Safety and efficacy of the selective progesterone receptor modulator asoprisnil for heavy menstrual bleeding with uterine fibroids: pooled analysis of two 12-month, placebo-controlled, randomized trials.

STUDY QUESTION: Can asoprisnil, a selective progesterone receptor modulator, provide clinically meaningful improvements in heavy menstrual bleeding (HMB) associated with uterine fibroids with an acceptable safety profile? SUMMARY ANSWER: Uninterrupted treatment with asoprisnil for 12 months effectively controlled HMB and reduced fibroid and uterine volume with few adverse events. WHAT IS KNOWN ALREADY: In a 3-month study, asoprisnil (5, 10 and 25 mg) suppressed uterine bleeding, reduced fibroid and uterine volume, and improved hematological parameters in a dose-dependent manner. STUDY DESIGN, SIZE, DURATION: In two Phase 3, double-blind, randomized, placebo-controlled, multicentre studies, women received oral asoprisnil 10 mg, asoprisnil 25 mg or placebo (2:2:1) once daily for up to 12 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Premenopausal women ≥18 years of age in North America with HMB associated with uterine fibroids were included (N = 907). The primary efficacy endpoint was the percentage of women who met all three predefined criteria at 12 months or the final month for patients who prematurely discontinued: (1) ≥50% reduction in monthly blood loss (MBL) by menstrual pictogram, (2) hemoglobin concentration ≥11 g/dL or an increase of ≥1 g/dL, and (3) no interventional therapy for uterine fibroids. Secondary efficacy endpoints included changes in other menstrual bleeding parameters, volume of the largest fibroids, uterine volume and health-related quality of life (HRQL). MAIN RESULTS AND THE ROLE OF CHANCE: In all, 90% and 93% of women in the asoprisnil 10-mg and 25-mg groups, respectively, and 35% of women in the placebo group met the primary endpoint (P < 0.001). Similar results were observed at month 6 (P < 0.001). The percentage of women who achieved amenorrhea in any specified month ranged from 66-78% in the asoprisnil 10-mg group and 83-93% in the asoprisnil 25-mg group, significantly higher than with placebo (3-12%, P < 0.001). Hemoglobin increased rapidly (by month 2) with asoprisnil treatment and was significantly higher versus placebo throughout treatment. The primary fibroid and uterine volumes were significantly reduced from baseline through month 12 with asoprisnil 10 mg (median changes up to -48% and -28%, respectively) and 25 mg (median changes up to -63% and -39%, respectively) versus placebo (median changes up to +16% and +13%, respectively; all P < 0.001). Dose-dependent, significant improvements in HRQL (Uterine Fibroid Symptom and Quality of Life instrument) were observed with asoprisnil treatment. Asoprisnil was generally well tolerated. Endometrial biopsies indicated dose- and time-dependent decreases in proliferative patterns and increases in quiescent or minimally stimulated endometrium at month 12 of treatment. Although not statistically significantly different at month 6, mean endometrial thickness at month 12 increased by ~2 mm in both asoprisnil groups compared with placebo (P < 0.01). This effect was associated with cystic changes in the endometrium on MRI and ultrasonography, which led to invasive diagnostic and therapeutic procedures in some asoprisnil-treated women. LIMITATIONS, REASONS FOR CAUTION: Most study participants were black; few Asian and Hispanic women participated. The study duration may have been insufficient to fully characterize the endometrial effects. WIDER IMPLICATIONS OF THE FINDINGS: Daily uninterrupted treatment with asoprisnil was highly effective in controlling menstrual bleeding, improving anemia, reducing fibroid and uterine volume, and increasing HRQL in women with HMB associated with uterine fibroids. However, this treatment led to an increase in endometrial thickness and invasive diagnostic and therapeutic procedures, with potential unknown consequences. STUDY FUNDING/COMPETING INTEREST(S): This trial was funded by AbbVie Inc. (prior sponsors: TAP Pharmaceutical Products Inc., Abbott Laboratories). E.A. Stewart was a site investigator in the Phase 2 study of asoprisnil and consulted for TAP during the design and conduct of these studies while at Harvard Medical School and Brigham and Women's Hospital. She received support from National Institutes of Health grants HD063312, HS023418 and HD074711 and research funding, paid to Mayo Clinic for patient care costs related to an NIH-funded trial from InSightec Ltd. She consulted for AbbVie, Allergan, Bayer HealthCare AG, Gynesonics, and Welltwigs. She received royalties from UpToDate and the Med Learning Group. M.P. Diamond received research funding for the conduct of the studies paid to the institution and consulted for AbbVie. He is a stockholder and board and director member of Advanced Reproductive Care. He has also received funding for study conduct paid to the institution from Bayer and ObsEva. A.R.W. Williams consulted for TAP and Repros Therapeutics Inc. He has current consultancies with PregLem SA, Gedeon Richter, HRA Pharma and Bayer. B.R. Carr consulted for and received research funding from AbbVie. E.R. Myers consulted for AbbVie, Allergan and Bayer. R.A. Feldman received compensation for serving as a principal investigator and participating in the conduct of the trial. W. Elger was co-inventor of several patents related to asoprisnil. C. Mattia-Goldberg is a former employee of AbbVie and may own AbbVie stock or stock options. B.M. Schwefel and K. Chwalisz are employees of AbbVie and may own AbbVie stock or stock options. TRIAL REGISTRATION NUMBER: NCT00152269, NCT00160381 (clinicaltrials.gov). TRIAL REGISTRATION DATE: 7 September 2005; 8 September 2005. DATE OF FIRST PATIENT'S ENROLMENT: 12 September 2002; 6 September 2002.

Inverse relation between low-density lipoprotein-cholesterol and dehydroisoandrosterone sulfate in human fetal plasma.

A striking inverse correlation was found in umbilical cord plasma between the concentrations of dehydroisoandrosterone sulfate and low-density lipoprotein (LDL)-cholesterol but not high-density lipoprotein-cholesterol or very low density lipoprotein-cholesterol. Dehydroisoandrosterone sulfate is a major secretory product of the human fetal adrenal and the principal precursor of placental estrogen production. The data suggest that the concentrations for LDL-cholesterol in fetal plasma are influenced by the rate of utilization of LDL-cholesterol by the fetal adrenal for steroidogenesis and are not necessarily related to a genetic predisposition for hypercholesterolemia or other lipoprotein disorders.

A 12-month extension study to evaluate the safety and efficacy of asoprisnil in women with heavy menstrual bleeding and uterine fibroids.

STUDY QUESTION: What is the safety and efficacy profile during long-term (12-24 months) uninterrupted treatment with the selective progesterone receptor modulator asoprisnil, 10 and 25 mg in women with heavy menstrual bleeding (HMB) associated with uterine fibroids? SUMMARY ANSWER: Uninterrupted treatment with asoprisnil should be avoided due to endometrial safety concerns and unknown potential long-term consequences. WHAT IS KNOWN ALREADY: Asoprisnil was well tolerated in shorter-term studies and effectively suppressed HMB and reduced fibroid volume. STUDY DESIGN SIZE DURATION: Women with uterine fibroids who had previously received placebo (n = 87) or asoprisnil 10 mg (n = 221) or 25 mg (n = 215) for 12 months in two double-blind studies entered this randomized uncontrolled extension study and received up to 12 additional months of treatment followed by 6 months of post-treatment follow-up. Women who previously received placebo were re-randomized to either asoprisnil 10 or 25 mg for the extension study. This report focuses on the 436 women who received asoprisnil in the double-blind studies and this extension study. Results for women who previously received placebo in the double-blind studies are not described. PARTICIPANTS/MATERIALS SETTING METHODS: Women ≥18 years of age who completed a 12-month, double-blind, placebo-controlled study, had estradiol levels indicating that they were not menopausal and had no endometrial hyperplasia or other significant endometrial pathology were eligible. The safety endpoints were focused on endometrial assessments. The composite primary efficacy endpoint was the proportion of women who demonstrated a response to treatment by meeting all three of the following criteria at the final month for participants who prematurely discontinued or at month 12 for those who completed the study: a reduction from initial baseline to final visit of ≥50% in the menstrual pictogram score, hemoglobin concentration ≥11 g/dl or an increase of ≥1 g/dl from initial baseline at the final visit, and no surgical or invasive intervention for uterine fibroids. Other efficacy endpoints included rates for amenorrhea and suppression of bleeding, changes in fibroid and uterine volume and changes in hematologic parameters. No statistical tests were planned or performed for this uncontrolled study. MAIN RESULTS AND ROLE OF CHANCE: Imaging studies revealed a progressive increase in endometrial thickness and cystic changes that frequently prompted invasive diagnostic procedures. Endometrial biopsy results were consistent with antiproliferative effects of asoprisnil. Two cases of endometrial cancer were diagnosed. At the final month of this extension study (total duration of uninterrupted treatment up to 24 months), the primary efficacy endpoint was achieved in 86 and 92% of women in the asoprisnil 10- and 25-mg groups, respectively. During each month of treatment, amenorrhea was observed in the majority of women (up to 77 and 94% at 10 and 25 mg, respectively). There was a progressive, dose-dependent decrease in the volume of the primary fibroid with asoprisnil 10 and 25 mg (-55.7 and -75.2% median decrease, respectively, from baseline [i.e. the beginning of the placebo-controlled study] to month 12 [cumulative months 12-24] of this extension study). These effects were associated with improvements in quality of life measures. LIMITATIONS REASONS FOR CAUTION: This study was uncontrolled, which limits the interpretation of safety and efficacy findings. The study also had multiple protocol amendments with the addition of diagnostic procedures and, because no active comparator was included, the potential place of asoprisnil in comparison to therapies such as GnRH agonists and surgery cannot be determined. WIDER IMPLICATIONS OF THE FINDINGS: Long-term, uninterrupted treatment with asoprisnil leads to prominent cystic endometrial changes that are consistent with the 'late progesterone receptor modulator' effects, which prompted invasive diagnostic procedures, although treatment efficacy is maintained. Although endometrial cancers were uncommon during both treatment and follow-up, these findings raise concerns regarding endometrial safety during uninterrupted long-term treatment with asoprisnil. This study shows that uninterrupted treatment with asoprisnil should be avoided due to safety concerns and unknown potential long-term consequences. STUDY FUNDING/COMPETING INTERESTS: AbbVie Inc. (prior sponsor, TAP Pharmaceutical Products Inc.) sponsored the study and contributed to the design and conduct of the study, data management, data analysis, interpretation of the data and the preparation and approval of the manuscript. Financial support for medical writing and editorial assistance was provided by AbbVie Inc. M. P. Diamond received research funding for the conduct of the study paid to the institution and is a consultant to AbbVie. He is a stockholder and board and director member of Advanced Reproductive Care. He has also received funding for study conduct paid to the institution for Bayer and ObsEva. E. A. Stewart participated as a site investigator in the phase 2 study of asoprisnil and served as a consultant to TAP Pharmaceuticals during the time of design and conduct of the studies while on the faculty of Harvard Medical School and Brigham and Women's Hospital, Boston, MA. In the last 3 years, she has received support from National Institutes of Health grants HD063312, HS023418 and HD074711. She has served as a consultant for AbbVie Inc., Allergan, Bayer HealthCare AG and Myovant for consulting related to uterine leiomyoma and to Welltwigs for consulting related to infertility. She has received royalties from UpToDate and the Med Learning Group. A.R.W. Williams has acted as a consultant for TAP Pharmaceutical Products Inc. and Repros Therapeutics Inc. He has current consultancies with PregLem SA, Gedeon Richter, HRA Pharma and Bayer. B.R. Carr has served as consultant and received research funding from AbbVie Inc. and Synteract (Medicines360). E.R. Myers has served as consultant for AbbVie Inc., Allergan and Bayer. R.A. Feldman received compensation for serving as a principal investigator and participating in the conduct of the trial. W. Elger was a co-inventor of several patents related to asoprisnil.C. Mattia-Goldberg is a former employee of AbbVie Inc. and owns AbbVie stock or stock options. B.M. Schwefel and K. Chwalisz are employees of AbbVie Inc. and own AbbVie stock or stock options. TRIAL REGISTRATION NUMBER: NCT00156195 at clinicaltrials.gov.

Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland.

A model proposed for regulation of steroidogenesis, lipoprotein utilization and cholesterol metabolism in HFA tissue is presented in Fig 17. We envision that the role of ACTH and cAMP in steroidogenesis and cholesterol metabolism is as follows. ACTH binds to specific receptors on the surface of the cells of the HFA gland and as a consequence, adenylate cyclase is activated, leading to increased formation of cAMP. cAMP causes activation of protein kinase that leads, presumably, to phosphorylation of specific proteins. This leads to the initiation of reactions that give rise to increased activity of key enzymes and levels of proteins involved in adrenal cholesterol metabolism. Presumably, the action of ACTH causes an increase in the activity of cholesterol side chain cleavage, the rate-limiting step in the conversion of cholesterol to steroid hormones. We suggest that once the mitochondrial cholesterol side-chain cleavage system is fully activated by ACTH, the supply of cholesterol to the mitochondria becomes rate-limiting for steroidogenesis. To meet this demand for cholesterol, a further action of ACTH results in an increase in the number of LDL receptors. LDL binds to specific receptors on the cell surface that are localized in coated pits. LDL is internalized by a process of adsorptive endocytosis and the internalized vesicles fuse with lysosomes and the protein component of LDL is hydrolyzed by lysosomal proteolytic enzymes to amino acids. The cholesteryl esters of LDL also are hydrolyzed to give rise to fatty acids and cholesterol. The liberated cholesterol is available for utilization in the biosynthesis of steroid hormones and other cellular processes. In addition, ACTH stimulates the activity of HMG CoA reductase and, thus, the rate of de novo cholesterol biosynthesis. In this way sufficient cholesterol is obtained to provide for precursor cholesterol to maintain the high rate of steroid synthesis by the HFA. HDL is not utilized as a source of cholesterol by the HFA. Because of the rapid rate of utilization of LDL by the HFA, fetal plasma levels of LDL are low and the activity of the HFA is a primary determinant of these levels. Thus, in the case of anencephaly, in which the activity of the adrenal is very low, plasma levels of LDL are 2--3 times higher than in normal fetuses, whereas plasma HDL levels are similar. In addition, in the normal neonate plasma LDL levels rise rapidly after birth, and this event is coincident with the involution of the fetal zone of the adrenal. The fetal liver is likely to be the major source ultimately of the LDL-cholesterol utilized by the HFA. Consequently, factors that regulate cholesterol and lipoprotein synthesis in the fetal liver may, in turn, affect the steroidogenic activity of the HFA through regulation of the supply of cholesterol precursor. Thus, if trophic factors for the HFA other than ACTH exist, an important site of their action might be the fetal liver, rather than a direct action to influence the rate of synthesis of steroids by the fetal adrenal.

3-Hydroxy-3-methylglutaryl coenzyme A reductase in anencephalic and normal human fetal liver.

In previous investigations, we have found that the liver appears to be the major source of cholesterol in the human fetus, and, in particular, a principal source of circulating low density lipo-protein-cholesterol (LDL-C). LDL-C plasma levels are low in the normal fetus, most likely due to the rapid uptake and metabolism by the fetal adrenal as precursor for steroid hormone biosynthesis. In contrast, in the anencephalic fetus the adrenals are atrophic, the rate of estrogen and glucocorticoid production is low, and the levels of LDL-C in fetal plasma are high. The purpose of the present investigation was to determine the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the primary rate-limiting enzyme of cholesterol biosynthesis, in anencephalic liver and normal fetal liver. We found that the specific activity of HMG-CoA reductase in normal fetal liver microsomes was 0.428 +/- 0.054 nmol mevalonate formed times mg-1 protein X min-1 (mean +/- SE, n = 9). The rate of HMG-CoA reductase in anencephalic liver microsome preparations was 10-fold less (0.040 +/- 0.003) (mean +/- SE, n = 7) P less than 0.001. Furthermore, we detected HMG-CoA reductase (97,000-mol wt protein) in normal human fetal liver after SDS PAGE and immunoblotting by using a monoclonal antibody directed against HMG-CoA reductase. We were unable to detect any significant quantity of HMG-CoA reductase protein in anencephalic fetal liver, which indicates that low reductase activity was due to low amounts of enzyme protein rather than inactive enzyme. In summary, we conclude that the low levels of cholesterol synthesis observed in anencephalic fetal liver are probably due to both the high levels of LDL-C in fetal plasma as well as the presence of low circulating levels of estrogens and glucocorticoids and that these factors regulate cholesterol synthesis both in vivo and in vitro in fetal liver. This occurs most probably by the modulation of the amount of HMG-CoA reductase, a primary rate-limiting and regulatory enzyme of the cholesterol biosynthetic sequence.

Effects of adrenocorticotropic hormone on low density lipoprotein receptors of human fetal adrenal tissue.

In the present investigation, the mechanism(s) whereby ACTH stimulates low density lipoprotein (LDL) metabolism in human fetal adrenal was evaluated. Plasma membrane fractions were prepared from fetal adrenal tissue fragments incubated in lipoprotein-poor serum with or without ACTH, and the binding of [125I]iodo-LDL to such membrane fractions was examined. The mean specific binding capacity for [125I]iodo-LDL by membrane fractions prepared from four separate fresh human fetal adrenal glands was 1370 +/- 168 ng mg-1 protein (mean +/- SE), and the concentration of [125I]iodo-LDL producing half-maximal binding was 20.8 +/- 1.2 ng ml-1. Thus, the presence of high affinity, low capacity binding sites for LDL in human fetal adrenal tissue was confirmed. When human fetal adrenal tissue was maintained in organ culture for 2 days in medium containing lipoprotein-poor serum in the absence of ACTH, and plasma membrane fractions were subsequently prepared, the binding capacity for LDL in such membrane fractions was the same as or slightly greater than that in membrane fractions prepared from fresh tissue. When ACTH was present in the culture medium, the binding capacity for LDL was doubled compared to that in membrane fractions prepared from tissues incubated in the absence of ACTH. The rate of [125I]iodo-LDL degradation by human fetal adrenal tissue maintained in medium containing ACTH was also twice that of tissue maintained in the absence of ACTH. These results demonstrate that ACTH causes an increase in the number of LDL-binding sites in human fetal adrenal tissue in vitro. This is one mechanism whereby ACTH stimulates LDL metabolism in this tissue.

Lipoprotein-binding sites in human corpus luteum membrane fractions.

In a previous report evidence was presented that plasma low density lipoprotein (LDL), but not high density lipoprotein (HDL), is the major source of cholesterol used by the human corpus luteum for progesterone biosynthesis and, that plasma LDL is taken up by corpus luteum tissues via a receptor-mediated endocytotic process. Using membrane fractions prepared from fresh corpus luteum tissue obtained from nine women at various phases of the menstrual cycle, we conducted the present investigation to characterize lipoprotein-binding sites and changes in such binding sites that might occur throughout the ovarian cycle. High affinity, low capacity binding sites for [125I]iodo-LDL and also for [125I]iodo-HDL were detected in membrane fractions prepared from fresh corpus luteum tissue. The interaction of [125I]iodo-LDL with the high affinity binding sites in fresh corpus luteum membrane fractions was prevented by incubation with heparin. Also, the binding capacity of corpus luteum membrane fractions for [125I]iodo-LDL was reduced by treatment with pronase. On the other hand, the specific binding capacity for [125I]iodo-HDL was unaffected by pronase treatment. The binding capacity for [125I]iodo-LDL in membrane fractions prepared from midluteal phase corpus luteum was significantly greater than that of membrane fractions from tissue obtained at any other phase of the cycle, a finding that suggests that changes in progesterone biosynthesis throughout the cycle are positively correlated with changes in the numbers of binding sites for LDL in the corpus luteum.

Low density lipoprotein binding and de novo synthesis of cholesterol in the neocortex and fetal zones of the human fetal adrenal gland.

The binding of low density lipoprotein (LDL) and the de novo synthesis of cholesterol in separated zones of human fetal adrenal (HFA) tissues were investigated. The number of LDL-binding sites was 2-fold greater in membrane fractions prepared from fresh fetal zone tissue than in those from neocortex tissue. The binding capacity for LDL in fetal zone and neocortex membrane preparations of HFA tissues maintained in culture in the presence of ACTH was 2-fold greater than that in membrane fractions of control tissues. The rates of de novo synthesis of cholesterol also were determined in separated zones of HFA tissue by measuring the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal fractions prepared from HFA tissues and by determining the rate of incorporation of tritium from [3H]water into cholesterol in HFA tissue fragments. The rate of de novo synthesis of cholesterol in fresh fetal zone tissue was twice that in neocortex tissue as estimated by these methods. When separated zones of HFA tissue were maintained in culture in the presence or absence of ACTH, the rates of de novo synthesis, as determined by the rate of incorporation of tritium from [3H]water into cholesterol, were stimulated to a similar extent by ACTH in both fetal zone and neocortex tissues. However, the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was increased to a greater extent by ACTH pretreatment in neocortex tissues than in fetal zone tissues. In summary, fetal zone tissues of the HFA gland have a larger number of LDL-binding sites and higher rates of de novo synthesis of cholesterol than do neocortex tissues, and ACTH stimulates LDL binding and de novo synthesis of cholesterol in both zones of the HFA gland.

The role of calmodulin antagonists on steroidogenesis by fetal zone cells of the human fetal adrenal gland.

The adrenal gland of the human fetus (HFA) is relatively large compared to that of the adult and exhibits an extremely high rate of steroidogenesis both in vivo and in vitro. The fetal zone cells make up 80-85% of the volume of the HFA and are the major site of steroid production during fetal development. We have recently demonstrated that calcium is involved in the regulation of steroidogenesis in fetal zone cells of the HFA. There is considerable evidence that many actions of calcium within cells are mediated by the calcium-binding protein calmodulin. The purpose of the present investigation was to determine if calmodulin also plays a role in HFA steroidogenesis. To investigate this possibility, the fetal zone was dissected from fetal adrenals of first and second trimester human abortuses. After collagenase digestion of the tissue, dispersed fetal zone cells were maintained in a Krebs-Ringers medium at 37 C for a 3-h incubation. Cells were incubated with and without ACTH (10(-8) M) in the presence of the calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and calmidazolium (CAL) at concentrations of 5-100 microM. The media were assayed for contents of dehydroepiandrosterone sulfate (DS), cortisol (F), pregnenolone, and cAMP by RIA. The addition of ACTH stimulated F secretion 5- to 10-fold compared to that in control fetal zone cells. DS secretion increased up to 5-fold and pregnenolone about 2-fold in the presence of ACTH compared to values in control cells. ACTH also stimulated cAMP secretion by 10-fold compared to that in control cells. The addition of TFP, CPZ, and CAL significantly inhibited ACTH-stimulated DS, F, and pregnenolone secretion in a dose-related fashion to near-control levels. We observed that TFP, CPZ, and CAL inhibited cAMP accumulation as well as Bu2cAMP-stimulated steroid secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by TFP and CPZ, but not by CAL. These studies suggest that calmodulin plays a role in regulating steroidogenesis in fetal zone cells of the HFA.

Metabolism of high density lipoprotein by human fetal adrenal tissue.

The role of lipoproteins as a source of the cholesterol utilized for steroidogenesis by human fetal adrenal (HFA) tissue was investigated previously. It was found that low density lipoprotein (LDL) was the lipoprotein preferred as a source of cholesterol for steroidogenesis by the HFA. [125I]Iodo-LDL was taken up and degraded by HFA tissue in organ culture, and the degradation of [125I]iodo-LDL was stimulated when ACTH (1 microgram X ml-1) was present in the culture medium. Others have shown that high density lipoprotein (HDL) is utilized as a source of cholesterol for steroidogenesis by rat adrenocortical cells in vitro and by the adrenals of the adult rat in vivo. In the present investigation we evaluated the metabolism of [125I]iodo-HDL by HFA tissue. [125I]iodo-HDL uptake by the HFA tissue increased in a linear manner with time and as the concentration of [125I]iodo-HDL in the culture medium was increased. However, there was little degradation of [125I]iodo-HDL by HFA. Moreover, preincubation of HFA tissue in medium containing ACTH (1 microgram X ml-1) or HDL, in various concentrations, did not affect the rate of uptake and degradation of [125I]iodo-HDL. The rate of degradation of [125I]iodo-LDL was found to decrease to low levels as the concentration of nonradiolabeled LDL in the culture medium was increased, whereas nonradiolabeled HDL had little effect on the degradation of [125I]iodo-LDL. HFA tissue fragments were incubated in medium containing ACTH plus lipoprotein-poor serum (LPPS) alone or LPPS plus HDL in various concentrations (50-1000 microgram X ml-1). The medium was changed daily and assayed for dehydroisoandrosterone sulfate and cortisol. In the presence of HDL, steroid secretion rates were no greater than those attained by HFA maintained in medium containing LPPS. It is concluded that the HFA utilizes cholesterol derived from LDL for steroidogenesis and that HDL is not metabolized efficiently by the human fetal adrenal.

Factors affecting the conversion of androstenedione to estrogens by human fetal hepatocytes in monolayer culture.

The purpose of the present investigation was to characterize and determine what hormones affect the activity of aromatase in human fetal hepatocytes maintained in primary monolayer culture. The major product of aromatization of androstenedione was estrone sulfate. Optimal conditions for assay of aromatase activity in fetal liver cells were determined. The apparent Km for androstenedione was 50 nM. Aromatase activity was stimulated by glucocorticoids in the presence of fetal calf serum. The concentration of dexamethasone required for half-maximal stimulation was 10(-8) M, similar to the concentration required for half-maximal binding to glucocorticoid receptors. This action of dexamethasone was inhibited by cortisol 21-mesylate, a glucocorticoid antagonist. Aromatase activity was also stimulated by (Bu)2cAMP and cholera toxin, and was inhibited by fetal calf serum. This effect of fetal calf serum was mimicked by epidermal growth factor. However, epidermal growth factor did not mimic the permissive action of serum to stimulate aromatase activity by dexamethasone. In these respects, the regulation of aromatase activity of human fetal hepatocytes is similar to that of human adipose stromal cells. A polycyclic hydrocarbon, benzo(a)pyrene, which causes induction of aryl hydrocarbon hydroxylase activity in fetal hepatocytes, inhibited the stimulation of aromatase activity by dexamethasone. Of a number of hormones tested, including glucagon, insulin, angiotensin II, ACTH, hCG, GH, PRL, and T3, only glucocorticoids were effective in stimulating aromatase activity of human fetal hepatocytes. These results emphasize the complex and multiparameter nature of the regulation of aromatase activity in this as in other tissues.

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.

Transforming growth factor-beta inhibits steroid 17 alpha-hydroxylase cytochrome P-450 expression in ovine adrenocortical cells.

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of ACTH to promote the synthesis of the various steroid-metabolizing cytochrome P-450 enzymes. In the steroidogenic pathway, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) is a key enzyme controlling the formation of cortisol and androgens. Recently, we demonstrated that transforming growth factor-beta (TGF beta) is a potent inhibitor of steroid production in ovine adrenocortical cells. In the present study we used a polyclonal antibody to P450(17) alpha to determine adrenal cell P-450(17) alpha enzyme content by Western analysis. In addition, we used a cDNA probe encoding for bovine P-450(17) alpha mRNA to determine levels of P-450(17) alpha mRNA in sheep ovarian adrenocortical cells in primary culture. When cells were cultured in a serum-free medium in the presence of ACTH for 48 h, P-450(17) alpha activity, enzyme content, and mRNA levels for P-450(17) alpha increased by 3- to more than 10-fold. TGF beta decreased the basal level and completely blocked the stimulatory action of ACTH on P-450(17) alpha enzyme activity. The effects of TGF beta on P-450(17) alpha enzyme content and mRNA levels were manifested in a dose-dependent manner, with maximal inhibition observed using 1 ng/ml TGF beta. Importantly, the inhibitory effects of TGF beta on P-450(17) alpha were not overcome by (Bu)2cAMP. These findings indicate that TGF beta is a potent negative regulator of P-450, and the inhibitory action appears to be at the level of P-450(17) alpha gene expression. The ability of TGF beta to suppress the positive stimulatory action of ACTH suggests that TGF beta could play a role in determining the pathway of steroidogenesis and, thereby, the specific steroids secreted by adrenocortical cells.

Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses.

There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH.

Synthesis of cholesterol in the human fetus: 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of liver microsomes.

The specific activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in microsome-enriched fractions prepared from normal human fetal liver tissue was assessed. The mean specific activity was 0.58 +/- 0.18 nmol mevalonate formed min-1 mg-1 protein. The activity of the enzyme was inhibited by preincubation of microsomes with ATP (4 mM) and was greatly reduced when microsomes were prepared from tissue homogenized in the presence of NaF (50 mM). It can be computed that the activity of HMG CoA reductase in human fetal liver microsomes is adequate to provide cholesterol to meet the requirements of the fetal adrenal for steroid precursor provided that cholesterol synthesized in the fetal liver appears in the plasma in the form of low density lipoprotein.

Cholesterol synthesis by human fetal hepatocytes: effects of hormones.

In previous investigations, we found high rates of cholesterol synthesis in human fetal liver tissue, second only to rates in fetal adrenal tissue. Previous estimates of the amount of cholesterol in the fetus derived from the maternal compartment are in the range of 20%. Thus, the liver may be the principal source of circulating lipoproteins in the human fetus, as is true in the human adult. Low density lipoprotein is the major source of cholesterol used for fetal adrenal steroidogenesis; therefore, it follows that factors regulating cholesterol synthesis in the human fetal liver may indirectly control the rate of steroid secretion by the adrenal cortex. The purpose of the present investigation was to determine if hormones, particularly those produced by the fetal-placental unit, might serve to stimulate cholesterol synthesis in the human fetal liver. The rate of cholesterol biosynthesis was determined by measuring the rate of incorporation of [3H]water into [3H]cholesterol in hepatocytes maintained in culture or by determination of the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal preparations from human fetal liver. The addition of dexamethasone (10(-10) - 10(-6)M) stimulated cholesterol synthesis up to 2- to 4-fold between days 2 and 6 of exposure. When human fetal liver cells were maintained in the presence of dexamethasone (10(-7)M), the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal fractions was stimulated 4-fold compared to that in control cells. Cortisol also stimulated cholesterol biosynthesis in a concentration-dependent manner. The addition of 17 beta-estradiol (E2) to the culture medium resulted in stimulation of cholesterol biosynthesis in a concentration-dependent manner from 10(-10) - 10(-7)M. The rate of cholesterol synthesis when E2 was present (10(-7)M) was 4-fold greater than that in untreated cells. Stimulation of cholesterol synthesis by E2 was maintained between 2-7 days of incubation with E2. Estrone, estriol, and E2 (10(-6)M) caused similar increases (3- to 4-fold) in the rates of cholesterol synthesis in human fetal hepatocytes. Finally, progesterone in concentrations greater than 10(-6) M significantly stimulated cholesterol synthesis in human fetal liver cells. In contrast, other hormones and factors, including insulin, glucagon, PRL, GH, dehydroepiandrosterone and its sulfate, epidermal growth factor, fibroblast growth factor, T3, (Bu)2cAMP, and cholera toxin, had no effect on the rate of cholesterol synthesis in human fetal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen 16 alpha-hydroxylase activity in human fetal tissues.

Estrogen 16 alpha-hydroxylase activity was measured in microsomes prepared from fetal tissues of first and second trimester human abortuses using [16 alpha-3H]estrone sulfate as substrate and NADPH as cofactor. Estrogen 16 alpha-hydroxylase activity was demonstrable in 13 of 14 fetal tissues examined in this study, viz. liver, adrenal fetal zone, adrenal neocortex, lung, kidney, intestine, heart, brain, skin, testis, spleen, pancreas, and stomach, and was either negligible or absent in placental tissue. The highest specific activity of the microsomal enzyme [pico-moles of product(s) formed per mg protein/h] was found in liver (mean +/- SEM, 338 +/- 62), and the next highest was found in the fetal zone of the adrenal cortex (70 +/- 20). The specific activities of estrogen 16 alpha-hydroxylase in adrenal neocortex, brain, skin, and testis were similar (25-53 pmol/mg protein X h) as were those in lung, kidney, intestine, heart, spleen and stomach (23-36 pmol/mg protein X h). The specific activity of the enzyme in the pancreas was 12 pmol/mg protein X h; the lowest specific activity, however, was in placental microsomes (0.2 +/- 0.1 pmol/mg protein X h).

Glucocorticoids inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by human fetal Kupffer cells.

Inflammatory mediators, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) are secreted by fixed tissue macrophages and exhibit local autocrine and paracrine effects as well as distant endocrine effects. Human fetal Kupffer cells, the fixed tissue macrophages of the liver, may play a role as modulators of immune and endocrine function in early embryonic and fetal development. In the present study we isolated human fetal Kupffer cells to greater than 90% purity and prepared short term cultures to investigate the effect of glucocorticoids on the secretion of the cytokine TNF alpha. Fetal Kupffer cells secreted TNF alpha and IL-1 beta after culture with bacterial lipopolysaccharide (LPS), indicating that these cells express mature macrophage function. Cortisol and dexamethasone dramatically suppressed the LPS-stimulated secretion of TNF alpha by fetal Kupffer cells. The inhibitory effects of glucocorticoids appeared to be specific, since estrogen, progesterone, and testosterone had no effect on LPS stimulation of TNF alpha production. None of the steroids tested altered basal production or enhanced the LPS-stimulated production of TNF alpha by fetal Kupffer cells. The inhibition by glucocorticoids could be reversed by the addition of RU 486, indicating that this effect was mediated by the glucocorticoid receptor. These results demonstrate that human fetal macrophages demonstrate mature macrophage function in early gestation; they can be activated to produce TNF alpha by a well characterized modulator of cellular function (LPS) and suppressed by glucocorticoids.

Effect of superovulation with human menopausal gonadotropins on growth hormone levels in women.

GH synthesis and secretion are influenced by several factors, including age, body weight, and sex steroid hormones. Endogenous and exogenous estrogens influence the circulating levels of GH. The purpose of the present investigation was to define the relationship between serum GH and estradiol levels during the follicular phase in women with normal ovulatory menstrual cycles compared with that in women undergoing superovulation with human menopausal gonadotropins (hMG) alone or hMG plus GnRH agonists during treatment for infertility. Serum GH and estradiol levels were determined by immunoassay in eight women during the follicular phase of a spontaneous natural cycle (group I). Thirty women underwent ovulation induction with hMG alone (group II), and 30 women received GnRH agonists followed by hMG (group III). During the follicular phase estradiol levels increased gradually in group I and reached a peak estradiol level of 1.19 +/- 0.2 nmol/L (mean +/- SEM). As expected, estradiol levels rose faster and reached higher levels in groups II and III (5.44 +/- 0.62 and 8.73 +/- 0.91 nmol/L, respectively). Whereas serum GH levels increased minimally in group I, reaching a peak level of 2.54 +/- 1.15 nmol/L, serum GH concentrations increased markedly after day 8 in groups II and III, reaching peak levels of 8.70 +/- 1.58 and 7.54 +/- 1.12 nmol/L, respectively (P less than 0.01). Basal to peak GH levels were higher in groups II and III than in group I. In summary, there are modest increases in GH levels during the follicular phase of the normal menstrual cycle, but the levels are markedly increased during superovulation with hMG or hMG plus GnRH agonists, and parallel increases in estradiol levels.

The role of cyclic adenosine 3',5'-monophosphate in cholesterol metabolism and steroidogenesis by the human fetal adrenal gland.

In the present investigation we studied the role of cAMP as a mediator of ACTH action in human fetal adrenal (HFA) tissue. We have characterized the response to ACTH, dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and cholera toxin (CT) with respect to steroidogenesis, low density lipoprotein (LDL) binding, degradation of LDL, and the rate of de novo synthesis of cholesterol. The rate of dehydroisoandrosterone sulfate secretion was similar in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT. In contrast, cortisol secretion by HFA tissue was more sensitive to dbcAMP and CT than to ACTH. In membrane preparations obtained from HFA tissue maintained in the presence of ACTH, dbcAMP, or CT, there was a 2 to 3-fold increase of specific binding of [125I]iodo-LDL. In HFA tissue maintained in the presence of ACTH or CT, the rate of degradation of LDL was significantly increased compared to tissue maintained in the lipoprotein-poor serum alone. Finally, in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT there was a 6- to 10-fold stimulation of the rate of incorporation of [14C]acetate into cholesterol. We conclude that steroidogenesis, LDL binding, and degradation, as well as de novo synthesis of cholesterol, are probably stimulated in HFA tissue via a cAMP-mediated pathway.