Testosterone (T) triggers aggressive behavior in males of many vertebrate species; however, the neural and hormonal basis of individual differences in the frequency or intensity of aggressive behavior is still debated. Using the Japanese quail (Coturnix coturnix japonica), a species in which individuals exhibit a wide range of aggressiveness in nature and the laboratory, together with a newly devised test procedure for quantifying aggressiveness, we recently demonstrated that aggression is estrogen dependent. Here we extend these studies by testing the hypothesis that aromatization in brain is a rate-limiting step in the expression of individual differences in aggressiveness. Using procedures previously validated for this species, aromatase and 5 alpha- and 5 beta-reductase activities were estimated in selected brain regions of reproductively active male quail by measuring conversion of [3H]<em>androstenedione</em> to [3H]estrone, [3H]5 alpha-androstanedione, and [3H]5 beta-androstanedione, respectively. In Exp 1, behaviorally inexperienced test birds were killed 90 sec after a single behavioral test. Aggressiveness of individuals in this group, as determined by pecking and locomotor activity in response to visualization of a conspecific, ranged 3- to <em>4</em>-fold from high to low. Aromatase activity in the posterior hypothalamus (PHYP) was significantly higher in males rated high for aggressiveness than in animals rated low (1.0<em>4</em> vs. 0.59 pmol/h.mg protein; P less than 0.02). Similar differences were observed in the anterior hypothalamus/preoptic area (AHPOA) but were not significant. In Exp 2, sexually mature males were behaviorally tested eight times over 22 days and killed 2<em>4</em> h after the final test. Aggressiveness varied 5-fold from high to low, although the rating in a given bird remained constant with time and repeat testing. Aromatase activity in the AHPOA was significantly greater in birds rated high for aggressiveness than in low aggressiveness birds (3.77 vs. 2.80 pmol/h.mg protein; P less than 0.02). In addition, when AHPOA aromatase in all birds was plotted against behavioral intensity, there was a 2-fold variation and a significant positive correlation (r = 0.556; P less than 0.02). Similar differences were observed in PHYP, but these were of borderline significance. By contrast, aromatase levels outside the AHPOA and PHYP were unrelated to behavior. Moreover, in both Exp 1 and 2, 5 alpha- and 5 beta-reductase activities in AHPOA, PHYP, and other brain regions; plasma T, 5 alpha-dihydrotestosterone, and total estrogens; and relative testicular weights were not consistently related to aggression.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

We have measured the plasma concentrations of sex steroids and sex hormone-binding globulin (SHBG) in twenty-three massively obese women and ten age-matched lean female volunteers. In the obese women increased plasma testosterone (obese 3.2 +/- 0.5 nmol/l controls 1.7 +/- 0.5 nmol/l, P less than 0.3) and <em>androstenedione</em> concentrations (obese 9.7 +/- 1.2 nmol/l, controls <em>4</em>.<em>4</em> +/- 0.6 nmol/l, P = less than 0.01) an increased ratio of oestrone:oestradiol (obese 2.<em>4</em> +/- 0.<em>4</em>, controls 1.0 +/- 0.1, P = less than 0.1) and decreased SHBG levels (obese 30 +/- <em>4</em> nmol/l, controls 60 +/- 8 nmol/l, P = less than 0.001) were found. Obesity differed from the polycystic ovary syndrome (in which a similar pattern of changes of sex steroid concentrations and binding are seen) in that it was associated with normal increases in serum luteinizing hormone (LH) follicle stimulating hormone (FSH) levels in response to the administration of LHRH. We conclude that the common occurrence of menstrual abnormalities in obesity results from abnormal secretion and binding of sex steroids. In addition, the unaltered secretion of LH and FSH in the presence of such changes is evidence for a disorder of hypothalamic function.

OBJECTIVE

What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles?

CONCLUSIONS

The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system.

BACKGROUND

For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers.

METHODS

Non-human primate model, n = <em>4</em>, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (<em>4</em>8 h) and isolated secondary follicle culture (6 weeks).

METHODS

Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/<em>4</em>, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a <em>4</em>0°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 2<em>4</em>/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A<em>4</em>), estradiol (E2)] production were analyzed weekly.

RESULTS

Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P>> 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (6<em>4</em> ± 8%) follicles. There was no significant change in levels in culture media of E2, P<em>4</em> and A<em>4</em> between fresh and VEG groups at any time point during culture.

CONCLUSIONS

Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues.

CONCLUSIONS

This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined.

BACKGROUND

National Institutes of Health (NIH) Oncofertility Consortium UL1 RR02<em>4</em>926 (1RL1-HD058293, HD058295, PL1 EB0085<em>4</em>2), the Eunice Kennedy Shriver NICHD/NIH (U5<em>4</em> HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

A loss of function mutation of the CYP19 aromatase gene leads to excess circulating androgens in the fetus and in the mother, resulting in ambiguous genitalia in the female fetus. Later on, lack of aromatase is responsible for sexual infantilism, primary amenorrhea, tall stature, and multicystic ovaries, even in preadolescent girls. Up to now, 11 CYP19 aromatase point mutations and 10 well-documented cases have been reported. In the present case, we are reporting the clinical and hormonal follow-up, from birth to 7 yr of age, of an affected girl with ambiguous genitalia. Gene analysis showed that she was a compound heterozygote for two new CYP19 aromatase point mutations. In the father's allele, there was a consensus 5' splice donor sequence mutation, GAA-AAA at cDNA position bp 655 in exon 5, which probably results in a cryptic donor site. In the mother's allele, there was a base A deletion in exon 9 (Delta A GLU <em>4</em>12X), causing a frame shift mutation, and a stop codon after 98 bp (33 codons) downstream, altering the critical heme-binding region. Basal serum LH and FSH levels were high at 8 d of age (<em>4</em>2.9 and 51.3 U/liter), 26 d of age (76.2 and 119 U/liter), and 60 d of age (58.7 and 150 U/liter, respectively). Both gonadotropins dropped dramatically between the second and fifth months of age (to 1.79 and 1<em>4</em>.9 U/liter) but remained higher than in normal control girls (0.6<em>4</em> and 8.5 U/liter, respectively). Serum testosterone (T) and <em>androstenedione</em> (Delta(<em>4</em>)A) levels were high during the first month, but Delta(<em>4</em>)A was normal at 2 months of age. However, at 5 months of age, along with significant decrements of serum LH and FSH levels and increments in serum Delta(<em>4</em>)A and T levels, a large ovarian cyst was removed from each gonad. Relatively high levels of T [27.3 ng/ml (9<em>4</em>.6 nmol/liter); control, 3<em>4</em>.9 ng/ml (121 nmol/liter)], but not of estradiol [1.8 ng/ml (6.6 nmol/liter); control 62.9 ng/ml (231 nmol/liter)], and a high T/estradiol ratio [15.2; control < 1] were found in the follicular fluid. Serum Delta(<em>4</em>)A and T levels remained normal from 1-5 yr of age, but they were high at the last visit (late prepuberty). A GnRH test was performed at 3.9, 6, and 7.1 yr of age. At 3.9 yr, a low prepubertal serum LH peak (2.12 U/liter) was found, but at the older ages, higher serum LH peaks (8.25 and 22.5 U/liter, respectively) were observed. Growth pattern and body mass index were normal, but after the age of 5.2 yr, delays in bone age greater than 2 yr were observed. We concluded that: 1) these two new CYP19 aromatase gene mutations are responsible for the phenotype of aromatase deficiency; 2) in girls, aromatase deficiency results in a decrease of the negative feedback of both serum LH and FSH, which can be detected as early as the second week after birth and persists up to the sixth month of life, and of FSH during the rest of prepuberty; and 3) because large ovarian cysts developed when serum LH and FSH dropped, aromatization of androgens might be required to prevent formation of cystic ovaries.

The effects of burn trauma in men on the production of adrenal and testicular steroids was investigated. Whereas there were significant increases in serum cortisol levels and urinary 17-hydroxycorticosteroid excretion soon after thermal injury, there were significant decreases in serum dehydroepiandrosterone sulfate, dehydroepiandrosterone, <em>androstenedione</em>, and testosterone concentrations during the first <em>4</em> weeks following burn trauma. Serum androstenediol and androstenediol sulfate levels also were reduced, though insignificantly, 10-23 days postburn. Serum LH levels were unchanged during the postburn interval. Since urinary 17-ketosteroid excretion was normal or below normal rather than increased, the decline in serum C19-steroid levels probably resulted from decreased glandular secretion rather than increased rates of metabolism and excretion. Low dehydroepiandrosterone sulfate and/or testosterone levels were found in some men several months after recovery from their burns. These data suggest that thermal injury leads to acute inhibition of adrenal and testicular C19-steroid secretion, but stimulation of adrenal glucocorticosteroid production, and that endocrine function in many instances is not normalized after complete healing of the burned surfaces. The mechanisms and physiological consequences of such changes in the steroid milieu of men after burn trauma are unknown.

We evaluated the comparative effects of aminoglutethimide (AG) on androgen and estrogen levels estrone ([E1], estradiol [E2], plasma dehydroepiandrosterone-sulfate [DHEA-S], testosterone [T], dihydrotestosterone [DHT], delta <em>4</em>-<em>androstenedione</em> [delta <em>4</em>-A]), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin in postmenopausal patients with breast cancer randomly allocated to either AG treatment or bilateral surgical adrenalectomy as a control group. In response to either treatment, the plasma levels of E1 fell 62-75% (P less than 0.001) and urine E1 85.7-88.7% (P less than 0.001) in all study days over a 12-wk period. Similarly, the concentrations of E2 in plasma and urine fell <em>4</em>0-72% without statistically significant differences between the two treatment modalities. The relatively weak androgen, DHEA-S, was reduced by 92% (877.3 +/- 18<em>4</em>.6 to 71.8 +/- 1<em>4</em>.5 ng/ml) at 12 wk in women treated with AG, but suppressed nearly 99% (1,151 +/- 262 to 5.8 +/- 3.3 ng/ml) in adrenalectomized women. At all time points after treatment, the DHEA-S levels were significantly higher in patients receiving AG. Plasma concentrations of the potent androgens, T and DHT, were also relatively preserved during AG treatment. T levels were never significantly reduced by AG, and DHT concentrations were decreased only at the <em>4</em>th wk to a maximum of 20%. delta <em>4</em>-A levels fell 56% in response to this drug only on the 12th wk of therapy (basal, 0.79 +/- 0.09 ng/ml; 12 wk, 0.35 +/- 0.07 ng/ml). In marked contrast, all androgens fell significantly at each time period in response to surgical adrenalectomy, with an 81% maximum suppression of T, 73% of DHT, and 97% of delta <em>4</em>-A. In response to estrogen suppression, plasma levels of FSH, LH, and prolactin did not change significantly throughout the treatment period in either therapy group. To examine possible contributions of the postmenopausal ovary to hormone levels during therapy, data from surgically castrate and spontaneously menopausal women were evaluated separately. No significant differences between the two groups were observed for E1, E2, T, DHT, DHEA-S, delta <em>4</em>-A, LH, FSH, and prolactin. We conclude that equivalent and highly significant estrogen suppression occurs with either AG or surgical adrenalectomy although androgen secretion is preserved during AG treatment but not after surgical adrenalectomy. The combined effects of estrogen deprivation associated with androgen preservation might be significant in the therapeutic action of AG in hormone-responsive neoplasms.

OBJECTIVE

The aim of this study was to assess the effects of metformin and rosiglitazone on insulin resistance and serum androgen levels in both obese and lean patients with polycystic ovary syndrome (PCOS).

METHODS

Forty lean [body mass index (BMI) < 25 kg/m2] and <em>4</em>0 overweight and obese (BMI>> 25 kg/m2) patients were included in the study. Waist and hip measurements, serum sex steroid levels, insulin response to 75-g oral glucose tolerance test, fasting insulin, fasting C-peptide levels and homeostasis model assessment of insulin resistance (HOMA-IR) were determined in all patients. The degree of hirsutism was determined by the Ferriman-Gallwey scoring system. Patients were divided into two groups, with <em>4</em>0 (20 overweight and obese; 20 non-obese) patients each. One group was treated with metformin (MET group) 850 mg bid while the other received rosiglitazone (ROSI group) <em>4</em> mg/day for 12 weeks. All measurements were repeated at the end of this period.

RESULTS

After the 12-week treatment period, HOMA-IR, area under the curve of insulin, fasting insulin and C-peptide levels were observed to have be decreased significantly in all groups. The decrease in the parameters mentioned above was similar in the four groups. The serum levels of free testosterone, androstenedione and DHEA-S decreased in all groups, but the decrease was statistically significant only in the ROSI groups. Within the lean MET group one patient became pregnant and was hence excluded from the final data analysis. Menstruations became regular after metformin therapy in <em>4</em>1.6% of lean and 35.7% of obese patients who had menstrual disturbance prior to the study. Rosiglitazone therapy improved menstrual disturbance in 61.5 % of lean and 53.8% of obese patients.

CONCLUSIONS

Our data showed that both metformin and rosiglitazone increased insulin sensitivity in obese patients with PCOS as expected, and in lean patients as well. Rosiglitazone seemed to be more effective in decreasing the androgen levels and in achieving slightly greater improvement in menstrual disturbance than metformin.

We longitudinally studied clinical endocrine and ultrasound parameters of the ovaries in 73 healthy adolescents having persistent menstrual irregularities. After the first examination, they were reexamined after a variable period ranging from 2 to 7 y. During the first examination, three basic features of the ovaries were observed: homogeneous (36%), multifollicular (23%), and polycystic (<em>4</em>1%). Polycystic ovaries were most frequent, and they generally exceeded the normal adult range. During the last examination, in the entire group of irregular adolescents, homogeneous ovaries decreased (-1<em>4</em>%), polycystic ovaries increased (+18%), and a further higher number of subjects exceeded the normal adult range (+10%). The subjects with enlarged ovaries had the highest values of LH, testosterone, and <em>androstenedione</em>. Fourteen subjects out of <em>4</em>6 (30%), with normal ovarian volume in the first examination, registered an ovarian enlargement in the last examination, exceeding the normal range. Moreover, a change from the homogeneous or multifollicular structure to the polycystic one was observed. Twenty-one subjects out of 27 (78%) with enlarged ovaries in the first examination confirmed the high ovarian volume and the unchanged structure in the last examination, whereas six subjects (22%) showed ovaries within the normal adult range; the polycystic structure was substantially confirmed. These results indicate the following. 1) Homogeneous, multifollicular, and polycystic ovaries can usually be found in the postmenarcheal period. 2) Enlarged ovaries, polycystic structure, hyperandrogenemia, and high LH values are strongly linked, and they are frequent in irregular cycles even in the absence of signs of hyperandrogenism. These characteristics may all persist or in various aggregations become a permanent feature. 3) Only a few subjects may lose ovarian enlargement and show a change in the polycystic structure; however, they frequently maintain hyperandrogenemia. <em>4</em>) During the postmenarcheal period, normal ovarian characteristics may suddenly change, and the ovaries may take on a polycystic structure and increase in volume. Moreover, some endocrine parameters may reach pathologic levels.

The serum levels of cortisol, progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, <em>androstenedione</em> (delta <em>4</em>), testosterone (T), estrone, and estradiol of HIV+ men and HIV- men were determined by radioimmunoassay. The cortisol, 17 alpha-hydroxyprogesterone, and estrone levels of all HIV+ subjects were 35-55% (p less than 0.01), 25-90% (p less than 0.01), and 30-50% (p less than 0.01) higher, respectively, than those of controls. Androgen levels were very high in Centers for Disease Control (CDC) groups II and III of HIV infection (DHEA, 85%, p less than 0.01; delta <em>4</em>, 60%, p less than 0.01; T, 30%, p less than 0.05), but much lower in group IVC1 and IVC2. The estradiol levels were significantly elevated only in group IVD (50%, p less than 0.01) and group IVC2 (25%, NS). These results indicate that serum hormone levels are correlated with HIV infection group. The changes in steroid hormone concentrations during the development of HIV infection may have important implications for the immune response of patients. The high cortisol and estrone levels of all groups, the elevated androgen levels in asymptomatic groups, and the low androgens in AIDS patients may form part of the complex network of immunomodulatory factors.

The aim of the present investigation was to compare the changes in plasma estradiol (E2), progesterone (P), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), <em>androstenedione</em> (delta <em>4</em>-A), dehydroepiandrosterone sulfate (DHEA-S), adrenocorticotropic hormone (ACTH), and prolactin (PRL) in standardized tests (15-min consecutive work loads of 60%, 70%, 80% VO2 max to exhaustion) in 13 eumenorrheic untrained (UT) and 8 highly trained women (MR). Blood was obtained 15 and 2 min before exercise and at the end of each work load or each 15 min period. The results showed a significant increase (0.05 greater than P less than 0.001, two-way ANOVA) in plasma E2 P, T delta <em>4</em>-A, PRL, and ACTH both in UT and MR irrespective of the phase of the menstrual cycle. DHEA S levels increased significantly in the MR, but not in the UT, PRL and ACTH increased linearly with exercise in MR and nonlinearly in UT. In the latter group, only the 80% VO2 max work load was able to elicit significant increments in the plasma levels of these hormones. In the MR plasma T and delta <em>4</em>-A levels increased relatively more pronounced (P less than 0.05) at comparable work loads and exercise times than in the UT. LH levels decreased with exercise both in the UT and MR, whereas FSH levels remained unchanged (MR) or decreased (UT). These findings suggest that during exercise the ovarian hormones are increased by more unspecific mechanisms such as a decreased metabolic clearance rate, whereas in the MR, adrenal secretion of androgens is enhanced.

The enzyme 5beta-reductase catalyzes the reduction of the <em>4</em>-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of <em>4</em>-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, <em>4</em>-<em>androstenedione</em>, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than <em>4</em>2 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, <em>4</em> and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, <em>androstenedione</em>, 17alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids.

We studied the time-course and steroid specificity for aromatase induction in the hypothalamus-preoptic area (HPOA) of the adult male rat. Aromatase activity (AA) was measured in tissue homogenates by using a radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H] <em>androstenedione</em>. We found that by <em>4</em>8 h after administration of testosterone, HPOA AA was significantly (p less than 0.01) greater than control values in castrated rats. In contrast, AA was significantly (p less than 0.01) reduced 12 h after castration, and reached its lowest levels by <em>4</em> days after castration. Several other steroids, administered in 3-cm Silastic capsules for 7 days, were tested for their capacity to induce hypothalamic AA. In addition to testosterone, only 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were effective. Neither the stereoisomers of these compounds nor several other steroids, including estradiol, progesterone, and corticosterone, were active. This profile of activity indicates that the induction of HPOA AA is androgen-specific and, together with the demonstrated time-course of induction, lends further support to the hypothesis that androgens regulate AA through a receptor mechanism and the synthesis of new protein.

We investigated the role of adrenal androgens, cortisol, testosterone and sex-hormone binding globulin (SHBG) as coronary risk factors using a nested case-control design. The study population consisted of 62 cases with cardiac end-points and 97 controls on placebo during the last <em>4</em> years in the Helsinki Heart Study. Serum concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), <em>androstenedione</em>, androstanediol glucuronide, cortisol, testosterone, and SHBG at the first annual visit of the 5-year study period were determined by radioimmunoassays. The only significant difference was found in DHEAS, with cases having higher levels than controls (P < 0.0<em>4</em>). DHEAS levels were positively associated with smoking (P < 0.001), alcohol consumption (P < 0.0<em>4</em>) and triglyceride levels (P < 0.002) and with systolic (P < 0.0<em>4</em>) and diastolic (P < 0.006) blood pressures, and negatively associated with age (P < 0.01) and HDL-cholesterol (P < 0.03). The association between DHEAS and the CHD risk was studied using logistic regression analyses with the classical risk factors--age, smoking, blood pressure, and lipid levels--as covariates in the models. Studies of the joint effects of age and DHEAS disclosed that the risk associated with elevated DHEAS was confirmed to older men (odds ratio (OR) 7.3, 95%, CI 2.3-23.3). A similar analysis with smoking revealed that the DHEAS-related risk was mainly found in smokers (OR 3.<em>4</em>, 95% CI 1.5-8.2). One possible explanation for these results is that some form of mild steroid biosynthetic defect of the adrenals or functional adrenal hyperplasia associated with high DHEAS levels increases the CHD risk in this population.

The influence of the prototype aromatase inhibitor Aminoglutethimide (AG) and its analogue Rogletimide (RG) on peripheral aromatisation were investigated in 13 postmenopausal women with advanced breast cancer. Seven patients received AG 1,000 mg daily plus Hydrocortisone (HC) cover and six received RG as dose escalation of 200 mg bd, <em>4</em>00 mg bd and 800 mg bd. In vivo aromatase inhibition was investigated using the double bolus injection technique with [<em>4</em>-1<em>4</em>C] oestrone ([<em>4</em>-1<em>4</em>C]E1) and [6,7-3H] <em>androstenedione</em> ([6,7-3H]<em>4</em>A) followed by a 96 h urine collection. The labelled urinary oestrogens were separated and purified by chromatography and HPLC. Plasma oestradiol (E2) was also measured. AG mean aromatase inhibition was 90.6% +/- 1.8 s.e.m. and E2 suppression 75.7% +/- 7.3 s.e.m. RG mean aromatase inhibition was 50.6% +/- 9.8 s.e.m. at 200 mg bd, 63.5% +/- 5.7 s.e.m. at <em>4</em>00 mg bd and 73.8% +/- 5.8 s.e.m. at 800 mg bd. E2 suppression was 30.7% +/- 9.5 s.e.m., <em>4</em>0.2% +/- 10.3 s.e.m. and 57.6% +/- 9.2 s.e.m. respectively. These results confirm the efficacy of AG as an aromatase inhibitor. RG produced dose dependent E2 suppression and aromatase inhibition, but even at the maximum tolerated dose of 800 mg bd had sub-optimal aromatase inhibition and oestradiol suppression compared with AG.

One of the hallmarks of polycystic ovary syndrome (PCOS) is increased ovarian androgen secretion that contributes to the ovarian, hormonal, and metabolic features of this condition. Thecal cells from women with PCOS have an enhanced capacity for androgen synthesis. To investigate whether this propensity is a potential cause, rather than a consequence, of PCOS, we used an ovine prenatal androgenization model of PCOS and assessed ewes at 11 months of age. Pregnant Scottish Greyface ewes were administered 100 mg testosterone propionate (TP) or vehicle control twice weekly from d 62 to 102 of gestation, and female offspring (TP = 9, control = 5) were studied. Prenatal TP exposure did not alter ovarian morphology or cyclicity, or plasma androgen, estrogen, and gonadotropin concentrations, at this stage. However, follicle function was reprogrammed in vivo with increased proportions of estrogenic follicles (P < 0.05) in the TP-exposed cohort. Furthermore, in vitro the thecal cells of follicles >><em>4</em> mm) secreted more LH-stimulated <em>androstenedione</em> after prenatal androgenization (P < 0.05), associated with increased basal expression of thecal StAR (P < 0.01), CYP11A (P < 0.05), HSD3B1 (P < 0.01), CYP17 (P < 0.05), and LHR (P < 0.05). This provides the first evidence of increased thecal androgenic capacity in the absence of a PCOS phenotype, suggesting a thecal defect induced during fetal life.

The objective of this work was to examine the effects of insulin-like growth factors (IGFs) on estradiol (E2) production by granulosa cells obtained from ovaries of patients with polycystic ovary disease (PCO). Granulosa cells, isolated from ovaries of three PCO patients, were cultured in serum-free medium containing either <em>androstenedione</em> alone (10(-7) M) or <em>androstenedione</em> plus graded doses of FSH, IGF-I, IGF-II, and/or insulin. At the end of the culture period (2, <em>4</em>, or 6 days) E2 levels in the medium were measured by RIA. The results from each patient were similar, and therefore, the data were pooled. In the 6-day time-course experiments, the control (untreated) cells produced relatively high levels of E2 at 2 days; however, none was detected thereafter. Treatment with FSH (30 ng/mL) stimulated E2 production <em>4</em>-fold at 2 days, but the stimulatory effects of FSH were not sustained during culture. IGF-I at 30 ng/mL mimicked the effects of FSH. Concomitant treatment with FSH and IGF-I caused synergistic increases in E2 production (3-, 13-, and 33-fold at 2, <em>4</em>, and 6 days, respectively). Dose-response studies revealed that FSH and IGF-I stimulated E2 production in a dose-dependent fashion (ED50 of FSH and IGF-I, were 1.1 +/- 0.3 and 7.6 +/- 7.2 ng/mL, respectively). In the presence of a maximally effective dose of FSH (30 ng/mL), the cells appeared to become more responsive to IGF-I (ED50 of IGF-I plus FSH, 1.09 +/- 0.29 ng/mL); however, this effect was not significant (P = 0.086). In the presence of a maximally effective dose of IGF-I (30 ng/mL), the stimulatory effect of FSH on E2 production was dramatically amplified, but the IGF-I did not significantly (P = 0.85) change the potency of FSH (ED50 of FSH plus IGF-I, 1.07 +/- 2.3 ng/mL). Treatment with IGF-II over the concentration range of 0.1-100 ng/mL had no effect on either control or FSH-stimulated E2 production. Treatment with insulin, either alone or together with FSH, increased the levels of E2, but the insulin effects were seen only at the highest doses tested (0.3-10 micrograms/mL). The results in these in vitro experiments with PCO granulosa cells indicate that 1) physiological concentrations of IGF-I are as effective as FSH in stimulating E2 production; 2) IGF-I and FSH act synergistically to control the level of E2 production; and 3) this synergy was not observed with insulin or IGF-II.

It is known that among women over the age of 65, bone mineral density is lower, and the risk of hip fracture higher, in smokers than non-smokers. We report a study in 133<em>4</em> health pre- and post-menopausal women aged 35-6<em>4</em> years, to determine whether this effect can be attributed to lower oestrogen levels in smokers. Among 676 premenopausal women forearm bone density was no lower in smokers (95% confidence interval 1% lower, <em>4</em>% higher). Among 5<em>4</em>3 postmenopausal women who had not used hormone replacement therapy (HRT) for more than a year there was no statistically significant difference, but the lower confidence interval was consistent with a lower bone density in older smokers (by 8% at age 55-59, 16% at age 60-6<em>4</em>). Measurements in 19<em>4</em> postmenopausal women not taking HRT showed that oestrone and oestradiol were similar in smokers and non-smokers, as were cortisol and FSH, LH and prolactin. Meta-analysis of the present study and previous studies confirmed significantly higher levels in smokers of the androgens DHEAS (by 37%) and <em>androstenedione</em> (by 3<em>4</em>%). Oestrogens were no lower in smokers, and the lower confidence limit excluded more than a trivial effect of smoking in lowering oestrogen. These results indicate that the recognised lower bone density in elderly smokers cannot be explained by an effect of smoking on oestrogen, since in premenopausal women bone density is no lower in smokers and in postmenopausal women oestrogens are no lower in smokers. The data suggest a balance between higher androgen levels but lower rates of conversion of androgens to oestrogens in smokers. The effect of smoking on bone may be due to impaired response of bone and other target organs to oestrogen, or to actions independent of oestrogen.

OBJECTIVE

Functional hypothalamic amenorrhoea (FHA) is a consequence of low dietary intake as observed in two major pathophysiological conditions, anorexia nervosa and/or intensive physical exercise. The aim of the present study was to assess in women with FHA and normal body mass index (BMI) and apparently normal daily activities, the degree of impairment of GnRH secretion, its nutritional origin and its reversibility.

METHODS

Twelve women (22-35 years) with FHA not related with exercise and 12 age and BMI matched menstruating controls (NC) were studied. Six women with congenital hypothalamic hypogonadism (CHH), representative of complete gonadotrophin deficiency, were also enrolled for comparison.

METHODS

Plasma oestradiol (E2) and androstenedione (A) levels were measured and the pulsatile profile of LH was studied. A GnRH agonist test, using 100 micrograms S/C of DTrp6 GnRH (Triptorelin) was performed (sampling every 2 h for 24 h). Dietary intake, body composition and nutritional markers (FT3, ferritin, retinol binding protein (RBP), SHBG, IGF-1 and leptin) were measured. All the women with FHA were advised to normalize their diet during four months. The same studies were performed if nutritional markers and body composition were normalized.

RESULTS

In FHA, mean plasma E2 and A levels were low. LH pulse frequency and amplitude were significantly reduced compared to NC (P < 0.005). FSH/LH ratio increased rapidly after triptorelin with a significant increase in plasma E2 levels between 18 and 24 h. In contrast, no response to triptorelin was observed in women with CHH. The fat body mass was lower and the lean body mass higher in FHA than in NC. Marked differences in nutritional intake were identified, with altered dietary composition. FHA consumed significantly less fat (P < 0.001) and less carbohydrate (P = NS) than the BMI-matched controls. Mean plasma levels of SHBG were increased whereas mean plasma levels of FT3, ferritin, RBP, IGF-1, and leptin were significantly decreased. Only three patients with FHA kept a balanced diet and improved their body composition after 4 months. LH pulsatile profile and response to triptorelin challenge were normalized in these patients.

CONCLUSIONS

Mild dieting, close to normal but prolonged and characterized by an important fat restriction, is able to interfere with gonadotrophin secretion. Assessment of nutritional markers allows recognition of mild nutritional insufficiency as a common cause of FHAs. The gonadotrophin deficiency is partial and may be reversible after improvement of nutritional intake and body composition.

This study was designed to investigate whether GH and insulin-like growth factor I (IGF-I) excess could lead to the development of benign prostatic hyperplasia and/or prostatic carcinoma. Prostatic diameters and volume as well as the occurrence of prostatic diseases were studied by ultrasonography in 10 untreated acromegalic patients less than <em>4</em>0 yr of age and 10 age- and body mass index-matched healthy males. Serum GH, IGF-I, PRL, testosterone, dihydrotestosterone, prostate-specific antigen, and prostatic acid phosphatase levels were assessed. All patients had secondary hypogonadism, as diagnosed by low testosterone levels, and <em>4</em> of 10 patients had hyperprolactinemia. After 1 yr of treatment with octreotide (0.3-0.6 mg/day), ultrasound scan and hormone parameters were repeated. The <em>4</em> hyperprolactinemic acromegalics were treated with octreotide and cabergoline (1-2 mg/week) to suppress PRL levels. Symptoms due to prostatic, seminal vesicle, and/or urethral disorders or obstruction were experienced by neither acromegalics nor controls. Digital rectal examination revealed no occurrence of prostatic nodules or other abnormalities. Compared to healthy subjects, a remarkable increase in transversal prostatic diameter and volume was observed in acromegalics. In healthy subjects, prostate volume ranged from 15.1-21.8 mL, whereas in acromegalics it ranged from 21.8-<em>4</em>1.8 mL. Similarly, an increased median lobe was observed. In fact, the transitional zone diameter was just detectable in 5 of 10 controls, whereas it was measurable in all acromegalics (18 +/- 1.2 vs. 2.8 +/- 0.3 mm; P < 0.001). The prevalence of periurethral calcifications was more than doubled in acromegalics (50%) compared to that in controls (20%). Treatment with octreotide for 1 yr produced normalization of circulating GH and IGF-I levels in 7 of 10 patients. In these 7 patients, ultrasound evaluation showed a significant reduction of the antero-posterior diameter (26.1 +/- 1 vs. 28.9 +/- 1.6 mm; P < 0.01), the transversal diameter (<em>4</em><em>4</em>.9 +/- 2 vs. <em>4</em>8 +/- 2 mm; P < 0.01), and the cranio-caudal diameter (36.5 +/- 1 vs. <em>4</em>1.3 +/- 1.5 mm; P < 0.001), whereas the transitional zone diameter was unchanged (16.<em>4</em> +/- 1.5 vs. 17.<em>4</em> +/- 1.7 mm). As a consequence, a significant decrease in prostate volume was recorded (22.1 +/- 1.1 vs. 29.8 +/- 2.5 mL; P < 0.001). Prostate volume increased in 2 of the 3 patients who did not achieve normalization of GH and IGF-I after octreotide treatment. Finally, after treatment, serum testosterone levels were significantly increased (from 1.5 +/- 0.3 to 3.5 +/- 0.3 microg/L), whereas dihydrotestosterone, dehydroepiandrosterone sulfate, delta<em>4</em>-<em>androstenedione</em>, 17beta-estradiol, prostate-specific antigen, and prostatic acid phosphatase were unchanged. Serum PRL levels were suppressed after cabergoline treatment in all <em>4</em> hyperprolactinemic patients throughout the study period. In conclusion, prostate enlargement occurs in young acromegalics with a higher than expected prevalence of micro- and macrocalcifications. This suggests that a careful prostate screening should be included in the work-up and follow-up of acromegalic males.

We studied the sex hormone status of 21 seropositive (IgM-RF) women with rheumatoid arthritis (RA), who were subdivided according to their premenopausal and postmenopausal status. Age matched women with secondary osteoarthritis were used as controls. The hormones evaluated were luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), 17-beta-estradiol (E2), progesterone (Pg), testosterone (T), delta <em>4</em>-<em>androstenedione</em> (A), dehydrotestosterone (DHT), dehydroepiandrosterone sulphate (DHEAS) and cortisol (C). Normal concentrations of all the hormones considered were found in premenopausal women with RA. Statistically higher concentrations of T (p less than 0.05), A (p less than 0.05) and DHEAS (p less than 0.01) were observed in postmenopausal women with RA when compared to controls, whereas no differences were found for all other hormones studied. Although the significance of observed relative hyperandrogenism in postmenopausal women with RA is not clear, our data seem to indicate that sex hormone levels are altered in patients with RA.

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to <em>androstenedione</em> or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-<em>4</em>-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D2<em>4</em>1N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D2<em>4</em>1N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).

BACKGROUND

Oral contraceptive pills (OC) are usually the first choice of treatment for polycystic ovarian syndrome (PCOS), when fertility is not desired. However, they do not improve, or may even further induce impairment of insulin sensitivity, which is already impaired in women with PCOS. In this prospective, randomized study, we analysed the additional benefits of adding metformin to the OC treatment in non-obese women with PCOS.

METHODS

After a baseline work-up including body mass index (BMI), waist:hip ratio (WHR), Ferriman-Gallwey score, ovarian volume, serum gonadotrophin, androgen and sex hormone-binding globulin (SHBG) levels, and fasting lipid, glucose and insulin levels, <em>4</em>0 non-obese women with PCOS were assigned either to the OC or to the OC + metformin treatment by computer-assisted randomization. At the end of the <em>4</em> month follow-up period, subjects were re-evaluated.

RESULTS

The two groups were similar at baseline. After treatment, women in the OC + metformin group had significant decreases in BMI and WHR, and a significant increase in insulin sensitivity, in contrast to those in the OC group, who had insignificant changes in these parameters. Adding metformin also caused significant improvements in serum androstenedione and SHBG levels compared with the OC treatment alone.

CONCLUSIONS

Adding metformin to the OC treatment may improve the insulin sensitivity, and may further suppress the hyperandrogenaemia in non-obese women with PCOS.

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and <em>androstenedione</em>, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a <em>4</em>-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)

We hypothesized that the administration of rosiglitazone, an insulin-sensitizing agent of the thiazolidinedione class, would improve the ovulatory dysfunction, hirsutism, hyperandrogenemia, and hyperinsulinemia of polycystic ovary syndrome (PCOS) patients. Forty women with PCOS and impaired glucose tolerance test (IGT) were randomly assigned to the 8-month treatment with rosiglitazone at either 2 mg/day or <em>4</em> mg/day. We compared changes in ovulatory function, hirsutism, hormonal levels (total and free testosterone, estradiol, estrone, <em>androstenedione</em>, LH and FSH), and measures of glycemic parameters (fasting and post-challenge levels of glucose and insulin, HOMA-IR, hemoglobin A1c), between the study groups. The patients' baseline characteristics were similar across all treatment arms. Fifteen of 20 women in the 2 mg group and 19 of 20 women in the <em>4</em> mg group achieved normal glucose tolerance; 1<em>4</em> of 20 women in the 2 mg group and 17 of 20 women in the <em>4</em> mg group achieved ovulatory menses at the end of the study period. The decreases of free testosterone levels were better in the <em>4</em> mg group than the 2 mg rosiglitazone group (-1.89+/-0.35 pg/ml vs. -2.21+/-0.39 pg/ml; P<0.01). There were neither any serious adverse events nor any liver enzyme elevations in our study patients during the treatment period. This study demonstrated that rosiglitazone improves the ovulatory dysfunction, hirsutism, hyperandrogenemia, and insulin resistance of PCOS in a dose-related fashion, with minimal adverse effects. This drug may be a good choice for lifetime treatment of patients with PCOS, especially for the ones who failed to show satisfactory results in metformin therapy.