Equine Estrogens Research Articles

Clinical Impact of Hormone Replacement Therapy on Atrial Fibrillation in Postmenopausal Women: A Nationwide Cohort Study.

Individuals with atrial fibrillation (AF), especially women, have an increased risk of stroke and death. Although hormone replacement therapy (HRT) is widely used in postmenopausal women, the association between HRT use and AF risk is unclear. We aimed to investigate the association between various types of HRT and AF. This was a population-based retrospective cohort study from The Korean National Health Insurance Service-National Sample Cohort (2004–2015). Participants were aged 45–60 years and were free from cardiovascular disease and AF at baseline. Overall, 13,452 (64.03%) women had never received HRT, 5671 (26.99%) had received HRT, and 1885 (8.98%) were currently receiving HRT. In multivariable analysis, the relative hazards for AF were significantly higher among current users (p < 0.001) and lower among past users (p = 0.069). Current users—except those using estradiol-only HRT—had significantly elevated AF risk. Among past users, only estradiol plus progestin HRT users had a reduced AF risk after adjusting for covariates (p = 0.027). Ongoing HRT posed an increased risk of AF. The degree of risk varied based on the specific type of estrogen and progestins co-administration. These findings indicate that, with respect to AF risk, oral estradiol-containing HRT is superior to HRT containing oral conjugated equine estrogen or tibolone.

Treatment of menopausal symptoms: concomitant modification of cortisol.

To evaluate whether change in menopausal symptoms is related to modification of 24-hour urinary cortisol. Sixty-nine postmenopausal women were treated for their menopausal symptoms with either estrogen progestin therapy (0.3 mg conjugate equine estrogens and 1.5 mg medroxyprogesterone acetate; n = 25), phytoestrogens (75 mg isoflavones, twice daily; n = 21) or acupuncture (once a week; n = 23). Baseline and treatment-induced changes of total and subscale scores (vasomotor, depression, anxiety, somatization, sexuality) of the Greene's Climacteric Scale and of 24-hour urinary cortisol were evaluated. At baseline, 24-hour urinary cortisol was related to Greene's Climacteric Scale score (P < 0.0001). Independent determinants (R2 = 0.319) were the Greene's subscales scores of depression (with a mean difference of 24-h cortisol for score unit expressed as beta coefficient of regression (b) of 4.91, 95% CI 2.14-7.7; P = 0.0007), and of somatization (b 3.04 95% CI 0.69-5.4; P = 0.012). The Greene's Climacteric Scale score (-5.67 ± 6.8; P = 0.0001) and 24-hour cortisol (-23.6 ± 45.7 μg/24 h; P = 0.0001) declined after 3 months of treatment. Changes of 24-hour cortisol values were linearly related to changes of total Greene's Climacteric Scale score with a mean change for unit score (b) of 2.10, 95% CI 0.47-3.73; P = 0.012). Present data indicate that greater reduction in menopausal symptoms is associated with a larger decrease in cortisol levels. Possible implication of this finding on the long-term consequences for women's health needs to be explored.

The Reactivity of Human and Equine Estrogen Quinones towards Purine Nucleosides

Conjugated estrogen medicines, which are produced from the urine of pregnant mares for the purpose of menopausal hormone replacement therapy (HRT), contain the sulfate conjugates of estrone, equilin, and equilenin in varying proportions. The latter three steroid sex hormones are highly similar in molecular structure as they only differ in the degree of unsaturation of the sterane ring “B”: the cyclohexene ring in estrone (which is naturally present in both humans and horses) is replaced by more symmetrical cyclohexadiene and benzene rings in the horse-specific (“equine”) hormones equilin and equilenin, respectively. Though the structure of ring “B” has only moderate influence on the estrogenic activity desired in HRT, it might still significantly affect the reactivity in potential carcinogenic pathways. In the present theoretical study, we focus on the interaction of estrogen orthoquinones, formed upon metabolic oxidation of estrogens in breast cells with purine nucleosides. This multistep process results in a purine base loss in the DNA chain (depurination) and the formation of a “depurinating adduct” from the quinone and the base. The point mutations induced in this manner are suggested to manifest in breast cancer development in the long run. We examine six reactions between deoxyadenosine and deoxyguanosine as nucleosides and estrone-3,4-quinone, equilin-3,4-quinone, and equilenin-3,4-quinone as mutagens. We performed DFT calculations to determine the reaction mechanisms and establish a structure–reactivity relationship between the degree of unsaturation of ring “B” and the expected rate of DNA depurination. As quinones might be present in the cytosol in various protonated forms, we introduce the concept of “effective barriers” to account for the different reactivity and different concentrations of quinone derivatives. According to our results, both equine estrogens have the potential to facilitate depurination as the activation barrier of one of the elementary steps (the initial Michael addition in the case of equilenin and the rearomatization step in the case of equilin) significantly decreases compared to that of estrone. We conclude that the appearance of exogenous equine estrogen quinones due to HRT might increase the risk of depurination-induced breast cancer development compared to the exposure to endogenous estrone metabolites. Still, further studies are required to identify the rate-limiting step of depurination under intracellular conditions to reveal whether the decrease in the barriers affects the overall rate of carcinogenesis.

Investigating the role of endogenous estrogens, hormone replacement therapy, and blockade of estrogen receptor-\u03b1 activity on breast metabolic signaling

PurposeMenopause is associated with an increased risk of estrogen receptor-positive (ER +) breast cancer. To characterize the metabolic shifts associated with reduced estrogen bioavailability on breast tissue, metabolomics was performed from ovary-intact and ovariectomized (OVX) female non-human primates (NHP). The effects of exogenous estrogen administration or estrogen receptor blockade (tamoxifen treatment) on menopause-induced metabolic changes were also investigated.MethodsBilateral ovariectomies were performed on female cynomolgus macaques (Macaca fascicularis) to model menopause. OVX NHP were then divided into untreated (n = 13), conjugated equine estrogen (CEE)-treated (n= 13), or tamoxifen-treated (n = 13) subgroups and followed for 3 years. Aged-matched ovary-intact female NHP (n = 12) were used as a premenopausal comparison group. Metabolomics was performed on snap-frozen breast tissue.ResultsChanges in several different metabolic biochemicals were noted, particularly in glucose and fatty acid metabolism. Specifically, glycolytic, Krebs cycle, acylcarnitines, and phospholipid metabolites were elevated in breast tissue from ovary-intact NHP and OVX + CEE in relation to the OVX and OVX + tamoxifen group. In contrast, treatment with CEE and tamoxifen decreased several cholesterol metabolites, compared to the ovary-intact and OVX NHP. These changes were accompanied by elevated bile acid metabolites in the ovary-intact group.ConclusionAlterations in estrogen bioavailability are associated with changes in the mammary tissue metabolome, particularly in glucose and fatty acid metabolism. Changes in these pathways may represent a bioenergetic shift in gland metabolism at menopause that may affect breast cancer risk.

Hormone therapy in the postmenopausal years: considering benefits and risks in clinical practice.

Menopausal symptoms can be very distressing and considerably affect a woman's personal and social life. It is becoming more and more evident that leaving bothersome symptoms untreated in midlife may lead to altered quality of life, reduced work productivity and, possibly, overall impaired health. Hormone therapy (HT) for the relief of menopausal symptoms has been the object of much controversy over the past two decades. At the beginning of the century, a shadow was cast on the use of HT owing to the concern for cardiovascular and cerebrovascular risks, and breast cancer, arising following publication of a large randomized placebo-controlled trial. Findings of a subanalysis of the trial data and extended follow-up studies, along with other more modern clinical trials and observational studies, have provided new evidence on the effects of HT. The goal of the following paper is to appraise the most significant clinical literature on the effects of hormones in postmenopausal women, and to report the benefits and risks of HT for the relief of menopausal symptoms. A Pubmed search of clinical trials was performed using the following terms: estrogens, progestogens, bazedoxifene, tibolone, selective estrogen receptor modulators, tissue-selective estrogen complex, androgens, and menopause. HT is an effective treatment for bothersome menopausal vasomotor symptoms, genitourinary syndrome, and prevention of osteoporotic fractures. Women should be made aware that there is a small increased risk of stroke that tends to persist over the years as well as breast cancer risk with long-term estrogen-progestin use. However, healthy women who begin HT soon after menopause will probably earn more benefit than harm from the treatment. HT can improve bothersome symptoms, all the while conferring offset benefits such as cardiovascular risk reduction, an increase in bone mineral density and a reduction in bone fracture risk. Moreover, a decrease in colorectal cancer risk is obtainable in women treated with estrogen-progestin therapy, and an overall but nonsignificant reduction in mortality has been observed in women treated with conjugated equine estrogens alone or combined with estrogen-progestin therapy. Where possible, transdermal routes of HT administration should be preferred as they have the least impact on coagulation. With combined treatment, natural progesterone should be favored as it is devoid of the antiapoptotic properties of other progestogens on breast cells. When beginning HT, low doses should be used and increased gradually until effective control of symptoms is achieved. Unless contraindications develop, patients may choose to continue HT as long as the benefits outweigh the risks. Regular reassessment of the woman's health status is mandatory. Women with premature menopause who begin HT before 50 years of age seem to have the most significant advantage in terms of longevity. In women with bothersome menopausal symptoms, HT should be considered one of the mainstays of treatment. Clinical practitioners should tailor HT based on patient history, physical characteristics, and current health status so that benefits outweigh the risks.

Breast Cancer Prevention: Time for Change.

Agency breast cancer prevention guidelines for other than hereditary cancers have not materially changed in 20 years; endocrine-targeted agents (then, tamoxifen; now, adding raloxifene and aromatase inhibitors) reduce good prognosis estrogen receptor (ER)-positive, progesterone receptor (PR)-positive cancers without reducing deaths from breast cancer. Across three tamoxifen placebo-controlled prevention trials (N = 23,360) begun almost 30 years ago, although there were 226 fewer breast cancer cases, there were nine more deaths from breast cancer in the tamoxifen groups. Following clinical advances, currently more than half of breast cancer cases are solved problems with extremely low risk of death. As endocrine-targeted agents commonly prevent these cancers, widespread implementation of current prevention strategies may not reduce deaths from breast cancer. Compared with other breast cancers, ER-positive, PR-negative cancers and triple-negative cancers have inferior survival (90.6% v 83.8% v 78.1%, respectively; P < .001). Against this background, in the Women's Health Initiative Dietary Modification randomized trial (N = 48,835), ER-positive, PR-negative cancers were statistically significantly reduced in the intervention group (hazard ratio, 0.77; 95% CI, 0.64 to 0.94) and deaths from breast cancer were reduced 21% (P = .02). In the Women's Health Initiative randomized, placebo-controlled trial evaluating conjugated equine estrogen (N = 10,739), ER-positive, PR-negative cancers were statistically significantly reduced in the intervention group (hazard ratio, 0.44; 95% CI, 0.27 to 0.74) and deaths from breast cancer were reduced 40% (P = .04). These findings suggest that reexamination of breast cancer risk reduction strategies and clinical practice is needed.

Comparative venous thromboembolic safety of oral and transdermal postmenopausal hormone therapies among women Veterans.

Hormone therapy (HT) is used by menopausal women to treat vasomotor symptoms. Venous thromboembolism (VTE) is an important risk of HT use, and more knowledge on the comparative safety of different estrogenic compounds is useful for women who use HT for these symptoms. The objective was to compare the risk of VTE among users of oral conjugated equine estrogen (CEE), oral estradiol (E2), and transdermal E2, in a cohort of women veterans. This retrospective cohort study included all women veterans aged 40 to 89 years, using CEE or E2, without prior VTE, between 2003 and 2011. All incident VTE events were adjudicated. Time-to-event analyses using a time-varying HT exposure evaluated the relative VTE risk between estrogen subtypes, with adjustment for age, race, and body mass index, with stratification for prevalent versus incident use of HT. Among 51,571 users of HT (74.5% CEE, 12.6% oral, and 12.9% transdermal E2 at cohort entry), with a mean age of 54.0 years, the incidence of VTE was 1.9/1,000 person-years. Compared with CEE use, in the multivariable regression model, there was no difference in the risk of incident VTE associated with oral E2 use (hazard ratio 0.96, 95% CI 0.64-1.46) or with transdermal E2 use (hazard ratio 0.95, 95% CI 0.60-1.49). Results were unchanged when restricting to incident users of HT. Among women veterans, the risk of VTE was similar in users of oral CEE, oral E2, and transdermal E2. These findings do not confirm the previously observed greater safety of transdermal and oral E2 over CEE.

Clinically Used Hormone Formulations Differentially Impact Memory, Anxiety-Like, and Depressive-Like Behaviors in a Rat Model of Transitional Menopause.

A variety of U.S. Food and Drug Administration-approved hormone therapy options are currently used to successfully alleviate unwanted symptoms associated with the changing endogenous hormonal milieu that occurs in midlife with menopause. Depending on the primary indication for treatment, different hormone therapy formulations are utilized, including estrogen-only, progestogen-only, or combined estrogen plus progestogen options. There is little known about how these formulations, or their unique pharmacodynamics, impact neurobiological processes. Seemingly disparate pre-clinical and clinical findings regarding the cognitive effects of hormone therapies, such as the negative effects associated with conjugated equine estrogens and medroxyprogesterone acetate vs. naturally circulating 17β-estradiol (E2) and progesterone, signal a critical need to further investigate the neuro-cognitive impact of hormone therapy formulations. Here, utilizing a rat model of transitional menopause, we administered either E2, progesterone, levonorgestrel, or combinations of E2 with progesterone or with levonorgestrel daily to follicle-depleted, middle-aged rats. A battery of assessments, including spatial memory, anxiety-like behaviors, and depressive-like behaviors, as well as endocrine status and ovarian follicle complement, were evaluated. Results indicate divergent outcomes for memory, anxiety, and depression, as well as unique physiological profiles, that were dependent upon the hormone regimen administered. Overall, the combination hormone treatments had the most consistently favorable profile for the domains evaluated in rats that had undergone experimentally induced transitional menopause and remained ovary-intact. The collective results underscore the importance of investigating variations in hormone therapy formulation as well as the menopause background upon which these formulations are delivered.

Menopausal Hormone Therapy and the Role of Estrogen.

Menopause is a universal experience for midlife women. The physiological decline in endogenous estrogen can be associated with vasomotor symptoms or hot flashes, sleep disruption, and mood disorders. Long-term concerns arise with sequelae of estrogen loss such as genitourinary syndrome of menopause and osteoporosis. Although the pendulum has swung widely since the 1942 approval of conjugated equine estrogens, estrogen therapy, now available in an ever-expanding menu of preparations, routes of administration, and dosing, remains the most effective means to collectively address these, and potentially, additional concerns. Refinement of knowledge of risks and benefits facilitates patient selection and counseling.

Effects of menopausal hormone therapy on erythrocyte n–3 and n–6 PUFA concentrations in the Women’s Health Initiative randomized trial

The factors other than dietary intake that determine tissue concentrations of EPA and DHA remain obscure. Prior studies suggested that, in women, endogenous estrogen may accelerate synthesis of DHA from ɑ-linolenic acid (ALA), but the effects of exogenous estrogen on RBC n-3 (ɷ-3) PUFA concentrations are unknown. We tested the hypothesis that menopausal hormone therapy (HT) would increase RBC n-3 PUFA concentrations. Postmenopausal women (ages 50-79 y) were assigned to HT or placebo in the Women's Health Initiative (WHI) randomized trial. The present analyses included a subset of 1170 women (ages 65-79 y) who had RBC PUFA concentrations measured at baseline and at 1 y as participants in the WHI Memory Study. HT included conjugated equine estrogens (E) alone for women without a uterus (n=560) and E plus medroxyprogesterone acetate (P) for those with an intact uterus (n=610). RBC n-3 and n-6 (ɷ-6) PUFAs were quantified. Effects of E alone and E+P on PUFA profiles were similar and were thus combined in the analyses. Relative to the changes in the placebo group after 1 y of HT, docosapentaenoic acid (DPA; n-3) concentrations decreased by 10% (95% CI: 7.3%, 12.5%), whereas DHA increased by 11% (95% CI: 7.4%, 13.9%) in the HT group. Like DHA, DPA n-6 increased by 13% from baseline (95% CI: 10.0%, 20.3%), whereas linoleic acid decreased by 2.0% (95% CI: 1.0%, 4.1%; P values at least <0.01 for all). EPA and arachidonic acid concentrations were unchanged. HT increased RBC concentrations of the terminal n-3 and n-6 PUFAs (DHA and DPA n-6). These findings are consistent with an estrogen-induced increase in DHA and DPA n-6 synthesis, which is consistent with an upregulation of fatty acid elongases and/or desaturases in the PUFA synthetic pathway. The clinical implications of these changes require further study. The Women's Health Initiative Memory Study is registered at clinicaltrials.gov as NCT00685009. Note that the data presented here were not planned as part of the original trial, and therefore are to be considered exploratory.

Conjugated equine estrogen used in postmenopausal women associated with a higher risk of stroke than estradiol

This study aimed to evaluate the risk of ischemic stroke (IS) in hormone therapy (HT) with oral conjugated equine estrogen (CEE) and estradiol (E2) in postmenopausal women in Taiwan. A retrospective cohort study was conducted using the Taiwan National Health Insurance Research Database, a population-based healthcare claims dataset. Eligible women, aged 40–65 years, who received HT with E2 and CEE orally were enrolled. The primary outcome was IS. Propensity score matching with menopausal age and comorbidities was used. Cox proportional hazard regression models were used to calculate the incidence and hazard ratios (HRs) for IS. The mean menopausal ages of the E2 and CEE groups were 50.31 ± 4.99 and 50.45 ± 5.31 years, respectively. After adjusting for age and comorbidities, the incidence of IS was 1.17-fold higher in the women treated with CEE than in those treated with E2 (4.24 vs. 3.61/1000 person-years), with an adjusted HR (aHR) of 1.23 (95% confidence interval [CI] 1.05–1.44). Moreover, HT with CEE initiated within 5 years of menopause had a higher HR than E2 (aHR = 1.20; 95% CI 1.02–1.42). In conclusion, HT with oral CEE might be associated with a higher risk of IS than E2 in postmenopausal Taiwanese women. The use of HT with CEE should be cautioned with the risk of IS.

Cardiovascular disease (CVD) risk scores, age, or years since menopause to predict cardiovascular disease in the Women's Health Initiative.

To assess the utility of cardiovascular disease (CVD) risk scores compared to age or years since menopause for prediction of CVD events in the WHI clinical trials. Briefly, in the randomized clinical trial 27,347 postmenopausal women age 50 to 79 years entered from 1993 to 1998. Women with a uterus (16,608) were randomized to receive daily oral conjugated equine estrogen (CEE) (0.625 mg) plus medroxyprogesterone acetate (2.5 mg) (5.7 years or placebo), while women with a hysterectomy (10,739) were randomized to receive daily oral CEE (0.625 mg) alone or placebo (7.2 y). CVD risk scores were assessed at baseline and CVD events were adjudicated throughout the follow-up period to the end of the main study phase and to the end of cumulative follow-up. The median follow-up time after the start of the randomized clinical trial to the end of the main study phase was 8.2 years. The median follow-up time to the end of cumulative follow-up was 17.6 years. We compared The American Heart Association/American College of Cardiology (AHA/ACC) and Framingham Heart Study risk scores to age or years since menopause all obtained at baseline to predict subsequent CVD events. The absolute event rates, hazard ratios, and C-statistics (Uno Concordance from Cox proportional models) were compared. Overall, the hazard ratios for CVD events were highest with calculated CVD scores calculated at trial onset both at the end of the main study (ranging from 2.02 to 10.8 for Q2-Q5, compared to Q1) and at cumulative follow-up (ranging from 1.76 to 8.86 for Q2-Q5, compared to Q1). While older age and years since menopause at baseline were also associated with higher CVD event rates, better risk prediction was accomplished by using CVD risk scores. The Framingham Heart Study BMI score had the highest C-statistic at the end of the main study (0.711) and after 17.6 years through the end of follow-up (0.689). CVD risk scores can help identify postmenopausal women at higher risk for CVD beyond age or time since menopause. Risk scoring that better estimates vascular aging may facilitate CVD risk prevention. When performed prior to initiation of menopausal hormone therapy, scores can better inform HT risk/benefit discussions.

Pituitary Stalk Interruption Syndrome in a 40-Year-Old Female With Absent Uterus and Ovaries

Background: A patient with multiple pituitary hormone deficiencies due to a rare congenital pituitary defect also presented with an absent uterus and ovaries. Coexisting Müllerian agenesis was suspected, however hormone replacement unraveled a different story. Case Presentation: A 40-year-old female presented with generalized body weakness and euvolemic hyponatremia. She also had cold intolerance, constipation, primary amenorrhea and dyspareunia. As a child, she was worked up for short stature but was lost to follow up. Physical examination showed Tanner stage 1 breast and pubic hair, and absence of axillary hair. She had no facial anomalies, visual abnormalities or anosmia. Hormone panel was consistent with panhypopituitarism: ACTH 5.69 pg/mL (<46 pg/mL), cortisol 1.9 ug/dL (4.30-22.40 μg/dL), TSH 3.375 uIU/mL (0.55-4.78 uIU/mL), FT4 0.51 ng/dL (0.55-4.78 uIU/mL), FT3 1.79 pg/mL (2.30-4.20 pg/mL), LH <0.07 mIU/mL (1.9-12.5 mIU/mL), FSH 0.95 mIU/mL (2.5-10.2 mIU/mL) and IGF-1 40.8 ng/dL (109-284 ng/mL). Bone age was delayed (16-year-old) but with most ossification centers fused. Pituitary MRI showed hypoplastic anterior pituitary, ectopic posterior pituitary and absent pituitary stalk suggestive of Pituitary Stalk Interruption Syndrome (PSIS). Low estradiol 19.35 pg/mL (19.5-144.2 pg/mL) in the setting of low LH and FSH was compatible with hypogonadotrophic hypogonadism. She had female-range serum testosterone level <0.007 ng/mL (0.1209-0.5946 ng/mL) and karyotype of 46XX. Transvaginal ultrasound revealed a blind vaginal pouch with absent uterus, fallopian tubes and ovaries; hence Müllerian agenesis was also considered. Hormonal replacement with prednisone, levothyroxine and conjugated equine estrogen was started. Secondary osteoporosis was treated with alendronate, calcium and vitamin D on top of estrogen therapy. Six months after initiation of estrogen, there was appearance of a small anteverted uterus and atrophic right ovary. Müllerian agenesis was ruled out and hypogonadotropic hypogonadism was proved to be the cause of the initial absence of the uterus and ovaries on imaging. Conclusion: Increased awareness of PSIS is important since early and accurate diagnosis is crucial for timely initiation of hormone replacement. This case also demonstrates the need for reassessment after hormonal replacement in patients with severe estrogen deficiency and apparent Müllerian agenesis.

Bias in animal studies of estrogen effects on cardiovascular disease: A systematic review and meta‐analysis

BackgroundRandomized controlled trials on menopausal hormone therapy in humans have not confirmed the benefit of estrogens on cardiovascular disease found in animal studies. Flawed methodology or publication bias in animal studies may explain the dicrepancy. ObjectivesThe aim of this study was to investigate whether publication of the randomized controlled trials Heart and Estrogen/Progestin Replacement Study and Women’s Health Initiative influenced study authors’ assessment of research findings (confirmation bias) as well as to investigate publication bias and small‐study effects in animal studies of estrogen effects on atherosclerosis. MethodsThe data source for this study was PubMed from inception to 2018. We selected animal studies with cardiovascular outcomes comparing 17‐β‐estradiol, its natural metabolites, or conjugated equine estrogen with control. Qualitative data were extracted on authors’ conclusions about estrogen effects on cardiovascular disease, as well as quantitative data for atherosclerosis outcomes. Fixed‐ and random‐effects meta‐analyses were used. Publication bias/small‐study effects were assessed using funnel plots and Egger’s regression. Trim‐and‐fill plots and extrapolation from Egger’s regression were used to adjust for publication bias. The main outcomes and measures were the primary study authors' interpretation of their own results, and estrogen effects on cardiovascular disease in general before and after publication of the Women’s health Initiative study (2003). The effects of estrogens on atherosclerosis were measured as standardized mean difference between intervention and control. ResultsOf 1925 studies retrieved, 360 were eligible for analyses. Study‐specific statements concluded that estrogens were protective against cardiovascular disease in 75% of studies before 2003 and 78% after, but the percentage of general statements about estrogens being cardioprotective changed from 70% to 40%. Meta‐analyses showed less atherosclerosis in estrogen‐treated animals. Extremely skewed funnel plots and P < .01 in Egger’s regression suggested publication bias and/or exaggerated effects in small studies, which was more pronounced after 2002. There was substantial heterogeneity of effects (I2 = 68%‐86%) overall and in all subgroups except cynomolgus monkeys (I2 = 9%), the only animal subgroup without clear bias. Adjusting for publication bias, overall estimates of estrogen effects on atherosclerosis were close to null effect. ConclusionsWe found substantial evidence of publication bias but not of confirmation bias. Publication bias and flawed small studies may partly explain why findings differ between animal studies and clinical trials on the effect of estrogens on cardiovascular disease.

Sleep quality and cortical amyloid-β deposition in postmenopausal women of the Kronos early estrogen prevention study.

Hormone therapy improves sleep in menopausal women and recent data suggest that transdermal 17β-estradiol may reduce the accumulation of cortical amyloid-β. However, how menopausal hormone therapies modify the associations of amyloid-β accumulation with sleep quality is not known. In this study, associations of sleep quality with cortical amyloid-β deposition and cognitive function were assessed in a subset of women who had participated in the Kronos early estrogen prevention study. It was a randomized, placebo-controlled trial in which recently menopausal women (age, 42-58; 5-36 months past menopause) were randomized to (1) oral conjugated equine estrogen (n = 19); (2) transdermal 17β-estradiol (tE2, n = 21); (3) placebo pills and patch (n = 32) for 4 years. Global sleep quality score was calculated using Pittsburgh sleep quality index, cortical amyloid-β deposition was measured with Pittsburgh compound-B positron emission tomography standard uptake value ratio and cognitive function was assessed in four cognitive domains 3 years after completion of trial treatments. Lower global sleep quality score (i.e., better sleep quality) correlated with lower cortical Pittsburgh compound-B standard uptake value ratio only in the tE2 group (r = 0.45, P = 0.047). Better global sleep quality also correlated with higher visual attention and executive function scores in the tE2 group (r = -0.54, P = 0.02) and in the oral conjugated equine estrogen group (r = -0.65, P = 0.005). Menopausal hormone therapies may influence the effects of sleep on cognitive function, specifically, visual attention and executive function. There also appears to be a complex relationship between sleep, menopausal hormone therapies, cortical amyloid-β accumulation and cognitive function, and tE2 formulation may modify the relationship between sleep and amyloid-β accumulation.

Peripheral Markers of Neurovascular Unit Integrity and Amyloid-β in the Brains of Menopausal Women.

Background:The identification of blood-borne biomarkers for the diagnosis and prognosis of Alzheimer’s disease and related dementias is more feasible at the population level than obtaining cerebrospinal fluid or neuroimaging markers.Objective:This study determined the association of blood microvesicles, derived from cells of the neurovascular unit, with brain amyloid-β deposition in menopausal women.Methods:A subset of women from the Kronos Early Estrogen Prevention Study underwent brain amyloid-β positron emission tomography three years following cessation of study treatment with placebo (PL, n = 29), transdermal 17β-estradiol (tE2; n = 21), or oral conjugated equine estrogen (oCEE; n = 17). Isolated peripheral venous blood microvesicles were analyzed by digital flow cytometry using fluorophore conjugated antibodies directed toward total tau, amyloid-β 1–42 (Aβ1–42), neuron specific class III β-tubulin (Tuj1), microglia ionized calcium -binding adaptor molecule 1(Iba1), glial fibrillary acid protein (GFAP), and low density lipoprotein receptor-related protein1 (LRP1). Principal components analysis reduced the dimensionality of these selected six markers to two principal components (PCs). Proportional odds ordinal logistic regression analysis was used with amyloid-β deposition regressed on these PCs.Results:Only the number of microvesicles positive for Aβ1–42 differed statistically among prior treatment groups (median [IQR]: 6.06 [2.11, 12.55] in PL; 2.49 [0.73, 3.59] in tE2; and 4.96 [0.83, 10.31] in oCEE; p = 0.032). The joint association between the 2 PCs and brain amyloid-β deposition was significant (p = 0.045).Conclusion:Six selected markers expressing peripheral blood microvesicles derived from cells of the neurovascular unit, when summarized into two principal components, were associated with brain amyloid-β deposition.

In vivo and in silico analyses of estrogenic potential of equine estrogens in medaka (Oryzias latipes)

Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17β-dihydroequilin (17β-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17β-dihydroequilenin (17β-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17β-Eq > Eq > 17β-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17β-Eq was lower than that of 17β-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERβ1, and ERβ2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17β-Eq > 17α-Eq > Eq > 17β-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.

Preservation of white matter integrity on DTI 3 years after early menopausal hormone therapies

AbstractBackgroundThe critical‐period theory of menopausal hormone therapy (mHT) suggests that if menopausal hormone therapy begins shortly after menopause, it may have beneficial effects on brain structure and cognitive health. We investigated white matter integrity with diffusion tensor MRI (DTI) 3 years after the end of mHT trial in recently menopausal women who were randomized shortly after menopause.MethodParticipants of this study were enrolled in the Kronos Early Estrogen Prevention Study (aged 42–56 years; 5–36 months past menopause). Participants received the following treatments: 0.45 mg/d oral conjugated equine estrogen (oCEE, n=19); 50 μg/d transdermal 17β‐estradiol (tE2, n=22); or placebo (n=33) for 4 years. Oral progesterone (200 mg/d) was given to hormone therapy groups for 12 days each month. DTI was performed 3 years after the end of 4 years of hormone therapy (n=75), which corresponded to 7 years after randomization. Voxel‐based analysis was performed on DTI to evaluate fractional anisotropy (FA) and mean diffusivity (MD) differences between treatment groups (uncorrected p&lt;0.001).ResultVoxel based analysis showed higher FA, a measure of white matter integrity, in the superior corona radiata, anterior limb of the internal capsule and splenium of the corpus callosum of participants treated with oCEE compared to placebo (p &lt;0.001, Figure 1A). Furthermore, higher FA was observed in the superior corona radiata, anterior limb of internal capsule and thalamus in participants treated with tE2 compared to placebo (p &lt;0.001, Figure 1B). There were no differences in FA findings between oCEE and tE2 groups, and no MD differences between treatments groups.ConclusionA relatively higher FA in a variety of white matter regions in the oCEE and tE2 groups compared to placebo suggest that mHT may have long‐term protective effects on white matter integrity. Previously, we had shown that women in the tE2 group preserved their dorsolateral prefrontal cortex volumes better than the placebo group over 7 years. Our findings suggest that the tracts that connect the dorsolateral prefrontal cortex and the thalamus such as the superior corona radiata and the anterior limb of internal capsule may particularly benefit from mHT.

Metabolomic Effects of Hormone Therapy and Associations With Coronary Heart Disease Among Postmenopausal Women.

In the WHI-HT trials (Women's Health Initiative Hormone Therapy), treatment with oral conjugated equine estrogens and medroxyprogesterone acetate (CEE+MPA) resulted in increased risk of coronary heart disease (CHD), whereas oral conjugated equine estrogens (CEE) did not. Four hundred eighty-one metabolites were measured at baseline and at 1-year in 503 and 431 participants in the WHI CEE and CEE+MPA trials, respectively. The effects of randomized HT on the metabolite profiles at 1-year was evaluated in linear models adjusting for baseline metabolite levels, age, body mass index, race, incident CHD, prevalent hypertension, and diabetes. Metabolites with discordant effects by HT type were evaluated for association with incident CHD in 944 participants (472 CHD cases) in the WHI-OS (Women's Health Initiative Observational Study), with replication in an independent cohort of 980 men and women at high risk for cardiovascular disease. HT effects on the metabolome were profound; 62% of metabolites significantly changed with randomized CEE and 52% with CEE+MPA (false discovery rate-adjusted P value<0.05) in multivariable models. Concerted increases in abundance were seen within various metabolite classes including triacylglycerols, phosphatidylethanolamines, and phosphatidylcholines; decreases in abundance was observed for acylcarnitines, lysophosphatidylcholines, quaternary amines, and cholesteryl/cholesteryl esters. Twelve metabolites had discordant effects by HT type and were associated with incident CHD in the WHI-OS; a metabolite score estimated in a Least Absolute Shrinkage and Selection Operator regression was associated with CHD risk with an odds ratio of 1.47 per SD increase (95% CI, 1.27-1.70, P<10-6). All twelve metabolites were altered in the CHD protective direction by CEE treatment. One metabolite (lysine) was significantly altered in the direction of increased CHD risk by CEE+MPA; the remaining 11 metabolites were not significantly changed by CEE+MPA. The CHD associations of a subset of 4 metabolites including C58:11 triacylglycerol, C54:9 triacylglycerol, C36:1 phosphatidylcholine and sucrose replicated in an independent dataset of 980 participants in the PREDIMED trial (Prevención con Dieta Mediterránea). Randomized treatment with oral HT resulted in large metabolome shifts that generally favored CEE alone over CEE+MPA in term of CHD risk. Discordant metabolite effects between HT regimens may partially mediate the differences in CHD risk between the 2 WHI-HT trials.

Randomized Trial Evaluation of the Benefits and Risks of Menopausal Hormone Therapy Among Women 50–59 Years of Age

The health benefits and risks of menopausal hormone therapy among women aged 50-59 years are examined in the Women's Health Initiative randomized, placebo-controlled trials using long-term follow-up data and a parsimonious statistical model that leverages data from older participants to increase precision. These trials enrolled 27,347 healthy postmenopausal women aged 50-79 years at 40 US clinical centers during 1993-1998, including 10,739 post-hysterectomy participants in a trial of conjugated equine estrogens and 16,608 participants with a uterus in the trial of these estrogens plus medroxyprogesterone acetate. Over a (median) 18-year follow-up period (1993-2016), risk for a global index (defined as the earliest of coronary heart disease, invasive breast cancer, stroke, pulmonary embolism, colorectal cancer, endometrial cancer, hip fracture, and all-cause mortality) was reduced with conjugated equine estrogens with a hazard ratio of 0.82 (95% confidence interval: 0.71, 0.95), and with nominally significant reductions for coronary heart disease, breast cancer, hip fracture, and all-cause mortality. Corresponding global index hazard ratio estimates of 1.06 (95% confidence interval: 0.95, 1.19) were nonsignificant for combined estrogens plus progestin, but increased breast cancer risk and reduced endometrial cancer risk were observed. These results, among women 50-59 years of age, substantially agree with the worldwide observational literature, with the exception of breast cancer for estrogens alone.