High E2 Research Articles

Patterns of sex hormone receptor expression in stimulated endometrium from oocyte donors

In patients with high serum E2 embryo transfer is often postponed, as high E2 levels adversely affect embryo transfer outcome. We aimed to determine if stratified serum oestradiol (E2) and progesterone (P4) levels differentially affect endometrial histology and endometrial oestrogen and progesterone receptor protein levels. Endometrial biopsies were collected from oocyte donors. Samples were divided based on peak serum E2 levels into three groups: (i) low-E2 (n = 33) E2≤2999pg/mL; (ii) mid-E2 (n = 40) E2 3000–4999 pg/mL; and (iii) high-E2 (n = 15) E2≥5000 pg/mL. Oestrogen receptor alpha (ERα) and progesterone receptors A and B (PR) protein levels in endometrial stroma (S), glandular (GE) and luminal (LE) epithelia were assessed by immunohistochemistry. Samples in high-E2 group demonstrated strongest association with accelerated endometrial maturation (2 (1–2); 2 (1–3); and 3 (2.8–3) median days of advancement of endometrial maturation respectively in low, mid, high-E2 groups, p = 0.046). There were significant differences in ERα and PR immunoexpression in S, GE and LE among the groups (p < 0.05). Higher E2 levels were associated with decreased ERα expression (p < 0.017) in GE and LE, and increased PR expression in S and GE (p < 0.011 and p < 0.0001, respectively). Higher serum E2 levels were associated with impaired endometrial steroid hormone receptor expression, higher serum P4 and more advancement of endometrial maturation.

Health status is related to testosterone, estrone and body fat: moving to functional hypogonadism in adult men with HIV.

Hypogonadism is common in HIV-infected men. The relationship between health status, sex steroids and body composition is poorly known in HIV. The aim was to investigate the association between health status (comorbidities/frailty), body composition, and gonadal function in young-to-middle-aged HIV-infected men. Prospective, cross-sectional, observational study. HIV-infected men aged <50 years and ongoing Highly Active Antiretroviral Therapy were enrolled. Serum total testosterone (TT), estradiol (E2), estrone (E1) were measured by liquid chromatography-tandem mass spectrometry, LH and FSH by immunoassay. Free testosterone (cFT) was calculated by Vermeulen equation. Body composition was assessed by dual-energy X-ray absorptiometry and abdominal CT scan. Multimorbidity (MM) and frailty were defined as ≥3 comorbidities and by a 37-item index, respectively. A total of 316 HIV-infected men aged 45.3 ± 5.3 years were enrolled. Body fat parameters were inversely related to cFT and TT, and directly related to E1 and E2/testosterone (TS) ratio. Patients with MM had lower cFT (P < 0.0001) and TT (P = 0.036), and higher E1 (P < 0.0001) and E2/TS ratio (P = 0.002). Frailty was inversely related to cFT (R2 = 0.057, P < 0.0001) and TT (R2 = 0.013, P = 0.043), and directly related to E1 (R2 = 0.171, P < 0.0001), E2 (R2 = 0.041, P = 0.004) and E2/TS ratio (R2 = 0.104, P < 0.0001). Lower TT and cFT, higher E1, E2/TS ratio and visceral fat were independently associated to poor health status and frailty, being possible hallmarks of unhealthy conditions in adult HIV-infected men. Overall, MM, frailty and body fat mass are strictly associated to each other and to sex steroids, concurring together to functional male hypogonadism in HIV.

Letrozole versus clomiphene in prevention of premature LH surge in women undergoing intrauterine insemination

Introduction Soft stimulation protocols were developed to impart a less stressful and less expensive mode of stimulation for patient insemination. Aim To evaluate the effect of letrozole versus clomiphene citrate (CC) in prevention of premature luteinizing hormone (LH) surge. Patients and methods This is a prospective comparative study held at Obstetrics and Gynecology Department, Alzahraa University Hospital. A total of 90 infertile patients between 18 and 35 years of age with unexplained infertility or mild male factor were included. Overall, 30 patients had induction by human menopausal gonadotropin and then CC from seventh day of the cycle; 30 patients had induction by human menopausal gonadotropin and then letrozole from seventh day of the cycle, and 30 patients had induction by human menopausal gonadotropin only and were evaluated according to LH and estradiol (E2) on triggering day, folliculometry, and pregnancy outcome. Results We found that the letrozole was associated with lesser LH on triggering day and higher endometrial thickness than the other two groups, and CC was associated with higher E2 on triggering day and number of mature follicles than the other two groups. The difference between the study groups in LH, E2, number of mature follicles, and endometrial thickness was statistically significant, whereas the differences in pregnancy rate was statistically insignificant. Conclusion The study shows that clomiphene and letrozole are available options to prevent LH surge.

High ovarian GDF-8 levels contribute to elevated estradiol production in ovarian hyperstimulation syndrome by stimulating aromatase expression.

Rationale: Growth differentiation factor-8 (GDF-8), also known as myostatin, belongs to the transforming growth factor-beta (TGF-β) superfamily. GDF-8 is expressed in the ovary and regulates various ovarian functions. Ovarian hyperstimulation syndrome (OHSS) is one of the most serious disorders during in vitro fertilization treatment. Aromatase, encoded by the CYP19A1 gene, is the enzyme that catalyzes the final step in estradiol (E2) biosynthesis. It has been demonstrated that high serum E2 levels are associated with the development of OHSS. However, the effects of GDF-8 on aromatase expression and its roles in the pathogenesis of OHSS remain unclear.Methods: The effect of GDF-8 on aromatase expression and the underlying mechanisms were explored by a series of in vitro experiments in primary human granulosa-lutein (hGL) and KGN cells. Rat OHSS model and human follicular fluid samples were used to examine the roles of the GDF-8 system in the pathogenesis of OHSS.Results: We demonstrate that GDF-8 stimulates aromatase expression and E2 production in hGL and KGN cells. In addition, TGF-β type I receptor ALK5-mediated SMAD2/3 signaling is required for GDF-8-induced aromatase expression and E2 production. Using a rat OHSS model, we show that the aromatase and GDF-8 levels are upregulated in the ovaries of OHSS rats. Blocking the function of ALK5 by the administration of its inhibitor, SB431542, alleviates OHSS symptoms and the upregulation of aromatase. Clinical results reveal that the protein levels of GDF-8 are upregulated in the follicular fluid of OHSS patients. Moreover, the expression of GDF-8 is increased in hGL cells of OHSS patients.Conclusions: This study helps to elucidate the mechanisms mediating the expression of aromatase in human granulosa cells, which may lead to the development of alternative therapeutic approaches for OHSS.

Neutrophil:lymphocyte and estradiol:progesterone ratios as predictive markers for ovarian hyperstimulation syndrome (OHSS).

This study was based on the pathophysiology of the disease and aimed at predicting ovarian hyperstimulation syndrome (OHSS) by determining the importance of ratios obtained from the inflammatory process associated with oestradiol and progesterone in recent years. Out of 242 infertile women who underwent assisted reproductive therapy, 59 patients who developed OHSS were taken as the study group, while the remaining 122 normo-responder (NR) and 61 hyper-responder (HR) patients constituted the control group. The neutrophil to lymphocyte (NLR) and oestradiol/progesterone (EPR) ratios were found to be statistically significantly higher in the OHSS group (P <0.001). A multivariate logistic regression analysis revealed that the NLR (OR=2.410, P =0.001) and EPR (OR=1.701, P =0.028) were independent predictors for the development of OHSS. In conclusion, in OHSS inadequate progesterone levels may inhibit suppression of the exaggerated inflammatory process caused by high E2 levels, and NLR and EPR can be used to predict the development of OHSS in patients undergoing controlled ovarian hyperstimulation during in vitro fertilisation cycles.

Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

OBJECTIVE: In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. STUDY DESIGN: In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. RESULTS: In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).CONCLUSION: In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.

Effect of follicle size on hormonal status of follicular fluid, oocyte ATP content, and in vitro embryo production in prepubertal sheep

The aim of this study was to assess the effect of follicular size on estradiol (E2) and progesterone (P4) levels in intrafollicular fluid, ATP content in oocytes, and the embryo development rate in prepubertal sheep. Slaughterhouse ovaries were dissected to recover the follicles, which were classified according to the follicle diameter as <3 mm (n = 20) and ≥3 mm (n = 17). Then, follicular fluid was obtained and analyzed by radioimmunoassay to determine the E2 and P4 concentrations. Another group of ovaries was used to recover cumulus-oocyte complexes according to follicle size. In vitro maturation (IVM), in vitro fertilization (IVF), and embryo culture were performed using standard procedures, and ATP level was assessed at 0 and 24 h of IVM. Intrafollicular concentrations of E2 and P4 and E2:P4 ratio were higher in ≥3 mm (18.7 ± 5.9 ng/mL, 7.8 ± 1.2 ng/mL, and 3.6 ± 1.3, respectively) than <3 mm (1.8 ± 0.4 ng/mL, 2.6 ± 0.3 ng/mL and 0.9 ± 0.3, respectively) follicles. The rate of ATP increased during IVM and was higher in oocytes from ≥3 mm than <3 mm (22.4 ± 0.7 and 8.6 ± 2.2-fold change; respectively) follicles. After IVF, the blastocyst development was higher in oocytes recovered from ≥3 mm (11.1 ± 0.9%) than from <3 mm (6.5 ± 0.7%) follicles. These results indicate an improvement in the competence and development of oocytes from ≥3 mm follicles with a higher E2:P4 ratio. Thus, this ratio could be used as reference to design IVM medium and to enhance the in vitro embryo production in lambs.

Estradiol correlates with erectile dysfunction and its severity in type 2 diabetic patients.

Erectile dysfunction (ED) is a frequent microvascular complication of type 2 diabetes mellitus (T2DM). Hormonal derangements such as hypogonadism and hyperestrogenism are common in T2DM. Our aim was to investigate the relationship between estrogens and ED in diabetic patients. We performed a retrospective study on 57 patients with T2DM suffering from ED. ED was assessed with the International Index of Erectile Function questionnaire (IIEF-5) and penile color-doppler ultrasound (PCDU). Blood tests included glycated hemoglobin, lipid profile, total testosterone (T), and estradiol (E2). E2 was negatively correlated with IIEF-5 score after correction for age, diabetes duration, BMI, HbA1c, LDL- and HDL-cholesterol, T and PSA (r = -0.457, p < 0.01). Patients in the higher E2 quartile, had statistically higher probability of severe ED (61.5%). In the same patients, the PCDU demonstrated a statistically longer Acceleration Time (120.0 ± 24.5, p = 0.048) indicating an impaired arterial flow. In diabetic patients, higher E2 is associated with worse erectile function and impaired cavernous arterial flow. Diabetic patients with high E2 are more prone to severe ED. It could be suggested to include estradiol measurement in the hormonal assessment of ED in patients with T2DM.

Further evidence that a supraphysiologic estradiol level during ovarian stimulation affects birthweight: findings of fresh and frozen embryo transfer with comparable estradiol levels on human chorionic gonadotropin trigger

Studies have reported a supraphysiologic estradiol (E2) level during ovarian stimulation may be related to low birthweight after fresh embryo transfer (ET), but whether a high E2 level in the stimulation cycle affects birthweight via frozen ET remained controversial. The study was to investigate whether a supraphysiologic E2 level in ovarian stimulation cycle affects birth weight in infants conceived via subsequent FET. In this retrospective cohort study, 2525 singletons conceived via FET and 2668 singletons conceived via fresh ET were analyzed. The birthweight and incidences of small for gestational age (SGA) in infants conceived via FET in groups with low and high levels of E2 did not differ significantly. Multivariate analyses showed that the peak E2 level was not predictive of SGA in infants conceived via FET. Singletons conceived via fresh ET had a higher risk of SGA than those conceived via FET even after comparable previous exposure to a high level of E2 > 3000 pg/mL. Our study results indicated that the increased risk of SGA was at least partly a result of the supraphysiologic E2 exposure to the intrauterine environment.

Estrogen Regulates Local Cysteine Metabolism in Mouse Myometrium.

Sulfur amino acid metabolism influences reproductive physiology, and transsulfuration in particular may be critical for normal cellular function. The sex hormone estrogen (E2) modulates gene expression and redox balance in some tissues by inducing the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The role of sex hormones in sulfur amino acid metabolism by uterine smooth muscle is not known. Here, we show that CBS and CSE proteins increase in the mouse myometrium during estrus and diestrus, respectively, suggesting that E2 reciprocally regulates myometrial CBS and CSE expression. In ovariectomized mice, exogenous E2 upregulates CBS and downregulates CSE levels. E2 promotes CBS mRNA and protein expression but attenuates CSE protein expression without affecting CSE mRNA. This pattern of E2-stimulated changes in transsulfuration enzyme expression is specific to the uterine smooth muscle. E2 does not change vaginal or cervical expression of CBS or CSE significantly, and E2 decreases expression of CSE in the liver without affecting CBS. E2 also downregulates myometrial cysteinesulfinic acid decarboxylase (CSAD) and decreases myometrial biochemical synthesis of the gaso-transmitter hydrogen sulfide (H2S). These findings suggest that myometrial sulfur amino acid metabolism may regulate uterine redox homeostasis, with implications for the source and metabolism of myometrial cysteine in high E2 states such as estrus and pregnancy.

Cyclin E2 Promotes Whole Genome Doubling in Breast Cancer

Genome doubling is an underlying cause of cancer cell aneuploidy and genomic instability, but few drivers have been identified for this process. Due to their physiological roles in the genome reduplication of normal cells, we hypothesised that the oncogenes cyclins E1 and E2 may be drivers of genome doubling in cancer. We show that both cyclin E1 (CCNE1) and cyclin E2 (CCNE2) mRNA are significantly associated with high genome ploidy in breast cancers. By live cell imaging and flow cytometry, we show that cyclin E2 overexpression promotes aberrant mitosis without causing mitotic slippage, and it increases ploidy with negative feedback on the replication licensing protein, Cdt1. We demonstrate that cyclin E2 localises with core preRC (pre-replication complex) proteins (MCM2, MCM7) on the chromatin of cancer cells. Low CCNE2 is associated with improved overall survival in breast cancers, and we demonstrate that low cyclin E2 protects from excess genome rereplication. This occurs regardless of p53 status, consistent with the association of high cyclin E2 with genome doubling in both p53 null/mutant and p53 wildtype cancers. In contrast, while cyclin E1 can localise to the preRC, its downregulation does not prevent rereplication, and overexpression promotes polyploidy via mitotic slippage. Thus, in breast cancer, cyclin E2 has a strong association with genome doubling, and likely contributes to highly proliferative and genomically unstable breast cancers.

Melatonin stimulates aromatase expression and estradiol production in human granulosa-lutein cells: relevance for high serum estradiol levels in patients with ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) is one of the most life-threatening and potentially fatal complications associated with controlled ovarian hyperstimulation (COH) during in vitro fertilization (IVF) treatment. Although the pathogenesis of OHSS remains unclear, elevated serum estradiol (E2) levels before human chorionic gonadotropin (hCG) administration are associated with the risk of OHSS. The pineal hormone melatonin and its receptors are expressed in human granulosa cells and have been shown to stimulate E2 production. However, the effect of melatonin on the expression of aromatase, an enzyme responsible for a key step in the biosynthesis of E2, in human granulosa cells remains to be determined. Here, we demonstrate that melatonin upregulates aromatase expression in primary cultured human granulosa-lutein (hGL) cells through the melatonin receptor-mediated PKA-CREB pathway. Using a mouse model of OHSS, we demonstrate that administration of the melatonin receptor inhibitor luzindole inhibits the development of OHSS. In addition, the expression of ovarian aromatase and serum E2 levels are upregulated in OHSS mice compared to control mice, but this upregulation is attenuated by inhibition of the function of melatonin. Moreover, clinical results reveal that aromatase expression levels are upregulated in hGL cells from OHSS patients. Melatonin and E2 levels in the follicular fluid are significantly higher in OHSS patients than in non-OHSS patients. Furthermore, melatonin levels are positively correlated with E2 levels in follicular fluid. This study helps to elucidate the mechanisms mediating the expression of aromatase in hGL cells and provides a potential mechanism explaining the high E2 levels in patients with OHSS.

Follicle stimulating hormone and estradiol trajectories from menopausal transition to late postmenopause in indigenous Chinese women

Objective The aim of this study was to examine follicle stimulating hormone (FSH) and estradiol (E2) trajectories in indigenous Chinese women through the ovarian aging process from 10 years before the final menstrual period (FMP) to 20 years after. Methods A prospective community-based longitudinal cohort study of 362 Chinese middle-aged women with a clearly determined FMP was performed. Results FSH and E2 trajectories were identified according to years from FMP and the Stages of Reproductive Aging Workshop + 10 (STRAW + 10), and further classified by body mass index. E2 decreases relatively steadily from Stage −2 to +1c, while FSH stabilizes quickly from Stage −2 to +1a. Obese women have higher E2 levels and lower FSH levels during menopausal transition, which last for decades after the FMP. There was no obvious decline in FSH levels for at least 20 years after the FMP. Conclusions The FSH and E2 trajectories in this indigenous Chinese cohort appear similar to those discussed in the Study of Women’s Health Across the Nation, with ethnic differences. STRAW + 10 criteria may be used to ascertain the ovarian aging process in Chinese women. Obese women may experience different levels of hormonal fluctuations during the menopause transition, while FSH levels in all women appear to remain high even at late postmenopause.

Reproducibility Of The Impact Of Menstrual Phase On Arterial Stiffness Over Two Consecutive Menstrual Cycles

Chronic increases in arterial stiffness are associated with increased risk of cardiovascular disease. Estrogen (E2), the primary female sex hormone, has been found to have cardioprotective effects, with several but not all, studies reporting a reduction in arterial stiffness in the late follicular (high E2; LF) phase compared to the early follicular (low E2; EF) phase of the menstrual cycle. Individual heterogeneity in the impact of phase on arterial stiffness and the consistency of these responses across consecutive cycles has yet to be explored. PURPOSE: to determine the impact of menstrual phase E2 fluctuations on arterial stiffness over two consecutive cycles. METHODS: 13 premenopausal women completed 4 experimental visits over 2 menstrual cycles (EFvisit 1, LFvisit 2, EFvisit 3, LFvisit 4). Central (CF) and peripheral (FF) PWV were measured twice during each visit using arterial tonometry. Participants were classified as positive, negative or non-responders, wherein positive responders experienced a reduction in PWV from EF to LF and negative responders experienced an increase in PWV from EF to LF that was greater than 2*typical error. RESULTS: At the group level, CF PWV did not differ between phases (p=0.11) or cycles (p=0.18; EFvisit 1=5.8±0.8, LFvisit 2=5.6±0.5, EFvisit 3=5.9±0.6, LFvisit 4=5.8±0.7). Likewise, FF PWV did not differ between phases (p=0.979) or cycles (p=0.58; EFvisit 1=8.1±1.0, LFvisit 2=8.1±1.1, EFvisit 3=7.9±1.4, LFvisit 4=8.0±1.3). Phase changes in E2 were not associated with phase changes in PWV in cycle 1 (CF: r=0.38, p=0.20; FF: r=0.11, p=0.73) or cycle 2 (FF: r=0.38, p=0.36). Classification of individuals as responders or non-responders revealed that no participants were consistently positive or negative responders for both cycles. CONCLUSION: At the group level, arterial stiffness was not impacted by menstrual phase over two cycles. Individual changes in arterial stiffness were inconsistent, with phase changes in cycle 1 not reflecting phase changes in cycle 2. Variability in phase changes in arterial stiffness does not appear to be driven by inter-individual differences in E2 fluctuation across phase. Future research is needed to investigate the mechanisms resulting in inter-individual variability in arterial stiffness and inconsistency between cycles. Funded by NSERC

Local sex steroid hormone milieu in the bovine oviduct ipsilateral and contralateral to preovulatory follicle or corpus luteum during the periovulatory phase

Estradiol-17β (E2) and progesterone (P4) regulate oviductal functions, providing a suitable environment for the transport and maturation of gametes, fertilization, and embryonic development. In addition to the E2 and P4 nuclear receptors, estrogen receptor (ESR) α and β, nuclear progesterone receptor (PGR), nongenomic mechanisms through G protein-coupled estrogen receptor (GPER1), and progesterone receptor membrane component (PGRMC) 1 and 2 mediate E2 and P4 actions. This study aimed to characterize the local endocrine environment of the oviduct by examining the oviductal E2 and P4 concentrations and their receptors' mRNA expression during the periovulatory phase. The bovine oviducts were collected in a slaughterhouse and the days postovulation were estimated according to state of the ovaries and the uterus. Samples of the ampulla and isthmus ipsilateral and contralateral to the preovulatory follicle or corpus luteum were collected on Days 19 to 21, Days 0 to 1, Days 2 to 4, and Days 5 to 7 of the estrous cycle. The effects of the estrous cycle phase and oviductal region (ampulla and isthmus) and side (ipsilateral and contralateral) were analyzed by 3-way ANOVA. Moreover, to clarify the regulatory mechanisms of the mRNA expression of hormone receptors, the effects of E2 and P4 on mRNA expression in the oviduct were examined by multiple linear regression. The oviductal endocrine milieu on Days 19 to 21 was characterized by an E2-dominant environment with high E2 and low P4, high ESR1 and PGR mRNA expression, and low ESR2, GPER1, and PGRMC2 mRNA expression, whereas the corresponding on Days 0 to 1 was characterized by the endocrine milieu without hormone dominance. The environment on Days 2 to 4 and Day 5 to 7 was characterized by opposite tendency of oviductal hormone concentrations and their receptors' mRNA expression to Days 19 to 21. Additionally, the ipsilateral oviduct had the more P4-dominant endocrine milieu, with lower E2 and higher P4 concentrations, and different expression of ESR1/2, GPER1, PGR, and PGRMC2 mRNA when compared with the contralateral oviduct on Days 2 to 4 and Days 5 to 7, except for PGRMC1. Although oviductal E2 and P4 influenced the mRNA expression of ESR1/2, GPER1, PGR, and PGRMC1/2, their effects were different between regions and sides. In summary, the oviductal endocrine milieu varies according to the estrous cycle phase and the oviductal region and side, which may be involved in the estrous cycle phase-specific and oviductal region-specific and side-specific functions.

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice.

Elevations in estrogen (17β-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERβ) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERβ into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERβ, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.SIGNIFICANCE STATEMENT Estrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances, such as alcohol. We investigated the mechanisms by which estrogen increases the response of neurons in the VTA to ethanol. We found that activation of the ERα increased the ethanol-induced excitation of VTA neurons. 17β-Estradiol-mediated enhancement of ethanol-induced excitation required the metabotropic glutamate receptor mGluR1. We also demonstrated that ERs in the VTA regulate binge-like alcohol drinking by female, but not male, mice. The influence of ERs on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

1589-P: Testosterone (T) in Early Puberty Predicts Visceral (VAT) and Subcutaneous Adipose Tissue (SAT) in the EPOCH (Exploring Perinatal Outcomes among Children) Cohort

Among adults, higher T predicts lower VAT in men and higher VAT in women, and higher E2 predicts greater SAT in women. The degree to which the association between adiposity and sex steroids is bidirectional is controversial. Using data from a cohort of adolescents, we examined whether sex steroid levels in early puberty influenced VAT and SAT deposition in adolescents. EPOCH participants (n=418, 209 girls and 209 boys) underwent sex steroid measures at visit 1 (mean age 10 years) and magnetic resonance imaging (MRI) assessments of VAT and SAT at visit 1 and visit 2 (mean age 17 years). Using mixed models, we examined whether T and E2 at visit 1 were associated with overall VAT and SAT levels from visit 1 to visit 2. Models adjusted for maternal education and income as well as children’s race, pubertal status, caloric intake, physical activity levels, insulin, leptin, and MRI-measured hepatic fat fraction. Among girls, each unit increase in T was associated with a higher overall VAT (0.16, 95% confidence interval [CI] 0.04, 0.28) and SAT (0.33, 95% CI 0.33, 1.52) levels. Each unit increase in E2 was associated with overall higher SAT (0.56, 95% CI 0.28, 0.83) levels. Among boys, undetectable T was associated with higher VAT (5.12, 95% CI 1.90, 8.36) and lower SAT (-18.85, 95% CI -36.23, -1.46) levels. We conclude that endogenous T and E2 during the pubertal transition significantly influence adolescent adipose tissue deposition in both boys and girls. Future investigations should examine how the use of exogenous sex hormones impacts adipose tissue deposition during this critical period. Additional follow-up is needed to determine how adolescent fat deposition impacts subsequent glucose tolerance in early adulthood. Disclosure C. Kim: Research Support; Spouse/Partner; Apple. K.K. Harrall: None. D.H. Glueck: None. D. Dabelea: None. Funding National Institutes of Health (R01DK068001)

Progesterone and Allopregnanolone Rapidly Attenuate Estrogen-Associated Mechanical Allodynia in Rats with Persistent Temporomandibular Joint Inflammation.

Temporomandibular joint disorder (TMD) is associated with pain in the joint (temporomandibular joint, TMJ) and muscles involved in mastication. TMD pain dissipates following menopause but returns in some women undergoing estrogen replacement therapy. Progesterone has both anti-inflammatory and antinociceptive properties, while estrogen’s effects on nociception are variable and highly dependent on both natural hormone fluctuations and estrogen dosage during pharmacological treatments, with high doses increasing pain. Allopregnanolone, a progesterone metabolite and positive allosteric modulator of the GABAA receptor, also has antinociceptive properties. While progesterone and allopregnanolone are antinociceptive, their effect on estrogen-exacerbated TMD pain has not been determined. We hypothesized that removing the source of endogenous ovarian hormones would reduce inflammatory allodynia in the TMJ of rats and both progesterone and allopregnanolone would attenuate the estrogen-provoked return of allodynia. Baseline mechanical sensitivity was measured in female Sprague–Dawley rats (150–175 g) using the von Frey filament method followed by a unilateral injection of complete Freund’s adjuvant (CFA) into the TMJ. Mechanical allodynia was confirmed 24 h later; then rats were ovariectomized or received sham surgery. Two weeks later, allodynia was reassessed and rats received one of the following subcutaneous hormone treatments over 5 days: a daily pharmacological dose of estradiol benzoate (E2; 50 μg/kg), daily E2 and pharmacological to sub-physiological doses of progesterone (P4; 16 mg/kg, 16 μg/kg, or 16 ng/kg), E2 daily and interrupted P4 given every other day, daily P4, or daily vehicle control. A separate group of animals received allopregnanolone (0.16 mg/kg) instead of P4. Allodynia was reassessed 1 h following injections. Here, we report that CFA-evoked mechanical allodynia was attenuated following ovariectomy and daily high E2 treatment triggered the return of allodynia, which was rapidly attenuated when P4 was also administered either daily or every other day. Allopregnanolone treatment, whether daily or every other day, also attenuated estrogen-exacerbated allodynia within 1 h of treatment, but only on the first treatment day. These data indicate that when gonadal hormone levels have diminished, treatment with a lower dose of progesterone may be effective at rapidly reducing the estrogen-evoked recurrence of inflammatory mechanical allodynia in the TMJ.

SUN-238 Estrogen Modulates Expression Levels of Gonadotropin-Releasing Hormone Receptor (GNRHR) in Immortalized Kisspeptin Neurons in Vitro

In female animals, ovarian estradiol (E2) can act as both a negative feedback inhibitor of GnRH secretion, as well as a positive feedback stimulator at the time of ovulation. Both of these E2-regulated mechanisms work via stimulation or repression of two distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While it is clear that AVPV KP neurons increase kiss1 expression during the preovulatory surge on proestrus, subsequent secretory mechanisms required for potentiation of GnRH surge release remain unclear. Two KP-secreting cell lines, KTaV-3, which demonstrate increased kiss1 expression under high E2 exposure, and KTaR-1, which exhibit kiss1 suppression under low E2 exposure, were used to probe the presence of GnRH receptor (GnRHR) expression under different E2 exposure conditions.KTaV-3 and KtaR-1 cells were treated with a range of doses of E2 (5-100pM) and/or progesterone (20nM) for varying durations (4-96h), exposed to steroid hormones either constitutively or via modulating levels over time, approximating concentration changes found during the murine estrous cycle. Following RNA isolation, cDNAs were probed with primers for gnrhr. Preliminary results in KTaV-3 cells reveal the expression of gnrhr is induced only following elevated (50-100pM) E2 treatment for 18-24h. These same E2 exposure conditions were also found to increase expression of the homeobox protein dlx3, a transcription factor required for GnRHR expression in pituitary gonadotropes. In Arc-derived KTaR-1 cells, gnrhr expression was observed only following decreases in E2 concentration, while dlx3 remained constitutively elevated in this cell line. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these observations suggest the potential for Kisspeptin neurons to respond to GnRH secretory changes under particular E2 exposure conditions, by modulating receptivity to GnRH at the level of the AVPV and/or Arcuate nuclei. We are continuing to explore the temporal parameters of this induction of GnRHR in KP cells, and if exposure of immortalized KP neurons to GnRH in vitro elicits expression and signaling changes in a time- and E2-dependent manner. Results will provide a more complete understanding of positive and negative feedback mechanisms required for normal neuroendocrine regulation of reproduction.

Combined effects of 17β-estradiol and copper on growth, biochemical characteristics and pollutant removals of freshwater microalgae Scenedesmus dimorphus

Contamination by estrogens and heavy metals can cause great environment concern and necessitate efficient approaches for their removals. In this study, the combined effects of 17β-estradiol (E2) and Cu(II) on microalgae growth and biochemical characteristics were investigated. Results showed that 1 mg/L Cu(II) promoted the growth of Scenedesmus dimorphus, while 2 mg/L Cu(II) exhibited growth inhibition, compared with the same concentration of E2. Biochemical characteristics including enzyme activities as well as the contents of chlorophyll, protein and carbohydrate were significantly affected by the coexistence of E2 and Cu(II) after 12 d of cultivation. S. dimorphus exhibited high E2 and Cu(II) removal efficiencies (89.9% of E2 and 76.6% Cu(II) under the coexistence of 0.5 mg/L E2 and 1 mg/L Cu(II), respectively). Lower concentration of Cu(II) might serve as a bridge during E2 removal by S. dimorphus while competitive adsorption of Cu(II) and E2 occurred under the condition of excessive Cu(II). Results could confirm that S. dimorphus was a potential bioresource for the effective removal of E2 and Cu(II).