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The effects of MPA and MGA on rat testicular steroidogenesis were examined by studying: 1) serum testosterone in hCG primed animals treated with MPA or MGA, 2) testosterone synthesis in rat Leydig cells cultured with MPA or MGA, 3) MPA and MGA binding to rat testis microsomal cytochrome P-450 and 4) MPA and MGA inhibition of enzymes of rat testicular steroidogenesis. In immature rats receiving 1.0 IU of hCG per day 20 mg/kg of MPA or MGA reduced serum testosterone by 57 and 56%, respectively. In mature male rats receiving 50.0 IU of hCG per day 20 mg/kg of MPA or MGA reduced serum testosterone by 40 and 29%, respectively. In rat interstitial cells cultured with 10 ng of rat LH, 1 microM MPA or MGA inhibited testosterone production by 32 and 23%, respectively. Addition of MPA or MGA to microsomal preparations resulted in a type I cytochrome P-450 difference spectrum. MPA and MGA inhibited rat testicular 17 alpha-hydroxylase, 17,20-lyase, and the 3 beta- and 17 beta-hydroxysteroid dehydrogenases. These findings suggest that MPA and MGA inhibit rat testicular steroidogenesis in vivo and in vitro.

The evidence that supports the hypothesis that insulin and LH both regulate ovarian androgen production was presented. The most dramatic clinical example of the association between hyperinsulinemia and hyperandrogenism is the HAIR-AN syndrome. Our hypothesis is that, in the HAIR-AN syndrome, the severe insulin resistance causes a compensatory hyperinsulinemia, which stimulates ovarian androgen production if adequate LH is present. The acanthosis nigricans is an epiphenomenon of the syndrome. Acanthosis nigricans is a dermatologic manifestation of severe insulin resistance. In vitro evidence suggests that insulin and IGF-I stimulate androgen production in incubations of human stroma and theca. The stromatropic effects of insulin may sensitize the stroma to the stimulatory effects of LH. In some hyperandrogenic-insulin-resistant women, a glucose load appears to produce an acute rise in circulating androgens. The magnitude of the rise in circulating androgens is proportional to the magnitude of the insulin response to the glucose load. These data suggest that hyperinsulinemia may play a central role in the development of ovarian hyperandrogenism.

Fourteen premenopausal women with uterine leiomyomata were randomized to receive a gonadotropin-releasing hormone agonist (GnRH-a), leuprolide, either by daily subcutaneous (SC) injection (500 micrograms/day) or by intranasal (IN) spray (1600 micrograms/day) for 24 weeks. In the SC group, a significant reduction in uterine volume occurred from a pretreatment volume of 368 +/- 60 (mean +/- standard error of the mean [SEM]) cm3 to 202 +/- 61 cm3 at 12 weeks of therapy (P less than 0.01) and to 172 +/- 49 cm3 at 24 weeks of therapy (P less than 0.005). In the IN group, no significant reduction in uterine volume occurred. In addition, there was a significant negative correlation between the serum estradiol concentration during treatment and the percent decrease in uterine volume (r = -0.55, P less than 0.05). Four months after completing therapy, uterine volume increased to 296 +/- 104 cm3 in the SC group, which was not significantly different from pretreatment volume. These findings suggest that reduction in uterine volume depends on the degree of hypoestrogenism induced and that uterine volume increases soon after cessation of GnRH-a therapy.

Hyperplasia of testicular Leydig cells and ovarian theca-lutein cells is a common histologic finding in infants of diabetic mothers. The functional correlates of this histologic finding were investigated by measurement of the beta-subunit of human chorionic gonadotropin, testosterone, dihydrotestosterone, androstenedione, estradiol, and estrone in the amniotic fluid compartment of gestations with male and female fetuses in diabetic mothers (N = 34) and control women (N = 34) at term. When compared with those of control subjects, gestations of diabetic mothers had significantly higher amniotic fluid concentrations of the beta-subunit of human chorionic gonadotropin. Gestations with either male or female fetuses in diabetic mothers had significantly higher amniotic fluid testosterone and dihydrotestosterone levels when compared with those of their respective gender controls. In gestations with male fetuses in diabetic mothers there was a significant positive correlation between the beta-subunit of human chorionic gonadotropin and testosterone. There was no significant difference in amniotic fluid androstenedione, estradiol, or estrone levels between the gestations of diabetic mothers and those of control women. These results suggest that the testicular Leydig cell and ovarian theca-lutein cell hyperplasia seen in infants of diabetic mothers is due, in part, to elevated levels of human chorionic gonadotropin and is associated with elevated testosterone and dihydrotestosterone concentrations in the amniotic fluid.

The effects of insulin and insulin-like growth factors (IGFs) on ovarian androgen production were examined in ovarian stroma obtained from four women with hyperandrogenism and three women without hyperandrogenism. In incubations of stroma obtained from all four hyperandrogenic patients, insulin alone (500 ng/ml) significantly stimulated androstenedione and testosterone release. LH alone (25 ng/ml) significantly stimulated androstenedione release in incubations of stroma obtained from three of the four hyperandrogenic patients and testosterone release in incubations of stroma obtained from one of the four hyperandrogenic patients. In stromal incubations from three of the four hyperandrogenic patients, insulin alone (500 ng/ml) resulted in a significantly greater release of androstenedione and testosterone than did LH alone (25 ng/ml). Dihydrotestosterone was released in measurable quantities in incubations of stromal tissue obtained from three of the four hyperandrogenic women. In all three instances in which dihydrotestosterone was detectable, insulin alone (500 ng/ml), but not LH alone (25 ng/ml), significantly stimulated dihydrostestosterone release. Incubations of stroma obtained from three nonhyperandrogenic, normally cycling women demonstrated low levels of androstenedione release and negligible testosterone and dihydrotestosterone release. Insulin alone (500 ng/ml) and LH alone (25 ng/ml) produced no significant increase in androstenedione release. Insulin (500 ng/ml) plus LH (25 ng/ml) significantly stimulated androstenedione accumulation in stroma obtained from two of the nonhyperandrogenic women. One insulin dose-response experiment was performed using stromal tissue obtained from a hyperandrogenic woman. In this experiment, insulin, at a dose of 50 ng/ml, was as effective as insulin at a dose of 500 ng/ml in stimulating androstenedione and testosterone release. In addition to insulin, IGF-I/somatomedin C (50 ng/ml) stimulated androstenedione and testosterone release. Relaxin (1 microgram/ml) and multiplication-stimulating activity (50 ng/ml) did not stimulate androstenedione and testosterone release. These studies suggest that human ovarian stroma may be a target tissue for insulin and IGF-I, and that hyperinsulinemia may be an important factor contributing to ovarian hyperandrogenism.

The effects of insulin on ovarian steroidogenesis were examined in four-day incubations of minced stroma and theca obtained from a woman with hyperandrogenism, insulin resistance, and acanthosis nigricans, and from a normally cycling woman. In incubations of theca obtained from the patient with hyperandrogenism, insulin resistance, and acanthosis nigricans, lutenizing hormone (LH) (25 ng/mL) alone stimulated androstenedione, testosterone, progesterone, and estradiol accumulation. Insulin (500 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not progesterone or estradiol accumulation. In incubations of stroma obtained from the hyperandrogenism, insulin resistance, and acanthosis nigricans patient, LH (25 ng/mL) alone stimulated androstenedione and testosterone accumulation, but not dihydrotestosterone accumulation. Insulin (500 ng/mL) alone stimulated androstenedione, testosterone, and dihydrotestosterone accumulation. In incubations of stroma from the normally cycling woman, LH plus insulin acted synergistically to stimulate androstenedione accumulation. These results suggest that insulin may be a regulator of steroid biosynthesis in the thecal and stromal compartments of the human ovary.

This study examined the effects of previous ovarian surgery on the clinical response to ovulation induction with clomiphene citrate-human menopausal gonadotropin in an in vitro fertilization program. Patients were divided into five clinical groups: group A (n = 63), no previous ovarian surgery; B (n = 9), unilateral cystectomy; C (n = 6), unilateral oophorectomy with no contralateral ovarian surgery; D (n = 7), bilateral ovarian surgery with both ovaries present; and E (n = 4), unilateral oophorectomy and contralateral cystectomy. Patients in group E demonstrated significantly lower serum estradiol on cycle days 9-11 (P less than or equal to .05) and fewer follicles on cycle days 11-12 (P less than or equal to .05) than did patients in groups A-D. The percentage of cancelled cycles increased with increasing amounts of ovarian surgery (P less than or equal to .03). The study suggests that one cause of a poor response to ovulation induction for in vitro fertilization may be prior extensive ovarian surgery.

Cigarette smoking has been reported to produce acute increases in plasma ACTH and cortisol, but the effect of chronic smoking on integrated adrenal steroid production has not been studied. The effects of chronic smoking on 24-hour urinary-free cortisol, 11-deoxycortisol, DHEAS, and 17-keto-steroids were studied in 10 premenopausal smokers, and their results were compared with 15 premenopausal nonsmokers. The 24-hour excretion of urinary-free cortisol (85.0 +/- 40.8 nmol/d in smokers versus 81.7 +/- 49.7 nmol/d in nonsmokers), 11-deoxycortisol (259 +/- 170 nmol/d in smokers versus 222 +/- 147 nmol/d in nonsmokers), DHEAS (3,140 +/- 2,909 nmol/d in smokers versus 2,890 +/- 1,960 nmol/d in nonsmokers), and 17-ketosteroids (17.4 +/- 8.3 mumol/d in smokers versus 23.4 +/- 19.9 mumol/d in nonsmokers) were similar in smokers and nonsmokers (all P values not significant). We conclude that chronic smoking does not result in abnormal levels of 24-hour urinary-free cortisol.

The potent gonadal steroids testosterone and estradiol are synthesized from the biologically weak precursors, androstenedione and estrone, by enzymatic reduction of the ketone group at carbon-17 of the steroid nucleus (17-ketosteroid reductase). To test the hypothesis that Leydig and granulosa cells may contain a distinct 17-ketosteroid reductase enzyme, the subcellular localization and the substrate specificity of the enzyme was examined in each cell type. In Leydig cells, the 17-ketosteroid reductase activity was concentrated in the microsomal fraction of the cell. In granulosa cells, the 17-ketosteroid reductase activity was concentrated in the cytosolic fraction of the cell. In Leydig cell microsomes, the apparent Michaelis-Menten constant for the conversion of androstenedione to testosterone was 0.41 mumol/L and for the conversion of estrone to estradiol it was 12 mumol/L. In granulosa cell cytosol, the apparent Michaelis-Menten constant for the conversion of estrone to estradiol was 1.1 mumol/L and for the conversion of androstenedione to testosterone it was 15 mumol/L. These results demonstrate that rat Leydig and granulosa cells each contain a 17-ketosteroid reductase enzyme with unique subcellular localization and substrate specificity.

The effects of danazol on steroidogenesis in vitro in the rat testis were examined by studying: 1) androgen synthesis in rat Leydig cells cultured with danazol, 2) danazol binding to rat testis microsomal cytochrome P-450, and 3) enzyme kinetics of danazol inhibition of the microsomal enzymes of testicular steroidogenesis. Concentrations of danazol as low as 1 micrometer suppressed LH-stimulated testosterone and androstenedione production in cultured Leydig cells. The addition of danazol to a preparation of testicular microsomes elicited a type I cytochrome P-450 binding spectrum, with an apparent spectral dissociation constant (Ks) of 4.8 micrometer. Danazol inhibited progesterone and 17alpha-hydroxy-progesterone binding to microsomal P-450 with apparent spectral inhibition constants of 2.4 micrometer and 2.8 micrometer, respectively. Danazol competitively inhibited 3beta-hydroxy-delta5-steroid dehydrogenase-isomerase (apparent enzymatic inhibition constant, KI = 5.8 micrometer), 17alpha-hydroxylase (KI = 2.4 micrometer), 17,20 lyase (KI = 1.9 micrometer), and 17beta-hydroxysteroid dehydrogenase (KI = 4.4 micrometer). These findings indicate that low concentrations of danazol directly inhibit steroidogenesis in the rat testis in vitro.

The effects of insulin on porcine thecal steroidogenesis were examined in long-term cultures of hyaluronidase-collagenase dispersed thecal cells. The thecal cultures made significant amounts of progesterone (P) and androstenedione (delta 4 A). Testosterone, dihydrotestosterone, estrone, and estradiol could not be detected in the media. Luteinizing hormone (LH) alone significantly increased P and delta 4 A accumulation. Insulin alone increased P accumulation on days 2 to 4 of culture. Insulin alone did not stimulate delta 4 A accumulation. Insulin plus LH resulted in a significantly greater accumulation of P and delta 4 A than LH alone. These results suggest that insulin may be a regulator of ovarian thecal steroidogenesis.

The interaction of 19-norethindrone [4-estren-17 alpha-ethinyl-17 beta-ol,3-one (NET)] with human placental microsomes was investigated using enzymatic and spectral techniques. The incubation of [6,7-3H]norethindrone with human placental microsomes, NADPH, and molecular oxygen resulted in the production of ethinyl estradiol [1,3,5-(10)estratrien-17 alpha-ethinyl-3,17 beta-diol (EE)]. The reaction was linear with respect to time and protein concentration. Androstenedione inhibited the enzymatic aromatization of NET to EE. The product was identified by thin layer chromatography, recrystallization to constant specific activity, and derivative formation. No acid or base was used in any step of product identification. To ensure that spontaneous aromatization of metabolites of NET did not contribute to our results, representative samples were treated with sodium borohydride before processing. Sodium borohydride reduces the 4-en-3-one grouping of the A-ring, thereby preventing chemical aromatization. Sodium borohydride treatment did not reduce our observed yields of EE from NET. The addition of NET to a preparation of solubilized, partially purified placental microsomal cytochrome P-450 yielded a type I cytochrome P-450 binding spectrum. The apparent spectral dissociation constant for NET binding to cytochrome P-450 was 28 microM. These results suggest that NET is enzymatically aromatized to EE by human placental microsomes.

CA-125 is a high-molecular-weight glycoprotein that is expressed on the cell surface of some derivatives of embryonic coelomic epithelium. Based on results of an immunoradiometric assay developed to detect CA-125 in peripheral blood, 82% of patients with ovarian cancer and less than 1% of apparently healthy controls have elevated peripheral blood levels of CA-125. Because endometriotic lesions are likely to be derivatives of embryonic coelomic epithelium, the authors investigated serum CA-125 levels in patients with endometriosis. Preoperative serum CA-125 concentrations were measured in 147 patients undergoing diagnostic laparoscopy or laparotomy. Serum CA-125 concentrations were elevated in patients with stage III or IV endometriosis, compared with controls with negative diagnostic laparoscopies (66.5 +/- 14.5 versus 8.20 +/- 0.59 U/ml, mean +/- standard error of the mean; P less than 0.001). Fifty-four percent of patients with stage III or IV endometriosis and 0% of the controls had CA-125 levels greater than 35 U/ml. Occasional patients with stage II endometriosis (13%), leiomyomata uteri (14%), and chronic pelvic inflammatory disease (5%) also had serum CA-125 concentrations greater than 35 U/ml. Immunocytochemical techniques demonstrated the presence of CA-125 on the cell surface of endometriotic lesions.

Danazol (17alpha-pregn-4-en-20-yno-[2,3-d]isoxazol-17-ol) was administered daily for 4 days to castrated female rats. As previously demonstrated, danazol lowered serum levels of luteinizing hormone (LH) in an apparent dose-dependent fashion. Animals which received danazol in a dose sufficient to lower serum LH responded to administered LH-releasing hormone (LHRH) with increases in serum LH levels which were not diminished as compared with those of control animals. Although these experiments do not preclude an effect of danazol directly on the pituitary, the results indicate that this agent probably lowers serum LH primarily by inhibition of hypothalamic LHRH secretion.

We monitored the estrogen metabolism and menstrual function of two groups of elite oarswomen as they progressed from a phase of low intensity training (phase I), to high intensity training (phase II), and back to low intensity training (phase III). Each phase lasted 3 months. The two groups of oarswomen included five oarswomen (group A) who experienced no menstrual dysfunction during the training year, even during the phase of high intensity training, and five oarswomen (group B) who experienced normal menses during phases of low intensity training but disrupted menses during the phase of high intensity training. Four nonathletic controls were also studied. Menstrual function was monitored throughout the training year by assay for pregnanediol glucuronide in overnight 12-h urine samples collected twice weekly. Repeated measures of the extent of estradiol metabolized by 2-hydroxylase oxidation, total body water, and nutrient intake of group A and B oarswomen were made at the three phases of the training year; the extent of estradiol metabolized by 2-hydroxylase oxidation was evaluated by radiometric analysis; total body water was measured by deuterium oxide dilution and bioimpedance analysis; and nutrient intake was evaluated by food frequency questionnaire. The group B oarswomen were found to metabolize a significantly greater fraction of administered [2-3H]estradiol by 2-hydroxylase oxidation than group A oarswomen (chi 2(1) = 6.57; P = 0.01). The extent of estradiol metabolized by 2-hydroxylase oxidation among group A oarswomen did not differ from that among nonathletic controls. The extent of 2-hydroxylase activity did not change significantly with the intensity of training among either group A or group B oarswomen. Oarswomen in groups A and B lost body weight and became leaner during the phase of high intensity training (phase II). Group A and B oarswomen did not differ in the degree of weight loss or in relative fatness during phase II. Over all subjects, the extent of estradiol metabolized by 2-hydroxylase oxidation was positively correlated with the extent of leanness. These data suggest that elevated estradiol 2-hydroxylase oxidation among elite oarswomen is associated with the occurrence of menstrual disturbances during phases of high intensity training and increased relative leanness.

The effects of nicotine, cotinine and anabasine on rat adrenal steroidogenesis were examined by spectral and enzymatic techniques. The addition of nicotine, cotinine or anabasine to preparations of rat adrenal mitochondria produced type II cytochrome P-450 difference spectra. The addition of nicotine or anabasine, but not cotinine, to rat adrenal microsomes yielded type II cytochrome P-450 difference spectra. Nicotine and anabasine competitively inhibited rat adrenal mitochondrial 11 beta-hydroxylase and microsomal 21-hydroxylase. Cotinine competitively inhibited mitochondrial 11 beta-hydroxylase, but did not inhibit microsomal 21-hydroxylase. The apparent enzymatic inhibition constants for cotinine, nicotine, anabasine and metyrapone inhibition of the mitochondrial 11 beta-hydroxylase were 32, 96, 120 and 74 microM respectively. These studies suggest that components of cigarette smoke may alter patterns of adrenal steroidogenesis.

Danazol was found to inhibit multiple enzymes of steroidogenesis directly in the pregnant mare serum (PMS)-treated hamster ovary and the rat testis and adrenal in vitro. In the PMS-treated hamster ovary, danazol inhibited 17alpha-hydroxylase, 17,20-lyase, and 3beta-hydroxysteroid dehydrogenase. In the rat testis, danazol inhibited 17alpha-hydroxylase, 17,20-lyase, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase. In the rat adrenal, danazol inhibited 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11beta-hydroxylase. Two hours after a subcutaneous injection of 5 mg/kg of danazol to adult male rats, serum luteinizing hormone levels were significantly increased and serum testosterone levels were significantly suppressed. These findings suggest that in the rodent one of danazol's major pharmacologic effects is the direct inhibition of steroidogenesis.

Infants of diabetic mothers are known to have a greater incidence of respiratory distress syndrome than normal control infants. Fetal lung maturation is modulated by a large number of hormones. To further investigate a possible role of hormonal modulators of lung maturation in infants of diabetic mothers, fetal cord prolactin, estrone, estradiol, thyroxine, triiodothyronine, and triiodothyronine-resin uptake index levels were measured in infants of diabetic mothers (n = 40) and nondiabetic mothers (n = 40) at term. Infants of diabetic mothers had significantly lower mixed-cord serum prolactin levels (p less than 0.0005) than control infants. There was no significant difference in cord serum thyroxine, triiodothyronine-resin uptake index, triiodothyronine, estrone, or estradiol levels between the infants of diabetic mothers and the infants of control mothers. These findings raise the possibility that decreased fetal prolactin levels may be associated with, or contribute to, the delayed lung maturation reported with diabetic pregnancies.

Clear ovarian follicular fluid and the follicular flush (bloody) of aspirated follicles from 18 women undergoing oocyte retrieval for in vitro fertilization were tested for the presence of T-lymphocyte subpopulations with the use of a panel of monoclonal antibodies in indirect immunofluorescence assay. A sample of peripheral venous blood from each patient was run as a control. There was no difference in the percentage of cells positive for the various markers between bloody follicular fluid (i.e., ovarian blood) and peripheral venous blood. T8-positive (suppressor/cytotoxic) lymphocytes were recovered from all clear follicular fluids; ten of the patients exhibited a dramatically decreased T4/T8 ratio (peripheral blood, 2:1; clear follicular fluid, 1:25; P less than 0.001). These lymphocytes may be involved in the suppression of autoimmune responses directed against ovarian antigens.