Health information needs of 1000 midlife Singaporean women.

OBJECTIVES: Due to demographic changes, aging is a health priority. We aimed to identify midlife women's perceived health information needs and the preferred method(s) of information delivery. METHODS: A questionnaire was offered to women, aged 45-69 years, attending gynecological clinics during April/May 2016, collecting age and ethnicity data. Participants were asked to indicate important midlife health topics out of 26 topics, including 'other'. For each topic, six delivery options were offered. Age was stratified by 5-year intervals. Associations with age and ethnicity were examined using Pearson's chi-square tests (p < 0.05); analyses were performed with SPSS version 22.0. RESULTS: The top health topics chosen were gynecological cancer (66.0%), joint/muscle aches and pain (64.4%), bone health (63.2%), breast screening (55.9%), and heart health (55.3%). Adjusted results from the logistic regression model found that the odds of choosing the topics gynecological cancer, cervical screening, and complementary and alternative medicine for menopausal symptoms were significantly lower in age groups 55-59, 60-64 and 65-69 years compared to age group 45-49 years. Both Malay and Indian women were less likely to report bone health as important (odds ratio = 0.59, 95% confidence interval = 0.41-0.86) and (odds ratio = 0.64, 95% confidence interval = 0.42-0.98), respectively. Written leaflets were chosen by the majority (84.7%). CONCLUSION: This study of over 1000 midlife Asian women found that holistic health information is desired and requires tailoring by age, not ethnicity. Written information was preferred over support groups. These findings will guide clinical health services in delivering patient-centered information resources for midlife women.

Fecundity among women with polycystic ovary syndrome (PCOS)-a population-based study.

STUDY QUESTION: Does the long-term fecundity of women with polycystic ovary syndrome (PCOS) differ from those without PCOS? SUMMARY ANSWER: Cumulative probability of childbirth is similar between women with and without PCOS. WHAT IS KNOWN ALREADY: PCOS is the main cause of anovulatory infertility in women after menarche. Previous studies indirectly suggest that fecundity in women with PCOS over the longer term may not be lower than in women without PCOS. STUDY DESIGN, SIZE, DURATION: This is a population-based study using four linked Swedish national registries. A total of 45 395 women with PCOS and 217 049 non-PCOS women were included. Follow-up began at the age of 18 years and continued for a maximum of 26 years, from 1989 to the end of 2015. Childbirth was the main outcome, as identified from the Medical Birth Register. PARTICIPANTS/MATERIALS, SETTING, METHODS: All women born between 1971 and 1997 who were identified with a PCOS diagnosis in the Swedish Patient Registry between 1 January 2001 and 31 December 2016 were included in the study population. Five controls per women with PCOS were randomly drawn from the Total Population Registry. The control women were born in the same year and living in the same municipality as the patient. The fecundity ratio (FR) was calculated by clustered Cox regression using a robust variance, adjusted for maternal birth period, country of birth and level of education. MAIN RESULTS AND THE ROLE OF CHANCE: The cumulative probability of childbirth was 80.2% (95% CI, 79.5-80.9%) in women with PCOS and 78.2% (95% CI, 77.9-78.5%) in those without PCOS. Adjusted FR was 0.81 (95% CI, 0.80-0.82) for first childbirth and 0.58 (95% CI, 0.57-0.60) for first childbirth following a spontaneous pregnancy. The FR for second childbirth was 0.79 (95% CI, 0.77-0.80). Women with PCOS had more than one child less frequently than the comparison group. Within the PCOS group, early age at diagnosis, later birth year, Nordic country of origin and low educational level positively influenced the FR. LIMITATIONS, REASONS FOR CAUTION: Results are not adjusted for BMI, and time from intention to conceive to first childbirth could not be captured. Data on pregnancies, miscarriages or abortions and fertility treatment are unknown for women who did not give birth during the study period. Women with PCOS who did not seek medical assistance might have been incorrectly classified as not having the disease. Such misclassification would lead to an underestimation of the true association between PCOS and outcomes. WIDER IMPLICATIONS OF THE FINDINGS: While cumulative probability of childbirth is similar between groups, women with PCOS need longer time to achieve their first childbirth. Women with PCOS have a lower FR and give birth to fewer children per woman than women without PCOS. Early diagnosis of and information about PCOS may improve affected women's reproductive potential. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Swedish Society of Medicine. Inger Sundström Poromaa has, over the past 3 years, received compensation as a consultant and lecturer for Bayer Schering Pharma, MSD, Gedeon Richter, Peptonics and Lundbeck A/S. The other authors declare no competing interests.

STAT-3 regulation of CXCR4 is necessary for the prenylflavonoid Icaritin to enhance mesenchymal stem cell proliferation, migration and osteogenic differentiation.

Mesenchymal stem cell (MSC) dysfunction has been implicated in the pathogenesis of osteoporosis. MSCs derived from osteoporotic subjects demonstrate significant impairment in proliferation, adhesion and chemotaxis, and osteogenic differentiation, leading to reduced functional bone-forming osteoblasts and ultimately nett bone loss and osteoporosis. Epimedium herbs and its active compound Icaritin (ICT) have been used in Chinese ethnopharmacology for the treatment of metabolic bone diseases. Using an in-vitro cell culture model, we investigated the benefits of ICT treatment in enhancing MSC proliferation, migration and osteogenic differentiation, and provide novel data to describe its mechanism of action. ICT enhances MSC proliferation, chemotaxis to stromal cell-derived factor-1 (SDF-1) and osteogenic differentiation through the activation of signal transduction activator transcription factor 3 (STAT-3), with a consequential up-regulation in the expression and activity of cysteine (C)-X-C motif chemokine receptor 4 (CXCR4). These findings provide a strong basis for future clinical studies to confirm the therapeutic potential of ICT for the prevention and treatment of osteoporosis and fragility fractures.

The prenylflavonoid Icaritin enhances osteoblast proliferation and function by signal transducer and activator of transcription factor 3 (STAT-3) regulation of C-X-C chemokine receptor type 4 (CXCR4) expression.

In this study, we examined the effects of a natural prenylflavonoid Icaritin (ICT), on human osteoblast proliferation and osteogenic function. We observed that ICT dose-dependently enhanced osteoblast proliferation by ~15% over a 7day period. This increase in cell proliferation was associated with corresponding increases in osteoblast functions as measured by ALP secretion, intracellular calcium ions influx and calcium deposition. These anabolic effects were associated with a 4-fold increase in CXCR4 mRNA and protein expression. Silencing of CXCR4 protein expression using small interfering RNA reversed ICT-induced increase in cell proliferation, ALP activity and calcium deposition. Interestingly, we observed that ICT dose-dependently increased STAT-3 phosphorylation; and this resulted in increased binding of phosphorylated STAT-3 to the promoter region of the CXCR4 gene, to increase CXCR4 protein expression. Furthermore, we found that inhibition of STAT-3 phosphorylation resulted in a decrease in CXCR4 protein expression; whilst increasing phosphorylation of STAT-3 using a constitutive active STAT-3 vector significantly increased CXCR4 levels. Moreover, the chemical inhibition of STAT-3 phosphorylation annulled our previously observed ICT-induced increases of osteoblast proliferation and function. Finally, in a rat model of estrogen-deficient osteoporosis, ICT restored both osteoblasts numbers and CXCR4 expression. Taken together, both cellular and animal models support the novel findings that ICT; through the phosphorylation of STAT-3, up-regulated CXCR4, to increase osteoblast proliferation and function.

Chronic joint pain and handgrip strength correlates with osteoporosis in mid-life women: a Singaporean cohort.

This study aimed to identify novel correlates which may relate to low bone mass at lumbar spine in mid-life Asian women. The possibility of developing a prediction model for osteoporosis (OP) was explored which resulted in a risk assessment tool that performed better than currently available tools. INTRODUCTION: In order to identify novel correlates associated with low spinal bone mineral density (BMD) in mid-life women, we examined a large number of lifestyle and medical and performance measurements and developed a prediction model for triage to BMD scanning. METHODS: Women (n = 512) aged 45-69 years (mean 57.0 ± 6.3) attending gynecology clinics for "well woman" visits were recruited for this cross-sectional study from 2014 to 2015. We assessed symptoms, medical history, anthropometry, and physical performance. Stepwise multinomial logistic regressions were performed to examine significant associated covariates for pre-specified outcomes (normal [T-score ≥ -1.0], low bone mass [T-score between -1 and -2.5], and OP [T-score ≤ -2.5] at the lumbar spine). A new screening model was developed, and its performance was compared with the OP Screening Tool for Asians (OSTA) and Fracture Risk Assessment Tool (FRAX®). RESULTS: Spinal OP was found in 6.8%. Multivariate analysis indicated that chronic joint pain, the most common symptom reported by 37.5% of the women, was significantly associated with OP. Only age (Relative Risk Ratio [RRR] 1.63; 95%CI, 1.03-2.60), weight (RRR 0.14; 95% CI, 0.07-0.27), postmenopausal status (RRR 11.59, 95%CI, 1.15-116.73), chronic joint pain (RRR, 4.12; 95% CI, 1.53-11.07), and right handgrip strength (RRR 0.50; 95% CI, 0.31-0.80) were independently associated with spinal OP. Combining these five variables, our final model's area under curve (AUC) was significantly higher at 84% than both the OSTA [AUC; 79% (p value < 0.0231 'c' statistics)] and FRAX® [AUC 58% (p value < 0.0001 'c' statistic)]. CONCLUSION: A novel screening tool that combines age, weight, and menopausal status with chronic joint pain and right handgrip strength more reliably predicts spinal OP in mid-life Singaporean women.

Phytoplankton blooms: an overlooked marine source of natural endocrine disrupting chemicals.

BACKGROUND: We had previously reported high androgenic and estrogenic activities in seawaters in confined clusters close to Singapore. Further investigations revealed a hitherto unsuspected link between estrogenic/androgenic activity and net phytoplankton count. OBJECTIVE: The primary objective of this study was to investigate the cause of a correlation between net phytoplankton and endocrine activity, and corroborate this observation, and rule out other possible confounding factors. Our secondary objective was to study if these estrogenic secretions can impact human health. METHODS: Five species of phytoplankton, Gymnodinium catenatum, Prorocentrum minimum, Alexandrium leei, Chattonella marina, and Fibrocapsa japonica, were isolated from Singapore waters and mass cultured and the cells and culture media screened for estrogenic and androgenic activity using human cell-based bioassays. RESULTS: The raphidophytes C. marina and F. japonica displayed significant estrogenic activity whilst the dinoflagellates G. catenatum and P. minimum displayed significant androgenic activity in both the cell extracts and the cell culture media extract. CONCLUSIONS: Our data shows that selected phytoplankton isolates are potent secretors of estrogenic and androgenic substances, which are potential endocrine disrupting chemicals (EDCs). As the harmful nature of EDCs is largely due to their bioaccumulation in the aquatic food chain our findings imply that the impact of these phytoplankton secretions needs to be investigated especially for seafoods, which are only a single trophic level away from phytoplankton. Alternatively, should these phytoplankton-origin EDCs not accumulate through marine food chains to significantly impact humans or marine mammals, our results indicate that functional assays could greatly over-estimate the risk from naturally occurring EDCs produced by marine phytoplankton. It remains to be determined if these EDCs affect zooplankton and other organisms that directly feed on marine phytoplankton, or if the secreted EDCs can directly impact other marine fauna.

Serum free estradiol and estrogen receptor-α mediated activity are related to decreased incident hip fractures in older women.

There is paucity of data from Asian women on the association between serum estrogens and osteoporotic hip fracture risk. We conducted a case-control study nested within a population-based prospective cohort, The Singapore Chinese Health Study, to evaluate serum estrogens levels, ERα-mediated estrogenic activity and hip fracture risk in postmenopausal Asian women. Among 35,298 women who were recruited between 1993 and 1998, 15,410 women donated blood for research between 1999 and 2004. From this subcohort, we identified 140 cases who subsequently suffered hip fracture after blood donation, and 278 age-matched controls. Serum levels of total estrone, estradiol and sex hormone binding globulin levels were measured in a blinded fashion among cases and controls. ERα-mediated estrogenic activity of serum samples was quantified using a sensitive ERα-driven cell bioassay. Women with hip fracture had lower serum estrogens than control women. Compared to the lowest quintile, women in the highest quintile of free estradiol exhibited a statistically significant 57% reduction in risk of hip fracture (95% confidence interval (CI), 6-80%), with a dose-dependent relationship (p for trend=0.021). High levels of ERα-mediated estrogenic activity were also associated with decreased risk of hip fracture (p for trend=0.048). Overall, women with relatively high levels of both free estradiol and ERα-mediated estrogenic activity had a 55% reduction in hip fracture risk (95% CI, 17-76%) compared to women with low levels of both. High levels of free estradiol and ERα-mediated estrogen activity in sera were associated with reduced hip fracture risk in Chinese postmenopausal women.

Pharmacokinetics of prenylflavonoids and correlations with the dynamics of estrogen action in sera following ingestion of a standardized Epimedium extract.

To explore pharmacokinetic properties of prenylflavonoids from the Traditional Chinese Medicinal plant Epimedium, three doses of a standardized extract (100, 300 and 600 mg/kg body weight), were administered to ovariectomized rats and serial blood samples were obtained. Serum concentrations of the Epimedium prenylflavonoids icariin, icariside I, icariside II, icaritin and desmethylicaritin were determined by LC-MS/MS. Aliquots of sera were also applied to human cell lines that permanently express ERalpha and ERbeta proteins for the ex vivo measurement of estrogenic activity. All five prenylflavonoids exhibit non-linear dose-dependent increases in the area under concentration versus time curves. Two distinct pharmacokinetic patterns were evident, an early phase wherein icariin and icariside II reached t(max) 0.5-1 h, and a late phase wherein icariside I, icaritin and desmethylicaritin peaked at t(max) 8h. Total concentrations of icaritin and desmethylicaritin reached C(max) approximately 2 microM and approximately 0.25 microM respectively. Estrogenic activity in Epimedium-treated rat sera lagged by several hours compared to animals treated with control drug estradiol benzoate, corresponding to the appearance of bioactive metabolites desmethylicaritin, icaritin and icariside I. Following glucuronidase/sulphatase treatment, prolonged estrogenic activity at higher Epimedium doses (300 and 600 mg/kg of body weight) was evident, and correlated with the persistence of micromolar levels of icaritin at the 48-72 h sampling period. The depot effect resulted in time-concentration bioactivity profiles at the three Epimedium doses (area under curve 374, 543, and 771pME2 h(-1)) that exceeded that observed for estradiol benzoate (148pME2 h(-1)). Our study correlated the pharmacokinetics of prenylflavonoids with the dynamics of their estrogenic effects and reveals the potential estrogenicity of this Epimedium extract. This study may aid the development of prenylflavonoids as drugs for menopause and other conditions requiring estrogenic action.

Bioassays for estrogenic activity: development and validation of estrogen receptor (ERalpha/ERbeta) and breast cancer proliferation bioassays to measure serum estrogenic activity in clinical studies.

Standard estrogenic prodrugs such as estradiol valerate (E2V) and increasingly popular phytoestrogen formulations are commonly prescribed to improve menopausal health. These drugs are metabolized to numerous bioactive compounds, known or unknown, which may exert combinatorial estrogenic effects in vivo. The aim of this study is to develop and validate estrogen receptor (ER) alpha/ERbeta reporter gene and MCF-7 breast cancer cell proliferation bioassays to quantify serum estrogenic activities in a clinical trial setting. We measured changes in serum estrogenicity following ingestion of E2V and compared this to mass spectrometric measurements of its bioactive metabolites, estrone and 17beta-stradiol. ERalpha bioactivity of the 192 serum samples correlated well (R = 79%) with 17beta-estradiol levels, and adding estrone improved R to 0.83 (likelihood ratio test, P < 0.0001), suggesting that the ERalpha assay reflects summated activity of compounds in serum. ERbeta correlated moderately (R = 0.52) with estrone and 17beta-estradiol, with an estrone/17beta-estradiol coefficient ratio that was twice that of ERalpha, indicating estrone was more active on a molar basis in the ERbeta assay. Unlike the ERalpha and ERbeta bioassays, MCF-7 cell proliferation was driven by 17beta-estradiol, and addition of estrone did not increase the predictive value of the model, suggesting that the driver or drivers for breast cancer cell proliferation were not the same as for ERalpha and ERbeta transactivation. In contrast, a decoction of the traditional Chinese medicinal herb Epimedium pubescens did not induce significant changes in estrogenic bioactivity over baseline. These data indicate that ERalpha/ERbeta reporter gene and MCF-7 breast cancer cell proliferation bioassays reflect different aspects of estrogenic activity and that these assays suggest that the Epimedium formulation tested is unlikely to exert significant estrogenic effects in humans.

Determination of breviflavone A and B in Epimedium herbs with liquid chromatography-tandem mass spectrometry.

Two new types of minor flavonoids, breviflavone A and B, have been recently isolated and identified from Epimedium brevicornu in our previous research. Breviflavone B is a novel flavonoid with potent and specific estrogen receptor (ER) bioactivity. Its positional isomer, breviflavone A, is not ER active. Therefore, it is important to determine the two minor components, breviflavone A and B, in Epimedium herbs. In this report, a robust method for measurement of the two breviflavones in Epimedium ethanolic extracts has been developed by using liquid chromatography tandem mass spectrometry via selected-reaction monitoring (m/z 437-->m/z 367 for breviflavone A and m/z 437-->m/z 351 for breviflavone B) under negative electrospray ionization mode. This method has been successfully used to determine the two breviflavones in ethanolic herbal extracts of five major Epimedium species (E. brevicornu, E. koreanum, E. pubescens, E. sagittatum, and E. wushanese) from various sources. The contents of the two breviflavones range from 0.0181 to 0.1791% for breviflavone A and 0.0026 to 0.0252% for breviflavone B in the dried ethanolic extracts of those Epimedium herbal samples.

A natural polymorphism in peroxisome proliferator-activated receptor-alpha hinge region attenuates transcription due to defective release of nuclear receptor corepressor from chromatin.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a central regulator of lipid metabolism. Fibrate drugs act on PPARalpha to modulate dyslipidemias. A natural variant (V227A) affecting the PPARalpha hinge region was associated with perturbations in blood lipid levels in Asian populations. In this study, we investigated the functional significance of the V227A substitution. The variant significantly attenuated PPARalpha-mediated transactivation of the cytochrome P450 4A6 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) genes in the presence of fibrate ligands. Screening of a panel of PPARalpha coregulators revealed that V227A enhanced recruitment of the nuclear corepressor NCoR. Transactivation activity of V227A could be restored by silencing NCoR or by inhibition of its histone deacetylase activity. Deletion studies indicated that PPARalpha interacted with NCoR receptor-interacting domain 1 (ID1) but not ID2 or ID3. These interactions were dependent on the intact consensus nonapeptide nuclear receptor interaction motif in NCoR ID1 and were enhanced by the adjacent 24 N-terminal residues. Novel corepressor interaction determinants involving PPARalpha helices 1 and 2 were identified. In hepatic cells, the V227A substitution stabilized PPARalpha/NCoR interactions and caused defective release of NCoR in the presence of agonists on the HMGCS2 promoter. These results provide the first indication that defective function of a natural PPARalpha variant was due, at least partially, to increased corepressor binding. Our data suggest that the PPARalpha/NCoR interaction is physiologically relevant and can produce a discernable phenotype when the magnitude of the interaction is altered by a naturally occurring variation.

Sensitive and rapid method to quantify icaritin and desmethylicaritin in human serum using gas chromatography-mass spectrometry.

The prenyl-flavones, icaritin and desmethylicaritin, are bioactive compounds from the traditional Chinese medicinal herb, Epimedium, extracts of which can enhance bone health in animal models. In order to examine their bioavailability in humans, we have developed and validated a sensitive method to quantify icaritin and desmethylicaritin in human sera, using gas chromatography-mass spectrometry. The serum samples were extracted with ethyl acetate and then derivatized with BSTFA in pyridine (4:1). With genistein as internal standard, calibration curves with good linearity (R(2)>0.99) within the concentration range of 0.15-10nM in the selective ion monitoring mode were obtained. The limits of detection and quantization were 11 and 33 pM for icaritin, and 23 and 70 pM for desmethylicaritin, respectively; inter- and intra-assay variabilities were <15%, and accuracies were between 89 and 110%. Icaritin, but not desmethylicaritin, was detected from 1h, increasing to a peak at 8h (1.51+/-1.6 nM) in sera of human volunteers after ingestion of an aqueous decoction of Epimedium. This sensitive method can be used to quantify serum levels of icaritin and desmethylicaritin for pharmacokinetic studies.

Molecular and pharmacodynamic properties of estrogenic extracts from the traditional Chinese medicinal herb, Epimedium.

The Chinese medicinal herb, Epimedium, used traditionally for bone health exerts estrogenic activity (EA) in vitro. A genetically characterized Epimedium brevicornum (EB) extract induced biphasic responses in the mRNA and protein expression of the estrogen-regulated progesterone receptor gene in breast cancer (MCF-7) cells. These changes were mirrored changes in estrogenic receptor (ERalpha) content. In male Sprague-Dawley rats, administration of the estrogenic prodrug, estradiol valerate increased area-under-curve of serum effects for ERalpha (AUC difference: 18,900EA(ERalpha) min; 95% CI: 0-37,800; p = 0.05) and breast cancer cell (MCF-7) growth (AUC difference: 30,200EA(MCF-7) min; 95% CI: 24,200-36,200; p<0.001), compared to placebo. Oral administration of Epimedium brevicornum increased ERalpha activity (1320EA(ERalpha) min, p<0.01). Our data indicate that estrogen-responsive bioassays can measure the pharmacokinetic/pharmacodynamics of estrogenic activity in serum. Epimedium brevicornum extract increases estrogenic activity in serum and human studies are required to evaluate whether Epimedium extracts have utility for estrogen replacement therapy.

Taxonomic, genetic, chemical and estrogenic characteristics of Epimedium species.

To understand the factors contributing to estrogenic properties of extracts from the genus Epimedium L. (Berberidaceae), we performed taxonomic, genetic and chemical characterization on 37 specimens from 18 species and related these to estrogen receptor (ERalpha and ERbeta) bioactivity, as measured by reporter genes in stable human cells. Boot strap values derived from amplified fragment length polymorphisms indicated that specimens of E. koreanum, E. brevicornum, E. myrianthum, E. leishanense, and E. membranaceum were genetically distinct and this was supported by their very similar ERalpha activities. In contrast, specimens from E. pubescens and E. sagittatum were diverse both genetically, chemically and in terms of ERalpha and ERbeta bioactivities. Strikingly, a genetic cluster comprising six rare Epimedium species exhibited strongest ERalpha and ERbeta activity, and this bioactivity was positively correlated with content of trace flavonoid aglycones (kaempferol, apigenin, quercetin, luteolin and breviflavone B). In contrast, there was no association between estrogenic activity and the major flavonol glycoside constituents (icariin and epimedin A-C). Although they exhibited equally strong ERalpha and ERbeta activity, E. koreanum can be clearly differentiated from E. pubescens and E. brevicornum by genetic distance and its significantly lower content of epimedin C. Our morphologic, genetic, chemical and bioactivity profiling provide the basis for the production of extracts with reproducible estrogenic properties. Such reproducibility will be critical for the standardization of Epimedium-based products.

Differential effects of isoflavones, from Astragalus membranaceus and Pueraria thomsonii, on the activation of PPARalpha, PPARgamma, and adipocyte differentiation in vitro.

Compounds that target the peroxisome proliferator-activated receptors PPARalpha and PPARgamma are used to correct dyslipidemia and to restore glycemic balance, respectively. Because the majority of diabetic patients suffer from atherogenic lipid abnormalities, in addition to insulin resistance, ligands are required that can activate both PPARalpha and PPARgamma. In this study, we used chimeric PPARalpha/gamma reporter-gene bioassays to screen herbal extracts with purported antidiabetic properties. Extracts of Astragalus membranaceus and Pueraria thomsonii significantly activated PPARalpha and PPARgamma. Bioassay-guided fractionation resulted in the isolation of the isoflavones, formononetin, and calycosin from Astragalus membranaceus, and daidzein from Pueraria thomsonii as the PPAR-activating compounds. We investigated the effects of these and 2 common isoflavones, genistein and biochanin A, using chimeric and full-length PPAR constructs in vitro. Biochanin A and formononectin were potent activators of both PPAR receptors (EC50 = 1-4 micromol/L) with PPARalpha/PPARgamma activity ratios of 1:3 in the chimeric and almost 1:1 in the full-length assay, comparable to those observed for synthetic dual PPAR-activating compounds under pharmaceutical development. There was a subtle hierarchy of PPARalpha/gamma activities, indicating that biochanin A, formononetin, and genistein were more potent than calycosin and daidzein in chimeric as well as full-length receptor assays. At low doses, only biochanin A and formononetin, but not genistein, calycosin, or daidzein, activated PPARgamma-driven reporter-gene activity and induced differentiation of 3T3-L1 preadipocytes. Our data suggest the potential value of isoflavones, especially biochanin A and their parent botanicals, as antidiabetic agents and for use in regulating lipid metabolism.

Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone.

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.

Clustering of sex hormone disruptors in Singapore's marine environment.

Abnormal sexual differentiation and other reproductive abnormalities in marine animals indicate the presence in seawater of endocrine-disrupting compounds (EDCs) that perturb the function of the sex hormone signaling pathways. However, most studies to date have reported on EDC effects in freshwater and sewage samples, and there is a paucity of bioassay data on the effects of EDCs in marine waters. Our aims in this study were to devise robust methodologies suitable for extracting potential EDCs and to measure their summated effects on activities of androgen receptors (ARs) and estrogen receptors (ER-alpha and ER-beta) in marine samples from Singapore's coastal waters. In this study, we examined the ability of C18, hydrophilic and lipophilic balance, and diol cartridges to extract potential EDCs from seawater samples. Extracts from C18 cartridges exhibited the highest sex hormone bioactivities in reporter gene assays based on a human cell line expressing AR, ER-alpha, and ER-beta. Examination of extracts from 20 coastal locations showed high androgenic and estrogenic agonist activities in confined clusters closest to the main island of Singapore. Sex hormone activity declined rapidly in clusters farther from the main coastline and in more open waters. Unexpectedly, surface and mid-depth samples from the confined high-activity clusters, in the presence of hormone, exhibited AR and ER-alpha activities that were 200-900% higher than those observed for the cognate hormone alone. This enhanced sex hormone activity suggests that analyses of complex seawater mixtures may uncover unusual bioactivities that may not be obvious by studying individual compounds. Our data present a "snapshot" of the sex hormone disruptor activity in Singapore's marine environment and indicate that C18 extraction for EDCs used in conjunction with reporter gene bioassays represents a robust and sensitive methodology for measuring summated androgenic and estrogenic activities in seawater.

Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions.

The androgen receptor (AR), a nuclear transcription factor, mediates male sexual differentiation, and its excessive action is associated with prostate cancer. We have characterized a negative regulatory domain in the AR hinge region, which interacted with filamin A (FLNa), an actin-binding cytoskeletal protein. FLNa interfered with AR interdomain interactions and competed with the coactivator transcriptional intermediary factor 2 to specifically down-regulate AR function. Although full-length FLNa was predominantly cytoplasmic, a C-terminal 100-kDa fragment of FLNa colocalized with AR to the nucleus. This naturally occurring FLNa fragment repressed AR transactivation and disrupted AR interdomain interactions and transcriptional intermediary factor 2-activated AR function in a manner reminiscent of full-length FLNa, raising the possibility that the inhibitory effects of cytoplasmic FLNa may be transduced through this fragment, which can localize to the nucleus and form part of the pre-initiation complex. This unanticipated role of FLNa adds to the growing evidence for the involvement of cytoskeletal proteins in transcription regulation.

Prostate-specific antigen, testosterone, sex-hormone binding globulin and androgen receptor CAG repeat polymorphisms in subfertile and normal men.

The aim of this study was to understand the androgen-related factors which may regulate concentrations of the tumour marker, prostate-specific antigen (PSA). We therefore measured the serum concentrations of total and free testosterone and of sex hormone-binding globulin (SHBG) and determined the androgen receptor (AR) gene CAG repeat length, then compared these values to total and free PSA concentrations in 91 subjects with proven fertility, and 112 subfertile men with defective spermatogenesis. Concentrations of free testosterone and total testosterone, adjusted for SHBG, were 17-20% lower in subfertile men compared with those in their fertile counterparts. This subtle, but highly significant (P < 0.001), difference in testosterone between fertile and subfertile men was accentuated by the positive correlation between testosterone and AR gene CAG repeat length in fertile, but not subfertile, subjects. In subfertile subjects, testosterone strongly correlated (r = 0.354, P < 0.001) with PSA concentrations, and independent of testosterone, total PSA negatively correlated (r = -0.229, P = 0.011) with AR CAG length. Overall our data suggest that, firstly, PSA correlates with testosterone only in an environment of relatively low androgenicity, such as in subfertile men. Secondly, in such a low androgenic environment, short CAG tracts (associated with high AR activity) correlate positively with PSA concentrations. These results suggest that interpretation of PSA is best made in conjunction with testosterone concentrations and AR CAG length.

Trinucleotide (CAG) repeat polymorphisms in the androgen receptor gene: molecular markers of risk for male infertility.

OBJECTIVE: To determine whether changes in the polymorphic trinucleotide (CAG) tract of the androgen receptor gene are associated with spermatogenic defects in patients with male infertility. DESIGN: Case-control study of two ethnic groups. SETTING: University referral centers for male infertility at Baylor College of Medicine, Houston, Texas, and National University Hospital, Singapore. PARTICIPANT(S): Two hundred and fifteen patients with male infertility and depressed spermatogenesis and 142 fertile controls. MAIN OUTCOME MEASURE(S): Size of androgen receptor CAG alleles according to fluorescent-labeled polymerase chain reaction and automated analysis using Genescan software (PE Biosystems Asia, Singapore), and statistical examination of its relation to clinical variables. RESULT(S): In U.S. patients, the mean androgen receptor CAG length was significantly longer in infertile patients than in fertile controls (21.95 +/- 0.31 vs. 20.72 +/- 0.52). Logistic regression showed that each unit increase in CAG length was associated with a 20% increase in the odds of being azoospermic. The odds ratio for azoospermia was sevenfold higher for patients with > or =26 CAG repeats than in those with <26 CAG repeats. Although mean CAG length in Singapore patients was longer than in the U.S. samples, long androgen receptor CAG alleles were significantly related to male infertility in both populations. CONCLUSION(S): Long (> or =26) androgen receptor CAG alleles, which are found in up to 25% of azoospermic men, are associated with male infertility and defective spermatogenesis. Conception in these men is possible with assisted reproductive technologies, as many have spermatozoa in their testes.