em>51659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×10(7) cells 2<em>4</em> h after an initial daily dose of testosterone propionate (TP) or vehicle. After <em>4</em> weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further <em>4</em> weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17β-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17β-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer.

OBJECTIVE

To investigate the adrenal response in terms of allopregnanolone secretion in a group of hyperinsulinemic patients with polycystic ovary syndrome (PCOS).

METHODS

Controlled clinical study.

METHODS

Patients with PCOS in a clinical research environment.

METHODS

Twenty-two overweight patients with PCOS with hyperinsulinism were enrolled after informed consent.

METHODS

All patients underwent hormonal evaluations, oral glucose tolerance test (OGTT) and adrenocorticotropic hormone (ACTH) test before and after <em>4</em> months of metformin administration (500 mg p.o. bi-daily). Ultrasound examinations and Ferriman-Gallway score were also performed. Main outcome measures. plasma luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), estradiol, 17-hydroxy-progesterone (17OHP), <em>androstenedione</em> (A), testosterone (T), allopregnanolone, glucose, insulin, C peptide concentrations, body mass index (BMI).

RESULTS

Metformin administration reduced significantly LH, A, T, insulin and BMI, while allopregnanolone was significantly increased with no change in progesterone plasma levels. Insulin response to OGTT decreased and allopregnanolone response to ACTH stimulation before while this was restored after the treatment interval. The Ferriman-Gallway score as well as the ovarian volume was significantly decreased after <em>4</em> months of metformin therapy.

CONCLUSIONS

In overweight patients with PCOS with hyperinsulinism, allopregnanolone secretion is impaired and metformin administration restored normal allopregnanolone concentrations modulating both steroid syntheses from the ovaries and from adrenal gland.

<em>Androstenedione</em> is a steroid hormone and an intermediate in the synthetic pathway of both testosterone and estradiol in men and women. It is available without prescription and taken with the expectation that it may have beneficial effects on strength, general well-being, libido, and quality of life. Although studies have shown that oral <em>androstenedione</em> increases serum testosterone and estradiol levels in men, the hormonal effects of <em>androstenedione</em> in postmenopausal women are unknown. We randomly assigned 30 healthy postmenopausal women to receive 0, 50, or 100 mg <em>androstenedione</em> as a single oral dose. After <em>androstenedione</em> administration, we made hourly measurements of serum <em>androstenedione</em>, estrone, estradiol, and testosterone concentrations during 12 h of frequent blood sampling. The mean change (+/-SD) in serum <em>androstenedione</em> area under the curve (AUC) was greater in both the 50-mg (79 +/- 39%) and 100-mg dose groups (2<em>4</em>2 +/- 18<em>4</em>%) than in the control group (-29 +/- 28%) (P < 0.0001 for controls vs. 50-mg group and controls vs. 100-mg group). The mean change in serum <em>androstenedione</em> AUC was also greater in the 100-mg than 50-mg dose group (P = 0.0026). The mean change in serum estrone AUC was greater in both the 50-mg (108 +/- 72%) and 100-mg dose groups (116 +/- 119%) than in the control group (-5 +/- 19%), although the control vs. 100-mg group comparison did not quite meet statistical significance (P < 0.0001 for controls vs. 50-mg group, P = 0.0631 controls vs. 100-mg group). The mean change in serum estradiol AUC remained stable after supplementation in all groups without any between-group differences observed (-11 +/- 17%, 2.8 +/- 3<em>4</em>%, -11 +/- 27%, for the control, 50-mg, and 100-mg groups, respectively). The mean change in serum testosterone AUC was greater in both the 50-mg (185 +/- 1<em>4</em>6%) and 100-mg dose groups (<em>4</em>57 +/- 601%) than in the control group (-27 +/- 13%) (P < 0.0001 for controls vs. 50-mg group and for controls vs. 100-mg group). The mean change in testosterone AUC was also greater in the 100-mg dose group than 50-mg dose group (P = 0.0257). There was considerable individual variability in the changes of serum <em>androstenedione</em>, estrone, and testosterone levels in the treated groups with peak serum testosterone levels exceeding the upper limit of normal in <em>4</em> of 10 women in the 50-mg dose group and 6 of 10 in the 100-mg dose group. We concluded that the acute administration of both 50-mg and 100-mg of <em>androstenedione</em> increases serum testosterone and estrone levels, but not estradiol levels, in postmenopausal women. If these hormonal effects are sustained during long-term administration, regular use of this supplement by postmenopausal women could thus cause both beneficial and adverse effects.

This study integrates all data obtained in women aged <em>4</em>0-80years enrolled with moderate to severe symptoms of vulvovaginal atrophy (VVA) who received daily intravaginal administration of 0.50% (6.5mg) dehydroepiandrosterone (DHEA; prasterone) for 12weeks (n=723; ITT-S population) as compared with placebo (n=266; ITT-S population). To this end, serum steroid levels (DHEA, DHEA-sulfate (DHEA-S), androst-5-ene-3β, 17β-diol (5-diol), testosterone, dihydrotestosterone (DHT), <em>androstenedione</em> (<em>4</em>-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17β-diol 17-glucuronide (3α-diol-17G)) were measured at Day 1 and Week 12 by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following validation performed according to the FDA guidelines [1-6]. In agreement with the mechanisms of intracrinology where DHEA is exclusively transformed intracellularly into active sex steroids which act and are inactivated locally before being released as glucuronided or sulfated metabolites for elimination by the kidneys and liver, all sex steroids remained well within normal postmenopausal values following administration of intravaginal DHEA. Serum estradiol, the most relevant sex steroid, was measured after 12weeks of treatment at 3.36pg/ml (cITT-S population) or 19% below the normal postmenopausal value of <em>4</em>.17pg/ml. On the other hand, serum E1-S, the best recognized marker of global estrogenic activity, shows an average value of 209pg/ml at 12 weeks compared to 220pg/ml in normal postmenopausal women. Moreover, serum ADT-G, the main metabolite of androgens, also remains well within normal postmenopausal values. The present data shows that a low daily intravaginal dose (6.5mg) of DHEA (prasterone) which is efficacious on the symptoms and signs of VVA, permits to achieve the desired local efficacy without systemic exposure, in agreement with the stringent mechanisms of menopause established after 500 million years of evolution where each cell in each tissue is the master of its sex steroid exposure.

Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(<em>4</em>) isomerase (3β-HSD) responsible for progesterone and <em>androstenedione</em> production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A(<em>4</em>) and P(<em>4</em>) by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A(<em>4</em>) by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P(<em>4</em>) did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P(<em>4</em>) in pregnant pigs on days 12 to 13. The release of P(<em>4</em>) by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

Acne is a multifactorial disorder reflecting the role of infection, abnormal keratinization and immunologic reaction, as well as hormonal influences, on the pilosebaceous unit. Clinical studies have correlated elevated levels of androgens, originating in both the adrenal glands and ovaries, with acne. These include total and free testosterone, delta <em>4</em>-<em>androstenedione</em>, dehydroepiandrosterone and its sulfate, and low levels of sex hormone binding globulin. The pathogenesis of acne initiation in childhood has been linked to rising serum levels of dehydroepiandrosterone sulfate. Hirsutism has been more directly correlated with increased levels of serum androgens, notably free testosterone. Underlying causes of elevated androgens in both disorders include very rare tumors, partial or late-onset forms of congenital adrenal hyperplasia, developmental adrenal abnormalities and, most commonly, polycystic ovary syndrome. Early acne treatment may include topical benzoyl peroxide, antibiotics, and tretinoin. More severe disease can be treated systemically (with antibiotics and/or isotretinoin). Very-low-dose corticosteroids can be used to eliminate the adrenal component of hyperandrogenism. Oral contraceptives, especially those that contain low-androgenic progestins, can reduce excessive androgens from any source and specifically suppress the ovary in polycystic ovary syndrome. Gonadotropin-releasing hormone agonists, with or without estrogen supplementation, and systemic or topical antiandrogens may play a more important role in the future.

The discrete effects of obesity on infertility in females remain undefined to date. To investigate obesity-induced ovarian dysfunction, we characterized metabolic parameters, steroidogenesis, and folliculogenesis in obese and lean female Ossabaw mini-pigs. Nineteen nulliparous, sexually mature female Ossabaw pigs were fed a high fat/cholesterol/fructose diet (n=10) or a control diet (n=9) for eight months. After a three-month diet-induction period, pigs remained on their respective diets and had ovarian ultrasound and blood collection conducted during a five-month study period after which ovaries were collected for histology, cell culture, and gene transcript level analysis. Blood was assayed for steroid and protein hormones. Obese pigs developed abdominal obesity and metabolic syndrome, including hyperglycemia, hypertension, insulin resistance and dyslipidemia. Obese pigs had elongated estrous cycles and hyperandrogenemia with decreased LH, increased FSH and luteal phase progesterone, and increased numbers of medium, ovulatory, and cystic follicles. Theca cells of obese, compared to control, pigs displayed <em>androstenedione</em> hypersecretion in response to in vitro treatment with LH, and up-regulated 3-beta-hydroxysteroid dehydrogenase 1 and 17-beta-hydroxysteroid dehydrogenase <em>4</em> transcript levels in response to in vitro treatment with LH or LH + insulin. Granulosa cells of obese pigs had increased 3-beta-hydroxysteroid dehydrogenase 1 transcript levels. In summary, obese Ossabaw pigs have increased transcript levels and function of ovarian enzymes in the delta <em>4</em> steroidogenic pathway. Alterations in LH, FSH, and progesterone, coupled with theca cell dysfunction, contribute to the hyperandrogenemia and disrupted folliculogenesis patterns observed in obese pigs. The obese Ossabaw mini-pig is a useful animal model in which to study the effects of obesity and metabolic syndrome on ovarian function and steroidogenesis. Ultimately, this animal model may be useful toward the development of therapies to improve fertility in obese and/or hyperandrogenemic females or in which to examine the effects of obesity on the maternal-fetal environment and offspring health.

In order to study the initial as well as the final steps in the aromatization of androgens to estrogens, high-specific activity [19-C3H3]<em>androstenedione</em> and testosterone were synthesized. Incubations of [19-C3H3]<em>androstenedione</em> with human placental microsomes resulted in the generation of [3H]water, as a result of the dual hydroxylation at C-19, and [3H]formic acid reflecting final aromatization. After an initial lag in the production of [3H]formic acid, the two radiolabeled products were formed linearly with time at a ratio of 2 to 1 under subsaturating conditions and 2.2 to 1 when saturating levels of substrate were present. Incubation of a mixture of [19-C3H3]- and [<em>4</em>-1<em>4</em>C]<em>androstenedione</em> with human placental microsomes yielded 19-hydroxy- and 19-oxo<em>androstenedione</em>, respectively, products of one and two hydroxylations at C-19. The isotope ratios of these derivatives revealed the presence of a tritium isotope effect in the first but not in the second hydroxylation at that site. When [19-C3H2]- and [<em>4</em>-1<em>4</em>C]19-hydroxy<em>androstenedione</em> were used as the substrate, the isotope ratio of the isolated 19-oxo<em>androstenedione</em> showed no evidence of any isotope effect in its formation. Thus, the second hydroxylation at C-19 exhibits no isotope effect irrespective of whether <em>androstenedione</em> or 19-hydroxy<em>androstenedione</em> are the substrates, and therefore, a concerted process and catalytic commitment are not responsible for the difference in isotope effects between the first and second C-19 hydroxylation by the placental aromatase complex. Radiometric kinetic analysis employing [19-C3H3]- and [1 beta,2 beta-3H]<em>androstenedione</em> as the comparative substrates provided evidence that the isotope effect is exerted solely through the Vmax component of the reaction. The distinction between the successive hydroxylations at C-19 in the aromatization sequence suggests, but does not prove, that different mechanisms, and hence different catalytic sites, may be involved in these steps.

BACKGROUND

Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is warranted.

METHODS

Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d(8)-17-OHP, d(7)-<em>androstenedione</em>, and d(<em>4</em>)-cortisol. The final extracts were analyzed for 17-OHP, <em>androstenedione</em> and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode.

RESULTS

Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9-9.9% to 3.6-10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%.

CONCLUSIONS

The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families.

OBJECTIVE

To investigate somatic symptom relief, gonadotropin secretion, and endogenous androgen bioavailability (protein-bound and free) during 3 months of estrogen-androgen therapy or matched estrogen-only replacement therapy.

METHODS

Ninety-three naturally menopausal outpatients with 6 or more months of amenorrhea, who were experiencing mild-to-moderate vasomotor symptoms, were randomized to receive one of five treatments: oral esterified estrogens (0.625 mg or 1.25 mg), oral esterified estrogens combined with methyltestosterone (0.625 mg combined with 1.25 mg methyltestosterone or esterified estrogens 1.25 mg combined with 2.5 mg methyltestosterone), or placebo for 12 weeks. All treatments were preceded by a <em>4</em>-week placebo lead-in period.

RESULTS

Patients receiving the lower dose of estrogen-androgen therapy had fewer somatic menopausal symptoms than patients receiving the lower dose estrogen (0.625 mg), and they experienced somatic symptom relief similar to those patients receiving the higher dose of estrogen (1.25 mg). Significantly greater luteinizing hormone suppression (p < or = 0.03) occurred in estrogen-androgen groups compared to estrogen groups, suggesting that added androgen might mediate a more pronounced negative feedback on the hypothalamic-pituitary axis. Sex hormone-binding globulin increased significantly in both estrogen-treated groups (p < or = 0.01), whereas decreases occurred in both estrogen-androgen groups (p < or = 0.006). The higher dose estrogen-only preparation significantly reduced androstenedione (p < or = 0.01) and dehydroepiandrosterone sulfate (p < or = 0.005).

CONCLUSIONS

The extent of relief with lower dose estrogen-androgen therapy was similar to higher dose estrogen-only treatment. The greater efficacy of combination therapy on somatic symptoms could be mediated by the same mechanism responsible for the suppressive effects of estrogen-androgen therapy on luteinizing hormone secretion. The marked differences in circulating levels of sex hormone building globulin, which were increased by estrogen and decreased by estrogen-androgen, and the resulting impact on bioavailable androgens and estrogens could also explain the differential somatic relief with both treatments. Endogenous adrenal androgens were lower in women treated with esterified estrogens 1.25 mg/day, suggesting that estrogen therapy can produce a significant hypoandrogenic state by inhibiting production or accelerating clearance of adrenal androgens.

OBJECTIVE

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder associated with infertility, cardiovascular disease and type 2 diabetes. Despite anecdotal evidence that lesbians may have higher PCOS rates than heterosexuals, little empirically based evidence supports this theory. To address this gap, we examined PCOS prevalence and associated factors among a community sample of lesbian and heterosexual women.

METHODS

Lesbian (n = 11<em>4</em>) and heterosexual (n = 97) women aged 35 to <em>4</em>5 who participated in The Epidemiologic STudy of HEalth Risk (ESTHER) Project (Pittsburgh, PA) were recruited into our PCOS exploratory study between April and October 2008. A reproductive endocrinologist, "blinded" to participant sexual orientation, identified women with PCOS using a modified version of the 2003 Rotterdam Diagnostic Criteria for PCOS. Sexual orientation was defined by self-reported sexual identity, behavior, and attraction. Fisher's exact, chi-square, and Wilcoxon rank-sum tests were used for analysis.

RESULTS

Approximately 6.2% (n = 13) of the total sample (n = 211) had PCOS. PCOS rates did not significantly differ between lesbian and heterosexual women ([7.9%, n = 9] vs. [<em>4</em>.1%, n = <em>4</em>]; p = .256). No significant differences in PCOS-related factors were found between lesbian and heterosexual women: polycystic ovaries ([10.5%, n = 12] vs. [6.2%, n = 6]; p = 0.261), hirsutism ([2<em>4</em>.6%, n = 28] vs. [15.5%, n = 15]; p = 0.102), oligomenorrhea ([3.6%, n = <em>4</em>] vs. [5.<em>4</em>%, n = 5]; p = 0.735), adult acne ([21.1%, n = 2<em>4</em>] vs. [2<em>4</em>.7%, n = 2<em>4</em>], p = 0.52<em>4</em>), and median testosterone ([1.69 ng/mL, n = 11<em>4</em>] vs. [1.52 ng/mL, n = 97]; p = 0.069) and <em>androstenedione</em> ([1.63 ng/mL, n = 11<em>4</em>] vs. [1.51 ng/mL, n = 97]; p = 0.079) concentrations, respectively.

CONCLUSIONS

PCOS and related factors did not differ by sexual orientation. Despite this, our observed rates warrant the need for additional studies to examine the relationship between PCOS diagnoses, PCOS-related factors, and sexual orientation.

The effects of Ketoconazole (600 mg/day) were evaluated in 10 patients with Cushing's syndrome during a mean period of <em>4</em>.5 weeks (range 1-12). The urinary free cortisol excretion (UFC) decreased by 21 +/- 15% (mean +/- SEM) (p less than 0.01) on day 1; 5<em>4</em> +/- 8% (p less than 0.0001) on day 2; 60 +/- 15% (p less than 0.0001) on day 3 and 87 +/- 3% (p less than 0.0001) on day 8 compared to baseline. Salivary cortisol at 0800 h decreased similarly. On day 3, 7 patients showed normal UFC values and on day 8, only 1 patient, with the ectopic ACTH syndrome, had persistent hypercortisolism. The cortisol decrease was associated with an increase in desoxycorticosterone values (p less than 0.01) and a decrease in dehydroepiandrosterone sulfate (p less than 0.001), delta <em>4</em> <em>androstenedione</em> (p less than 0.05) and testosterone (p less than 0.05). No significant variations were observed in ACTH, 11 desoxycortisol, aldosterone, plasma renin activity, corticosteroid-binding globulin and sex hormone-binding globulin. Side effects were few: mild clinical adrenal insufficiency (n = 5), oedema (n = 3) and reversible hepatic toxicity (n = 1). We conclude that Ketoconazole is an effective inhibitor of cortisol and androgens synthesis. It is well tolerated, rapidly effective and its efficacy persists unchanged for at least one month in all forms of Cushing's syndromes. For these reasons Ketoconazole may be a valuable drug for preoperative treatment of Cushing's syndrome.

This study was conducted to determine the distribution of oocytes in meiotic arrest as a function of follicle maturation, atresia status, and follicular fluid steroid concentrations. Oocytes (n = 138) from>> or = 3 mm follicles were recovered from gilts (n = 3/d) on Days 1, 3, 5, and 7 of the follicular phase initiated by withdrawal of altrenogest treatment. They were fixed in <em>4</em>% paraformaldehyde, stained with Hoechst 333<em>4</em>2, and examined by laser scanning confocal microscopy using combined bright field Nomarski optics and ultraviolet laser illumination. The number of oocytes in complete meiotic arrest increased (P < 0.05) as a function of the stage of maturation from 29% on Day 1 to 79 and 67% on Days 3 and 5, respectively. Oocytes showing complete germinal vesicle breakdown (GVBD) were found only on Day 7 (2<em>4</em> to 36 h after the preovulatory LH surge). The distribution of GV stages on Days 1 to 5 did not differ between atretic (n = 27) and nonatretic follicles (n = 81). In nonatretic follicles, GV stage was inversely related to the concentration of estradiol on Day 7 and to the concentrations of progesterone and <em>androstenedione</em> (P < 0.05) on Days 5 and 7 indicating that meiotically arrested oocytes were likely to be found in follicles with highest levels of steroidogenesis. In conclusion, a large proportion of oocytes present in 3 to 5 mm follicles had begun GVBD. The follicles in the ovulatory cohort may be recruited or selected from preexisting 3 to 5 mm follicles, or younger population with oocytes that are in complete meiotic arrest.

Groups of Sprague-Dawley rats were exposed, by inhalation, to n-hexane (900 ppm, 3,2<em>4</em>0 mg/m3), xylene (600 ppm, 2,625 mg/m3), methyl ethyl ketone (800 ppm, 2,3<em>4</em>5 mg/m3) and methylchloroform (800 ppm, <em>4</em>,3<em>4</em>5 mg/m3) for four weeks. Increased liver weights and liver to body weight ratios were observed for all the solvents except n-hexane. An increased in vitro formation of certain metabolites of all the investigated substrates was found only in the rats exposed to xylene. The in vitro microsomal metabolism of biphenyl, benzo(a)pyrene, <em>4</em>-androstene-3,17-dione and <em>4</em> alpha-androstane-3 alpha, 17 beta-diol in combination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that n-hexane was without effect on rat liver microsomal cytochrome P-<em>4</em>50 and that methyl ethyl ketone and methylchloroform depressed the formation of two metabolites of <em>androstenedione</em> but did not alter the concentration of cytochrome P-<em>4</em>50 under the experimental conditions used. Xylene was shown to be a phenobarbital-like inducer of rat liver microsomal cytochrome P-<em>4</em>50.

BACKGROUND

Prostate-specific antigen (PSA) is expressed in many female tissues and its concentrations were higher in hirsute subjects. We aimed to determine serum PSA level in hirsute women and evaluate the effect of flutamide+desogestrel/ethynil estradiol combination.

METHODS

Thirty patients with polycystic ovary syndrome (PCOS) and 30 healthy controls were studied. Hirsutism was defined by modified Ferriman-Gallwey score (FGS). Free androgen index (FAI) was used for hyperandrogenism. Patients received flutamide (500 mg/d) and oral contraceptive (desogestrel+ethinyl estradiol) for 9 months.

RESULTS

Mean FGS (p<0.0001), insulin (p<0.01), FAI (0.0001), androstenedione (p<0.0001), LH (p<0.05), and free testosterone (p<0.003) levels of patients with PCOS were higher than the control group. Mean serum total and free PSA level of PCOS patients were higher than the control group (p<0.0001 and p<0.0001). We found a positive correlation between total PSA levels and FGS (r=0.568, p<0.001), FAI and FGS (r=0.456 and p<0.01). There was also a positive correlation between FAI and total PSA (r=0.503 and p<0.005). At the end of treatment, FGS, androstenedione, free and total testosterone, FAI, serum PSA and LH levels decreased significantly [serum total PSA was 0.0208 +/- 0.0178 ng/ml at baseline and 0.0061 +/- 0.0044 ng/ml after treatment (p<0.0001)].

CONCLUSIONS

1. Serum prostate specific antigen level is higher in patients with PCOS; 2. There is a positive correlation among FGS, FAI and PSA levels; 3. Serum PSA levels decrease with antiandrogen treatment; 4. Serum PSA measurement might be a marker for hirsutism.

OBJECTIVE

To ascertain an association between the a priori known insulin resistance caused by antipsychotic agents and divalproex and adrenal hyperandrogenism and to determine whether the associated hyperandrogenism is reversible with insulin sensitizers.

METHODS

We studied 26 consecutive psychiatric inpatients (22 women and <em>4</em> men) receiving the aforementioned medications, who were referred to us for a consultation. They ranged in age from 19 to 79 years and had a mean body mass index (SEM) of 32.35 +/- 1.26 kg/m2. Between 8 AM and 9 AM, blood samples were collected for 17-hydroxyprogesterone, 17-hydroxypregnenolone, <em>androstenedione</em>, dehydroepiandrosterone (DHEA), DHEA sulfate, 11-deoxycortisol, luteinizing hormone and follicle-stimulating hormone (in reproductive age women), estrone, estradiol (in reproductive age women), free testosterone (in women), deoxycorticosterone, and sex hormone-binding globulin (SHBG), which were measured by radioimmunoassay, after chromatography if necessary. For intact, premenopausal women, measurement of the abnormal steroid metabolite or SHBG level was repeated during prednisone therapy (5 mg at bedtime) to document the likely adrenal origin of the abnormality. Men, women who had undergone bilateral oophorectomy, and postmenopausal women had hyperandrogenism of adrenal origin by default. Clinical features included central obesity, acanthosis, hirsutism, alopecia, type 2 diabetes mellitus, and oligomenorrhea.

RESULTS

We found reversed estrone/estradiol ratios in <em>4</em> patients, decreased SHBG in <em>4</em>, increased 17-hydroxy-pregnenolone in 8, increased 17-hydroxyprogesterone in 2, increased deoxycorticosterone in 2, increased DHEA sulfate in 1, increased 11-deoxycortisol in <em>4</em>, increased <em>androstenedione</em> in 1, and reversed ratios of luteinizin hormone to follicle-stimulating hormone in 2. The bio-chemical abnormalities were corrected in 8 of 8 patients receiving metformin and in 2 of 2 patients receiving rosiglitazone.

CONCLUSIONS

Insulin resistance caused by antipsychotic agents and divalproex is associated with adrenal hyperandrogenism. Metformin and rosiglitazone correct the biochemical abnormalities detected without compromising their psychotropic effect. Adrenal androgen synthesis may be increased by hyperinsulinemia-induced hyperphosphorylation of P<em>4</em>50c17 alpha, resulting in an increase in its 17,20-lyase activity, which magnifies the effects of any distal steroidogenic enzyme defects. Treatment with metformin or rosiglitazone prevents excess adrenal androgen synthesis.

Preovulatory bovine follices (n = 73) were collected at different times after the onset of oestrus until shortly before ovulation, which occurred at 2<em>4</em> +/- 1 X <em>4</em> h after the peak concentration of LH in the peripheral blood. Non-atretic antral follicles (n = 9) of 15-19 mm were also collected from cows during the luteal phase of the oestrous cycle. Follicular fluid concentrations of dehydroepiandrosterone, <em>androstenedione</em> and oestrone, and of LH, FSH and prolactin were compared in 2-h periods relative to the LH plasma peak. Before the LH surge the concentrations of the steroids were much higher than in non-atretic luteal-phase follicles of similar size. From 0 to 6 h after the LH peak the steroid concentrations decreased sharply to remain low until ovulation; only that of <em>androstenedione</em> increased again after 1<em>4</em> h to remain constant. The ratio between the concentrations of <em>androstenedione</em> and dehydroepiandrosterone remained constant until 1<em>4</em> h after the LH peak; at 1<em>4</em> h it increased about <em>4</em>-fold and remained high until ovulation. The ratio between the oestrone and <em>androstenedione</em> concentration increased gradually to a 10-fold higher value until at 1<em>4</em> h an abrupt decrease was observed. These changes indicate that after the LH peak androgen production is directly inhibited and, at a slower rate, the aromatizing activity. <em>Androstenedione</em> appeared to be the major aromatase substrate. Before the plasma LH peak the follicular fluid concentration of FSH was higher than in luteal-phase follicles; the concentrations of LH and prolactin were not different from those in luteal-phase follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Eight male subjects (2<em>4</em> +/- 1 years old) performed graded ergocycle exercises in normoxic (N) and acute hypoxic (H) conditions (1<em>4</em>.5% O2). VO2max decreased from 55.5 +/- 1.3 to <em>4</em>5.8 +/- 1.<em>4</em> ml . kg-1 . min-1 in H condition. Plasma glucose and free fatty acid concentrations remained unchanged throughout exercise in both conditions. Increase in blood lactate concentration was associated with relative workload in both conditions. At VO2max lactate concentrations were similar in the two conditions, plasma insulin, glucagon, and LH concentrations did not significantly change in either. Plasma delta <em>4</em>-<em>androstenedione</em> and testosterone increased in a similar manner in both conditions. Finally plasma norepinephrine concentration reached at VO2max was significantly lower in hypoxia. These results suggest that acute moderate hypoxia does not affect metabolic and hormonal responses to short exercise performed at similar relative workloads, i.e. when the reduction of VO2max due to hypoxia is taken into consideration. The lower catecholamine response to maximal exercise under acute hypoxia might suggest that the sympathetic response could be related to relative as well as absolute workloads.

Sebaceous glands were isolated by manual dissection under a microscope from surgical specimens of scalp skin with male pattern baldness and skin specimens of hairy and bald scalp obtained at autopsy. The 800 X g pellet (nuclear fraction) and the 16<em>4</em>,000 X g supernatant fraction (cytosol) of homogenates of the sebaceous glands were used for measurements of androgen binding characteristics, using dextran-coated charcoal and sucrose gradient methods. Scatchard plots showed high affinity binding for [3H]dihydrotestosterone (DHT) and [3H]methyltrienolone (R1881). Nuclei prepared from bald scalp contained greater total androgen binding capacity than nuclei of hairy scalp, although Kd values of type I binding were similar (0.68 vs 0.56 nM, respectively). On sucrose gradient, the binding protein from cytosol was found in the 7 to 8S density range. Androgen binding by cytosol of sebaceous glands of hairy scalp had Kd of 1.89 +/- .79 and 2.05 +/- .56 nM for DHT and R1881, respectively, and Bmax of 18.7 +/- <em>4</em>.<em>4</em> and 20.0 +/- <em>4</em>.6 fmol/mg protein for DHT and R1881, respectively. Cytosol from sebaceous glands of bald scalp had Kd values approximately half those of hairy scalp, and Bmax values 50%-100% higher. The bound 3H labeled DHT and R1881 could be partially displaced by testosterone (<em>4</em>0-50%), moxestrol (28-32%), promegestone (19-26%), and delta <em>4</em>-<em>androstenedione</em> (6-12%), but not by dehydroepiandrosterone. These data demonstrate the presence of specific androgen binding protein in sebaceous glands, and that sebaceous glands of bald scalp have greater binding affinity and capacity for androgens than those in hairy scalp. This difference may explain the greater androgenic response in androgenic alopecia.

Metabolism of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstene-3,17-dione (delta(<em>4</em>)) was performed at their physiological plasma concentrations in MCF-7 cell cultures (1 microM, 10 and 2 nM, respectively). Final metabolic products of these steroids were separated by HPLC-radioactive flow detection and identified by LC/MS or MS/MS. Typical and specific mass fragmentation spectra identified the presence of estrone (E(1)), 17beta-estradiol (E(2)), delta(<em>4</em>), DHEA, 5-androstene-3beta,17beta-diol (delta(5)), and testosterone as principal DHEAS metabolites. Other steroids, such as <em>androstenedione</em>, androsterone, and DHEA fatty acid esters at very low concentrations (from pM to nM), were also obtained after steroid incubation. This highly specific method allowed us to conclude whether a metabolite and enzymatic activity of interest were present in MCF-7 cells or not. We also showed that DHEAS at its physiological plasma concentration may be converted into estrogens and estrogen-like compounds in breast cancer cells. The estrogenic action of DHEAS on breast cancer cells was also measured by bioluminescence in a stably transfected human breast cancer MCF-7 cell line with a reporter gene that allowed expression of the firefly luciferase enzyme under the control of an estrogen regulatory element.