Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up.

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.

Steroid hormone related male biased parasitism in chamois, Rupicapra rupicapra rupicapra

Parasites are linked with their host in a trophic interaction with implications for both hosts and parasites. Interaction stretches from the host's immune response to the structuring of communities and the evolution of biodiversity. As in many species sex determines life history strategy, response to parasites may be sex-specific. Males of vertebrate species tend to exhibit higher rates of parasites than females. Sex-associated hormones may influence immunocompetence and are hypothesised to lead to this bias. In a field study, we tested the prediction of male biased parasitism (MBP) in free ranging chamois (Rupicapra rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminths. We further investigated sex differences in faecal androgen (testosterone and epiandrosterone), cortisol and oestrogen metabolites using enzyme immunoassays (EIA) to evaluate the impact of these hormones on sex dependent parasite susceptibility. Non-invasive methods were used and the study was conducted throughout a year to detect seasonal patterns. Hormone levels and parasite counts varied significantly throughout the year. Male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. The hypothesis of MBP originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. The faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. Our study shows that sex differences in steroid levels play an important role to explain MBP, although they alone cannot fully explain the phenomenon.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Gestation-specific reference intervals for comprehensive spot urinary steroid hormone metabolite analysis in normal singleton pregnancy and 6 weeks postpartum

BACKGROUND: Normal pregnancy depends on pronounced adaptations in steroid hormone concentrations. Although in recent years, the understanding of these hormones in pregnancy has improved, the interpretation is hampered by insufficient reference values. Our aim was to establish gestation-specific reference intervals for spot urinary steroid hormone levels in normal singleton pregnancies and 6 weeks postpartum. METHODS: Cross-sectional multicentre observational study. Women recruited between 2008 and 2013 at 3 University Hospitals in Switzerland (Bern), Scotland (Glasgow) and Austria (Graz). Spot urine was collected from healthy women undergoing a normal pregnancy (age, 16-45 years; mean, 31 years) attending routine antenatal clinics at gestation weeks 11, 20, and 28 and approximately 6 weeks postpartum. Urine steroid hormone levels were analysed using gas-chromatography mass spectrometry. Creatinine was also measured by routine analysis and used for normalisation. RESULTS: From the results, a reference interval was calculated for each hormone metabolite at each trimester and 6 weeks postpartum. Changes in these concentrations between trimesters and postpartum were also observed for several steroid hormones and followed changes proposed for index steroid hormones. CONCLUSIONS: Normal gestation-specific reference values for spot urinary steroid hormones throughout pregnancy and early postpartum are now available to facilitate clinical management and research approaches to steroid hormone metabolism in pregnancy and the early postpartum period.

Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays

BACKGROUND: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. METHODS: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. RESULTS: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. CONCLUSIONS: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

[Urinary hormone analysis]

Urinary hormone analysis is applied to detect an altered steroid hormone metabolism, an elevated production of biogenic amines and to non-invasively determine the protein hormone human beta-choriogonadotropin indicating a pregnancy. Occasionally, these determinations need to be complemented by plasma- or serum hormone analysis. Clinical data including current drug therapy and urinary creatinine as reference are required to interpret any urine analysis. Diseases to be investigated by steroid hormone analysis are excess production of a typical or atypical mineralocorticoid active steroid hormones, the hormonal activity of adrenal or ovarian tumors, acne of unknown origin, hirsutism, a PCO-, an adrenogenital or a suspected Cushing syndrome. Biogenic amines should be determined in suspected secondary or refractory arterial hypertension, in case of pheochromocytoma- or paraganglioma-associated symptoms or if a serotonin-producing tumor is suspected. In children genetically determined diseases are the primary background to perform an analysis.

Anti-Müllerian hormone and inhibin B as predictors of pregnancy after treatment by in vitro fertilization/intracytoplasmic sperm injection

OBJECTIVE: To evaluate anti-Müllerian hormone (AMH) as a marker of reproductive outcome after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Longitudinal study. SETTING: University hospital. PATIENT(S): Two hundred seventy-six consecutive women undergoing IVF/ICSI. INTERVENTION(S): Ovarian stimulation, oocyte retrieval, IVF, ICSI, embryo transfer, AMH, and inhibin B determinations in serum and follicular fluid (FF). MAIN OUTCOME MEASURE(S): The AMH and inhibin B concentrations in 276 matched FF/serum pairs have been determined. Different outcome groups have been compared and set in relation to the oocyte count, morphological parameters, and steroid hormone levels. RESULT(S): The concentrations of AMH and inhibin B in both serum and FF were significantly higher in the group of women who became pregnant in the corresponding treatment cycle than in those who did not conceive. Positive correlations were observed between serum inhibin B concentrations and embryo morphology (r = 0.126, 95% confidence interval 0.026-0.284). Serum and FF AMH or inhibin B correlated positively with the oocyte count and negatively with the pretreatment cycle day 3 FSH level and the total administered gonadotropin dose. CONCLUSION(S): The AMH and inhibin B levels on the day of oocyte retrieval are correlated to reproductive outcome.

Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.

STUDY QUESTION Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

No scope for social modulation of steroid levels in a year-round territorial damselfish.

Both latitude and mating system have been proposed to shape relationships between steroid hormone levels and social behavior. Recently it has been postulated that species with long lasting non-seasonal territorial behavior have low androgen responsiveness. Tropical damselfishes are an ideal family to test this proposition because they show a large variety in mating systems. Here we contribute to the comparative dataset by measuring the response in steroid levels after social modulation in the banded sergeant, Abudefduf septemfasciatus, a species with non-seasonal territoriality. In highly territorial and brooding males, we found low androgen and cortisol levels that did not increase after experimental intraspecific simulated territorial intrusions (STI tests). No relationship was found between the variation in steroid hormone levels and territorial responses to naturally occurring territorial intrusions. Although steroid levels were low, male A. septemfasciatus were highly territorial both to STI challenges and to fishes that passed the territory. They often chased intruders for several meters away from the territory. This indicates that during nest defence in a non-seasonal territorial damselfish species, territorial behaviors are shown independent of variation in androgen and cortisol levels.

FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

Monogenic forms of hypertension

Arterial hypertension in childhood is less frequent as compared to adulthood but is more likely to be secondary to an underlying disorder. After ruling out more obvious causes, some patients still present with strongly suspected secondary hypertension of yet unknown etiology. A number of these children have hypertension due to single gene mutations inherited in an autosomal dominant or recessive fashion. The finding of abnormal potassium levels (low or high) in the presence of suppressed renin secretion, and metabolic alkalosis or acidosis should prompt consideration of these familial diseases. However, mild hypertension and the absence of electrolyte abnormalities do not exclude hereditary conditions. In monogenic hypertensive disorders, three distinct mechanisms leading to the common final pathway of increased sodium reabsorption, volume expansion, and low plasma renin activity are documented. The first mechanism relates to gain-of-function mutations with a subsequent hyperactivity of renal sodium and chloride reabsorption leading to plasma volume expansion (e.g., Liddle's syndrome, Gordon's syndrome). The second mechanism involves deficiencies of enzymes that regulate adrenal steroid hormone synthesis and deactivation (e.g., subtypes of congenital adrenal hyperplasia, apparent mineralocorticoid excess (AME)). The third mechanism is characterized by excessive aldosterone synthesis that escapes normal regulatory mechanisms and leading to volume-dependent hypertension in the presence of suppressed renin release (glucocorticoid remediable aldosteronism). Hormonal studies coupled with genetic testing can help in the early diagnosis of these disorders.

The discovery of new 11beta-hydroxysteroid dehydrogenase type 1 inhibitors by common feature pharmacophore modeling and virtual screening

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the conversion of biologically inactive 11-ketosteroids into their active 11beta-hydroxy derivatives and vice versa. Inhibition of 11beta-HSD1 has considerable therapeutic potential for glucocorticoid-associated diseases including obesity, diabetes, wound healing, and muscle atrophy. Because inhibition of related enzymes such as 11beta-HSD2 and 17beta-HSDs causes sodium retention and hypertension or interferes with sex steroid hormone metabolism, respectively, highly selective 11beta-HSD1 inhibitors are required for successful therapy. Here, we employed the software package Catalyst to develop ligand-based multifeature pharmacophore models for 11beta-HSD1 inhibitors. Virtual screening experiments and subsequent in vitro evaluation of promising hits revealed several selective inhibitors. Efficient inhibition of recombinant human 11beta-HSD1 in intact transfected cells as well as endogenous enzyme in mouse 3T3-L1 adipocytes and C2C12 myotubes was demonstrated for compound 27, which was able to block subsequent cortisol-dependent activation of glucocorticoid receptors with only minor direct effects on the receptor itself. Our results suggest that inhibitor-based pharmacophore models for 11beta-HSD1 in combination with suitable cell-based activity assays, including such for related enzymes, can be used for the identification of selective and potent inhibitors.

Age-dependent decrease in 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in hypertensive patients

BACKGROUND: The prevalence of arterial hypertension lacking a defined underlying cause increases with age. Age-related arterial hypertension is insufficiently understood, yet known characteristics suggest an aldosterone-independent activation of the mineralocorticoid receptor. Therefore, we hypothesized that 11beta-HSD2 activity is age-dependently impaired, resulting in a compromised intracellular inactivation of cortisol (F) with F-mediated mineralocorticoid hypertension. METHODS: Steroid hormone metabolites in 24-h urine samples of 165 consecutive hypertensive patients were analyzed for F and cortisone (E), and their TH-metabolites tetrahydro-F (THF), 5alphaTHF, TH-deoxycortisol (THS), and THE by gas chromatography-mass spectroscopy. Apparent 11beta-HSD2 and 11beta-hydroxylase activity and excretion of F metabolites were assessed. RESULTS: In 72 female and 93 male patients aged 18-84 years, age correlated positively with the ratios of (THF + 5alphaTHF)/THE (P = 0.065) and F/E (P < 0.002) suggesting an age-dependent reduction in the apparent 11beta-HSD2 activity, which persisted (F/E; P = 0.020) after excluding impaired renal function. Excretion of F metabolites remained age-independent most likely as a consequence of an age-dependent diminished apparent 11beta-hydroxylase activity (P = 0.038). CONCLUSION: Reduced 11beta-HSD2 activity emerges as a previously unrecognized risk factor contributing to the rising prevalence of arterial hypertension in elderly. This opens new perspectives for targeted treatment of age-related hypertension.

Prematurity is related to high placental cortisol in preeclampsia

Fetal growth is compromised in animal models with high cortisol availability. In healthy pregnancies, the fetus is protected from high circulating cortisol levels by the placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which is reduced in preeclampsia. We hypothesized increased placental cortisol availability in preeclampsia as missing link to fetal growth restriction and prematurity. Placental tissue was obtained from 39 pregnant women dichotomized normotensive (n = 16) or preeclamptic (n = 23). Placental steroid hormone metabolites were analyzed by gas chromatography-mass spectrometry. Apparent 11beta-HSD2 enzyme activity was calculated as substrate to product ratio. Estradiol and pregnandiol positively correlated with gestational age. Cortisol was virtually absent in 93.8% of controls, yet detectable in 79.3% of preeclamptic samples resulting in an odds ratio (OR) of 0.019 (95% CI 0.002-0.185) for the presence of placental cortisol. Apparent 11beta-HSD2 activity directly correlated with birth weight (R2 = 0.16; p < 0.02) and gestational age (R2 = 0.11; p < 0.04) ensuing a reduced risk of premature delivery (OR 0.12; 95% CI 0.02-0.58). We conclude that normotensive pregnancies are characterized by an almost completely inactivated placental cortisol. In line with our hypothesis, reduced 11beta-HSD2 activity in preeclampsia is unable to abolish placental cortisol, a finding clearly associated with prematurity and low birth weight.