Estradiol Response Research Articles

Androstenedione acts on the coeliac ganglion and modulates luteal function via the superior ovarian nerve in the postpartum rat

Androstenedione can affect luteal function via a neural pathway in the late pregnant rat. Here, we investigate whether androstenedione is capable of opposing to regression of pregnancy corpus luteum that occurs after parturition, indirectly, from the coeliac ganglion. Thus, androstenedione was added into the ganglionar compartment of an ex vivo coeliac ganglion–superior ovarian nerve–ovary system isolated from non-lactating rats on day 4 postpartum. At the end of incubation, we measured the abundance of progesterone, androstenedione and oestradiol released into the ovarian compartment. Luteal mRNA expression and activity of progesterone synthesis and degradation enzymes, 3β-hydroxysteroid-dehydrogenase (3β-HSD) and 20α-hydroxysteroid-dehydrogenase (20α-HSD), respectively, as well as the aromatase, Bcl-2, Bax, Fas and FasL transcript levels, were also determined. Additionally, we measured the ovarian release of norepinephrine, nitric oxide and luteal inducible nitric oxide synthase (iNOS) mRNA expression. The presence of androstenedione in the ganglion compartment significantly increased the release of ovarian progesterone, androstenedione and oestradiol without modifying 3β-HSD and 20α-HSD activities or mRNA expression. The ovarian release of oestradiol in response to the presence of androstenedione in the ganglion compartment declined with time of incubation in accord with a reduction in the aromatase mRNA expression. Androstenedione added to the ganglion compartment decreased FasL mRNA expression, without affecting luteal Bcl-2, Bax and Fas transcript levels; also increased the release of norepinephrine, decreased the release of nitric oxide and increased iNOS mRNA. In summary, on day 4 after parturition, androstenedione can mediate a luteotropic effect acting at the coeliac ganglion and transmitting to the ovary a signaling via a neural pathway in association with increased release of norepinephrine, decreased nitric oxide release, and decreased expression of FasL.

Clinical Evidence for Predominance of Delta-5 Steroid Production in Women with Polycystic Ovary Syndrome

In women with polycystic ovary syndrome (PCOS), the basis for ovarian androgen overproduction involves an overall increase of steroidogenesis, notably in the delta-4 pathway. However, in vitro studies have suggested that excessive androgen production occurs predominantly through the delta-5 pathway. This study was performed to assess androgen dose-responses after human chorionic gonadotropin (hCG) stimulation in PCOS and normal women. We conducted a prospective study to compare androgen production after iv hCG in PCOS and normal women. The study was conducted in a General Clinical Research Center in an academic medical center. Women with PCOS (age, 18-37 yr; n = 10) and normal ovulatory controls (age, 18-37 yr; n = 11) were recruited. For dose-response studies, blood samples were obtained before and at 0.5, 24, and 48 h after iv recombinant hCG (1, 10, 25, 100, and 250 μg). A subset of subjects underwent frequent blood sampling over 24 h after iv injection of 25 μg of recombinant hCG. We measured basal and stimulated serum 17-hydroxyprogesterone (17-OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone, estradiol, and progesterone responses after hCG administration. In PCOS women, maximal A and T production was observed at the lowest doses of hCG, whereas responses were minimal in normal women. Incremental responses of 17-OHP, estradiol, and progesterone were greater in PCOS compared to normal women. In PCOS women, maximal A and T responses to hCG relative to those of 17-OHP are consistent with ovarian androgen overproduction via the delta-5 pathway.

Preconception Folic Acid Use Modulates Estradiol and Follicular Responses to Ovarian Stimulation

Background: Folate is a methyl donor. Availability of folate affects DNA methylation profiles and thereby gene expression profiles. We investigated the effects of low-dose folic acid use (0.4 mg/d) on the ovarian response to mild and conventional ovarian stimulation in women. Methods: In a randomized trial among subfertile women, 24 and 26 subjects received conventional and mild ovarian stimulation, respectively. Blood samples were taken during the early follicular phase of the cycle prior to treatment and on the day of human chorionic gonadotropin administration for determination of serum total homocysteine, anti-Müllerian hormone (AMH), estradiol, and folate. Folic acid use was validated by questionnaire and serum folate levels. Preovulatory follicles were visualized, counted, and diameters recorded using transvaginal ultrasound. The relation between folic acid use and ovarian response was assessed using linear regression analysis. Results: Folic acid use modified the ovarian response to ovarian stimulation treatment. The estradiol response was higher in nonfolic acid users receiving conventional treatment [βinteraction = 0.52 (0.07–0.97); P = 0.03], and this effect was independent of serum AMH levels and the preovulatory follicle count. In the conventional treatment, the mean follicle number was also greater in nonusers compared with the users group (14.1 vs. 8.9, P = 0.03). Conclusion: Low-dose folic acid use attenuates follicular and endocrine responses to conventional stimulation, independent of AMH and follicle count. The nature of this observation suggests that the effect of folic acid is most prominent during early follicle development, affecting immature follicles. Deleterious effects of folate deficiency, like DNA hypomethylation and oxidative stress, can help to explain our observations.

Increased preconception omega-3 polyunsaturated fatty acid intake improves embryo morphology

The association between preconception dietary intake of the polyunsaturated fatty acids (LC-PUFAs) omega-6 and omega-3 and the E(2) levels and IVF/intracytoplasmic sperm injection (ICSI) outcome were investigated in women in a prospective study. It revealed that high intakes of omega-3 LC-PUFA alpha-linolenic acid increase baseline E(2), high intakes of eicosapentaenoic acid and docosahexaenoic acid reduce E(2) response and the number of follicles after ovarian stimulation, and total omega-3 intake, in particular alpha-linolenic acid and docosahexaenoic acid, improve embryo morphology.

Preconception Folic Acid Use Modulates Estradiol and Follicular Responses to Ovarian Stimulation

Folate is a methyl donor. Availability of folate affects DNA methylation profiles and thereby gene expression profiles. We investigated the effects of low-dose folic acid use (0.4 mg/d) on the ovarian response to mild and conventional ovarian stimulation in women.In a randomized trial among subfertile women, 24 and 26 subjects received conventional and mild ovarian stimulation, respectively. Blood samples were taken during the early follicular phase of the cycle prior to treatment and on the day of human chorionic gonadotropin administration for determination of serum total homocysteine, anti-Müllerian hormone (AMH), estradiol, and folate. Folic acid use was validated by questionnaire and serum folate levels. Preovulatory follicles were visualized, counted, and diameters recorded using transvaginal ultrasound. The relation between folic acid use and ovarian response was assessed using linear regression analysis.Folic acid use modified the ovarian response to ovarian stimulation treatment. The estradiol response was higher in nonfolic acid users receiving conventional treatment [β(interaction) = 0.52 (0.07-0.97); P = 0.03], and this effect was independent of serum AMH levels and the preovulatory follicle count. In the conventional treatment, the mean follicle number was also greater in nonusers compared with the users group (14.1 vs. 8.9, P = 0.03).Low-dose folic acid use attenuates follicular and endocrine responses to conventional stimulation, independent of AMH and follicle count. The nature of this observation suggests that the effect of folic acid is most prominent during early follicle development, affecting immature follicles. Deleterious effects of folate deficiency, like DNA hypomethylation and oxidative stress, can help to explain our observations.

Withholding Gonadotropins Until Human Chorionic Gonadotropin Administration

Withholding gonadotropins in women who exhibit high estradiol responses before follicles reach full maturation is called "coasting." Coasting, or suspending gonadotropin administration, can be an effective strategy for decreasing the risk of ovarian hyperstimulation syndrome (OHSS) while reducing cancelation rates. In in vitro fertilization cycles, mechanistically it is believed that withholding gonadotropins starves smaller follicles, induces apoptosis, and decreases the potential for these follicles to elaborate vascular endothelial growth factor, a known mediator of OHSS. It is generally accepted that coasting should be initiated when the estradiol (E₂) level is >3000 pg/mL in the setting of immature follicles. The human chorionic gonadotropin (hCG) trigger should be administered when the E₂ level subsequently drops to a "safe" level. Cycle cancellation should be considered if, after 3 to 4 days of coasting, the E₂ level remains excessively elevated. Oocyte retrieval may also be cancelled if the E₂ level on the day after hCG trigger drops precipitously. In gonadotropin-releasing hormone agonist (GnRHa)-based protocols, one can consider withholding GnRHa administration if the E₂ level continues to increase after a few days of coasting. Current data seem to show that the coasting period is short and/or is less likely to be required in GnRH-antagonist protocols as compared with GnRHa-based protocols. Large randomized control trials are still needed to establish the relative efficacy of coasting versus embryo cryopreservation in the context of OHSS prevention.

Raloxifene inhibits tumor growth and lymph node metastasis in a xenograft model of metastatic mammary cancer.

BackgroundThe effects of raloxifene, a novel selective estrogen receptor modulator, were studied in a mouse metastatic mammary cancer model expressing cytoplasmic ERα.MethodsMammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879luc2 cells, were subsequently treated with raloxifene at 0, 18 and 27 mg/kg/day using mini-osmotic pumps.ResultsIn vitro study demonstrated that the ERα in BJMC3879luc2 cells was smaller (between 50 and 64 kDa) than the normal-sized ERα (66 kDa) and showed cytoplasmic localization. A statistically significant but weak estradiol response was observed in this cell line. When BJMC3879luc2 tumors were implanted into mice, the ERα mRNA levels were significantly higher in females than in males. In vitro studies showed that raloxifene induced mitochondria-mediated apoptosis and cell-cycle arrest in the G1-phase and a decrease in the cell population in the S-phase. In animal experiments, tumor volumes were significantly suppressed in the raloxifene-treated groups. The multiplicity of lymph node metastasis was significantly decreased in the 27 mg/kg group. Levels of apoptosis were significantly increased in the raloxifene-treated groups, whereas the levels of DNA synthesis were significantly decreased in these groups. No differences in microvessel density in tumors were observed between the control and raloxifene-treated groups. The numbers of dilated lymphatic vessels containing intraluminal tumor cells were significantly reduced in mammary tumors in the raloxifene-treated groups. The levels of ERα mRNA in mammary tumors tended to be decreased in the raloxifene-treated groups.ConclusionThese results suggest that the antimetastatic activity of raloxifene in mammary cancer expressing cytoplasmic ERα may be a crucial finding with clinical applications and that raloxifene may be useful as an adjuvant therapy and for the chemoprevention of breast cancer development.

Curcumin counteracts the proliferative effect of estradiol and induces apoptosis in cervical cancer cells

Cervical cancer is the most common cancer in Indian females and is associated with infection with high-risk Human papilloma viruses (HPVs) which encode viral oncoprotein E6 and E7. Estradiol has been established as a risk factor for cervical cancer and has been shown to play a synergistic role with viral oncoproteins. Curcumin (Diferuloyl methane), a chemopreventive agent, is a natural compound extracted from Curcuma longa that allows suppression and retardation of carcinogenesis in many types of cancer and is currently being tested in various human clinical trials as it has been found to be well tolerated at higher doses with a relatively well established safety profile. The objective of this study was to test the effect of curcumin on HPV-positive and negative cervical cancer cell lines HeLa, SiHa, CaSki, and C33A pretreated with estradiol. It was found that HPV-positive cells pretreated with estradiol show reduced apoptosis as compared to curcumin by itself. However, curcumin was able to counteract the proliferative response of estradiol, and induce apoptosis. There was no difference in percentage apoptosis as compared to estradiol pretreatment in HPV-negative cell line C33A. Molecular studies showed elevation of Telomerase, viral oncoproteins E6 and E7, PCNA, p16, Cyclin D1 in HPV-positive cell lines on treatment with estradiol but after treatment with curcumin the level of E7, PCNA, and Cyclin D1 was reduced but the level of E6, Telomerase, and p16 was unaltered. Furthermore, estradiol-pretreated HPV-negative cell line C33A showed reduction in level of Telomerase, PCNA, p16, and activation of both p53 and p73 tumor suppressor proteins, thus, demonstrating the importance of E6 in estradiol-mediated protective effect.

Do ovarian reserve tests predict miscarriage in women undergoing assisted reproduction treatment?

Objective To evaluate the role of ovarian reserve tests for the prediction of miscarriage among pregnancies resulting from assisted reproduction treatment. Study design Cohort study in a large IVF centre. Three hundred and fourteen women aged <43 years with basal FSH <12 IU/L who conceived following their first cycle of IVF/ICSI were included. Sixty-seven participants experienced miscarriage while the remaining 247 had ongoing pregnancies. Intervention included transvaginal scan and venepuncture on day 3 of the menstrual cycle immediately prior to treatment. Ovarian reserve parameters assessed were antral follicle count (AFC), FSH, basal oestradiol and oestradiol response. The main outcome measure was miscarriage rate (pregnancy loss at <12 weeks). Results While the baseline clinical characteristics including age and duration of subfertility were comparable, BMI was significantly higher in women who experienced miscarriage compared to those having ongoing pregnancies. Among the ovarian reserve markers studied, only the AFC was significantly different between the miscarriage and pregnant groups. Logistic regression analysis revealed that only these two markers (BMI and AFC) were the significant predictors of clinical miscarriage. However, the discriminative ability of both BMI and AFC for the prediction of miscarriage was low as indicated by AUCs of 0.617 and 0.588 respectively on ROC curve analysis. Conclusion While AFC and BMI are significantly predictive of first trimester miscarriage, the ability of these markers individually or in combination to discriminate women who experience miscarriage from those who continue the pregnancy beyond 12 weeks of gestation is poor.

ORIGINAL ARTICLE: Leuprolide stimulation testing for the evaluation of early female sexual maturation

SummaryContext Low concentrations of serum LH and/or oestradiol (E2) in girls with early physical signs of precocious puberty pose a diagnostic challenge.Objective To assess the diagnostic value of the leuprolide stimulation test in female precocious puberty.Design Retrospective Chart Review.Setting Outpatient clinic.Patients and intervention Thirty‐nine girls, 6·9 (1·4) years, with premature stage II–III breast development, with or without pubarche, underwent stimulation testing with subcutaneous leuprolide (20 μg/kg) with the following hormonal measurements in serum: FSH, LH, oestradiol at baseline; FSH and LH at 1 and 2 h; oestradiol at 24 h. Twelve girls with isolated pubarche were also tested with leuprolide.Main outcome measure A pubertal hormonal pattern was defined as at least one of the following: a baseline serum level of LH ≥ 0·3 U/l, a baseline oestradiol ≥ 37 pmol/l (10 ng/l), a stimulated (peak) LH ≥ 5·0 U/l, a stimulated oestradiol ≥ 184 pmol/l (50 ng/l) to leuprolide. The hormonal response was related to the clinical course during a period of observation of at least 6 months.Results Following leuprolide stimulation, the hormonal response was concordant with pubertal progression (n = 23) or lack thereof (n = 16) in all children. At baseline, pubertal serum concentrations of LH and/or oestradiol were associated with pubertal progression in all, while serum prepubertal LH and/or oestradiol concentrations were associated with pubertal progression in approximately 50% of the patients.Conclusions In girls with early clinical signs of precocious puberty and low serum concentrations of LH and oestradiol in random samples, the LH and oestradiol responses to leuprolide stimulation accurately predict pubertal progression.

Relevance of mating-associated stimuli, ovulation, and the progesterone receptor for the post-coital inhibition of estrous behavior in the female rabbit

Estrous female domestic rabbits ( Oryctolagus cuniculus) display scent marking (“chinning”) and sexual receptivity. Mating induces ovulation, which occurs approximately 12 h later, and also decreases chinning and receptivity. In the present study, we explored the participation of mating-associated stimuli, ovulation, and the progesterone receptor (PR) in mediating such behavioral effects. We found that copulatory stimuli were not necessary, and that ovulation alone was sufficient, as these behavioral changes were replicated in unmated females by intravenous administration of human chorionic gonadotropin (hCG). The post-mating administration (s.c.) of 5 μg/day estradiol benzoate (EB), prevented the decline in chinning and receptivity. A lower dose of EB (1 μg/day) had no effect, nor did the antiprogestin RU486 (20 mg, s.c., administered 3 h before mating). However, the combination of a single pre-mating administration of RU486 plus the post-mating administration of 1 μg/day EB completely blocked the decline in estrous behavior. We propose that PR activation around the time of mating and a post-mating decline in ovarian estradiol secretion and/or estradiol responsiveness act in parallel to terminate estrus in this species.

Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk

BackgroundInterpreting proteomic and genomic data is a major challenge in predictive ecotoxicology that can be addressed by a systems biology approach. Mathematical modeling provides an organizational platform to consolidate protein dynamics with possible genomic regulation. Here, a model of ovarian steroidogenesis in the fathead minnow, Pimephales promelas, (FHM) is developed to evaluate possible transcriptional regulation of steroid production observed in microarray studies.ResultsThe model was developed from literature sources, integrating key signaling components (G-protein and PKA activation) with their ensuing effect on steroid production. The model properly predicted trajectory behavior of estradiol and testosterone when fish were exposed to fadrozole, a specific aromatase inhibitor, but failed to predict the steroid hormone behavior occurring one week post-exposure as well as the increase in steroid levels when the stressor was removed. In vivo microarray data implicated three modes of regulation which may account for over-production of steroids during a depuration phase (when the stressor is removed): P450 enzyme up-regulation, inhibin down-regulation, and luteinizing hormone receptor up-regulation. Simulation studies and sensitivity analysis were used to evaluate each case as possible source of compensation to endocrine stress.ConclusionsSimulation studies of the testosterone and estradiol response to regulation observed in microarray data supported the hypothesis that the FHM steroidogenesis network compensated for endocrine stress by modulating the sensitivity of the ovarian network to global cues coming from the hypothalamus and pituitary. Model predictions of luteinizing hormone receptor regulation were consistent with depuration and in vitro data. These results challenge the traditional approach to network elucidation in systems biology. Generally, the most sensitive interactions in a network are targeted for further elucidation but microarray evidence shows that homeostatic regulation of the steroidogenic network is likely maintained by a mildly sensitive interaction. We hypothesize that effective network elucidation must consider both the sensitivity of the target as well as the target's robustness to biological noise (in this case, to cross-talk) when identifying possible points of regulation.

LH Dynamics in Overweight Girls with Premature Adrenarche and Slowly Progressive Sexual Precocity

Background. Excess adiposity and premature adrenarche (PA) are risk factors for the development of polycystic ovary syndrome (PCOS). Methods. Girls with slowly progressive precocious breast development, who were overweight and had PA (SPPOPA, 6.2–8.2 years, n = 5), overweight PA (6.6–10.8 years, n = 7), and overweight premenarcheal controls (OW-PUB, 10.6–12.8 years, n = 8) underwent hormonal sleep testing and GnRH agonist (GnRHag) and ACTH tests. Results. Despite an insignificant sleep-related increase in LH and prepubertal baseline hormone levels, SPPOPA peak LH and estradiol responses to GnRHag were intermediate between those of PA and OW-PUB, the LH being significantly different from both. Conclusions. GnRHag tests indicate that SPPOPA is a slowly progressive form of true puberty with blunted LH dynamics. These results argue against the prepubertal hyperandrogenism of excess adiposity or PA enhancing LH secretion or causing ovarian hyperandrogenism prior to menarche. Excess adiposity may contribute to both the early onset and slow progression of puberty.

Assessment of theca cell function prior to controlled ovarian stimulation: the predictive value of serum basal/stimulated steroid levels

Serum androgen levels correlate with ovarian sensitivity to follicle-stimulating hormone (FSH) but in practice, standard baseline serum testosterone (T) levels prior to in-vitro fertilization (IVF) may not be the most appropriate marker for determination. Infertile women enrolled in an IVF programme were included in this study. Serum T and Delta4-androstenedione (A), and the androgen precursor 17-hydroxyprogesterone (17-OHP) were measured before and 24 h after a gonadotrophin-releasing hormone agonist stimulation test (GAST). An early follicular phase antral follicle count (AFC) was also performed. Patients were subsequently enrolled in a long gonadotrophin-releasing hormone agonist protocol with a standard FSH dose (150 IU) for 7 days to assess the association between androgen levels and ovarian responsiveness to FSH. The GAST elicited a significant increase in serum androgen levels that was well correlated with AFC. 17-OHP showed the greatest response to GAST and strongest correlation with AFC. The 17-OHP response to GAST differentiated patients with high ovarian reserve (OR) from those with low or normal OR as assessed by AFC, whereas only the estradiol response could differentiate those with low AFC. GAST-stimulated serum levels of 17-OHP were also correlated with ovarian response to FSH. Using receiver operating characteristic curve analysis, stimulated 17-OHP levels were predictive of the ovarian response to controlled ovarian stimulation, with similar power to that observed with AFC but lower power than with anti-Müllerian hormone. Serum androgen levels following GAST are correlated with AFC and ovarian response to FSH. Serum T is a less sensitive marker of theca cell function than 17-OHP.

Comparison of inhibin B and estradiol responses to intravenous FSH in women with polycystic ovary syndrome and normal women

Inhibin B (Inh B) is produced by pre-antral and early antral follicles whereas estradiol (E(2)) is a product of follicles undergoing antrum formation. This temporal distinction is evident in the patterns of Inh B and E(2) release earlier and later during the follicular phase of the menstrual cycle, respectively. However, in previous studies of women with polycystic ovary syndrome (PCOS) and normal controls, release of these granulosa cell (GC) products appears to be simultaneous in response to FSH stimulation. In order to reconcile these disparate findings, we conducted dose-response studies in both PCOS women and normal controls to determine whether GC product responses were due to the amount of FSH administered. In addition, we compared FSH-stimulated responses in PCOS women at various stages of recovery following ovarian suppression with a long-acting GnRH agonist to examine whether Inh B and E(2) responses reflected the level of ovarian follicle activity (i.e. circulating E(2) levels). Women with PCOS, 18-35 years (n = 23), and normal ovulatory controls, 18-35 years (n = 10) were recruited for study. Dose-responses were assessed over 24 h following intravenous administration of 0 (saline), 37.5, 75 and 150 IU of recombinant human FSH (r-hFSH) in PCOS and normal women. In addition, E(2) and Inh B responses to 150 IU of r-hFSH were assessed at baseline and 4, 6 and 8 weeks following suppression of ovarian steroidogenesis by a long-acting GnRH agonist in PCOS women. In PCOS women and normal controls, serum Inh B and E(2) exhibit similar and simultaneous dose-responsiveness to FSH stimulation. During recovery from ovarian suppression, basal and stimulated Inh B release appear to be restored earlier than that of E(2) in PCOS women. These findings are consistent with the notion that, in PCOS women, the level of ovarian follicle activity largely determines the earlier release of Inh B compared with E(2).

Ovulation Induction for In-Vitro-Fertilization and Embryo Transfer Applying Decapeptyl (DTRP-6 LH/RH) in Combination with HMG or FSH

47 patients out of the IVF-program of the Department of Obstetrics and Gynecology University of Kiel, who demonstrated in previous stimulation cycles premature LH surges, were treated in two modalities with a down regulation applying the GnRN-analogue decapeptyl (DTRP-6 LH/RH) and a concomitant HMG- or FSH-stimulation. The down-regulation was started after ovulation up to a negative LH/RH test followed by a concomitant gonadotropin stimulation in group 1. In group 2 a parallel treatment with decapeptyl and HMG or FSH was performed from day 2 of the cycle. 10 husbands of the punctured patients had pathological sperm. In both groups 6 patients were discarded from of the stimulation protocol as their oestradiol responses were not adequate, sperm contamination was detected late in one case, and in one patient a premature LH surge occurred once again. In 36 patients vaginal follicular punctures were performed. With respect to pregnancies group 1 revealed a much higher pregnancy rate than group 2. It seemed better to start the down-regulation with the GnRH-analogue decapeptyl in the luteal phase of the previous cycle. The treatment with decapeptyl should not only be applied in patients with previous LH surges but also in order to establish a synchronous follicular maturation in ovulating patients treated for in-vitro fertilization, or gamete-intra-Falloppian-tube-transfer.

Estradiol-regulated microRNAs control estradiol response in breast cancer cells

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). E2-inducible proteins c-Myc and E2Fs are required for optimal ERα activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERα-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERα-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

Coactivator Function Defines the Active Estrogen Receptor Alpha Cistrome

Proper activation of transcriptional networks in complex organisms is central to the response to stimuli. We demonstrate that the selective activation of a subset of the estrogen receptor alpha (ERalpha) cistrome in MCF7 breast cancer cells provides specificity to the estradiol (E2) response. ERalpha-specific enhancers that are subject to E2-induced coactivator-associated arginine methyltransferase 1 (CARM1) action are critical to E2-stimulated gene expression. This is true for both FoxA1-dependent and independent enhancers. In contrast, a subset of E2-suppressed genes are controlled by FoxA1-independent ERalpha binding sites. Nonetheless, these are sites of E2-induced CARM1 activity. In addition, the MCF7 RNA polymerase II cistrome reveals preferential occupancy of E2-regulated promoters prior to stimulation. Interestingly, E2-suppressed genes tend to lie in otherwise silent genomic regions. Together, our results suggest that the transcriptional response to E2 in breast cancer cells is dependent on the interplay between polymerase II pre-occupied promoters and the subset of the ERalpha cistrome associated with coactivation.

Characterization of Functionally Typical and Atypical Types of Polycystic Ovary Syndrome

The typical polycystic ovary syndrome (PCOS) phenotype includes 17-hydroxyprogesterone (17OHP) hyperresponsiveness to GnRH agonist (GnRHag) testing. Functionally atypical PCOS lacks this feature. The hypothesis was tested that the typical PCOS ovarian dysfunction results from intrinsically increased sensitivity to LH/human chorionic gonadotropin (hCG) due to a flaw in FSH action. PARTICIPANTS/DESIGN/INTERVENTIONS/MAIN OUTCOME MEASURES: After phenotyping a cohort of 60 women, steroid and inhibin-B responses to gonadotropins were evaluated in representative typical (n = 7) and atypical (n = 5) PCOS and healthy controls (n = 8). Submaximal hCG testing before and after an FSH test dose was performed in random order before and after prolonged ovarian suppression by depot GnRHag. The study was performed at a Clinical Research Center. Of our PCOS cohort, 68% were the typical type. Typical PCOS had 17OHP hyperresponsiveness and, unlike controls, significant androgen and estradiol responses to hCG. FSH increased inhibin-B and did not inhibit free testosterone or enhance estradiol responsiveness to hCG, all unlike controls. After ovarian suppression, 17OHP, androstenedione, and inhibin-B responsiveness to gonadotropin testing persisted. Atypical PCOS had significantly higher body mass index but lower ovarian volume and plasma free testosterone than typical PCOS. Steroid responses to hCG were insignificant and similar to controls. FSH suppressed free testosterone but stimulated inhibin-B. The estradiol level after combined hCG-FSH was subnormal. Free testosterone was less GnRHag suppressible than in typical PCOS. Typical PCOS is characterized by intrinsic ovarian hypersensitivity to hCG to which excessive paracrine FSH signaling via inhibin-B may contribute. Atypical PCOS is due to a unique type of ovarian dysfunction that is relatively gonadotropin hyposensitive.

Blunted Sleep-Related Luteinizing Hormone Rise in Healthy Premenarcheal Pubertal Girls with Elevated Body Mass Index

Our objective was to determine whether excessive adiposity is associated with alteration of the normal hormonal changes of early pubertal girls. Healthy 6.4- to 9.5-yr-old, prepubertal (PRE, n = 20) and 9.4- to 13.0-yr-old pubertal premenarcheal volunteers (PUB, n = 20) were divided into excessive-weight (EW) or normal-weight (NW) groups according to the 85th percentile body mass index. INTERVENTIONS AND SETTING: Overnight blood sampling; GnRH agonist (GnRHag), low-dose ACTH, oral glucose tolerance tests, and pelvic ultrasonograms were performed in our Clinical Research Center. EW girls were similar in age and baseline and ACTH- and GnRHag-stimulated androgen levels to stage-matched NW girls. However, the sleep-related LH rise was blunted in EW-PUB girls compared with NW-PUB girls. The sleep-related rise of mean LH in EW-PUB [0.68 +/- 0.35 (sem) U/liter] was insignificant, less than that of NW-PUB (2.1 +/- 0.45, P < 0.05) and not significantly different from that of PRE girls (0.08+/-0.03). EW-PUB had slower LH pulse frequency and a lower rise in LH pulse amplitude during sleep than NW-PUB girls (both P < 0.05). Overnight FSH patterns paralleled LH patterns, whereas estradiol levels were similar in stage-matched NW and EW groups, differing between stages as expected. Early morning and peak LH, FSH, and estradiol responses to GnRHag were similar in EW-PUB and NW-PUB and significantly greater than those of PRE girls. Healthy EW-PUB girls have significantly blunted sleep-related LH production. These data suggest that excess adiposity, in the absence of sex steroid excess, may subtly suppress hypothalamic-pituitary-gonadal function in premenarcheal pubertal girls.