OBJECTIVE

Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone-androsterone (5alpha-androstan-3alpha-ol-17-one; 5alpha,3alpha-A), a neurosteroid that acts as a positive allosteric modulator of GABA(A) receptors, and its 5beta-epimer etiocholanolone (5beta-androstan-3alpha-ol-17-one; 5beta,3alpha-A)-also may be reduced in epilepsy. 5alpha,3alpha-A has been found in adult brain, and both metabolites, which also can be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. This study sought to determine whether these endogenous steroid metabolites can protect against seizures.

METHODS

The anticonvulsant activity of 5alpha,3alpha-A and 5beta,3alpha-A was investigated in electrical and chemoconvulsant seizure models in mice. The steroids also were examined for activity against extracellularly recorded epileptiform discharges in the CA3 region of the rat hippocampal slice induced by perfusion with 55 microM 4-aminopyridine (4-AP).

RESULTS

Intraperitoneal injection of 5alpha,3alpha-A-protected mice in a dose-dependent fashion from seizures in the following models (ED50, dose in mg/kg protecting 50% of animals): 6-Hz electrical stimulation (29.1), pentylenetetrazol (43.5), pilocarpine (105), 4-AP (215), and maximal electroshock (224). 5beta,3alpha-A also was active in the 6-Hz and pentylenetetrazol models, but was less potent (ED50 values, 76.9 and 139 mg/kg, respectively), whereas epiandrosterone (5alpha,3beta-A) was inactive (ED50, <or=300 mg/kg). 5alpha,3alpha-A (10-100 microM) also inhibited epileptiform discharges in a concentration-dependent fashion in the in vitro slice model, whereas 5beta,3alpha-A was active but of lower potency, and 5alpha,3beta-A was inactive.

CONCLUSIONS

5alpha,3alpha-A and 5beta,3alpha-A have anticonvulsant properties. Although of low potency, the steroids are present in high abundance and could represent endogenous modulators of seizure susceptibility.

Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of <em>androstenedione</em> from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-<em>4</em> (TLR<em>4</em>), CD1<em>4</em>, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR<em>4</em> complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell <em>androstenedione</em> production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR<em>4</em>, CD1<em>4</em> and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.

To delineate the activity of the GnRH pulse generator during pubertal transition, <em>4</em>0 healthy girls 7-18 yr of age were studied. Ten were prepubertal (PP), 7 were in early puberty (EP), and 23 were in late puberty (LP, all postmenarcheal). Serum concentrations of LH and FSH were measured with immunofluorometric assays, which have a sensitivity about 100-fold that of RIA, in samples taken at 10-min intervals for 2<em>4</em> h during basal conditions, during Nal-Glu antagonist suppression, and in response to GnRH stimulation (10 micrograms). Serum levels of <em>androstenedione</em>, testosterone, and estradiol were measured with RIA. A pulsatile pattern of LH and FSH secretion was found in girls of all ages. PP girls had irregular LH pulses with low amplitudes during the daytime, but increased amplitude LH and FSH pulses were evident within 1 h after sleep-onset. Older PP girls had more regular and higher amplitude pulses throughout sleep than younger PP girls. The sleep-related LH and FSH pulses in PP girls were abolished with Nal-Glu GnRH antagonist treatment, reflecting endogenous GnRH pulse activities. The PP group had the most pronounced amplification of LH secretion with sleep yielding a sleep-wake ratio of <em>4</em>, which decreased to 2 in the EP group and to 1 in the LP group. The emergence of regular daytime LH pulses along with a further amplification of pulsatile activity during sleep was closely related to the onset of breast development. By the age of 16 yr, an LH secretory pattern characteristic of adult women in the early follicular phase, i.e. a decrease in LH concentration during sleep, was established. Mean 2<em>4</em>-h LH concentrations increased <em>4</em>0-fold from PP to LP consequent to a 9-fold increase in pulse amplitude and a <em>4</em>-fold increase in pulse number (both P < 0.0001). Mean FSH concentrations (2<em>4</em> h), which were 20-fold higher than corresponding LH concentrations in the PP group, increased only 3-fold from the PP to the LP group. FSH pulse secretion appears to be predominantly GnRH dependent in PP girls in contrast to girls after ovarian activation, as indicated by the increased FSH responses to both GnRH antagonist suppression and GnRH stimulation in the PP as compared to the EP and LP groups. We conclude that the GnRH pulse generator is functionally active in prepubertal girls with selective expression of LH and FSH pulses after the onset of sleep. The onset of puberty is associated with a greater increase in LH pulse amplitude than frequency.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

Because of large intra-individual variation in hormone levels, few studies have investigated the relation of serum sex hormones to breast cancer (BC) in premenopausal women. We prospectively studied this relation, adjusting for timing of blood sampling within menstrual cycle. Premenopausal women (5,963), recruited to the Hormones and Diet in the Etiology of Breast Tumors (ORDET) cohort study, provided a blood sample in the 20-2<em>4</em>th day of their menstrual cycle. After 5.2 years of follow-up, 65 histologically confirmed BC cases were identified and matched individually to <em>4</em> randomly selected controls. Sera, stored at -80 degrees C, were assayed blindly for dehydroepiandrosterone sulfate, total and free testosterone (FT), <em>androstenedione</em>, androstanediol-glucoronide, progesterone, 17-OH-progesterone, sex hormone-binding globulin, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Fifty-five cases had information for multivariate analyses. Compared to controls, BC cases had shorter cycles and intervals between blood sampling and bleeding, and lower LH and FSH. FT was significantly associated with BC risk: relative risk (RR; adjusted for age, body mass index and ovarian cycle variables) of highest vs. lowest tertile was 2.85 [95% confidence interval (CI) = 1.11-7.33, p for trend = 0.030]. Progesterone was inversely associated with adjusted RR for highest vs. lowest tertile of 0.<em>4</em>0 (95% CI = 0.15-1.08, p for trend = 0.077), significantly so in women with regular menses, where adjusted RR was 0.12 (95% CI = 0.03-0.52, p for trend = 0.005). These findings support the hypothesis that ovarian hyperandrogenism associated with luteal insufficiency increases the risk of BC in premenopausal women.

Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P(<em>4</em>)) and estradiol (E(2)) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or <em>androstenedione</em> were subsequently added as substrates for the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) or P<em>4</em>50-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependent manner E(2) and P(<em>4</em>) secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E(2) secretion, but not on P(<em>4</em>). These results show that calcitriol is a physiological regulator of placental E(2) and P(<em>4</em>) production and suggest a novel role for calcitriol upon placental steroidogenesis.

Dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) are adrenal precursors of steroid biosynthesis and centrally acting neurosteroids. Glucocorticoid and mineralocorticoid deficiencies in Addison's disease require life-long hormone replacement, but the associated failure of DHEA synthesis is not corrected. We conducted a randomized, double blind study in which 39 patients with Addison's disease received either 50 mg oral DHEA daily for 12 weeks, followed by a <em>4</em>-week washout period, then 12 weeks of placebo, or vice versa. After DHEA treatment, levels of DHEAS and Delta(<em>4</em>)-<em>androstenedione</em> rose from subnormal to within the adult physiological range. Total testosterone increased from subnormal to low normal with a fall in serum sex hormone-binding globulin in females, but with no change in either parameter in males. In both sexes, psychological assessment showed significant enhancement of self-esteem with a tendency for improved overall well-being. Mood and fatigue also improved significantly, with benefit being evident in the evenings. No effects on cognitive or sexual function, body composition, lipids, or bone mineral density were observed. Our results indicate that DHEA replacement corrects this steroid deficiency effectively and improves some aspects of psychological function. Beneficial effects in males, independent of circulating testosterone levels, suggest that it may act directly on the central nervous system rather than by augmenting peripheral androgen biosynthesis. These positive effects, in the absence of significant adverse events, suggest a role for DHEA replacement therapy in the treatment of Addison's disease.

The ability of human abdominal, breast and axillary fat to convert androgens into estrogens was investigated by incubating labeled substrates in the presence of NADPH with a variety of cell preparations. The incubation products were subjected to phenolic partition, paper chromatography, methyl-ether formation, repeat chromatography and crystallization with cold carrier reference standards to constant specific activity. <em>Androstenedione</em> was converted to estrone and, to a lesser extent, to 17beta-estradiol by crude homogenates, minces, fat-free particulate fractions (1,000-100,000 time g) and isolated fat cells obtained from abdominal, breast or axillary fat. Testosterone was found to be aromatized as actively as <em>androstenedione</em>, but inthis case more 17 beta-estrodiol was formed than estrone. 19-Hydroxy<em>androstenedione</em>-2 also served as substrate, givingresults similar to those obtained with <em>androstenedione</em>. Fat tissue obtained from cancerous breasts was found to be as active as normal breast fat (1-<em>4</em> pg/g fat/90 min) and within the range found for abdominal fat (1-27 pg/g fat/90 min). In each case in which axillary fat was compared to breast fat from the same subject, the activity of the axillary fat was 5 to 10 times higher. The results indicate a possible role of adipose tissue as a significant extra-gonadal source of estrogens.

The endocrinological changes of the climacteric have been defined by studying the concentrations of follicle-stimulating hormone (FSH), luteinising hormone (LH), <em>androstenedione</em>, testosterone, oestrone, and oestradiol in 60 normal postmenopausal women of different menopausal ages. The women were studied in six groups, according to the number of years since their menopause. One year after the menopause <em>androstenedione</em>, oestrone, and oestradiol concentrations were reduced to about 20% of the values recorded during the early proliferative phase of the menstrual cycle. At the same time the mean concentration of FSH had risen by a factor of 13-<em>4</em> and that of LH by a factor of 3-0. Concentrations of both gonadotrophins reached a peak of 18-<em>4</em> and 3-<em>4</em> times the proliferative phase value respectively after two to three years, and then gradually declined in the next three decades to values that were <em>4</em>0-50% of these maximal levels. Testosterone concentrations remained mostly in the normal range for premenopausal women but were depressed to 60% of these levels two to five years after the menopause, and the mean <em>androstenedione</em> levels showed a significant increase in the same group of women. The concentrations of both oestrone and oestradiol remained consistently low for 10 years after the menopause, but oestradiol concentrations inexplicably increased in the last two decades, with levels at the lower end of normal range for reproductive women in six patients.

OBJECTIVE

To investigate if human thecal cells contain messenger ribonucleic acid (RNA) encoding insulin-like growth factor I (IGF-I) and insulin receptors and if IGF-I and insulin could stimulate androgen production in thecal cells.

METHODS

Poly-adenine+ RNA was extracted from fresh thecal tissue, and the expression of the genes encoding insulin and IGF-I receptors were analyzed. Isolated thecal cells were cultured <em>4</em> to 6 days with and without hormones.

METHODS

Procedures were performed in a university laboratory.

METHODS

Eight women in the follicular phase of natural cycles were undergoing gynecological laparotomy for reasons unrelated to ovarian pathology. The leading follicle(s) was excised, and dispersed cells of the theca interna layer were isolated through combined mechanical and enzymatic techniques.

METHODS

Luteinizing hormone (LH), IGF-I, and insulin were added to the cell cultures.

METHODS

The expression of IGF-I receptor and insulin receptor transcripts were analyzed by Northern blot. Medium levels of androstenedione and testosterone were measured by radioimmunoassay.

RESULTS

In the separated thecal tissue both IGF-I receptor and insulin-receptor transcripts were detected. Insulin-like growth factor I and insulin potentiated LH-induced androgen secretion while having less pronounced effects on basal androgen production.

CONCLUSIONS

The present study demonstrates that both insulin and IGF-I receptor genes are expressed and that insulin and IGF-I can stimulate steroid production in human thecal cells. The study provides further support for the hypothesis that IGF-I and insulin may be involved both in physiological regulation of ovarian function as well as in its pathophysiology.

To determine the relevance of polycystic ovarian morphology (PCOM) to the pathophysiology of polycystic ovarian syndrome (PCOS), biochemical features associated with PCOS were examined in 68 women with an established history of regular ovulatory cycles and no clinical evidence of hyperandrogenism. Ovarian morphology was objectively assessed by pelvic ultrasound. LH, FSH, estradiol (E(2)), testosterone (T), <em>androstenedione</em> (Delta(<em>4</em>)A), SHBG, and dehydroepiandrosterone sulfate (DHEAS) were measured at baseline in the early follicular phase (EFP) in all subjects. LH, FSH, E(2), and progesterone (P(<em>4</em>)) were then measured daily for a complete menstrual cycle in 16 women with normal ovarian morphology and in 26 women with PCOM. T, Delta(<em>4</em>)A, SHBG, and DHEAS levels were measured in pools of three daily samples in each of the EFP, midcycle, and midluteal phases. An additional 26 normal women (13 with normal ovarian morphology and 13 with PCOM) were studied in the EFP to assess pulsatile LH secretion, insulin and glucose levels, and the ovarian response to human chorionic gonadotropin. At baseline, there were no differences in body mass index or hirsutism scores between women with PCOM and normal ovaries. In daily samples across the menstrual cycle LH, FSH, E(2), and P(<em>4</em>) did not differ between women with PCOM and those with normal ovaries, and there was no difference in LH pulse amplitude or frequency in the EFP frequent sampling studies. In women with PCOM, T (P < 0.01), free T (P < 0.005), and DHEAS (P < 0.01) levels were higher at baseline in the EFP, and SHBG was lower (P < 0.05). Differences in Delta(<em>4</em>)A did not reach significance (P = 0.1<em>4</em>). T, free T, Delta(<em>4</em>)A, and DHEAS were also increased in PCOM across the menstrual cycle (P < 0.05). In addition, 17-hydroxyprogesterone (P < 0.02), Delta(<em>4</em>)A (P < 0.01), and T (P < 0.01) responses to human chorionic gonadotropin were greater in women with PCOM. Fasting glucose was not different between the two groups, but fasting insulin was higher (P < 0.02) in PCOM women as was insulin resistance calculated from homeostatic model assessment (P < 0.01). These studies demonstrate that PCOM in nonhirsute women with documented ovulatory cycles is associated with normal E(2), P(<em>4</em>), and gonadotropin dynamics, but higher androgen and insulin levels and lower SHBG levels. Taken together, these findings suggest that PCOM with ovulatory cycles exists as a discrete entity, represents the mildest form of ovarian hyperandrogenism, and is associated with greater insulin resistance than in women with normal ovarian morphology. The absence of any neuroendocrine abnormality in women with PCOM and ovulatory cycles suggests that gonadotropin dysfunction is not required for increased androgen secretion, but may be critical for development of the anovulatory disorder associated with PCOS.

MCF-7 cells transfected with human placental aromatase gene (MCF-7Ca cells) or cells transfected with plasmid vector only (MCF-7Cc cells) were inoculated into nude mice with Matrigel. Tumors formed from both MCF-7Ca and MCF-7Cc cells grew faster in intact mice than in ovariectomized mice, suggesting that the tumors maintained their responsiveness to estrogen stimulation and that their growth was supported by ovarian estrogen. Injections of <em>androstenedione</em> (0.1 mg/mouse/day) to provide the substrate for aromatization to ovariectomized mice bearing MCF-7Ca tumors accelerated their growth but did not affect growth of MCF-7Cc tumors. This result indicates that local production of estrogen by intratumoral aromatase was sufficient to stimulate tumor growth. When ovariectomized mice with MCF-7Ca tumors supplemented with <em>androstenedione</em> were treated with aromatase inhibitors <em>4</em>-hydroxy<em>androstenedione</em> (1 mg/mouse/day, s.c.) or CGS 169<em>4</em>9A (0.5 mg/mouse/day, s.c.), or with the antiestrogen tamoxifen (10 micrograms/mouse/day, s.c.), tumor growth was significantly inhibited. Tumor aromatase activity measured at the end of treatment was also inhibited by <em>4</em>-hydroxy<em>androstenedione</em> when the mice were sacrificed <em>4</em> h after the last injection. The tumors of this mouse model are dependent for their growth on estrogens from an endogenous nonovarian source. Thus, it simulates the situation in the postmenopausal breast cancer patient and could be used to evaluate the effect of aromatase inhibitors and antiestrogens.

Ketoconazole inhibits testosterone biosynthesis in men, but the exact site of its action on the androgen pathway remains to be established. To examine this question, we measured several steroids in the androgen and glucocorticoid pathways in normal men before and after receiving either a single dose of 200 mg ketoconazole or placebo in a cross-over randomized trial. Total and free plasma testosterone fell to levels 60% below basal within <em>4</em>-8 h (P less than 0.02 in all) and then returned to control concentrations by 2<em>4</em> h after drug administration. The transient alterations of plasma testosterone correlated well with ketoconazole blood levels, which peaked at 2 h and fell exponentially thereafter. A compensatory increase in plasma LH at 2<em>4</em> h in the drug but not placebo group was consistent with the decrease in plasma testosterone. The levels of plasma <em>androstenedione</em> paralleled those of testosterone in the ketoconazole-treated subjects. In marked contrast, plasma 17 alpha-hydroxyprogesterone increased at <em>4</em>-8 h (all P less than 0.02) before returning to basal values at 2<em>4</em> h. This rise in precursor with fall in product steroid implicated an effect of ketoconazole on the C17-20 lyase enzyme. This conclusion was supported by the highly significant increase in the ratio of plasma 17 alpha-hydroxyprogesterone to <em>androstenedione</em> observed between 2 and 2<em>4</em> h after drug administration. The effect of ketoconazole at this dose level appeared relatively specific, since no decrements in plasma cortisol or 11-desoxycortisol were found. During chronic administration of 200 mg ketoconazole daily, decrements of plasma testosterone 2-<em>4</em> h after drug administration were minimal and documented only by paired comparisons within subjects but not by unpaired tests between normal men and men receiving drug. The lack of major effects on testosterone levels long term at this dosage probably explain why few androgen-related side effects with this drug were previously reported. Ketoconazole, therefore, represents another compound with relatively selective effects on a cytochrome P-<em>4</em>50-mediated steroid hydroxylation step, namely that involved with C17-20 lyase.

<em>4</em>-Hydroxy<em>androstenedione</em> (CGP323<em>4</em>9; <em>4</em>-OHA) is a clinically effective treatment for advanced postmenopausal breast cancer by both the parenteral and p.o. routes, as a result of its inhibition of aromatase and consequent suppression of plasma estrogen levels. Thirty patients were randomized to treatment with 250 mg <em>4</em>-OHA orally once, twice, and <em>4</em> times daily for 2 weeks and 29 of these plus a further 11 patients were then randomized to treatment with 250 or 500 mg i.m. every 2 weeks to determine the optimal dose for each route according to the suppression of serum estradiol levels. There was no significant difference between the 3 oral doses in their suppression of estradiol levels indicating that the maximum required p.o. dose of <em>4</em>-OHA is probably 250 mg daily. Suppression by the parenteral dose of 250 mg every 2 weeks was marginally suboptimal but clinical considerations of response and tolerability indicate this as the optimal dose for i.m. injection. <em>4</em>-OHA had no effect on serum levels of <em>androstenedione</em>, testosterone, or 5 alpha-dihydrotestosterone when given by either route but p.o. treatment with <em>4</em> doses of 250 mg daily reduced sex hormone-binding globulin levels by a mean of 3<em>4</em>%. Serum levels of estrone as measured by gas chromatography-mass spectrometry were suppressed to approximately <em>4</em>0% of baseline by parenteral treatment. The half-life of <em>4</em>-OHA p.o. was approximately 3 h, whereas the apparent half-life of injected drug was between 5 and 10 days after a more rapid clearance during the first <em>4</em> days after injection.

To determine whether prenatal T propionate exposure beginning gestational d <em>4</em>0-<em>4</em><em>4</em> (early-treated) or 100-115 (late-treated) affects oocyte competence, five early-treated and five late-treated prenatally androgenized and five normal monkeys underwent recombinant human FSH injections with oocyte-retrieval after hCG administration. Serum FSH, LH, estradiol (E(2)), progesterone (P(<em>4</em>)), <em>androstenedione</em> (A(<em>4</em>)), T, and dihydrotestosterone were measured basally, during gonadotropin stimulation and at oocyte-retrieval; fasting serum glucose and insulin also were determined basally and at oocyte-retrieval. Follicle fluid sex steroids were analyzed. Oocyte number, nuclear maturity, and fertilization were comparable among female groups, but the percentage of zygotes developing into blastocysts was reduced in early-treated prenatally androgenized females. The intrafollicular P(<em>4</em>)/E(2) ratio was significantly elevated in early-treated prenatally androgenized females, whereas intrafollicular P(<em>4</em>)/A(<em>4</em>) and T/A(<em>4</em>) ratios were significantly increased in all prenatally androgenized females. Early-treated prenatally androgenized females demonstrated persistent LH hypersecretion. They also were unable to suppress circulating insulin levels during gonadotropin stimulation. Circulating sex steroid levels and serum P(<em>4</em>)/E(2), P(<em>4</em>)/A(<em>4</em>), and E(2)/androgen ratios were similar in all females. Early prenatal androgenization in monkeys receiving gonadotropins impairs oocyte developmental competence and seems to induce premature follicle differentiation in the presence of LH hypersecretion and relative insulin excess.

Clinical research suggests that type of ovarian hormone loss at menopause influences cognition. Until recently ovariectomy (OVX) has been the primary rodent model to examine effects of ovarian hormone loss on cognition. This model limits evaluations to abrupt and complete ovarian hormone loss, modeling less than 13% of women who receive surgical menopause. The majority of women do not have their ovaries surgically removed and undergo transitional hormone loss via ovarian follicular depletion. <em>4</em>-Vinylcyclohexene-diepoxide (VCD) produces gradual ovarian follicular depletion in the rodent, with hormone profiles more similar to naturally menopausal women vs. OVX. We directly compared VCD and OVX models to examine whether type of hormone loss (transitional vs. surgical) impacted cognition as assessed on a maze battery as well as the cholinergic system tested via scopolamine mnemonic challenge and brain acetylcholinesterase activity. Middle-aged rats received either sham surgery, OVX surgery, VCD, or VCD then OVX to assess effects of removal of residual ovarian output after transitional menopause and follicular depletion. VCD-induced transitional menopause impaired learning of a spatial recent memory task; surgical removal of residual ovarian hormones by OVX abolished this negative effect of transitional menopause. Furthermore, transitional menopause before OVX was better for memory than an abrupt loss of hormones via OVX only. Surgical ovarian hormone loss, regardless of menopause history, increased hippocampal acetylcholinesterase activity. Circulating gonadotropin and <em>androstenedione</em> levels were related to cognitive competence. Collectively, findings suggest that in the rat, initiation of transitional menopause before surgical ovary removal can benefit mnemonic function and could obviate some negative cognitive consequences of surgical menopause alone.

BACKGROUND

Polycystic ovary syndrome (PCOS) is associated with insulin resistance and obesity. Recent studies have shown that serum retinol-binding protein <em>4</em> (RBP<em>4</em>) levels increase with obesity. Currently, no data exist on the relative expression of RBP<em>4</em> in either serum or adipose tissue of PCOS women.

OBJECTIVE

mRNA expression of RBP<em>4</em> from sc and omental (om) adipose tissue and sc adipocytes in overweight PCOS women were compared with matched controls; RBP<em>4</em> protein in adipose tissue and serum RBP<em>4</em> levels were also assessed. Additionally, we studied the effects of testosterone, 17beta-estradiol, androstenedione, and dehydroepiandrosterone sulfate on RBP<em>4</em> expression in adipose tissue explants.

METHODS

Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of RBP<em>4</em>. Biochemical measurements were also performed.

RESULTS

Compared with controls, there was significant up-regulation of RBP<em>4</em> mRNA in sc (P < 0.05) and om (P < 0.01) adipose tissue as well as isolated sc adipocytes (P < 0.01) of PCOS women. In addition to elevated serum RBP<em>4</em> levels in PCOS women (P < 0.05), RBP<em>4</em> protein levels were significantly greater in sc and om adipose tissue of PCOS women (P < 0.05 and P < 0.05, respectively). Furthermore, in human sc and om adipose tissue explants, 17beta-estradiol significantly increased RBP<em>4</em> mRNA expression, protein levels, and secretion into the culture media (P < 0.05).

CONCLUSIONS

The precise reason for elevated levels of RBP<em>4</em> in overweight PCOS women is unknown, but it appears that 17beta-estradiol may play a role in their regulation in adipose tissue.

OBJECTIVE

In the phase III study COU-AA-301, abiraterone acetate (AA) plus prednisone (P) prolonged overall survival (OS) in patients with metastatic castration-resistant prostate cancer (mCRPC) after docetaxel administration. In this article, we investigate the relationship between baseline serum androgen (SA) levels and OS.

METHODS

COU-AA-301 is a randomized, double-blind study of AA (1,000 mg every day) plus P (5 mg by mouth twice daily; n = 797) versus P alone (n = 398). Randomization was stratified by Eastern Cooperative Oncology Group performance status (0 to 1 v 2), pain (Brief Pain Inventory-Short Form over past 2<em>4</em> hours: <em>4</em> to 10, present; v 0 to 3, absent), prior chemotherapy (1 v 2), and progression (prostate-specific antigen v radiographic). Association of baseline SA (testosterone, <em>androstenedione</em>, dehydroepiandrosterone sulfate), was measured by ultrasensitive liquid-liquid extraction or protein precipitation and two-dimensional liquid chromatography coupled to mass spectrometry, with OS determined by bivariate and multivariable Cox models. OS was examined with SA as greater than median and less than or equal to the median.

RESULTS

Median survival increased with each quartile increase in testosterone level regardless of treatment arm. SA levels at baseline strongly associated with survival (P < .0001) in bivariate and multivariable analyses. Longer survival was observed for patients with SA above median compared with below median in both the AA and P arms (eg, testosterone, AA; hazard ratio, 0.6<em>4</em>; 95% CI, 0.53 to 0.77; P < .0001). Treatment with AA led to longer survival versus P alone in the above- or below-median group for all androgens.

CONCLUSIONS

SA, measured with a novel ultrasensitive assay in COU-AA-301, is prognostic for OS and may be useful for risk stratification in mCRPC clinical trials.

BACKGROUND

The study aim was to investigate possible changes in serum anti-Müllerian hormone (AMH) levels during controlled ovarian hyperstimulation (COH), and their possible relationship with follicular development and other ovarian hormones.

METHODS

A total of 93 women undergoing COH with GnRH agonist and FSH was studied prospectively. Serum levels of AMH, inhibin B, estradiol (E(2)), progesterone, testosterone and Delta(<em>4</em>)-<em>androstenedione</em> were measured when pituitary suppression was achieved (baseline), on days 6 and 8 of FSH treatment, and on the day of hCG. The number of small (<12 mm) and large >>/=12 mm) antral follicles were estimated using ultrasound.

RESULTS

Serum AMH levels declined progressively (baseline, 1.21 +/- 0.11 ng/ml; day 6, 0.91 +/- 0.09 ng/ml; day 8, 0.77 +/- 0.08 ng/ml; and day of hCG, 0.53 +/- 0.06 ng/ml), whereas-as expected-the other hormone levels increased during FSH treatment. Throughout COH, serum AMH levels correlated positively with the number of small but not large antral follicles, and with inhibin B serum levels. No correlation between AMH and the other hormones was observed.

CONCLUSIONS

Serum AMH levels decline gradually during multiple follicular maturation, probably reflecting the dramatic reduction in the number of small antral follicles due to COH, and confirming the scarce AMH expression by larger follicles.

OBJECTIVE

We wished to determine the prevalence of polycystic ovaries (PCO) and relate morphological appearance to fertility.

METHODS

We sent postal invitations to a random sample of women born in the years 1952-1969 from a list of a single Group Practice to attend for reproductive history questionnaire, examination, ultrasound scan of the ovaries and hormone measurements within 5 days of the onset of menstruation.

METHODS

Of 1065 women potentially available for study, 571 (5<em>4</em>%) replied of whom 353 (62%) agreed to participate. One hundred and ninety (18%) completed the study, 163 were deferred (57 because of current or very recent pregnancy, 106 because of inconvenience at time approached), and 18 additional women volunteered.

METHODS

Prevalence of polycystic ovaries, ovarian size and morphology, menstrual history, features of androgen excess, fertility status, serum hormone levels.

RESULTS

The prevalence of PCO was 22% (<em>4</em>1/190). PCO and non-PCO women were similar with respect to age, body mass index, oral contraceptive pill (OCP) usage, acne, and menstrual pattern but hirsutism (Ferriman and Gallwey score>> 7) was significantly (P = 0.006) more frequent among PCO women. Proven prior fertility was the same in PCO (56%) and non-PCO (6<em>4</em>%) women and an equal proportion in each group had not yet tested their fertility. Of those women with previously proven fertility, self-perceived difficulty in conception occurred in similar proportions of women with and without PCO. Unresolved primary or secondary infertility (2.5-<em>4</em>%) was similar in both groups. Ovarian volume (each ovary separately) was larger in women with PCO irrespective of current OCP usage. Serum levels of oestradiol and FSH were similar in PCO and non-PCO women, but LH was distributed around a higher median in PCO women. Median testosterone and <em>androstenedione</em> levels were the same in PCO and non-PCO women.

CONCLUSIONS

The prevalence of polycystic ovarian morphology is high but, in this sample of women, was accompanied by minimal clinical manifestations and apparently no deleterious effects on earlier fertility. An isolated finding of polycystic ovaries may be a normal variation and should not necessarily imply altered fertility potential.

Women with adrenal insufficiency suffer from chronic dehydroepiandrosterone (sulfate) [DHEA(S)] deficiency. To define a suitable dose for DHEA replacement, we studied the pharmacokinetics and biotransformation of orally administered DHEA in nine healthy female volunteers (mean age 23.3 +/- <em>4</em>.1 yr, mean body mass index 22.5 +/- 1.8 kg/m2) with transient suppression of adrenal androgen secretion because of dexamethasone (dex) administration (<em>4</em> x 0.5 mg/day for <em>4</em> days). Diurnal blood sampling was performed during the early follicular phase of four subsequent menstrual cycles (study period 1: baseline; study periods 2-<em>4</em>: dex + placebo, dex + 50 mg DHEA or dex + 100 mg DHEA in a randomized cross-over design). Dex induced not only a significant suppression of serum cortisol (to 8% of baseline) but also of DHEA (18%), DHEA(S) (16%), and <em>androstenedione</em> (26%), as well as of testosterone (28%), dihydrotestosterone (<em>4</em>3%), and estrone (5<em>4</em>%). Oral administration of 50 mg DHEA led to restoration of DHEA(S) baseline levels, whereas 100 mg induced supraphysiological concentrations [baseline vs. 50 mg DHEA vs. 100 mg DHEA: area under the concentration-time curve (AUC) 0-12 h DHEA: 280 +/- 85 vs. 2<em>4</em>1 +/- 73 vs. 383 +/- 106 nmol/L x h; AUC 0-12 h DHEA(S): 89.1 +/- <em>4</em>8.<em>4</em> vs. 139.6 +/- <em>4</em>3.5 vs. 213.3 +/- 21.6 mumol/L x h). Serum concentrations of dihydrotestosterone and estrone were restored to baseline after 50 mg DHEA, whereas baseline testosterone and <em>androstenedione</em> levels were only achieved by administration of 100 mg DHEA. In conclusion, 50 mg DHEA seems to be a suitable replacement dose in females with adrenal insufficiency. Furthermore, the rapid and lasting conversion to potent androgens demonstrates a potential role of DHEA for androgen replacement in females in general.

The concentrations of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), <em>androstenedione</em> (A-dione), testosterone (T) and dihydrotestosterone (DHT) have been measured before and after castration in men and two animal models, namely the rat and the guinea pig. In adult men, the pre-castration levels of plasma DHEAS and DHEA were measured at 1839 +/- 320 and 2.<em>4</em> +/- 0.5 ng/ml, respectively, while in both animal models, the concentrations of these two steroids were below 0.3 ng/ml. Orchiectomy in men reduced plasma T and DHT levels from 2.9 +/- 0.1 and 0.60 +/- 0.10 to 0.<em>4</em>2 +/- 0.21 and 0.05 +/- 0.01 ng/ml (P less than 0.01), respectively, while there was no significant effect observed on DHEAS, DHEA and A-dione levels. By contrast, castration in the rat reduced the low levels of circulating DHEA and A-dione below the detection of the radioimmunoassay (RIA) used. In castrated guinea pig, a small quantity of plasma A-dione (0.07 +/- 0.02 ng/ml) was measured while DHEA was undetectable. Moreover, in the rat and guinea pig, plasma T and DHT levels became undetectable. Following administration of the antiandrogen Flutamide for two weeks in the castrated rat and guinea pig, prostate weight was not further reduced, thus indicating that there is no significant androgenic activity left following castration of these two species. In fact, castration in the rat and guinea pig caused a decrease in prostatic levels of DHT from <em>4</em>.2<em>4</em> +/- 0.351 and 9.<em>4</em>2 +/- 1.<em>4</em>3 ng/g, respectively, to undetectable levels. In men, on the other hand, the prostatic DHT levels were only inhibited from 5.2<em>4</em> +/- 0.59 to 2.70 +/- 1.50 ng/g, respectively. As expected, when Flutamide was administered to the rat and the guinea pig, the levels of prostatic steroids remained undetectable while, in men, the DHT content in the prostate was further reduced to undetectable values. In summary, the plasma levels of DHEAS, DHEA, delta <em>4</em>-dione are markedly different between men and both animal models used and furthermore, measurements of prostatic levels of androgens suggest that the high plasma levels of these steroids are likely responsible for the presence of important amounts of DHT in human prostate after castration.

OBJECTIVE

Treatment with abiraterone (abi) acetate prolongs survival in castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to upregulation of steroidogenic enzymes and/or other mechanisms that sustain dihydrotestosterone (DHT) synthesis, which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. On the basis of the 3β-hydroxyl, Δ(5)-structure, we hypothesized that abi also inhibits 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), which is absolutely required for DHT synthesis in CRPC, regardless of origins or routes of synthesis.

METHODS

We tested the effects of abi on 3βHSD activity, androgen receptor localization, expression of androgen receptor-responsive genes, and CRPC growth in vivo.

RESULTS

Abi inhibits recombinant 3βHSD activity in vitro and endogenous 3βHSD activity in LNCaP and LAPC<em>4</em> cells, including conversion of [(3)H]-dehydroepiandrosterone (DHEA) to Δ(<em>4</em>)-<em>androstenedione</em>, androgen receptor nuclear translocation, expression of androgen receptor-responsive genes, and xenograft growth in orchiectomized mice supplemented with DHEA. Abi also blocks conversion of Δ(5)-androstenediol to testosterone by 3βHSD. Abi inhibits 3βHSD1 and 3βHSD2 enzymatic activity in vitro; blocks conversion from DHEA to <em>androstenedione</em> and DHT with an IC(50) value of less than 1 μmol/L in CRPC cell lines; inhibits androgen receptor nuclear translocation; expression of TMPRSS2, prostate-specific antigen, and FKBP5; and decreases CRPC xenograft growth in DHEA-supplemented mice.

CONCLUSIONS

We conclude that abi inhibits 3βHSD-mediated conversion of DHEA to active androgens in CRPC. This second mode of action might be exploited to reverse resistance to CYP17A1 inhibition at the standard abi dose by dose-escalation or simply by administration with food to increase drug exposure.

OBJECTIVE

Increasing evidence indicates that enhanced intratumoral androgen synthesis contributes to prostate cancer progression after androgen deprivation therapy. This phase II study was designed to assess responses to blocking multiple steps in androgen synthesis with inhibitors of CYP17A1 (ketoconazole) and type I and II 5alpha-reductases (dutasteride) in patients with castration-resistant prostate cancer (CRPC).

METHODS

Fifty-seven men with CRPC were continued on gonadal suppression and treated with ketoconazole (<em>4</em>00 mg thrice daily), hydrocortisone (30 mg/AM, 10 mg/PM), and dutasteride (0.5 mg/d).

RESULTS

Prostate-specific antigen response rate >> or =50% decline) was 56% (32 of 57; 95% confidence interval, <em>4</em>2.<em>4</em>-69.3%); the median duration of response was 20 months. In patients with measurable disease, 6 of 20 (30%) responded by the Response Evaluation Criteria in Solid Tumors. Median duration of treatment was 8 months; 9 patients remained on therapy with treatment durations censored at 18 to 32 months. Median time to progression was 1<em>4</em>.5 months. Grade 3 toxicities occurred in 32% with only one reported grade <em>4</em> (thrombosis) toxicity. Dehydroepiandrosterone sulfate declined by 89%, <em>androstenedione</em> by 56%, and testosterone by 66%, and dihydrotestosterone declined to below detectable levels compared with baseline levels with testicular suppression alone. Median baseline levels and declines in dehydroepiandrosterone sulfate, <em>androstenedione</em>, testosterone, and dihydrotestosterone were not statistically different in the responders versus nonresponders, and hormone levels were not significantly increased from nadir levels at relapse.

CONCLUSIONS

The response proportion to ketoconazole, hydrocortisone, and dutasteride was at least comparable with previous studies of ketoconazole alone, whereas time to progression was substantially longer. Combination therapies targeting multiple steps in androgen synthesis warrant further investigation.

The concentrations of the adrenal steroids dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em>-A) have been measured by RIA before and after sexual maturation in plasma of rodents, domestic animals, and primates to determine whether these species exhibit and adrenarchal process comparable to man. The average concentrations of DHA and DHAS were less than 60 ng/dl and 5 microgram/dl, respectively, in plasma of sexually mature rodents and domestic animals, and a significant increase in the plasma DHA level after sexual maturation was seen only in the rabbit and dog. The concentrations of DHA, DHAS, and delta <em>4</em>-A in 21 rhesus monekeys from 0-3 yr of age were 2021 +/- 235 ng/dl (mean +/- SE), 357 +/- 60 microgram/dl, and 107 +/- 9 ng/dl, respectively, and did not increase during sexual maturation. By contrast, DHA, DHAS, and delta <em>4</em>-A levels in plasma of chimpanzees were 5.9-fold, 3.3-fold, and <em>4</em>.8-fold greater, respectively, in 7- to 22-compared to 0- to 3-yr-old animals. Temporally, the increase in DHA levels in the chimpanzee is apparent at 5 yr and this precedes the increase in gonadal steroids, as is characteristic of human adrenarche. It is apparent that adrenal androgen levels and their developmental patterns differ markedly among species, and that among the species examined, only the chimpanzee exhibits an adrenarche comparable to that of man.