The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.

STUDY QUESTION: What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries? SUMMARY ANSWER: Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS. WHAT IS KNOWN ALREADY: In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women. STUDY DESIGN, SIZE, DURATION: Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor ß (ERß) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERß. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability. PARTICIPANTS/MATERIALS, SETTING, METHODS: AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERß was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis. MAIN RESULTS AND THE ROLE OF CHANCE: Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7 M (P < 0.05; P < 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P = 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERß produced a corresponding increase in AMH mRNA expression (P = 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P < 0.05, one-way ANOVA) and FSHR (P < 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n = 7, PCOM n = 5, PCOS n = 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P < 0.05, normal n = 6, PCOS n = 3). AMH at 20 and 50 ng/ml decreased KGN-GC cell proliferation but not viability after 8 days of treatment (P < 0.005, two-way ANOVA). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT. WIDER IMPLICATIONS OF THE FINDINGS: Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose. TRIAL REGISTRATION NUMBER: N/A.

Metformin inhibits follicle-stimulating hormone (FSH) action in human granulosa cells: relevance to polycystic ovary syndrome.

BACKGROUND: Women with anovulatory polycystic ovary syndrome (PCOS) are generally insulin-resistant and as a consequence are often treated with the biguanide metformin. Results with metformin have, however, been variable with some studies demonstrating induction of regular cycles and an increase in ovulation, whereas others do not. Hence more understanding is needed regarding the mechanism of metformin's actions in ovarian granulosa cells especially in light of previous demonstrations of direct actions. OBJECTIVE: The aim of this study was to investigate metformin's interaction with the FSH/cAMP/protein kinase A pathway, which is the primary signaling pathway controlling CYP19A1 (aromatase) expression in the ovary. METHODS: The effect of metformin on FSH and forskolin-stimulated aromatase expression in human granulosa cells was measured by quantitative real-time PCR. Activity was assessed after transfection with a promoter II-luciferase construct, and by an RIA measuring conversion of androgen to estrogens. The effect on FSH receptor (FSHR) mRNA was assessed by quantitative PCR. Levels of phosphorylated cAMP response element binding protein (CREB) and CREB-regulated transcription coactivator 2 (CRTC2) were measured by Western blotting and cAMP by a bioluminescent assay. RESULTS: Metformin markedly reduced FSH but not forskolin-stimulated aromatase expression and activity. This effect was exerted by inhibition of basal and ligand-induced up-regulation of FSHR expression. Metformin also reduced FSH-induced phosphorylation of CREB and hence CRE activity, which could potentially disrupt the CREB-CREB-binding protein-CRTC2 coactivator complex that binds to CRE in promoter II of the aromatase gene. This is mediated in an AMP-activated protein kinase-independent manner, and does not involve alteration of cAMP levels. CONCLUSION: These finding have implications for the use of metformin in the treatment of anovulation in women with PCOS.

Role of Insulin-like growth factors in initiation of follicle growth in normal and polycystic human ovaries.

CONTEXT: Polycystic ovary syndrome (PCOS), the commonest cause of anovulatory infertility, is characterized by disordered follicle development including increased activation and accelerated growth of preantral follicles. Data from experimental animals and preliminary results from studies of human ovarian tissue suggest that IGFs affect preantral follicle development. OBJECTIVE: Our objectives were to investigate the expression of the type-1 IGF receptor (IGFR-1) in the human ovary and to determine whether IGFs are involved in stimulating the transition of follicles from primordial to primary stage in normal and polycystic ovaries. DESIGN: We used archived ovarian tissue for protein expression studies and small cortical biopsies for follicle isolation and for tissue culture. SETTING: This was a laboratory-based study, using clinical tissue samples. PATIENTS: A total of 54 women, 33 with normal ovaries and 21 with polycystic ovaries, were classified by reference to menstrual cycle history and ultrasonography. MAIN OUTCOME MEASURES: We evaluated expression of IGFR-1 mRNA in isolated preantral follicles and of IGFR-1 protein in archived ovarian tissue samples from normal and polycystic ovaries and effects of exogenous IGF-1 on preantral follicle development and survival in cultured fragments of normal and polycystic ovaries. RESULTS: IGFR-1 mRNA and protein was expressed in preantral follicles at all stages of development and enhanced expression was noted in PCOS follicles during early preantral development. IGF-1 stimulated initiation of follicle growth in normal tissue but had little effect on preantral follicle growth in polycystic ovaries in which, characteristically, there was a higher proportion of follicles that had entered the growing phase even before culture. CONCLUSIONS: IGFs are plausible candidates in regulation of initiation of human follicle growth, and accelerated preantral follicle growth in PCOS may be due to increased activity of endogenous IGFs.

Aromatase expression in abdominal omental/visceral and subcutaneous fat depots: a comparison of pregnant and obese women.

OBJECTIVE: To investigate total and promoter expression of aromatase in subcutaneous and omental (visceral) fat and compare this expression in pregnant and obese women. DESIGN: Cross-sectional study. SETTING: Academic hospital. PATIENT(S): Six women undergoing elective cesarean section and three women undergoing bariatric surgery. INTERVENTION(S): Subcutaneous and omental fat obtained during surgery. MAIN OUTCOME MEASURE(S): Total aromatase and promoter expression was measured by polymerase chain reaction and protein levels by Western blotting. RESULT(S): Total aromatase expression was significantly higher in omental compared with subcutaneous fat from pregnant women; this pattern was reversed in obese women. Aromatase messenger RNA in omentum was significantly higher in pregnancy than obesity, and this was linked to an up-regulation of promoter II (PII). Promoter 1.4 (P1.4) expression was lower than PII, and there was no difference in P1.4 expression between the two fat depots from pregnant subjects. In obese women both P1.4 and PII were up-regulated in subcutaneous compared with omental depots, with P1.4 expression greater than that of PII. Aromatase protein levels were extremely low in fat depots of pregnant women and undetectable in obese women. CONCLUSION(S): There are differences between total aromatase and promoter expression in subcutaneous and omental fat from pregnant compared with obese women. These differences support the evidence that the fat depots are derived from different cell lineages and that the promoter-derived aromatase translation varies according to physiologic/pathophysiologic status.

Anti-Müllerian hormone reduces follicle sensitivity to follicle-stimulating hormone in human granulosa cells.

OBJECTIVE: To determine that anti-Müllerian hormone (AMH) has been shown to inhibits E(2) production in rodents and in luteinized granulosa cells (GC). We determined whether this occurs in human cells most highly expressing AMH (i.e., from small antral follicles) and whether this is an effect on aromatase promoter activity. We also investigated the effects of AMH on other factors determining FSH sensitivity. DESIGN: Granulosa cells were exposed to AMH with and without gonadotropins for 48 hours. SETTING: University laboratory. PATIENT(S): Not applicable. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Aromatase and FSH receptor messenger RNA expression measured using real time quantitative polymerase chain reaction (PCR). Aromatase promoter II activity measured using a luciferase assay. Estradiol, inhibin A and B, and vascular endothelial growth factor production were measured in the conditioned medium. RESULT(S): The AMH decreased gonadotropin-stimulated aromatase expression and decreased forskolin-stimulated aromatase in KGN cells and this effect was through a dose-dependent inhibition of promoter II. Surprisingly, AMH also reduced FSH receptor mRNA expression. High AMH doses had no effect on inhibin B, whereas a low dose stimulated production. There was no effect on inhibin A or vascular endothelial growth factor. CONCLUSION(S): The AMH inhibits factors affecting FSH sensitivity. As AMH levels decrease with follicle growth, this inhibition would be removed. The AMH overproduction in anovulatory polycystic ovaries (PCO) may therefore restrict folliculogenesis by an inhibitory effect on FSH sensitivity, thereby contributing to anovulation.

Phosphorylation and activation of AMP-activated protein kinase (AMPK) by metformin in the human ovary requires insulin.

Metformin is commonly used to treat women with polycystic ovary syndrome, but its precise mechanism of action is unclear, and it even appears to have direct ovarian effects. At the cellular level, it may act either via an insulin-dependent pathway or an independent pathway by activating AMP-activated protein kinase (AMPK). In the ovary, metformin directly decreased estradiol and progesterone production by human granulosa cells, and inhibition of progesterone production by metformin in rat granulosa cells caused an increase in phosphorylated AMPK (pAMPK). We investigated whether metformin activates AMPK in the human ovary by looking for changes in phosphorylation of AMPK and its downstream target acetyl CoA carboxylase (ACC). mRNA and protein for α1 and α2 AMPK subunits were present in all human ovarian tissue. Neither 100 nm nor 2 mm of metformin affected subunit expression. After 1 or 4 h, neither dose of metformin increased pAMPK or pACC, although after 1 h, the addition of insulin significantly enhanced pAMPK, whereas insulin alone had no effect on phosphorylation of either AMPK or ACC. The addition of compound C, an inhibitor of AMPK, negated the effect of metformin in the presence of insulin on pAMPK. This effect on AMPK was not due to a change in the ADP/ATP ratio measured by HPLC. In summary, the presence of insulin was required to cause a metformin-induced increase in pAMPK in these human ovarian cells. Although previous data suggest that metformin may act via an insulin-independent pathway, our results therefore imply that insulin may be required to initiate an effect.

Action of metformin on the insulin-signaling pathway and on glucose transport in human granulosa cells.

CONTEXT: Hyperinsulinemia in polycystic ovary syndrome is widely treated with the insulin sensitizer metformin, which, in addition to its systemic effects, directly affects the ovarian insulin-stimulated steroidogenesis pathway. OBJECTIVE: Our aim was to investigate the interaction of metformin with the other insulin-stimulated ovarian pathway, namely that leading to glucose uptake. DESIGN: Human granulosa-luteal cells were cultured with metformin (10(-7) M), insulin (10 ng/ml) or metformin and insulin (met + ins) combined. Insulin receptor (IR) involvement was assessed by culture with an (anti)-insulin receptor (IR) antibody. MAIN OUTCOME MEASURES: The effect of metformin on insulin-receptor substrate proteins 1 and 2 (IRS-1 and -2) mRNA and protein expression was determined. The KGN granulosa-cell line was used to investigate the effect of insulin and metformin on Akt activation and glucose transporter-4 (Glut-4) expression. Glut-4 translocation from the cytosol to the membrane was determined in cytoplasmic and membrane-enriched fractions of protein lysates. RESULTS: IRS-1 mRNA and protein increased with all treatments. In contrast, basal IRS-2 mRNA levels were barely detectable, but transcription was up-regulated by metformin. The anti-IR antibody reduced treatment-stimulated IRS-1 to basal levels and IRS-2 expression to an even greater extent than IRS-1, showing greater dependence on the IR than IRS-1. Metformin in the presence of insulin activated Akt and this was dependent on phosphoinositide-3 kinase, as was translocation of Glut-4 to the membrane. Metformin was able to substantially enhance the insulin-stimulated translocation of Glut-4 transporters from the cytosol to the membrane. CONCLUSION: This net increase in Glut-4 transporters in the plasma membrane has the potential to increase glucose uptake and metabolism by granulosa cells of the insulin-resistant polycystic ovary, thereby facilitating follicle growth.

Anti-Müllerian hormone and polycystic ovary syndrome: a mountain too high?

Anti-Müllerian hormone (AMH) was initially thought to be produced solely by the foetal male during sexual differentiation to promote regression of the Müllerian ducts. Over the last decade, however, a new and interesting role has emerged for AMH in the ovary. In human ovaries, AMH is produced by granulosa cells from 36 weeks of gestation until menopause, with the highest expression being in small antral follicles. AMH production gradually declines as follicles grow; once follicles reach a size at which they are dominant, it has largely disappeared. Its removal from these larger follicles appears to be an important requirement for dominant follicle selection and progression to ovulation as AMH has an inhibitory role in the ovary, reducing both primordial follicle initiation and follicle sensitivity to FSH by inhibition of aromatase. It is for this reason that AMH is a focus of interest in polycystic ovary syndrome (PCOS). Serum levels are doubled, and granulosa cell production is greatly increased. Interestingly, there appear to be two groups of women with PCOS who can be distinguished by their AMH level: one group consists of those who have high levels which do not reduce with treatment and who respond less well to induction of ovulation, and a second group consists of those in whom the level is less elevated and reduces on treatment and who seem to respond rather better. Understanding the reason for the raised AMH in PCOS may give clues as to the mechanism of anovulation. To conclude, AMH appears to have a major inhibitory role during folliculogenesis, which may contribute to anovulation in PCOS.

Metformin inhibits aromatase via an extracellular signal-regulated kinase-mediated pathway.

Metformin treatment, now widely prescribed in polycystic ovary syndrome, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms, however, by which metformin inhibits estradiol production in human granulosa cells remains unknown. Granulosa luteal cells were incubated with metformin, insulin, or combined metformin and insulin treatment, and aromatase mRNA expression was quantified using real-time RT-PCR. Enzyme activity was assessed by the conversion of (3)H-androstenedione to estrone and estradiol. Metformin's effect on the expression of specific untranslated first exon aromatase promoters was analyzed using semiquantitative PCR. The involvement of MAPK kinase (MEK)/ERK pathway was investigated by immunoblotting for aromatase, phosphorylated, and total ERK-1,2 from cells cultured as above with/without the MEK inhibitor PD98059. Metformin significantly inhibited basal and insulin-stimulated aromatase mRNA expression, with parallel results from the aromatase activity assay and protein assessment. This suppression was via down-regulation of aromatase promoter II, I.3, and 1.4 expression and was reversed by the addition of PD98059. Involvement of the ERK signaling pathway was demonstrated by the significant increase in phosphorylated ERK-1,2 with the combined metformin and insulin treatment. We have shown for the first time in human granulosa cells that metformin signficantly attenuated basal and insulin-stimulated P450 aromatase mRNA expression and activity, via silencing of key promoters. This occurred by activation of MEK/ERK pathway, which negatively regulated aromatase production. This is an important consideration given metformin's widespread use in polycystic ovary syndrome and may further support a possible therapeutic indication in estrogen-dependent breast tumors.

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells.

Glucose concentrations of normal human airway surface liquid are approximately 12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5-10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

Human ovarian biopsies as a viable source of pre-antral follicles.

BACKGROUND: Our knowledge of the early pre-antral stage of human folliculogenesis is still poor due to small follicle size and the limited availability of human ovarian tissue. Our aim was to determine the utility of ovarian biopsy for pre-antral follicle research. METHODS: Ovarian cortical biopsies were obtained from women (28-46 years old) undergoing elective Caesarean sections or total abdominal hysterectomy/bilateral salpingo-oophorectomy for a variety of benign gynaecological conditions. Follicle isolation and staging was performed according to a well-established protocol, involving enzymatic digestion, isolation using fine needles and image capture analysis software. RNA was also isolated for reverse transcription. RESULTS: More than 351 follicles were retrieved from 19 patients and 249 were classifiable into follicle stages: 80 primordial, 53 transitional, 82 primary, 26 secondary and 8 multilaminar. All samples, except two from women aged over 40 years, yielded follicles. The average yield of classifiable follicles/patient was 13. There was an age-related decline in mean follicle numbers/patient (r(2) = -0.986). Microgram quantities of complementary DNA per follicle were synthesized. CONCLUSIONS: Despite the heterogeneous distribution of follicles throughout the cortex and the significant age-related decline in the numbers of follicles retrieved, biopsy samples of ovarian cortical tissue provide a useful source of pre-antral follicles. This, coupled with the sensitivity of genomic technology, makes this method a viable research approach.

Phytoestrogens oestrogen synthesis and breast cancer.

Phytoestrogens are used as 'natural' alternatives to HRT and, although epidemiological evidence implies that diets rich in phytoestrogens reduce the incidence of breast cancer, their weak oestrogenicity is also known to stimulate growth in experimental models of breast cancer. This review addresses the question as to how phytoestrogens may protect against breast cancer through their ability to bind preferentially to oestrogen receptor beta, inhibit enzymes that convert circulating steroid precursors into oestradiol and inhibit cell signalling pathways of growth factors.

Stage-specific expression of androgen receptor, follicle-stimulating hormone receptor, and anti-Müllerian hormone type II receptor in single, isolated, human preantral follicles: relevance to polycystic ovaries.

CONTEXT: Recent evidence indicates that the increase in follicle numbers seen in polycystic ovary syndrome occurs early in folliculogenesis, with androgens being a likely causative candidate. In primates and sheep, androgen excess in utero results in ovarian changes similar to those in polycystic ovary syndrome. There is also increasing interest in the role of anti-Müllerian hormone (AMH) in early folliculogenesis because AMH knockout mice have an early depletion of their stock of primordial follicles. Initiation and early folliculogenesis may therefore be under negative control by AMH and positive control by androgens. OBJECTIVE: Because AMH signals exclusively through its type II receptor (AMHRII), the aim of this study was to determine and colocalize the stage-specific expression of AMHRII, androgen receptor (AR), and FSH receptor (FSHR) mRNA in individual, well-characterized preantral follicles. METHOD: Follicles were isolated from human ovarian cortex obtained from either oophorectomies or cortical biopsies at cesarean section. Expression of AR, FSHR, and AMHRII mRNA was determined using a nested RT-PCR protocol. RESULTS: AR mRNA was not detected in any primordial follicles but was from the transitional stage onward. The number of AR-positive follicles increased at each progressive growth stage. The expression of AR preceded that of FSHR, and only a small percentage of primary follicles expressed FSHR. AMHRII expression was rarely detected. CONCLUSIONS: This is the first study to identify the expression of AR in human transitional follicles. Results suggest a role for androgens in promoting early follicle growth and challenging the hypothesis that AMH exerts a direct, inhibitory effect on follicles at this stage.

Ethanolic extracts of black cohosh (Actaea racemosa) inhibit growth and oestradiol synthesis from oestrone sulphate in breast cancer cells.

Extracts of black cohosh (Actaea racemosa) and soy are used as 'natural' alternatives to conventional hormone replacement therapy (HRT) and there is some evidence that soy may protect against breast cancer by inhibiting the production of active oestrogens. This study compares the action of ethanolic extracts of black cohosh (BCE) and genistein on growth and enzyme activity in MCF-7 and MDA-MB-123 breast cancer cells. BCE inhibited growth at the two highest doses tested, i.e. 50 and 100 microg/ml, whilst genistein stimulated growth in the oestrogen receptor positive (ER(+)) MCF-7 cells, but at high doses it inhibited growth in both cell lines. BCE did not affect the conversion of androstenedione to oestradiol and only the highest doses (50 and 100 microg/ml) significantly inhibited the conversion of oestrone to oestradiol in MDA cells. In contrast, BCE induced a dose-dependent inhibition of the conversion of oestrone sulphate to oestradiol in both cell lines, whilst in human granulosa lutein (GL) cells enzyme activity was only inhibited at the highest dose of BCE. Genistein had no significant effect on enzyme activity in breast cancer cells and like BCE only the highest doses (10 and 50 microM) inhibited enzyme activity in human GL cells. In vivo genistein may have growth stimulatory effects on breast tissue but BCE not only inhibits growth but inhibits the conversion of oestrone sulphate to active oestradiol, considered by some, to be the preferred pathway of oestradiol synthesis in breast tissue.

Phytoestrogens and breast cancer--promoters or protectors?

The majority of breast cancers are oestrogen dependent and in postmenopausal women the supply of oestrogens in breast tissue is derived from the peripheral conversion of circulating androgens. There is, however, a paradox concerning the epidemiology of breast cancer and the dietary intake of phytoestrogens that bind weakly to oestrogen receptors and initiate oestrogen-dependent transcription. In Eastern countries, such as Japan, the incidence of breast cancer is approximately one-third that of Western countries whilst their high dietary intake of phytoestrogens, mainly in the form of soy products, can produce circulating levels of phytoestrogens that are known experimentally to have oestrogenic effects. Indeed, their weak oestrogenicity has been used to advantage by herbalist medicine to promote soy products as a natural alternative to conventional hormone replacement therapy (HRT). Such usage could increase in light of recent evidence that long-term HRT usage may be associated with an increased risk of breast cancer with a consequent reduction in prescription rates. So, are phytoestrogens safe as a natural alternative to HRT and could they be promoters or protectors of breast cancer? If they are promoters, then we must assume that it is due to their oestrogenic effect. If they are protectors, then other actions of phytoestrogens, including their ability to inhibit enzymes that are responsible for converting androgens and weak oestrogens into oestradiol, must be considered. This paper addresses these questions by reviewing the actions of phytoestrogens on oestrogen receptors and key enzymes that convert androgens to oestrogens in relation to the growth of breast cancer cells. In addition, it compares the experimental and epidemiological evidence pertinent to the potential beneficial or harmful effects of phytoestrogens in relation to the incidence/progression of breast cancer and their efficacy as natural alternatives to conventional HRT.

Phytoestrogens and their low dose combinations inhibit mRNA expression and activity of aromatase in human granulosa-luteal cells.

There is evidence that certain phytoestrogens inhibit aromatase, the enzyme that converts androgens to oestrogens. Kinetic studies in cell-free preparations show that they may inhibit aromatase by competitive binding to the enzyme, but there is a paucity of studies investigating longer-term effects of phytoestrogens on the expression of steroidogenic enzymes. This study tested the hypothesis that phytoestrogens could reduce aromatase activity by down-regulation of its expression. Experiments were carried out on primary cultures of human granulosa-luteal (GL) cells after they had been exposed to phytoestrogens for 48 h. Aromatase activity was measured by the ability of cells to convert testosterone to estradiol over a 4h period and aromatase mRNA expression (mRNA(arom)) was subsequently measured from the same cells using quantitative real-time PCR. The compounds investigated were the flavones, apigenin and quercetin, and the isoflavones, genistein, biochanin A and daidzein at doses of 10 microM and 100 nM. Combinations of these compounds at the lower dose were also investigated. All compounds tested dose-dependently reduced mean mRNA(arom) compared with controls. Apigenin was the most potent inhibitor with significant inhibition of mRNA(arom) seen at both 10 microM and 100 nM, whilst other flavonoids (except biochanin A) only induced significant inhibition (p

Endocrine-disrupting chemicals as modulators of sex steroid synthesis.

Endocrine-disrupting chemicals (EDCs) are typically identified as compounds that can interact with oestrogen or androgen receptors and thus act as agonists or antagonists of endogenous hormones. Growing evidence shows that they may also modulate the activity/expression of steroidogenic enzymes. These are expressed not only in the adrenal glands and gonads but also in many tissues that have the ability to convert circulating precursors into active hormones. In this way, EDCs may impact both on sexual differentiation and development and on hormone-dependent cancers. This review summarizes the evidence for EDCs as modulators of steroidogenic enzymes, identifies the structure/activity relationship in terms of inhibiting specific enzyme activity, questions whether experimental observations can equate with natural in vivo exposure or dietary intake of EDCs, and finally looks at the mechanisms through which these chemicals may disrupt normal steroidogenesis. In summarizing the evidence, the question of whether or not the dietary intake of these endocrine disrupters could pose a threat to human sexual development and health will be addressed.

Future developments in assisted reproduction in humans.

The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.