E2 Therapy Research Articles

Tamoxifen Like Estradiol Attenuates Systemic Inflammation in Young and Aged Female Mice Fed with High-Fat Diet

Background: Consumption of a high-fat diet (HFD) is associated with an increased incidence of inflammatory diseases and metabolic disorders. Also, these disorders will increase in women with aging and menopause, which is probably due to the reduced role of estradiol (E2). Selective estrogen modulators including tamoxifen (TAM), which acts through estrogen receptors, have important metabolic effects. This study aimed to determine whether TAM and E2 have protective effects on inflammation caused by HFD in young and aged mice. Methods: Four-month-old (Sham and ovariectomized [OVX]) and 20-month-old female C57BL/6J mice were used in this study. After feeding them with HFD for 12 weeks, they were divided into nine groups consisting of Sham+Oil, Sham+TAM, Sham+E2, OVX+Oil, OVX+TAM, OVX+E2, Aged+Oil, Aged+TAM, and Aged+E2. TAM and E2 were injected subcutaneously every four days for four weeks. At the end of the experiments, the mice’s blood was sampled. The serum cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) were also determined using ELISA kits. Results: The results revealed that HFD increased inflammation by reducing IL-10 and increasing TNF-a/IL-10 and IL-6/IL-10 ratio in young and aged mice, and TAM and E2 therapy resulted in a significant decrease in TNF-α and IL-6, and an increase in IL-10 in young and aged mice. Conclusion: In conclusion, the results of this study indicated that TAM, in addition to being used as an anticancer drug, can reduce HFD-induced inflammation in both young and aged mice. Therefore, probably it is a good candidate to substitute E2.

The effect of daidzein on renal injury in ovariectomized rats: interaction of angiotensin receptors and long non-coding RNAs H19, GAS5, MIAT, and Rian.

Renin-angiotensin system (RAS) is prominently associated with renal pathophysiology in postmenopausal women. Long non-coding RNAs (lncRNAs) H19, GAS5, MIAT, and Rian have been linked to the pathogenesis of renal injury. This study aimed to evaluate the beneficial effects of daidzein on unilateral ureteral obstruction (UUO) induced-renal injury in ovariectomized (OVX) rats through interaction with angiotensin AT1, Mas receptors, and lncRNAs. 84 female rats were ovariectomized (OVX) two weeks before performing obstruction of the left kidney ureter (UUO). The animals were then randomly divided into four main groups (n=21): Sham+DMSO, UUO+DMSO, UUO+17β-Estradiol (E2) (positive control), and UUO+daidzein. Each main group comprised three subgroups (n=7) and were treated with saline, A779 (MasR antagonist), or losartan (AT1R antagonist) for 15 days. On day 16, the animals were euthanized, and the left kidneys were harvested for histopathology and lncRNAs expression assays. UUO significantly increased kidney tissue damage score (KTDS) in the UUO rats, increased the expression of H19 and MIAT, and decreased the expression of GAS5 and Rian. Daidzein alone and in co-treatment with losartan or A779 reversed these effects. Daidzein with 1 mg/kg dose was more effective than E2. Daidzein alone and in co-treatment with A779 and losartan improved renal injury in UUO rats and recovered dysregulated expression of UUO-related lncRNAs through modulating MasR and AT1R receptors, associating with modulation of the expression of lncRNAs. Daidzein could be considered a renoprotective phytoestrogen substitute for E2 therapy in postmenopausal women suffering from renal diseases.

Timing Matters: Effects of Early and Late Estrogen Replacement Therapy on Glucose Metabolism and Vascular Reactivity in Ovariectomized Aged Wistar Rats.

Cardiovascular disease incidence increases after menopause due to the loss of estrogen cardioprotective effects. However, there are conflicting data regarding the timing of estrogen therapy (ERT) and its effect on vascular dysfunction associated with impaired glucose metabolism. The aim of this work was to evaluate the effect of early and late ERT on blood glucose/insulin balance and vascular reactivity in aged ovariectomized Wistar rats. Eighteen-month-old female Wistar rats were randomized as follows: (1) sham, (2) 10-week postovariectomy (10 w), (3) 10 w postovariectomy+early estradiol therapy (10 w-early E2), (4) 20-week postovariectomy (20 w), and (5) 20-week postovariectomy+late estradiol therapy (20 w-late E2). Early E2 was administered 3 days after ovariectomy and late therapy after 10 weeks, in both groups. 17β-Estradiol (E2) was administered daily for 10 weeks (5 μg/kg/day). Concentration-response curves to angiotensin II, KCl, and acetylcholine (ACh) were performed. Heart rate (HR), diastolic and systolic blood pressure (DBP and SBP), glucose, insulin, HOMA-IR, and nitric oxide (NO) levels were determined. Higher glucose levels were found in all groups compared to the sham group, except the 20 w-late E2 group. Insulin was increased in all ovariectomized groups compared to sham. The HOMA-IR index showed insulin resistance in all ovariectomized groups, except for the 10 w-early E2 group. The 10 w-early E2 group increased NO levels vs. the 10 w group. After 10 w postovariectomy, the vascular response to KCl and Ach increases, despite early E2 administration. Early and late E2 treatment decreased vascular reactivity to Ang II. At 20-week postovariectomy, DBP increased, even with E2 administration, while SBP and HR remained unchanged. The effects of E2 therapy on blood glucose/insulin balance and vascular reactivity depend on the timing of therapy. Early ERT may provide some protective effects on insulin resistance and vascular function, whereas late ERT may not have the same benefits.

Predictors of Alzheimer’s disease risk in women with bilateral oophorectomy from the UK Biobank

AbstractBackgroundTwo‐thirds of individuals with Alzheimer’s disease (AD) are women and at age 45, the lifetime risk for AD is twice as high in women compared to men (Alzheimer’s Association, 2019). Early life events affecting 17β‐estradiol (E2) may be a mechanism behind this sex difference. Bilateral oophorectomy (BO) predicts cognitive decline, and neurodegeneration, and increased risk of AD in later life—adverse outcomes that are mitigated with E2 therapy (Gervais et al., 2020; Kantarci et al., 2018; Rocca et al., 2007; Zeydan et al., 2019). Our aim was to investigate compounding risk factors for AD in women with BO using UK Biobank data.MethodWe studied women aged 60 or older who had BO prior to age 49 with an AD diagnosis (n = 53) and without an AD diagnosis (n = 5418) from the UK Biobank (Sudlow et al. 2015). Data were analyzed using Firth’s bias‐reduced logistic regression in R 3.6.1 (R Core Team, 2019).ResultParticipants were on average 63.7 years old and 43.4 years old at BO. Women with BO who developed AD: (a) were significantly older, (b) had fewer years of education, (c) had an earlier age at BO, (d) less frequently used any form of hormone therapy (HT), and (e) were more frequently apolipoprotein E4 (APOE4) carriers. As age increased, AD risk increased by 25%; it deceased by 10% with greater years of education. Women with BO and an APOE4 allele were 5.2 times as likely to develop AD than those without an APOE4 allele. Finally, ever using any form of HT decreased AD risk by 54%.ConclusionThis research fills a gap in understanding predictors of AD in a high‐risk group of women with early E2 loss. The heightened AD risk for women with BO and APOE4 is of note, as prior work suggests women with APOE4 may benefit less from HT (MacLusky, 2004). Further, the reduction in AD risk with any HT provides added support for HT in mitigating cognitive decline. Although details on HT formulation were unavailable, the specific use of E2‐based therapy may further decrease AD risk (Sherwin & Henry, 2008).

Evaluation of pharmacokinetics and safety of a long-term estradiol-releasing stent in rat uterine.

PurposeIntrauterine adhesion (IUA), often leading to gynecological complications including amenorrhea, abdominal pain and infertility, is frequently induced by injuries to the endometrium. Hence it would be of great benefit to take efforts to prevent adhesion after intrauterine operations. Orally administration of 17β-estradiol (E2) is commonly used to promote endometrium regeneration, but is limited by low concentrations at the injured sites. We aim at preparing an E2-releasing uterine stent, which could improve the efficiency of E2 therapy and be utilized for IUA prevention.MethodsWe designed a silicone rubber stent, which could be implanted in the uterine cavity and continuously release E2 in long term. Stents were placed in rodent uterine, and removed at different time points. Remaining E2 in stent was measured by high performance liquid chromatography (HPLC), and organ E2 concentrations were detected by enzyme-linked immuno sorbent assay (ELISA). Endometrium morphology was examined by histological staining of paraffin sections.ResultsOur stent showed a controlled release of E2 in rodent uterine for over 60 days, and significantly increased E2 concentration in serum and in situ uterine. After the stent was removed from uterine, E2 rapidly reverted to a normal level. Also, the stent did not induce pathological changes in endometrium.ConclusionsThe uterine stent provided abundant local E2 in uterine cavity with satisfactory safety. The silicone rubber based E2-releasing uterine stent could be further advanced by adjusting its shape and E2 load for its clinical application, and might promisingly help lowering the incidence of IUA.

Selective estrogen receptor α and β antagonist aggravate cardiovascular dysfunction in type 2 diabetic ovariectomized female rats.

Type 2 diabetes (T2D) is a major risk factor for cardiovascular disorders (CVD), characterized by pathological diastolic as well as systolic dysfunction, ventricular dilation, and cardiomyocyte hypertrophy. CVD is the main cause of death in postmenopausal women. Estradiol (E2) has protective effects on cardiovascular function. The biological effects of E2 are mainly mediated by classical estrogen receptors (ERs). The present study aimed to investigate the cardioprotective effects of classical ERs in ovariectomized (OVX) diabetic female rats. T2D was induced in female rats by high-fat diet feeding along with a low dose of streptozotocin. Then diabetic animals were divided into eight groups: Sham-control, OVX, OVX+Vehicle (Veh), OVX+E2, OVX+E2+MPP (ERα antagonist), OVX+E2+PHTPP (ERβ antagonist), OVX+E2+Veh, OVX+E2+MPP+PHTPP. Animals received E2, MPP, and PHTPP every four days for 28days. At the end blood was collected, serum separated, and used for biochemical parameters. Heart tissue was used for cardiac angiotensin II and cytokines measurement. E2 treatment improved the metabolic disorders caused by T2D, and its receptor antagonists intensified the effects of T2D on the metabolic status. Also, E2 therapy decreased cardiac inflammatory cytokines, and MPP and PHTPP increased cardiac inflammation by increasing TNF-α and IL-6 and decreasing IL-10. Classical ERs have protective effects on diabetic hearts by improving the metabolic status and inflammatory balance.

Abstract PD1-01: Estrogen therapy induces R-loop-dependent DNA damage that can be enhanced by PARP inhibition to improve response in ER+ breast cancer

Abstract Treatment with estrogens can elicit anti-cancer effects in a subset of patients with anti-estrogen-resistant ER+ breast cancer. Clinical studies suggest that ~30% of postmenopausal patients with advanced ER+ breast cancer are likely to respond to estrogen, translating into thousands of patients who could benefit from this treatment each year. Despite the proven efficacy of estrogen therapy, it is under-utilized in the clinic due in large part to the unknown mechanism of action. We found that overexpression of ER confers resistance to estrogen deprivation through upregulation of estrogen-independent ER transcriptional activity. Further, this overexpression of ER confers sensitivity to treatment with the natural estrogen 17β-estradiol (E2) through stimulation of high levels of ER transcriptional activity. Using cell line- and patient-derived xenograft (PDX) models, including the novel PDX model CTG-3346, we aimed to determine the mechanism through which high levels of ER transcriptional activation elicit anti-cancer effects. RNA sequencing revealed a DNA damage response and G2/M checkpoint activation upon treatment with estrogen therapy. We further determined that treatment with E2 induces DNA damage specifically in ER-overexpressing cells, and knockdown of ER prevents E2-induced DNA damage. We performed a genome-wide CRISPR/Cas9 knockout screen to determine gene essentiality in the context of estrogen therapy. This revealed that knockout of CDK2/4/7/9, as well as MYC target genes, rescues from the cytotoxic effects of E2. This suggests that proliferation is required prior to apoptosis from E2 treatment. In agreement, E2 therapy initially induces a proliferative burst which precedes apoptosis. Blocking proliferation through pharmacological inhibition of CDK4/6 or knockdown of MYC prevented induction of DNA damage upon E2 treatment. MYC is an ER target gene, so high levels of ER transcriptional activity induce overexpression of MYC. Both ER and MYC are transcription factors that stimulate increased proliferation and transcription, so we hypothesized that E2/ER-induced DNA damage is a result of replication stress in ER-overexpressing cells. We found that E2 induces the formation of DNA:RNA hybrids called R-loops. R-loops occur when transcribed RNA anneals to the DNA template, leaving a displaced single-stranded DNA prone to breakage and replication stress. Reversal of R-loops through overexpression of the endonuclease RNase H1, which specifically degrades RNA in DNA:RNA hybrids, prevented the accumulation of DNA damage upon E2 treatment. Poly (ADP-ribose) polymerase-1 (PARP) has been implicated in the repair of R-loop-associated DNA damage. We therefore hypothesized that inhibition of PARP with FDA-approved drugs would enhance the effects of E2 therapy. Indeed, treatment with olaparib enhanced E2-induced DNA damage and growth-inhibition in vitro. In two PDX models, olaparib had minimal effects as a single agent but synergized with E2 to enhance anti-cancer effects. Collectively, this work implicates estrogen-induced DNA damage through R-loop formation and replication stress as the mechanism of action of estrogen therapy. Importantly, these results suggest that estrogen therapy should not be used in combination with CDK4/6 inhibitors. Our finding that E2 therapy synergizes with olaparib even in models that do not respond to olaparib monotherapy presents a novel use for PARP inhibitors outside of the setting of germline BRCA1/2 mutations. Citation Format: Nicole A. Traphagen, Steven Tau, Amanda Jiang, Jason D. Wells, Sarah R. Hosford, Abigail E. Goen, Eugene Demidenko. Estrogen therapy induces R-loop-dependent DNA damage that can be enhanced by PARP inhibition to improve response in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-01.

Abstract PD1-04: Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level

Abstract Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Methods: Characterization of estrogen regulation in the two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer. We succeeded in establishing estrogen-stimulating (SC31) and estrogen-suppressing (GS3) PDX models. In vivo tumor promotion or suppression by estrogen was confirmed through experiments by implanting E2 pellets in mice carrying SC31/GS3, and then single cell analysis was performed using SC31/GS3 tumors. To investigate whether GS3 would change its response to E2, we performed intermittent E2 treatment every 28 days (E2 pellet on/off every 28 days) and whole-genome RNA sequencing using GS3 tumors. Results: SC31 and GS3 behaved oppositely regarding estrogen-mediated tumor growth. Immunohistochemistry indicated that the number of ERα+ cells and Ki-67+ cells were increased in SC31 and decreased in GS3 after E2 treatment, but progesterone receptor+ cells appeared in both SC31 and GS3 after E2 treatment at the protein level. The E2-induced suppression of GS3 involves ERα, not ERβ, which was wild-type and not amplified. Single cell RNA sequencing analysis of these PDXs had revealed that E2 upregulated the expression of estrogen-regulated genes (e.g., PGR and AREG) in both SC31 and GS3. However, E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. These gene-expression changes occurred in both ESR1+ cells and ESR1- cells, demonstrating for the first time the influence of estrogen on ESR1- cells in ER+breast tumors. This result suggests that 100% ER positivity is not essential for endocrine response. E2 also upregulated a tumor suppressor gene, IL24, only in GS3. More IL24+ cells were ESR1+ and in G1 phase than IL24- cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. After three rounds of intermittent E2 treatment on GS3, an E2 independent growth developed. Approximately 60% of genes in the intermittent E2-treated sample had the same trend as E2-treated samples (mainly ER target genes), in which 40% of the genes behaved similarly to the placebo-treated sample (mainly cell cycle progression genes). Furthermore, lower levels of IL24 were linked to estrogen independence. Conclusions: Estrogen/ERα signaling increases the expression of estrogen-regulated genes, but it can modulate HR+ tumor growth in different manners. While E2 should activate ERα and regulate ESR1+ cells, our findings regarding ESR1- cells were new and suggested crosstalk between ESR1+ cells and ESR1- cells in both estrogen-stimulating and -suppressing ER+ tumors. Furthermore, our studies demonstrate the potential roles of tumor suppressor gene, IL24 in HR+ cancer. Our findings point to the need to identify biomarkers for patients with estrogen-suppressing tumors who can benefit from E2 treatment after AI resistance; measurements of ER and PR expression are insufficient. Expression of IL24 in AI-resistant tumors may be one such indicator for favorable response to E2 therapy. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Masafumi Nakamura, Andrea Bild, Shiuan Chen. Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-04.

Use of oral estradiol plus vaginal progesterone in healthy postmenopausal women

ObjectivesTo compare the effect of oral estradiol (E2) plus vaginal progesterone (P4) against placebo on endometrial thickness, endometrial biopsy pathology, cervical cytology and total cancer incidence among healthy postmenopausal women. Study designThis study is a sub-analysis of the Early versus Late Intervention Trial with Estradiol (ELITE), a randomized, double-blinded, placebo-controlled trial that previously demonstrated that hormone therapy (HT) was associated with less progression of subclinical atherosclerosis than placebo when therapy was initiated within 6 years after menopause but not when it was initiated 10 or more years after menopause. This sub-analysis included only ELITE participants with an intact uterus, who were randomized to either daily oral micronized 17-beta-E2 1 mg/day with 4% vaginal micronized P4 gel 45 mg/day for 10 days each month or placebo. Main outcome measuresParticipants were evaluated at baseline and annually during a median follow-up of 4.8 years for endometrial thickness as determined by pelvic transvaginal ultrasound followed by an endometrial biopsy when indicated, and cervical cytology and cancer incidence. ResultsOver up to 80 months of follow-up, participants randomized to oral E2 plus vaginal P4 had progressive and statistically significant increases in endometrial thickness (p<0.001), underwent more endometrial biopsies and had a higher rate of endometrial hyperplasia on endometrial biopsy compared with the placebo group. Due to the close follow-up of participants in the trial protocol, these abnormal findings were effectively treated. ConclusionOur results suggest that 10 days of vaginal P4 45 mg/day is insufficient to completely oppose the effect of oral E2 1 mg/day on the endometrium. Further studies are needed to test alternative doses or frequencies of administration of vaginal P4 for adequate endometrial protection from E2 therapy among postmenopausal women.ClinicalTrials.gov registration NCT00114517

Efficacy of Sex Steroid Therapy Without Progestin or GnRH Agonist for Gonadal Suppression in Adult Transgender Patients.

Testosterone (T) or estradiol (E2) are administered to suppress gonadal function in female-to-male (FTM) and male-to-female (MTF) transgender patients. How often sex steroids cause adequate suppression without GnRH agonist (GnRHa) or progestin therapy has not been reported. (1) To determine how often T and E2 therapy alone can effectively suppress gonadal function in MTF and FTM transgender patients, and (2) to determine the frequency and range of serum E2 levels above the normal male range in FTM patients receiving T therapy. Retrospective cohort study. Outpatient reproductive endocrinology clinic at an academic medical center. A total of 65 FTM and 33 MTF patients were included who were > 18 years of age and not receiving progestin or GnRHa therapy. Female-to-male patients were receiving T through injections or gel. Male-to-female patients were receiving oral or subcutaneous E2. In FTM patients the indicator of ovary suppression was amenorrhea. In MTF patients, the indicator of testes suppression was T levels <50 ng/dL. Median serum total T level for FTM patients was 712 ng/dL (range, 370-1164 ng/dL). On T therapy alone, 90.8% of patients achieved amenorrhea and 49.2% of patients had serum E2 levels above the normal range for women. For MTF patients, the median serum E2 level was 129.2 pg/mL (range, 75-197 pg/mL). On E2 therapy alone, 84.8% of MTF patients had adequate suppression of testicular function. Testosterone and E2 therapy are usually effective without progestin or GnRHa therapy to suppress gonadal function in transgender patients. Progestin and/or GnRHa therapy should only be initiated in those patients who do not have adequate gonadal suppression on optimized doses of T or E2 alone.

Unlike estrogens that increase PCSK9 levels post-menopause HSP27 vaccination lowers cholesterol levels and atherogenesis due to divergent effects on PCSK9 and LDLR

AimsThe estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE−/− mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. Methods and ResultsApoE−/− mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. ConclusionThe reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women – are there effects beyond cholesterol?

INITIAL EVALUATION OF SAFETY AND EFFICACY OF ADMINISTRATION OF ESTRADIOL (E2) BY SUBCUTANEOUS (SC) INJECTIONS TO MALE-TO-FEMALE (MTF) TRANSGENDER PATIENTS

The primary aim of this study was to determine if SC injection of E2 valerate could produce serum E2 levels within our clinic’s target therapeutic range for MTF patients (75-200 pg/mL) without causing significant adverse effects. Secondary aims were to assess: 1) if SC E2 can suppress serum total testosterone (T) levels (<50 ng/dL) as effectively as oral (PO) E2 , 2) if SC administration has less of a hepatic effect than PO, 3) if serum E2 levels remain within the target range between weekly injections and 4) if SC E2 is acceptable to patients. Retrospective chart review of MTF patients treated in Maine Medical Center Reproductive Endocrinology clinic. All charts of MTF patients seen from 2011-2020 were reviewed. Patients were allowed to choose PO, SC or transdermal (TD) administration. Inclusion criteria were age 18-79 years, PO or SC E2 therapy, and serum E2 and T levels measured by LabCorp (Calabasas CA). E2 valerate was injected SC using a 5/8’ 25g needle. 17β-E2 was administered orally. Serum E2, T, estrone (E1), and sex hormone binding globulin (SHBG) levels and fasting lipid panels were measured in SC and PO patients and values compared by Mann Whitney U test. Peak and nadir serum E2 concentrations were measured in a subgroup of SC patients. Serum sex steroid concentrations were measured by liquid chromatography/mass spectrometry. Local and systemic adverse effects were assessed by history and physical exam. The study was approved by the Maine Medical Center IRB. 102 charts were reviewed. Of 65 patients who were offered PO, TD or SC therapy (the SC option became available in May 2017), 23 choose SC. 61 patients were included in the final analysis, 23 in the SC group and 38 in the PO group. Median dose of E2 for the PO group was 4 mg/day [full range=1-12]; for SC E2 it was 4 mg/week [full range=2-6]. For the SC and PO groups the median E2 were 155.5 [75-291] (n=18) and 122 [76-208] pg/mL (n=38) respectively. Median and range of serum T levels in the SC and PO groups were 16 [4.5-82] (n=14) and 13 [4.8-515] (n=38) ng/dL respectively. E1 serum levels in the SC and PO groups were 49.0 [31.0-104.0] (n=10) and 495 [198-926] (n=12) pg/mL (p=0.003). SHBG and lipid levels did not vary significantly between the two groups (not shown). In a subgroup of SC patients, the median serum E2 decreased from 212 [158-245] to 112 [87-150] pg/mL from the day after to the day before an injection (n=12). 22/23 patients in the SC group chose to continue this method, 0/23 patients experienced a systemic reaction and 1/23 experienced a local site reaction. These preliminary data suggest that SC E2 injection is a safe and effective option for administration of E2 that appears to be well accepted by patients. It is well absorbed with minimal adverse effects and suppresses serum T levels as effectively as PO E2. SC E2 has less hepatic effect as reflected by serum E1 levels (but not SHBG or lipids in this small sample size). Serum E2 levels decrease between weekly injections and additional studies are required to refine dosing to maintain E2 values within the target range. Additional pharmacodynamic studies are also indicated.

Effects of Estradiol Dose and Serum Estradiol Levels on Metabolic Measures in Early and Late Postmenopausal Women in the REPLENISH Trial.

Background: To identify the association of estradiol (E2) dose and serum E2 levels with metabolic measures in early (<6 years) compared with late (≥10 years) postmenopausal women from the REPLENISH trial. Material and Methods: This is a post hoc analysis of a multicenter randomized clinical trial in the United States. Four doses of TX-001HR, an oral combination of E2 and progesterone (P4), and placebo were tested. This analysis included a total of 1,216 early and 297 late postmenopausal women. Linear mixed-effects models tested the association of E2 dose and serum E2 levels with changes in metabolic parameters; total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and glucose (GLUC) levels from six visits over 12 months, adjusted for the serum P4 level. Results: A higher E2 dose was significantly associated with lower TC (p = 0.02) and LDL-C (p = 0.002) and higher HDL-C (p = 0.04) levels in early, but not late, postmenopause. With longer time since menopause, the inverse association of E2 dose with TC and LDL-C and positive association with HDL-C were attenuated (interaction p < 0.05). Higher serum E2 levels were significantly associated with lower TC (p = 0.004), LDL-C (p = 0.0001), and fasting blood GLUC (p = 0.003) and higher TG (p = 0.002) levels in early postmenopause. Conclusion: E2 dose differentially affects metabolic measures among early compared with late postmenopausal women. No significant main effect of the serum P4 level was found. As the metabolic parameters studied are risk factors for cardiovascular events, these results support the timing hypothesis of E2 therapy and its cardiovascular benefits.

SUN-LB61 Differential and Opposite Regulation of Acid Labile Subunit (ALS) Versus Insulin-Like Growth Factor I (IGF-I) by Oral Estrogens in Premenopausal Women

Background: The impact of estrogens (E2) on the growth hormone (GH)/IGF-I axis is known to depend on route of administration: While oral E2 increases GH and decreases IGF-I, transdermal E2 has only limited or no effect. However, data concerning the impact of E2 on IGF binding protein 3 (IGFBP 3) and ALS are less clear. One study in girls demonstrated higher ALS with oral E2, while the opposite was suggested for postmenopausal women. No data are available for healthy premenopausal women.Methods: We measured IGF-I, IGFBP 3 and ALS in fasted healthy adults (93 males (M), 35 premenopausal women without E2-containing oral contraception (FPRE), 37 premenopausal women with E2-containing oral contraception (FPREOC) and 34 postmenopausal women (FPOST)). IGF-I and IGFBP 3 were measured using the IDS-iSYS chemiluminescence immunoassay, and ALS by an in-house immunofluorometric assay (limit of quantification (LoQ) < 50 mU/ml, range 50 - 4000 mU/mL).Results: Median age (range) was 33 (20 - 76), 28 (20 - 44), 24 (21 - 36) and 56 (49 - 70) years for M, FPRE, FPREOC and FPOST, respectively. As expected, IGF-I was lower in FPREOC compared to FPRE (median IGF-I xULN (IQR) 0.56 (0.45 - 0.73) and 0.72 (0.63 – 0.80), P = 0.0017, Kruskal-Wallis). ALS was significantly higher in FPREOC compared to all other groups (mean ALS in M, FPRE, FPREOC and FPOST: 636, 708, 861 and 648 mU/mL, respectively, ANOVA P < 0.0001, Dunnett’s post-hoc test: M vs FPREOC: P < 0.0001, FPRE vs FPREOC: P = 0.0007, FPOST vs FPREOC: P < 0.0001). IGFBP 3 was not different in females with and without oral E2 (median IGFBP 3 xULN (IQR) FPREOC vs FPRE: 0.62 (0.54 - 0.67) vs 0.60 (0.49 – 0.76), Kruskal-Wallis P = 0.295, Dunn’s post-hoc test: P > 0.9999). This was also true between all other groups (Dunn’s post-hoc test: P ≥ 0.4). In our adult cohort, ALS exhibited negative correlation with age (Pearson r = -0.282, P = 0.0003), similar to IGF-I and IGFBP 3. While IGF-I exhibited a moderate negative correlation to BMI (Pearson r = -0.25, P = 0.0013), IGFBP 3 and ALS were not significantly related to BMI.Conclusion: While IGF-I, IGFBP 3 and ALS all are known to be secreted in response to GH, and IGF-I and ALS are assumed to be produced by the same cells in the liver (hepatocytes), the three GH dependent biomarkers appear to be differently regulated by metabolic factors and oral E2. Only IGF-I has some modest association with BMI. Oral E2 is associated with reduced IGF-I, unchanged IGFBP 3 but increased ALS. While the mechanism behind the differential regulation remains to be uncovered, E2 therapy must be taken into account when interpreting IGF-I and ALS concentrations.

17β-Estradiol improves insulin signalling and insulin resistance in the aged female hearts: Role of inflammatory and anti-inflammatory cytokines

Aging effects in energy balance in all tissues and organs, including the cardiovascular. The risk of cardiovascular disease is drastically higher in postmenopausal women than in premenopausal women. Estrogen plays an important role in the cardiac function and body's metabolism. The aim of this study was to determine whether 17β-estradiol (E2) has beneficial effects on insulin resistance and some key stages of the insulin signalling pathway in the aged hearts. Young and aged female Wistar rats were ovariectomized and were randomly divided into three groups: young (YS) and aged (AS) sham, young (YV) and aged (AV) vehicle, and young (YE2) and aged (AE2) E2 treatment groups. E2 (1 mg/kg) was administrated every four days for four weeks. Results showed that ovariectomy increased fasting blood glucose, insulin, and HOMAIR in young, while none of these parameters was affected in aged animals. On the other hand, aging itself increased these variables. Furthermore, E2 therapy alleviated these changes in both young and aged animals. Moreover, aging also decreased the p-IRS1, p-Akt level, and translocation of GLUT4 to the plasma membrane. E2 reduced the negative impact of menopause and aging on insulin sensitivity by favoring increase in the level of IL-10 and decrease in the levels of TNF-α and IL-1β. Our results indicated that the heart response to E2 depended on age, and E2 increased insulin sensitivity in the heart of both young and aged animals by altering inflammatory conditions. Determining the exact mechanism of this action is suggested in future studies.

Association of oral estradiol dose/levels with coagulation measures in early/late postmenopausal women

Objective: This study evaluated associations of estradiol (E2) dose and serum E2 levels with coagulation/anti-coagulation measures in early (<6 years) compared with late (≥10 years) postmenopausal women.Methods: Postmenopausal women from the REPLENISH trial tested four formulations of oral combined E2 and progesterone compared with placebo. Mixed-effects linear models tested the association of E2 dose and serum E2 levels with the prothrombin time (PT), the activated partial thromboplastin time (APTT), antithrombin (ATHRM), fibrinogen (FIBRINO), protein C (PROTC), and protein S (PROTS), assessed five times over 12 months.Results: Among 1215 early and 297 late postmenopausal women, the E2 dose was statistically significantly inversely associated with the APTT in early postmenopause, PROTC in late postmenopause, and with the PT, ATHRM, and PROTS in both groups. Serum E2 levels were statistically significantly inversely associated with the APTT, PROTS, and FIBRINO in early postmenopause, the PT in late postmenopause, and ATHRM and PROTC in both groups. With longer time since menopause, the inverse E2 dose effect and serum E2 effects became stronger.Conclusion: Increasing E2 dose and serum E2 levels were associated with changes in coagulation/anti-coagulation measures. The associations were stronger among women ≥10 years since menopause when initiating E2. The timing of E2 therapy, E2 dose, and serum E2 levels relative to time since menopause may modify the venous thromboembolism risk.

SUN-292 Adolescent Transgender Females Have an Intermediate Body Composition Between Female and Male Controls

Background and Objectives: Among youth, 0.7% identify as transgender. Markers of cardiometabolic health and body composition in transgender youth undergoing gender-affirming hormone therapy have not been rigorously studied. Our objectives were to evaluate differences in body composition, insulin sensitivity, lipid profile, and aspartate and alanine aminotransferases (AST, ALT) in male-to-female (MTF) youth treated with estradiol (E2) compared to non-transgender female (FC) and male controls (MC). Methods: Fourteen MTF E2-treated (mean + SD E2 dose 1.5 + 1.0 mg/day, duration 1 + 0.8 years) participants (aged 14.5-19.4 years) were compared to BMI percentile-category-matched and age-matched (within 1 year) FC (n=23, aged 14.2-18.3) and MC (n=24, aged 14.5-19.8). Three MTF participants were also on a gonadotropin releasing-hormone analogue (GnRHa) for suppression of endogenous testicular function. Participants had fasted morning laboratory testing and body composition measured by dual-energy X-ray absorptiometry (DXA). Differences (mean + SD) in body composition, insulin sensitivity (estimated from 1/[fasting insulin]), lipids, leptin, AST, ALT and blood pressure (BP) were evaluated using a mixed linear regression model with a random effect for the matched set. A spearman correlation was performed to evaluate for a correlation between 1/[fasting insulin] and % lean mass. Results: MTF vs. FC: Total testosterone was higher in MTF vs. FC (224 + 182 vs. 43 + 10 ng/dL, respectively, p<0.001); however, the serum E2 was not different between the groups (98 + 135 vs. 96 + 127 pg/mL, respectively, p=0.8). MTF had a lower % fat (31 + 7 vs. 35 + 8%, p=0.03) and higher % lean mass (66 + 6 vs. 62 + 7%, p=0.03) than FC. MTF had a higher AST (37 + 4 vs. 23 + 6 U/L, p<0.001) and higher 1/[fasting insulin] (0.135 + 0.055 vs. 0.098 + 0.045, p=0.04) than FC. MTF vs. MC: Serum E2 was higher (124 + 162 vs. 23 + 9 pg/mL, p=0.005) and total testosterone was lower (252 + 214 vs. 412 + 168, p=0.012) in MTF than MC. MTF had higher % fat (28 + 6 vs. 20 + 10%, p=0.003) and lower % lean mass (69 + 5 vs. 77 + 9%, p=0.001) than MC. MTF had higher HDL (50 + 10 vs. 43 + 6 mg/dL, p=0.02) and leptin (14 + 12 vs. 6 + 9 ng/mL, p<0.001) and lower systolic BP (106 + 11 vs. 116 + 8 mmHg, p=0.007) than MC. 1/[fasting insulin] correlated with % lean mass (r=0.58 [95% CI: 0.37, 0.74], p<0.0001). Conclusions: MTF body composition differences may be explained by a male lean mass accrual pattern during puberty, followed by a gain in % fat and concomitant rise in leptin due to E2 treatment. This is similar to what is seen in adults, but adolescents have not been studied. The higher insulin sensitivity in MTF vs. FC may be explained by differences in lean mass. Longitudinal studies with and without pubertal blockade are now needed to understand the impact of E2 therapy in MTF youth in the absence of exposure to a male pubertal hormonal pattern on body composition and cardiometabolic health.

Sexual Function Under Adequate Estrogen Therapy in Women After Oophorectomy Versus Natural Menopause.

Background: There is scarce evidence regarding endogenous postmenopausal ovarian testosterone (T) production and estrogen replacement roles in different sexual domains. This study aimed to determine whether lower endogenous T in oophorectomized women that were estradiol (E2)-treated influenced global or specific domains of sexual function. Depressive and cognitive symptoms were evaluated to exclude potential confounders. Materials and Methods: Eighty-one recently postmenopausal women treated with transdermal E2, 36 with bilateral oophorectomy (O), and 45 controls (C) were investigated through hormonal profile, Female Sexual Function Index, Mini Mental, and Beck Depression Inventory. Results: T levels, as expected, were lower in O than in C (p = 0.001); nonetheless, O presented a lower risk of sexual dysfunction (55.6% vs. 85.7%, p = 0.037), due to less pain (p = 0.005), increased lubrication (p = 0.012), and satisfaction (p = 0.042). O, however, required 50% higher E2 gel doses to control vasomotor symptoms (VMS) than did C. In O, all T measurements were positively, although weakly, correlated with desire (r = 0.374-0.381, p = 0.016-0.024). E2 levels were positively correlated with arousal in all women (r = 0.338, p = 0.038) and in O (r = 0.521, p = 0.032). Depression and cognition scores did not differ between the groups. Conclusions: Despite lower T levels, O women receiving E2 therapy had better global sexual function. Earlier onset and longer E2 treatment could have prevented vulvovaginal atrophy in O. Oophorectomized patients may require higher doses of E2 replacement. E2 levels, achieved by appropriate hormone therapy for VMS control, and very low T levels correlated with distinct sexual domains and may act in complementary areas of sexuality in postmenopausal women.

Impact of 17β-estradiol on complex I kinetics and H2O2 production in liver and skeletal muscle mitochondria

Naturally or surgically induced postmenopausal women are widely prescribed estrogen therapies to alleviate symptoms associated with estrogen loss and to lower the subsequent risk of developing metabolic diseases, including diabetes and nonalcoholic fatty liver disease. However, the molecular mechanisms by which estrogens modulate metabolism across tissues remain ill-defined. We have previously reported that 17β-estradiol (E2) exerts antidiabetogenic effects in ovariectomized (OVX) mice by protecting mitochondrial and cellular redox function in skeletal muscle. The liver is another key tissue for glucose homeostasis and a target of E2 therapy. Thus, in the present study we determined the effects of acute loss of ovarian E2 and E2 administration on liver mitochondria. In contrast to skeletal muscle mitochondria, E2 depletion via OVX did not alter liver mitochondrial respiratory function or complex I (CI) specific activities (NADH oxidation, quinone reduction, and H2O2 production). Surprisingly, in vivo E2 replacement therapy and in vitro E2 exposure induced tissue-specific effects on both CI activity and on the rate and topology of CI H2O2 production. Overall, E2 therapy protected and restored the OVX-induced reduction in CI activity in skeletal muscle, whereas in liver mitochondria E2 increased CI H2O2 production and decreased ADP-stimulated respiratory capacity. These results offer novel insights into the tissue-specific effects of E2 on mitochondrial function.

Estrogen Therapy Worsens Cardiac Function and Remodeling and Reverses the Effects of Exercise Training After Myocardial Infarction in Ovariectomized Female Rats.

There is an increase in the incidence of cardiovascular events such as myocardial infarction (MI) after menopause. However, the use of estrogen therapy (E2) remains controversial. The aim of this study was to evaluate the effects of E2, alone and combined with exercise training (ET), on cardiac function and remodeling in ovariectomized (OVX) rats after MI. Wistar female rats underwent ovariectomy, followed by MI induction were separated into five groups: S; MI; MI+ET; MI+E2; and MI+ET+E2. Fifteen days after MI or sham surgery, treadmill ET and/or estrogen therapy [17-β estradiol-3-benzoate (E2), s.c. three times/week] were initiated and maintained for 8 weeks. After the treatment and/or training period, the animals underwent cardiac hemodynamic evaluation through catheterization of the left ventricle (LV); the LV systolic and diastolic pressures (LVSP and LVEDP, respectively), maximum LV contraction and relaxation derivatives (dP/dt+ and dP/dt−), and isovolumic relaxation time (Tau) were assessed. Moreover, histological analyses of the heart (collagen and hypertrophy), cardiac oxidative stress [advanced oxidation protein products (AOPPs)], pro- and antioxidant protein expression by Western blotting and antioxidant enzyme activity in the heart were evaluated. The MI reduced the LVSP, dP/dt+ and dP/dt− but increased the LVEDP and Tau. E2 did not prevent the MI-induced changes in cardiac function, even when combined with ET. An increase in the dP/dt+ was observed in the E2 group compared with the MI group. There were no changes in collagen deposition and myocyte hypertrophy caused by the treatments. The increases in AOPP, gp91-phox, and angiotensin II type 1 receptor expression induced by MI were not reduced by E2. There were no changes in the expression of catalase caused by MI or by the treatments, although, a reduction in superoxide dismutase (SOD) expression occurred in the groups subjected to E2 treatment. Whereas there were post-MI reductions in activities of SOD and catalase enzymes, only that of SOD was prevented by ET. Therefore, we conclude that E2 therapy does not prevent the MI-induced changes in cardiac function and worsens parameters related to cardiac remodeling. Moreover, E2 reverses the positive effects of ET when used in combination, in OVX infarcted female rats.