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.

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.

The human placenta expresses CYP17 and generates androgens de novo.

INTRODUCTION: The human placenta is believed to have insignificant CYP17 expression, rendering it dependent on the maternal and fetal compartments for the necessary androgenic precursors to yield the high levels of estrogens seen in pregnancy. OBJECTIVE: The aim of the study was to analyze whether the human trophoblast is capable of expressing CYP17 and producing androgens de novo. METHODS: Human trophoblasts from fresh placentas and JEG-3 cells were used for all experiments. CYP17 mRNA analysis was performed via RT-PCR, and protein detection by Western blot and immunohistochemical staining. Steroid products were quantified using RIAs. RESULTS: CYP17 mRNA was expressed in both cell types. CYP17 protein was detected by Western blotting and localized by immunostaining mainly to the cytoplasm of syncytiotrophoblasts. Measurement of 17α-hydroxyprogesterone, androstenedione, and their aromatized products in the media further demonstrated CYP17 expression and activity in the human trophoblast. Baseline levels of CYP17 steroid products were higher in primary cells and significantly increased in the presence of 22-hydroxycholesterol. CONCLUSIONS: We have demonstrated CYP17 mRNA and protein expression and activity in human trophoblasts. Considering the precursor concentration, blood flow, and mass of the placenta, we suggest that its contribution of androgens is an important source of estrogen production in pregnancy.

Inhibition of Src tyrosine kinase stimulates adrenal androgen production.

A unique characteristic of the primate adrenal is the ability to produce 19-carbon steroids, often called the adrenal androgens. Although it is clear that the major human adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), are produced almost solely in the adrenal reticularis, the mechanisms regulating production are poorly understood. Herein, we tested the hypothesis that the Src family of tyrosine kinases are involved in the regulation of adrenal androgen production. The NCI-H295R human adrenal cell line and primary human adrenal cells in culture were used to study adrenal androgen production and expression of enzymes involved in steroidogenesis. To examine the role of Src tyrosine kinase, cells were treated with PP2, a specific Src inhibitor. Alternatively, adrenal cells were transfected with an expression vector containing a dominant-negative form of Src. PP2 treatment inhibited basal cortisol production while significantly increasing the production of DHEA and DHEA-S (together referred to as DHEA(S)) in both adrenal cell models. The effect of PP2 on steroidogenesis occurred along with a rapid induction of steroidogenic acute regulatory (StAR) protein synthesis as revealed by Western analysis. Treatment with PP2 also increased mRNA levels for StAR, and cholesterol side-chain cleavage (CYP11A) and 17alpha-hydroxylase/17,20-lyase (CYP17) enzymes. Treatment of adrenal cells with the cAMP agonist dibutyryladenosine cyclic monophosphate (dbcAMP), stimulated the production of cortisol and DHEA(S). However, treatment of adrenal cells with a combination of PP2 and dbcAMP enhanced the production of DHEA(S) while inhibiting cortisol production. During dbcAMP treatment PP2 was able to augment the expression of CYP17 and to inhibit the induction of 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) levels. Increasing the CYP17 to HSD3B2 ratio is likely to promote the use of steroid precursors for the production of DHEA(S) and not for cortisol. Taken together these data suggest that the inhibition of Src tyrosine kinases causes adrenal cells to adopt a reticularis phenotype both by the production of DHEA(S) and by the steroidogenic enzymes expressed.

Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes.

In the current study we test the hypothesis that liver receptor homologue-1 (LRH; designated NR5A2) is involved in the regulation of steroid hormone production. The potential role of LRH was assessed by first examining expression in human steroidogenic tissues and second by examining effects on transcription of genes encoding enzymes involved in steroidogenesis. LRH is closely related to steroidogenic factor 1 (SF1; designated NR5A1), which is expressed in most steroidogenic tissues and regulates expression of several steroid-metabolizing enzymes. LRH transcripts were expressed at high levels in the human ovary and testis. Adrenal and placenta expressed much lower levels of LRH than either ovary or liver. To examine the effects of LRH on steroidogenic capacity we used reporter constructs prepared with the 5'-flanking region of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A1), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), 11beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). Co-transfection of these reporter constructs with LRH expression vector demonstrated that like SF1, LRH enhanced reporter activity driven by flanking DNA from StAR, CYP11A1, CYP17, HSD3B2, and CYP11B1. Reporter constructs driven by CYP11A1 and CYP17 were increased the most by co-transfection with LRH and SF1. Of the promoters examined only HSD3B2 was more sensitive to LRH than SF1. The high level of ovarian and testicular LRH expression make it likely that LRH plays an important role in the regulation of gonadal function.

Gene profiling of human fetal and adult adrenals.

The mechanisms that lead to the steroidogenic differences in the human fetal adrenal (HFA) and adult adrenal gland are not known. However, gene expression clearly plays a critical role in defining their distinct steroidogenic and structural phenotypes. We used DNA microarrays to compare expression levels of several thousand transcripts between the HFA and adult adrenal gland. Total RNA was isolated from 18 HFA and 12 adult adrenal glands. Samples of total RNA were used to make five pools of poly A+ RNA (mRNA). Gene profiling was done using five independent microarrays that contained between 7075 and 9182 cDNA elements. Sixty-nine transcripts were found to have a greater than 2.5-fold difference in expression between HFA and adult adrenals. The largest differences were observed for transcripts that encode IGF-II (25-fold higher in HFA) and 3beta-hydroxysteroid dehydrogenase (24-fold higher in adult). Among the other genes, transcripts related to sterol biosynthesis or to growth and development were higher in the HFA than adult adrenals. Transcripts concerned with cellular immunity and signal transduction were preferentially expressed in the adult adrenal. The vast majority of the 69 transcripts have not been studied with regard to adrenal function. Thus, these gene profiles provide valuable information that could help define the mechanisms that control adrenal function.

Metformin directly inhibits androgen production in human thecal cells.

OBJECTIVE: To examine the direct effect of metformin on thecal cell androgen production. SETTING: Basic science research laboratory, University of Texas Southwestern, Dallas, Texas. INTERVENTION(S): Human ovarian theca-like tumor cells were treated with various concentrations of metformin in the presence and absence of forskolin for 48 hours. MAIN OUTCOME MEASURE(S): Media were collected, and radioimmunoassay (RIA) for progesterone, 17 alpha-hydroxyprogesterone (17OHP), androstenedione, and testosterone was performed. The effect of metformin on the expression of various enzymes involved in theca cell steroidogenesis was examined. RESULT(S): Metformin (50 microM and 200 microM) significantly inhibited androstenedione production from both forskolin-stimulated and unstimulated theca cells. Testosterone production was also significantly inhibited in forskolin-treated cells in the presence of 200 microM of metformin-treated compared with forskolin-only-treated cells. Western blot analysis revealed that metformin significantly inhibited the expression of steroidogenic acute regulatory (StAR) protein and 17 alpha-hydroxylase (CYP17) expression in cells stimulated with forskolin compared with forskolin treatment alone. There was no significant change in either 3beta-hydroxysteroid dehydrogenase (3 beta HSD) or cholesterol side-chain cleavage (CYP11A1) protein expression. Northern analysis revealed a significant decrease in the expression of CYP17 mRNA in forskolin-stimulated cells treated with metformin (200 microM) compared with forskolin-only-treated cells, however, there was no significant change in steroidogenic acute regulatory protein mRNA expression. CONCLUSION(S): Our results suggest that metformin may have a direct effect on thecal cells' androgen production.

Effects of lower doses of conjugated equine estrogens and medroxyprogesterone acetate on plasma lipids and lipoproteins, coagulation factors, and carbohydrate metabolism.

OBJECTIVE: To determine the effects of lower doses of conjugated equine estrogens (CEE) alone or CEE and medroxyprogesterone acetate (MPA) on lipoproteins, carbohydrate metabolism, and coagulation/fibrinolytic factors. DESIGN: Randomized, double-blind, placebo-controlled study. SETTING: Multicenter substudy of the Women's HOPE trial. PATIENT(S): Seven hundred and forty-nine healthy, postmenopausal women. INTERVENTION(S): Women were randomized to receive the following doses in milligrams per day: 0.625 CEE; 0.625 CEE/2.5 MPA; 0.45 CEE; 0.45 CEE/2.5 MPA; 0.45 CEE/1.5 MPA; 0.3 CEE; 0.3 CEE/1.5 MPA; or placebo. MAIN OUTCOME MEASURE(S): Assessment of lipids, lipoproteins, glucose tolerance, and coagulation/fibrinolytic factors at baseline, cycle 6, and year 1. RESULT(S): One year of treatment with any of the CEE or CEE/MPA regimens studied increased high-density lipoprotein cholesterol (HDL-C); the 10% increase in HDL-C for the CEE 0.45/MPA 1.5 group was similar to the CEE 0.625/MPA 2.5 group. Low-density lipoprotein cholesterol was significantly reduced in all of the active treatment groups except the CEE 0.3/MPA 1.5 group at cycle 13. Apolipoprotein A-I and triglyceride levels increased and apolipoprotein B levels decreased in all groups. The lipoprotein (a) level was reduced in the CEE 0.45/MPA 2.5, CEE 0.45/MPA 1.5, and CEE 0.625/MPA 2.5 groups. Minimal changes were observed in carbohydrate metabolism for all groups. Fibrinogen and PAI-1 activity decreased and plasminogen activity increased in all groups. Decreases in antithrombin III and protein S activities were significant for all active treatment groups except the CEE 0.3/MPA 1.5 group. CONCLUSION(S): Lower doses of CEE and CEE/MPA induce favorable changes in lipids, lipoproteins, and hemostatic factors with minimal changes in carbohydrate metabolism.

A role for src tyrosine kinase in regulating adrenal aldosterone production.

Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regulators of aldosterone production are angiotensin II (Ang IotaIota) and potassium (K(+)). Ang IotaIota stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang IotaIota activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 microM-10 microM) inhibited basal, Ang IotaIota, K(+) and dibutyryladenosine cyclic monophosphate (dbcAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 microM) for 6, 12, 24, and 48 h revealed a time-dependent inhibition of aldosterone production. Inhibition by PP2 (0.3-10 microM) was also observed for the metabolism of 22R-hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non-specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3beta-hydroxysteroid dehydrogenase (3betaHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 3betaHSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [(14)C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17alpha-hydroxyprogesterone. To determine if this apparent increase in 17alpha-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3betaHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechanism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.

Bone morphogenetic protein inhibits ovarian androgen production.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, were recently shown to be expressed and to regulate steroidogenesis in rat ovarian tissue. The purpose of this study was to investigate the effect of BMP-4 on androgen production in a human ovarian theca-like tumor (HOTT) cell culture model. We have previously demonstrated the usefulness of these cells as a model for human thecal cells. HOTT cells respond to protein kinase A agonists by increased production of androstenedione and with an induction of steroid-metabolizing enzymes. In this investigation, HOTT cells were treated with forskolin or dibutyryl cyclic AMP (dbcAMP) in the presence or absence of various concentrations of BMP-4. The accumulation of androstenedione, progesterone, and 17alpha-hydroxyprogesterone (17OHP) in the incubation medium was measured by RIA. The expression of 17alpha-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cholesterol side-chain cleavage (CYP11A1), and steroidogenic acute regulatory (StAR) protein was determined by protein immunoblotting analysis using specific rabbit polyclonal antibodies. We also examined the expression of BMP receptor subtypes in our HOTT cells using RT-PCR. In cells treated with medium alone, steroid accumulation and steroid enzyme expression was unchanged. In cells treated with BMP alone there was a modest decrease in androstenedione secretion. In the presence of forskolin, HOTT cell production of androstenedione, 17OHP, and progesterone increased by approximately 4.5-, 35-, and 3-fold, respectively. In contrast, BMP-4 decreased forskolin-stimulated HOTT cell secretion of androstenedione and 17OHP by 50% but increased progesterone production 3-fold above forskolin treatment alone. Forskolin treatment led to an increase in CYP17, CYP11A1, 3betaHSD, and StAR protein expression. BMP-4 markedly inhibited forskolin stimulation of CYP17 expression but had little effect on 3betaHSD, CYP11A1, or StAR protein levels. Similar results were observed with the cAMP analog dbcAMP. In addition, BMP-4 inhibited basal and forskolin stimulation of CYP17 messenger RNA expression as determined by RNase protection assay. Other members of the transforming growth factor beta superfamily, including activin and inhibin, had minimal effect on androstenedione production in the absence of forskolin. In the presence of forskolin, activin inhibited androstenedione production by 80%. Activin also inhibited forskolin induction of CYP17 protein expression as determined by Western analysis. We identified the presence of messenger RNA for three BMP receptors (BMP-IA, BMP-IB, and BMP-II) in the HOTT cells model. In conclusion, BMP-4 inhibits HOTT cell expression of CYP17, leading to an alteration of steroidogenic pathway resulting in reduced androstenedione accumulation and increased progesterone production. These effects of BMP-4 seem similar to those caused by activin, another member of the transforming growth factor-beta superfamily of proteins.

Progesterone receptor isoform A but not B is expressed in endometriosis.

We previously demonstrated that 17beta hydroxysteroid dehydrogenase type 2, the enzyme that inactivates estradiol to estrone, is expressed in luteal eutopic endometrium in response to progesterone but not in simultaneously biopsied peritoneal endometriotic tissue. This molecular evidence of progesterone resistance, together with the clinical observation of resistance of endometriosis to treatment with progestins, led us to determine the levels of progesterone receptor (PR) isoforms PR-A and PR-B in eutopic endometrial and extra-ovarian endometriotic tissues. It was proposed that progesterone action on target genes is mediated primarily by homodimers of PR-B, whereas the truncated variant PR-A acts as a repressor of PR-B function. Immunoprecipitation, followed by Western blot analysis, was performed to detect bands specific for PR-A and PR-B in paired samples of endometriotic and eutopic endometrial tissues simultaneously biopsed from 18 women undergoing laparoscopy during various phases of the menstrual cycle. PR-B was present in 17 of 18 eutopic endometrial samples, and its level increased in the preovulatory phase, as expected, whereas PR-A was detected in all samples (n = 18) with a similar, but less prominent, cyclic variation in its levels. In endometriotic samples, however, no detectable PR-B could be demonstrated, whereas PR-A was detected in all samples (n = 18), albeit in much lower levels and without any cyclic variation in contrast with the eutopic endometrium. Levels of PR-A and PR-B in endometriotic and eutopic endometrial tissues were determined and compared after normalization to total protein and estrogen receptor-alpha levels. Using RNase protection assay, we also demonstrated indirectly that only PR-A transcripts were present in endometriotic tissue samples (n = 8), whereas both PR-A and PR-B transcripts were readily detectable in all eutopic endometrial samples (n = 8). This was indicative that failure to detect PR-B protein in endometriotic tissues is due to the absence of PR-B transcripts. We conclude that progesterone resistance in endometriotic tissue from laboratory and clinical observations may be accounted for by the presence of the inhibitory PR isoform PR-A and the absence of the stimulatory isoform PR-B.

Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex: immunohistochemical studies.

Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.

Transforming growth factor beta inhibits steroidogenic acute regulatory (StAR) protein expression in human ovarian thecal cells.

In this study, we investigated the effects of TGFbeta1 on steroidogensis and expression of the steroidogenic acute regulatory (StAR) protein which regulates an important early step in the steroidogenic pathway. We utilized a human ovarian thecal like tumor (HOTT) cell model and investigated the effects of activin-A, inhibin-A, or TGFbeta1 in the presence of forskolin and the effect of dibutyryl cyclic AMP (dbcAMP) on steroid accumulation in the culture medium. Cells were also treated with different concentration of TGFbeta1 in the presence of forskolin, combined steroid production was measured at the end of 48 h and after 3 h incubation with 22R-hydroxycholesterol. In the presence of TGFbeta1 there was a dose-dependent inhibition of androstenedione production. Inhibition in combined steroid production was apparent at the highest concentration of TGFbeta1 tested. In the presence of 22R-hydroxycholesterol, combined steroid production was significantly inhibited at lower concentrations. TGFbeta1 inhibited StAR protein expression in a concentration dependent manner. There was also a similar inhibition in StAR mRNA. These results suggest that the effect of TGFbeta1 on steroid production and possibly follicular development may be in part due to its effects on StAR expression.

Is there an increased risk of stroke associated with oral contraceptives?

In the last few years several studies were published about the relationship between oral contraceptive use, estrogen dose, different types of progestogens, cigarette smoking and the risk of stroke. There is a persistent association between the use of oral contraceptives containing more than 50microg of ethinylestradiol and the risk of stroke. Also, cigarette smoking seems to be a strong additive risk factor, especially in women >35 years old even with lower doses (< or =30microg) of estrogen. Unlike oral contraceptives containing >50microg of estrogen, currently there is no convincing evidence that the use of oral contraceptives containing <50microg in the absence of smoking is associated with any meaningful increase in the risk of ischaemic or haemorrhagic stroke. Progestogen-only pills are not associated with an increased risk of stroke. A specific type of progestogen in combined pills was associated with an increased risk of stroke in a few studies. Data regarding this issue is, however, inconsistent and controversial and needs further investigation. There were few if any studies that have addressed the effects of new types of progestogens (i.e. norgestimate, norgestrel or gestodene) and formulations containing 20microg of ethinylestradiol. At the present time we find no reason to alter the current practice in prescribing oral contraceptives. We do concede, however, that there might be a slight causal relationship between use of oral contraceptives containing <50microg of ethinylestradiol and stroke that did not reach statistical significance. This relationship is rare and should be viewed in context with the many benefits of oral contraceptives. Underlying risk factors for stroke such as factor V Leiden mutation and other thrombophilias might explain the role of oral contraceptive-induced stroke.

Uniqueness of oral contraceptive progestins.

PIP: Gonane progestins are more potent and, except for levonorgestrel, less androgenic than the estrane progestins that preceded them. The current, informal method for classifying oral contraceptives (OCs) as old, first generation, or second generation on the basis of their date of introduction obscures the fact that each of the gonane progestins has unique biologic properties. Recommended, instead, is the classification of OCs according to their level of androgenicity. Under such a system, norgestimate, desogestrel, and monophasic norethindrone (0.4-0.5 mg) would fall into the low category. Triphasic levonorgestrel, norethindrone (1.0 mg), norethindrone acetate (1.0 mg), and ethynodiol diacetate (1.0 mg) would be classified as having medium androgenicity, while norgestrel (0.3 mg), norethindrone acetate (1.5-2.5 mg), and levonorgestrel (0.15 mg) would fall into the high androgenicity category.^ieng

Adrenarche results from development of a 3beta-hydroxysteroid dehydrogenase-deficient adrenal reticularis.

Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

Regulation of biologically active dimeric inhibin A and B from infancy to adulthood in the male.

Inhibins are glycoprotein members of the transforming growth factor-beta family that have been implicated in the control of spermatogenesis by exerting a negative feedback on FSH secretion. In addition, locally produced inhibins may play a role in paracrine regulation of testicular function. Immunoassays were used to measure the two biologically active dimeric forms of inhibin (inhibin A and B) in serum, seminal plasma, and urine. To better define their actions, inhibins were measured in the male during infancy, sexual maturation, and senescence. Inhibin B but not A was measurable in the serum of male newborns, infants, children, and adults. In adult males, measurable levels of inhibin B were detected in the seminal plasma but not the urine. The circulating levels of inhibin B increased shortly after birth and peaked at 4-12 months of age (210 +/- 31 pg/mL). The concentration measured in the serum then decreased to a low of 81 +/- 12 pg/mL of inhibin B from 3-9 yr of age followed by a gradual increase beginning with the onset of puberty and reaching another peak of 167 +/- 20 pg/mL in males who were 20-30 yr of age. Inhibin B levels then gradually declined with increasing age up through 90 yr of age. Serum levels of gonadotropins and total testosterone production were also measured in these same males. There was a brief increase in the gonadotropins (FSH and LH) during the few months of postnatal development, followed by a decrease to basal levels until the onset of puberty at 10-14 yr of age. Testosterone was also increased in the serum of infants from day 1 through 12 months of age, which decreased in young children but increased again following the elevation of gonadotropins during puberty. In adults aged 20-90 yr, serum levels of inhibin B were inversely proportional to levels of FSH but not LH or testosterone. In males in which a semen analysis was performed, those males with normal semen analysis had a significantly higher inhibin B levels, sperm production, and lower FSH levels than males with either oligospermia or nonobstructive azoospermia. The levels of Inhibin B found in circulation were a good marker for testicular function and could be useful in the diagnosis of patients with semen abnormalities or a complete absence of spermatogenesis. Because this glycoprotein is secreted in high amounts in the prepubertal testis up to 3 yr of age, inhibin B could potentially be used as a marker in the diagnosis of cryptorchidism and precocious puberty.

Effect of estradiol on DHEAS production in the human adrenocortical cell line, H295R.

OBJECTIVE: To determine if estradiol regulates DHEA and DHEAS production in a human adrenocortical (H295R) cell line and to determine if this effect is receptor mediated. METHODS: NCI-H295 (H295R) cells were rinsed and placed in phenol red free Dulbecco's Modified Eagle's-F12 medium supplemented with 0.1% charcoal-stripped serum. After 24 hours, cells were rinsed and treated based on experimental design. The effects of estradiol were investigated by: 1) treatment of cells with increasing concentrations of estradiol (300-3000 nmol/L) with or without forskolin (10 mumol/L), 2) treatment of cells with the nonsteroidal synthetic estrogen diethylstilbestrol (DES) (300-3000 nmol/L) with or without forskolin (10 mumol/L), and 3) treatment of cells with an estradiol antagonist (ICI 182, 780) in the presence of estradiol. RESULTS: Estradiol alone increased the basal production of DHEAS in H295R cells in a concentration-dependent manner with a maximal effect at 1000 nmol/L. Forskolin treatment increased the basal production of DHEAS ten-fold. Estradiol also increased the forskolin stimulation of DHEAS production two-fold. In contrast, DES alone or DES in addition to forskolin did not stimulate DHEAS production. Estradiol, in contrast, inhibited H295R adrenal cell production of cortisol whereas DES exhibited a similar inhibition. The estrogen receptor antagonist ICI 182,780 was unable to inhibit the stimulatory effect of estradiol. Finally, estradiol in a concentration-dependent manner suppressed 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in H295R adrenal cells. CONCLUSION: These experiments support the role of estradiol in regulation DHEAS production by inhibiting 3 beta HSD activity; however, the mechanism appears to require high concentrations of estradiol and appears to be independent of the estrogen receptor.

Treatment of severe postmenopausal endometriosis with an aromatase inhibitor.

OBJECTIVE: To treat an unusually aggressive case of recurrent postmenopausal endometriosis. DESIGN: Case report. SETTING: University of Texas Southwestern Medical Center (Dallas, Texas). PATIENT(S): A 57-year-old woman who presented with recurrent severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy. INTERVENTION(S): Oral administration of anastrozole (an aromatase inhibitor) (1 mg/d) and elemental calcium (1.5 g/d) for 9 months. Alendronate (a nonestrogenic inhibitor of bone resorption), 10 mg/d, was added to this regimen. MAIN OUTCOME MEASURE(S): Reduction in size of endometriotic lesion, pain relief, tissue levels of aromatase P450 messenger RNA, bone density. RESULT(S): Circulating levels of estradiol-17beta were reduced to approximately 50% of the baseline value after the onset of treatment with anastrozole. Pain rapidly decreased and completely disappeared after the 2nd month of treatment. The 30 x 30 x 20-mm bright red polypoid vaginal lesion was reduced to a 3-mm gray tissue by the end of 9 months of treatment. Markedly high pretreatment levels of aromatase P450 messenger RNA in the endometriotic tissue became undetectable in a specimen obtained from a repeated biopsy after 6 months of treatment. Bone density of lumbar spine decreased by 6.2% after 9 months of treatment. CONCLUSION(S): This is the first description of the use of an aromatase inhibitor in the treatment of endometriosis. The short-term results were extraordinarily successful in elimination of pain and near-complete eradication of implants associated with severe endometriosis not responsive to other therapy. We conclude that the recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis that is resistant to standard regimens.

Expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal.

OBJECTIVE: To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. METHODS: Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. RESULTS: The RNase protection assay was designed to distinguish specific bands that corresponded to CYP11B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. CONCLUSION: The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C19 steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.