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Les femmes occidentales passent maintenant plus d’un tiers de leur vie ménopausées. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne de troubles fonctionnels qui affectent la qualité de vie. La ménopause est également associée à un risque de fractures élevé, ainsi qu’à une augmentation des événements cardiovasculaires et des troubles métaboliques. Ces désordres fonctionnels peuvent être limités par l’administration d’un traitement hormonal de la ménopause (THM) à base d’œstrogènes. Cependant ces traitements ont des effets délétères, comme l’augmentation du risque de thrombose veineuse et de cancer du sein. L’amélioration du THM représente donc un enjeu majeur pour la santé des femmes. Une des stratégies d’optimisation du THM repose sur la modulation du récepteur alpha des œstrogènes (ERα) qui appartient à la famille des récepteurs nucléaires. En plus de son action classique comme facteur de transcription, il peut également agir à la membrane en induisant des voies de signalisation cytoplasmique. Le découplage des effets membranaire/nucléaire pourrait améliorer la balance bénéfice/risque dans l’action de ce récepteur. Le but de mon projet a été d’évaluer l’impact de ce découplage dans le développement des maladies cardiovasculaires et métaboliques. En combinant des modèles d’animaux invalidés pour les fonctions membranaires ou nucléaires de ERα et des approches pharmacologiques, ce travail a permis de montrer que la majorité des effets protecteurs des œstrogènes sur le plan vasculaire étaient relayés par le ERα nucléaire, avec un rôle minime des effets membranaires. Dans ce contexte, nous avons pu mettre en évidence que l’Estétrol (E4), un œstrogène naturel et endogène ciblant spécifiquement le ERα nucléaire, était capable de prévenir l’obésité en réponse à un régime gras chez la souris ovariectomisée et les désordres vasculaires et métaboliques qui lui sont associés. Cette étude s’inscrit dans une démarche plus globale de caractérisation des effets de l’Estétrol puisque cette molécule est développée en clinique comme nouveau THM. Contrairement aux œstrogènes utilisés actuellement, l’E4 n’induit pas les facteurs de la coagulation hépatique aux doses utilisées, ce qui pourrait limiter le risque thrombotique. Mes résultats montrent un effet bénéfique à la fois sur le plan vasculaire et métabolique de cet œstrogène. Une meilleure compréhension des mécanismes cellulaires et moléculaires impliqués dans ces effets pourrait permettre de positionner l’E4 comme le premier œstrogène naturel agissant comme un modulateur sélectif du ERα.

Objective The aim of this study was to select the minimum effective dose of estetrol (E4) for the treatment of vasomotor symptoms in postmenopausal women. Methods This was a multicenter, randomized, double-blind, placebo-controlled study. Postmenopausal women (n = 257, of whom 32 were hysterectomized) aged 40 to 65 years, with ≥7 moderate to severe hot flushes (HFs) per day, or 50 or more moderate to severe HFs weekly, received 2.5, 5, 10, or 15 mg E4, or placebo once-daily for a period of 12 weeks. Efficacy was assessed by recording the frequency and severity of HFs. Overall safety was assessed by recording adverse events, measuring endometrial thickness, and monitoring bleeding patterns. Treatment groups were compared using analysis of covariance. Results The frequency of moderate to severe HFs decreased with all E4 doses. The difference in the percentage change of weekly HF frequency was significant for 15 mg E4 versus placebo at both W4 (-66% vs -49%, P = 0.032) and W12 (-82% vs -65%, P = 0.022). The decrease in severity of HFs was significantly more pronounced for 15 mg E4 than for placebo at both W4 (-0.59 vs -0.33, P = 0.049) and W12 (-1.04 vs -0.66, P = 0.049); the other doses failed to achieve statistical significance. In nonhysterectomized women, endometrial thickness increased during treatment and normalized following progestin treatment at study completion. No endometrial hyperplasia was observed. Conclusions Estetrol 15 mg is considered to be the minimum effective daily oral dose for treatment of vasomotor symptoms. Its current seemingly favorable safety profile is further to be confirmed in phase 3 clinical development. : Video Summary:http://links.lww.com/MENO/A591.

Long-term estrogen deprivation (LTED) with tamoxifen (TAM) or aromatase inhibitors leads to endocrine-resistance, whereby physiologic levels of estrogen kill breast cancer (BC). Estrogen therapy is effective in treating patients with advanced BC after resistance to TAM and aromatase inhibitors develops. This therapeutic effect is attributed to estrogen-induced apoptosis via the estrogen receptor (ER). Estrogen therapy can have unpleasant gynecologic and nongynecologic adverse events. Here, we study estetrol (E4) and a model Selective Human ER Partial Agonist (ShERPA) BMI-135. Estetrol and ShERPA TTC-352 are being evaluated in clinical trials. These agents are proposed as safer estrogenic candidates compared with 17β-estradiol (E2) for the treatment of endocrine-resistant BC. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, docking and molecular dynamics simulations, human unfolded protein response (UPR) RT2 PCR profiler arrays, live cell microscopic imaging and analysis, and annexin V staining assays were conducted. Our work was done in eight biologically different human BC cell lines and one human endometrial cancer cell line, and results were compared with full agonists estrone, E2, and estriol, a benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. Our study shows the pharmacology of E4 and BMI-135 as less-potent full-estrogen agonists as well as their molecular mechanisms of tumor regression in LTED BC through triggering a rapid UPR and apoptosis. Our work concludes that the use of a full agonist to treat BC is potentially superior to a partial agonist given BPTPE's delayed induction of UPR and apoptosis, with a higher probability of tumor clonal evolution and resistance. SIGNIFICANCE STATEMENT: Given the unpleasant gynecologic and nongynecologic adverse effects of estrogen treatment, the development of safer estrogens for endocrine-resistant breast cancer (BC) treatment and hormone replacement therapy remains a priority. The naturally occurring estrogen estetrol and Selective Human Estrogen-Receptor Partial Agonists are being evaluated in endocrine-resistant BC clinical trials. This work provides a comprehensive evaluation of their pharmacology in numerous endocrine-resistant BC models and an endometrial cancer model and their molecular mechanisms of tumor regression through the unfolded protein response and apoptosis.

Estetrol (E4) is a natural fetal estrogenic steroid discovered in 1965 at the Karolinska Institutet in Stockholm (Sweden) [1]. It was studied for a period of 20 years originally as a biomarker of f...

Les femmes occidentales passent maintenant plus d’un tiers de leur vie ménopausées. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne de troubles fonctionnels qui affectent la qualité de vie. La ménopause est également associée à un risque de fractures élevé, ainsi qu’à une augmentation des événements cardiovasculaires et des troubles métaboliques. Ces désordres fonctionnels peuvent être limités par l’administration d’un traitement hormonal de la ménopause (THM) à base d’œstrogènes. Cependant ces traitements ont des effets délétères, comme l’augmentation du risque de thrombose veineuse et de cancer du sein. L’amélioration du THM représente donc un enjeu majeur pour la santé des femmes. Une des stratégies d’optimisation du THM repose sur la modulation du récepteur alpha des œstrogènes (ERα) qui appartient à la famille des récepteurs nucléaires. En plus de son action classique comme facteur de transcription, il peut également agir à la membrane en induisant des voies de signalisation cytoplasmique. Le découplage des effets membranaire/nucléaire pourrait améliorer la balance bénéfice/risque dans l’action de ce récepteur. Le but de mon projet a été d’évaluer l’impact de ce découplage dans le développement des maladies cardiovasculaires et métaboliques. En combinant des modèles d’animaux invalidés pour les fonctions membranaires ou nucléaires de ERα et des approches pharmacologiques, ce travail a permis de montrer que la majorité des effets protecteurs des œstrogènes sur le plan vasculaire étaient relayés par le ERα nucléaire, avec un rôle minime des effets membranaires. Dans ce contexte, nous avons pu mettre en évidence que l’Estétrol (E4), un œstrogène naturel et endogène ciblant spécifiquement le ERα nucléaire, était capable de prévenir l’obésité en réponse à un régime gras chez la souris ovariectomisée et les désordres vasculaires et métaboliques qui lui sont associés. Cette étude s’inscrit dans une démarche plus globale de caractérisation des effets de l’Estétrol puisque cette molécule est développée en clinique comme nouveau THM. Contrairement aux œstrogènes utilisés actuellement, l’E4 n’induit pas les facteurs de la coagulation hépatique aux doses utilisées, ce qui pourrait limiter le risque thrombotique. Mes résultats montrent un effet bénéfique à la fois sur le plan vasculaire et métabolique de cet œstrogène. Une meilleure compréhension des mécanismes cellulaires et moléculaires impliqués dans ces effets pourrait permettre de positionner l’E4 comme le premier œstrogène naturel agissant comme un modulateur sélectif du ERα.

Context Luteinizing hormone–releasing hormone (LHRH) agonists have replaced estrogens for endocrine treatment of advanced prostate cancer (PC) because of cardiovascular side effects. The fetal estrogen estetrol (E4) may be safer for PC treatment and is expected to decrease testosterone (T) and prevent estrogen deficiency. Objective To investigate the safety and T-suppressive effect of E4 in healthy men. Design Double-blind, randomized, placebo-controlled, dose-escalating study. Setting The study was conducted at a phase I clinical unit (QPS, Netherlands). Participants Healthy male volunteers aged 40 to 70 years. Intervention(s) Three treatment cohorts of 15 volunteers with placebo (n = 5) and E4 (n = 10). Estetrol doses tested were 20, 40, and 60 mg/d. Subjects were treated for 4 weeks. Main Outcome Measures Subjective side effects, pharmacodynamic effects on hemostatic variables, lipids, glucose, bone parameters, and endocrine parameters related to T metabolism. Results Total and free T decreased dose-dependently and significantly. Nipple tenderness occurred in 40% and decrease of libido occurred in 30% of E4-treated men. The unwanted estrogenic effects on hemostasis were small, dose dependent, and in some cases significant. Lipid and bone parameters showed a favorable trend. Conclusion The effect of E4 on testosterone levels is insufficient for standalone PC treatment. Taking all clinical and pharmacodynamic variables into consideration, a daily dose of 40 mg E4 seems safe for further evaluation of endocrine PC treatment in combination with LHRH analogs.

Background information Estrogen therapy was the endocrine treatment of choice in postmenopausal women with advanced breast cancer for several decades since 1944. In the 1970s, estrogen therapy was replaced by tamoxifen. Tamoxifen showed similar regression rates but less toxicity as compared to estrogen therapy and was therefore considered preferred. Recently, estrogen therapy has gained new interest as several clinical studies showed clinical benefit in heavily pre-treated patients with advanced breast cancer after long-term estrogen deprivation. Estrogen therapy is an effective treatment for breast cancer but it has a negative safety reputation, especially related to the cardiovascular (CV) system. The fetal estrogen estetrol (E4) might be a new treatment option for patients with advanced breast cancer. It has less interference with liver function and is expected to be less harmful for the CV system compared to other estrogens whereas data from non-clinical and clinical studies suggest anti-tumor effects of E4 in breast tumors. Hypothesis The fetal estrogen E4 may have anti-tumor effects in patients with advanced ER+ breast cancer. It will most likely also reduce symptoms and signs of estrogen deficiency such as hot flushes, arthralgia, sleep disturbances, bone loss and cognition and improve patient's quality of life. Trial design and methods This is a multi-center, open-label, phase I/IIa, dose-escalation study with a 3 + 3 cohort design to determine the recommended dose of E4 for the treatment of patients with advanced breast cancer. Cohorts of at least 3 patients will receive doses of 20 mg, 40 mg and 60 mg E4 respectively, until the non-tolerable dose is determined or the maximum dose of 60 mg is reached. Patients will be treated once daily by oral administration for 12 weeks. In total 9-18 patients with advanced ER+ breast cancer will be enrolled at three centers in Germany. Objectives The main objectives of this study are to evaluate the safety, the effects on estrogen deficiency symptoms and the preliminary anti-tumor activity of E4 in patients with advanced breast cancer. Eligibility criteria Women who are more than 5 years postmenopausal, with ER+ and HER2-negative locally advanced and/or metastatic breast cancer are eligible for inclusion. In addition, patients should have failed on anti-estrogen treatment with tamoxifen and aromatase inhibitors. Patients with a history of venous or arterial thromboembolic disease or a known defect in the blood coagulation system will be excluded as well as patients who have used treatment with fulvestrant within 6 months prior to start of E4 treatment. Statistical methods Statistical tests used in this study are exploratory only. No confirmatory statistical test procedure is envisaged. Contact information For further information regarding the study, please contact Dr Carole Verhoeven (cv@pantarheibio.com). Citation Format: Verhoeven C, Schmidt M, Hönig A, Dünnebacke J, Dutman E, Krijgh J, Coelingh Bennink H. Estetrol for treatment of advanced ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-04-01.

Estetrol (E4) is a natural estrogenic steroid that is normally produced by human fetal liver. Recent research has demonstrated that it is a potent, orally bioavailable, natural selective estrogen receptor modulator; it has a moderate affinity for both human estrogen receptor alpha (ERα) and ERβ, with a preference for ERα. Clinical studies have demonstrated possible use as an estrogen in combined oral contraceptives (COC). COCs containing E4 and drospirenone (DRSP) showed a high acceptability, tolerability, and user satisfaction also when compared to COCs containing ethinylestradiol (EE). E4/DRSP effectively inhibits ovulation, with a similar effect on endometrium thickness than that of EE-containing COCs. Low doses (15 mg) of E4 with DRSP (3 mg) showed promising results in term of bleeding pattern and cycle control, also when compared to other COCs containing synthetic estrogens. Moreover, the association has limited effects on serum lipids, liver, SHBG levels, and carbohydrate metabolism. This combination also could drive a lower risk of venous thromboembolism than EE-containing COCs. In this review, we will summarize the actual knowledge about the new E4-containing contraceptive. Further large-scale studies in the full target population are needed to provide more insights into the cardiovascular safety profile and user satisfaction of E4/DRSP.

Objective: \ud Review evidence to guide management of menopausal signs and symptoms in women after breast cancer and make recommendations accordingly.\ud Evidence: \ud Randomized controlled clinical trials, observational studies, evidence-based guidelines, and expert opinion from professional societies.\ud Background: \ud Symptoms and clinical problems associated with estrogen depletion—sleep disorders, vulvovaginal atrophy (VVA), vasomotor symptoms (VMS), mood changes, depressive symptoms, cardiovascular disease, osteopenia, and osteoporosis—confront the estimated 9.3 million breast cancer survivors globally.\ud Recommendations: \ud Following breast cancer, women should not generally be treated with menopausal hormone therapy or tibolone but should optimize lifestyle. Women with moderate to severe symptoms may benefit from mind–brain behavior or nonhormone, pharmacologic therapy. The selective serotonin/noradrenaline reuptake inhibitors and gabapentenoid agents improve VMS and quality of life. For osteoporosis, nonhormonal agents are available. Treatment of VVA remains an area of unmet need. Low-dose vaginal estrogen is absorbed in small amounts with blood levels remaining within the normal postmenopausal range but could potentially stimulate occult breast cancer cells, and although poorly studied, is not generally advised, particularly for those on aromatase inhibitors. Intravaginal dehydroepiandrosterone and oral ospemiphene have been approved to treat dyspareunia, but safety after breast cancer has not been established. Vaginal laser therapy is being used for VVA but efficacy from sham-controlled studies is lacking. Therapies undergoing development include lasofoxifene, neurokinin B inhibitors, stellate ganglion blockade, vaginal testosterone, and estetrol.\ud Conclusions: \ud Nonhormone options and therapies are available for treatment of estrogen depletion symptoms and clinical problems after a diagnosis of breast cancer. Individualization of treatment is essential.

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20μl of whole blood from tested subjects. In this study, we present the development and validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof-of-concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

Estetrol (E4) is an estrogen produced exclusively by the human fetal liver during pregnancy. In this study the pharmacodynamic effects of escalating doses of E4 in postmenopausal women were investigated.This was a partly randomized, open-label, multiple-rising-dose study in 49 postmenopausal women. Participants were randomized to receive either 2 mg E4 or 2 mg estradiol-valerate (E2 V) for 28 days. Subsequent dose-escalation groups were (non-randomized): 10, 20 and 40 mg E4. Blood samples were collected regularly for measuring endocrine and hemostasis variables, lipids and lipoproteins, fasting glucose and bone turnover markers.Estetrol treatment resulted in a decrease of follicle-stimulating hormone and luteinizing hormone and an increase of sex-hormone binding globulin. Changes in hemostasis variables were small. A lowering effect on low-density lipoprotein cholesterol was accompanied with an increase in high-density lipoprotein cholesterol and no or minimal changes in triglycerides. The considerable decrease in osteocalcin levels in the three highest E4 dose groups and the small decrease in C-telopeptide levels were comparable to the E2 V control group and suggest a preventive effect on bone loss. All changes observed were dose-dependent.In this study, estetrol treatment showed dose-dependent estrogenic effects on endocrine parameters, bone turnover markers, and lipids and lipoproteins. The effect on triglycerides was small as were the effects on hemostatic variables. These results support the further investigation of estetrol as a candidate for hormone therapy. Quantitatively, the effects of 10 mg estetrol were similar to the study comparator 2 mg estradiol valerate.

BACKGROUND: Estetrol (E4) is a natural estrogen produced solely during human pregnancy. E4 is suitable for clinical use since it acts as a selective estrogen receptor modulator. In clinical trials E4 has been seen to have little or no effect on coagulation. Hence, it is interesting to investigate whether E4 alters endothelial-dependent fibrinolysis. OBJECTIVES: We studied the effects of E4 on the fibrinolytic system and whether this could influence the ability of endothelial cells to migrate. In addition, we compared the effects of E4 with those of 17β-estradiol (E2). STUDY DESIGN: Human umbilical vein endothelial cells (HUVEC) were obtained from healthy women. Expression of plasminogen-activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) proteins was evaluated by Western blot analysis. Endothelial cell migration was studied by razor-scrape horizontal and multiwell insert systems assays. RESULTS: E4 increased the expression of t-PA, u-PA and PAI-1 in HUVEC, but less so than did equimolar amounts of E2. The effects of E4 on t-PA, u-PA and PAI-1 were mediated by the induction of the early-immediate genes c-Jun and c-Fos. E4 in combination with E2 antagonized the effects induced by pregnancy-like E2 concentrations but did not impair the effects of postmenopausal-like E2 levels. We also found that the increased synthesis of PAI-1, u-PA and t-PA induced by E2 and E4 is important for horizontal and three-dimensional migration of HUVEC. CONCLUSIONS: These results support the hypothesis that E4 acts as an endogenous selective estrogen receptor modulator (SERM), controlling the fibrinolytic system and endothelial cell migration. Fil: Montt Guevara, Maria Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Università degli Studi di Pisa; Italia Fil: Palla, Giulia. Università degli Studi di Pisa; Italia Fil: Spina, Stefania. Università degli Studi di Pisa; Italia Fil: Bernacchi, Guja. Università degli Studi di Pisa; Italia Fil: Cecchi, Elena. Università degli Studi di Pisa; Italia Fil: Campelo, Adrián Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina. Università degli Studi di Pisa; Italia Fil: Shortrede, Jorge Eduardo. Università degli Studi di Pisa; Italia Fil: Canu, Alessio. Università degli Studi di Pisa; Italia Fil: Simoncini, Tommaso. Università degli Studi di Pisa; Italia

Estetrol (E4) has strong antioxidative, neurogenic and angiogenic effects in neural system resulting in the attenuation of neonatal hypoxic–ischemic encephalopathy. We aimed to define the role of estrogen receptors in E4-dependent actions in neuronal cell cultures and prove the promyelinating effect of E4. In vitro the antioxidative and cell survival/proliferating effects of E4 on H2O2-induced oxidative stress in primary hippocampal cell cultures were studied using different combinations of specific inhibitors for ERα (MPP dihydrochloride), ERβ (PHTTP), GPR30 (G15) and palmytoilation (2-BR). LDH activity and cell survival assays were performed. In vivo the promyelinating role of different concentrations of E4 (1 mg/kg/day, 5 mg/kg/day, 10 mg/kg/day, 50 mg/kg/day) was investigated using the hypoxic–ischemic brain damage model in the 7-day-old immature rats before/after the induction of hypoxic–ischemic insult. Myelin basic protein (MBP) immunostaining was performed on brain coronal sections. Our results show that LDH activity is significantly upregulated in cell cultures where the E4’s effect was completely blocked by concomitant treatment either with ERα and ERβ inhibitors (MPP and PHTPP, respectively), or ERα and ERβ inhibitors combined with 2-BR. Cell survival is significantly downregulated in cell cultures where the effect of E4 was blocked by ERβ inhibitor (PHTTP) alone. The blockage of GRP30 receptor did affect neither LDH activity nor cell survival. MBP immunostaining is significantly upregulated in E4-pretreated groups at a concentration of 5 mg/kg/day and 50 mg/kg/day E4, whereas the MBP-positive area OD ratio is significantly increased in all the E4-treated groups. E4’s antioxidative actions mostly depend on ERα and ERβ, whereas neurogenesis and possibly promyelinating activities might be realized through ERβ.

Estetrol (E4) acts as a typical estrogen (E) agonist in stimulating the endometrium, and when administered to postmenopausal women in various doses, reverses vaginal atrophy and decreases vasomotor symptoms. However, in contrast to other Es administered orally, E4 demonstrates a minimal hepatic effect and does not stimulate breast proliferation. Data in pre and postmenopausal women show minimal stimulatory effects on triglycerides, sex hormone binding globulin, corticosteroid-binding globulin, and angiotensinogen. Similarly, E4 exerts minimal effects on coagulation markers, and when combined with drospirenone as a combined oral contraceptive does not increase the generation of thrombin as measured by the activated protein C resistance ratio, unlike the use of ethinylestradiol. Because of the tissue selectivity of E4, this study sought to better delineate the molecular mode of action of E4. Selective E Receptor Modulators (SERMs) exhibit specific cell and tissue estrogenic, neutral or anti-estrogenic activities. SERMs interact with the ligand binding domain of the E Receptor alpha (ERα) in a way that is distinct from that of E. By preventing the kinking of helix 12 in ERα, SERMs recruit other co-receptor activators and inhibitors that are responsible for SERM action. We compared in replicates the binding pattern and affinities of 154 motifs of ERα co-regulator activators and repressors to complexes of ERα with Estradiol (E2), Estriol (E3) or E4 in a specific Pam Gene assay (1).The pattern of co-receptor binding in the presence of E4 was identical to that elicited by E2 and E3, but with a 50-fold lower potency. This pattern is strikingly distinct from that observed with 4-hydroxytamoxifen or raloxifen. These data together with previously published studies of the crystalline structures of ERα with E2, E3 or E4 complexes confirm that E4 interacts with nuclear ERα in a manner identical to that of the other Es (2). Previous studies have shown that E4 inhibits the activity of the membrane ERα, and blocks the stimulatory action of E2 in this membrane ERα subpopulation. In conclusion, E4 is a unique E with specific actions in tissues, demonstrating absence of specific membrane receptor effects, and no interaction with ERα in the way characteristic of SERMs. The unique profile of E4 activity with lower hepatic effects (minimal or no effects on triglycerides and coagulation) may ultimately translate into safer clinical use. 1) Broekema et al. Endocrinology. 2018; 159:2397. 2) Abot et al. EMBO Mol Med. 2014; 6:1328 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Menopausal symptoms have a substantial effect on the quality of life of many women; hence, investigations for the amelioration of menopausal symptoms continue to be necessary. The two main approaches to the amelioration of symptoms are hormone therapy (HT) and non-hormonal therapy.This review provides a background for understanding the types of menopausal symptoms and their underlying physiology. The early clinical development of natural estrogen (estetrol, E4), neurokinin 3 receptor (NK3R) antagonists, and other non-hormonal therapies are covered. The status and outcome of these novel treatment modalities are also discussed.The recent observation in the Women's Health Initiative (WHI) trials that HT was not associated in the long-term with all-cause mortality, brings renewed interest in the development of new treatment modalities in postmenopausal women. Estetrol (E4), a native estrogen with selective action in tissues, is a potential next-generation HT with improved cardiovascular and breast safety. NK3R antagonists may become an interesting new modality for the amelioration of hot flushes in women with contraindications to estrogens.

Estrogens (E), in oral contraceptives (COCs) and hormone replacement therapy (HRT) drugs used for the relief of climacteric symptoms of menopause, increase the synthesis of clotting factors, decrease the levels of coagulation inhibitors, and increase the risk of venous thromboembolic events (VTE). Ischemic stroke incidence in postmenopausal women during HRT use is also increased and is probably due to a thrombotic event. This suggests that a safer estrogen may reduce stroke and VTE incidence, with lower impact on hemostasis.

Hematopoietic stem cells provide continuous supply of all the immune cells, through proliferation and differentiation decisions. These decisions are controlled by local bone marrow environment as well as by long-range signals for example endocrine system. Sex dependent differential immunological responses have been described under homeostasis and disease conditions. Females show higher longevity than male counterpart that seems to depend on major female sex hormone, estrogen. There are four estrogens - Estrone (E1), estradiol (E2), Estriol (E3) and Estetrol (E4) that spatially and temporarily present during different female reproductive phases. In this review, we discussed recent updates describing the effects of estrogen on HSC, immune cells and in bone biology. Estradiol (E2) being a major/abundant estrogen is extensively investigated, while effects of other estrogens E1, E3 and E4 are started to unravel recently. Furthermore, clinical effect of estrogen as hormone therapy is discussed in HSC and immune cells perspectives. The data presented in this review is compiled by searches of PubMed, database of American Cancer Society (ACS). We have included article from September 1994 to March 2020 as covering all article in chronological order is not fissile so we included relevant article with substantial information in this specific area of research by using the search term (alone or in combination) estrogen, hematopoietic stem cell, immune cells, gender difference, estrone, estriol, estetrol, therapeutic application, pregnancy, effect on bone.

Objective Estetrol (E4) is a natural fetal estrogen. The safety of increasing doses of E4 and its preliminary effects on the vagina, endometrium and menopausal vasomotor symptoms were investigated. Study design This was a partly randomized, open-label, multiple-rising-dose study in 49 postmenopausal women. Subjects with an intact uterus were randomized to receive either 2 mg E4 or 2 mg estradiol-valerate (E2V) for 28 days. Subsequent dose-escalation groups (non-randomized) were: 10 mg E4 (intact uterus and ≥35 hot flushes/week); and 20 mg and 40 mg E4 (hysterectomized subjects). Main outcome measure Adverse events (AEs) and vaginal cytology were evaluated in all treatment groups; hot flushes/sweating and endometrial proliferation were analyzed with 2 and 10 mg E4 and 2 mg E2V. Results Estetrol appeared to be safe, without serious drug-related AEs. In all the groups there was a clear shift from parabasal to superficial vaginal cells, indicating an estrogenic effect and a potential for the treatment of vulvovaginal atrophy. The endometrial thickness remained stable in the 2 mg E4 group and increased with E2V and 10 mg E4. A decrease in the mean number of hot flushes and sweating was seen with 2 and 10 mg E4 and 2 mg E2V. Conclusions Estetrol in a dose range of 2–40 mg per day improved vaginal cytology and vasomotor symptoms in postmenopausal women. Endometrial proliferation occurred with the 10 mg dose. Estetrol seems a safe and suitable candidate to develop further for hormone therapy.

// Ekaterine Tskitishvili 1 , Christel Pequeux 1 , Carine Munaut 1 , Renaud Viellevoye 2 , Michelle Nisolle 3 , Agnes Noel 1 and Jean-Michel Foidart 1 1 Laboratory of Development Biology and Tumor, GIGA-Cancer, Department of Obstetrics and Gynecology/Department of Clinical Sciences, University of Liege, Liege 1, Belgium 2 Neonatal Intensive Care Unit, Department of Pediatrics, University of Liege, Liege 1, Belgium 3 Department of Obstetrics and Gynecology, University of Liege, Liege1, Belgium Correspondence to: Ekaterine Tskitishvili, email: // Keywords : neonatal hypoxic-ischemic encephalopathy, hippocampus, cortex, estetrol, estradiol, Pathology Section Received : March 16, 2016 Accepted : May 17, 2016 Published : May 25, 2016 Abstract Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatal hypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated.The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival. In vivo pretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.

Modern contraception began in the 1960s with the introduction of combined oestrogen and progestogen (COC) pills and intrauterine devices made of thermoplastic. By the end of that decade, it had become apparent that COCs containing over 50 µg of mestranol or ethinyl-estradiol carried a risk of producing venous thrombosis and thromboembolism

Objective: Inhibition of GH signaling has been associated with increased longevity and cancer resistance. As the fetal estrogen estetrol was also linked to longevity and cancer resistance, we hypothesized that these effects of estetrol are mediated by inhibition of GH activity. Design: The CNMm-6TR mammary cell line, stably transfected with doxycycline (DOX)-inducible GHR (CNMm-cGHR cells), HEK293 cell line transfected with rGHR and the human T47D cell line were used to assess effects of estetrol on GH signal transduction. Proliferation was measured using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT). Spi-luciferase reporter activity, responsive to phosphorylated Stat5, was used to assess GH signaling. GHR mRNA expression was normalized to a reference gene. Results: Treatment of CNMm-cGHR cells with GH resulted in decreased proliferation rates. No effect was seen by the co-inhibition with estetrol. Using the spi-luc reporter activity, it was shown that GH activates the GHR/Jak2/ Stat5 pathway. Estetrol partially inhibited this activation. In the HEK-rGHR cells, estetrol was able to inhibit the GH induced activation of the spi-luc reporter, but only in the presence of an ERα expression plasmid. Effects of estetrol on the expression of GHR mRNA was assessed in T47D cells, a cell-line expressing ER, PR and GHR under natural promoters. After incubation with estetrol, a mean 4-fold inhibition of GHR mRNA expression was observed. Conclusion: Using different cell systems, it was shown that estetrol is able to inhibit GH signaling, a process dependent on the presence of the Estrogen Receptor (ER). Estetrol may therefore be used to inhibit GH-induced effects on breast cancer proliferation.

Objective: Inhibition of GH signaling has been associated with increased longevity and cancer resistance. As the fetal estrogen estetrol was also linked to longevity and cancer resistance, we hypothesized that these effects of estetrol are mediated by inhibition of GH activity. Design: The CNMm-6TR mammary cell line, stably transfected with doxycycline (DOX)-inducible GHR (CNMm-cGHR cells), HEK293 cell line transfected with rGHR and the human T47D cell line were used to assess effects of estetrol on GH signal transduction. Proliferation was measured using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT). Spi-luciferase reporter activity, responsive to phosphorylated Stat5, was used to assess GH signaling. GHR mRNA expression was normalized to a reference gene. Results: Treatment of CNMm-cGHR cells with GH resulted in decreased proliferation rates. No effect was seen by the co-inhibition with estetrol. Using the spi-luc reporter activity, it was shown that GH activates the GHR/Jak2/ Stat5 pathway. Estetrol partially inhibited this activation. In the HEK-rGHR cells, estetrol was able to inhibit the GH induced activation of the spi-luc reporter, but only in the presence of an ERα expression plasmid. Effects of estetrol on the expression of GHR mRNA was assessed in T47D cells, a cell-line expressing ER, PR and GHR under natural promoters. After incubation with estetrol, a mean 4-fold inhibition of GHR mRNA expression was observed. Conclusion: Using different cell systems, it was shown that estetrol is able to inhibit GH signaling, a process dependent on the presence of the Estrogen Receptor (ER). Estetrol may therefore be used to inhibit GH-induced effects on breast cancer proliferation.

Background Although estrogen receptor α ( ER α) acts primarily as a transcription factor, it can also elicit membrane‐initiated steroid signaling. Pharmacological tools and transgenic mouse models previously highlighted the key role of ER α membrane‐initiated steroid signaling in 2 actions of estrogens in the endothelium: increase in NO production and acceleration of reendothelialization. Methods and Results Using mice with ER α mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ER α plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear versus membrane‐initiated steroid signaling in the arterial protection conferred by ER α. ERaC451A mice were fully responsive to estrogens to prevent atheroma and angiotensin II –induced hypertension as well as to allow flow‐mediated arteriolar remodeling. By contrast, ER α AF 20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ER α with estetrol was able to prevent hypertension and to restore flow‐mediated arteriolar remodeling. Conclusions Altogether, these results reveal an unexpected prominent role of nuclear ER α in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ER α agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women.

Neonatal Hypoxic-Ischemic Encephalopathy (HIE), a brain disease due to brain hypoxia along with ischemia and reduced cerebral blood flow, is one of the primary reasons of severe injury among babies prematurely born. No efficacy treatment is available to the present day. Estetrol (E4), a major estradiol metabolite, has an important role in the brain development and protection. The aim of this study is to develop new injectable liposome and drug-in-cyclodextrin-in-liposome (DCL) formulations, encapsulating E4 in order to enhance its crossing through the blood-brain barrier (BBB). Liposome and DCL formulations were prepared and were physiochemically characterized. Stability in foetal bovine serum (FBS) was evaluated. LDH and MTS tests on endothelial, neuronal and BBB model cells, as well as hemocompatibility of the nanovectors were performed in vitro. In vitro BBB passage was evaluated using human BBB cell line (hCMEC/D3). All the formulations had average particle size below 150 nm, polydispersity index below 0.10 and ζ potential around +30 mV. The encapsulation efficacy for liposomes was between 3% and 10% while those of DCL are between 15% and 35%. The effect of liposome and DCL formulations on cell viability and integrity was evaluated. The results showed no toxic effects on all the tested cell lines. Hemocompatibility tests showed no hemolysis, platelet aggregation or effects on coagulation, confirming the possibility of the formulations to be intravenously administrated. BBB passage tests highlighted the capability of the formulations to pass the BBB and reach the brain. Therefore, the formulations are promising drug delivery system to target estrogens to the brain, due to their physiochemical characteristics.