Low-dose Estradiol Research Articles

Comparative effects of estradiol, methyl-piperidino-pyrazole, raloxifene, and ICI 182 780 on gene expression in the murine uterus

Selective estrogen receptor modulators (SERMs) are potentially useful in treating various endometrial disorders, including endometrial cancer, as they block some of the detrimental effects of estrogen. It remains unclear whether each SERM regulates a unique subset of genes and, if so, whether the combination of a SERM and 17beta-estradiol has an additive or synergistic effect on gene expression. We performed microarray analysis with Affymetrix Mouse Genome 430 2.0 short oligomer arrays to determine gene expression changes in uteri of ovariectomized mice treated with estradiol (low and high dose), methyl-piperidino-pyrazole (MPP), ICI 182 780, raloxifene, and combinations of high dose of estradiol with one of the SERM and dimethyl sulfoxide (DMSO) vehicle control. The nine treatments clustered into two groups, with MPP, raloxifene, and high dose of estradiol in one, and low dose of estradiol, ICI + estradiol, ICI, MPP + estradiol, and raloxifene + estradiol in the second group. Surprisingly, combining a high dose of estradiol with a SERM markedly increased (P<0.02) the number of regulated genes compared with each individual treatment. Analysis of expression for selected genes in uteri of estradiol and SERM-treated mice by quantitative (Q)RT-PCR generally supported the microarray results. For some cancer-associated genes, including Klk1, Ihh, Cdc45l, and Cdca8, administration of MPP or raloxifene with estradiol resulted in greater expression than estradiol alone (P<0.05). By contrast, ICI 182 780 suppressed more genes governing DNA replication compared with MPP and raloxifene treatments. Therefore, ICI 182 780 might be superior to MPP and raloxifene to treat estrogen-induced endometrial cancer in women.

Mechanism of collagen biosynthesis up-regulation in cultured leiomyoma cells.

Uterine leiomyoma is the most common tumour in women with a reported incidence of 25-30%. The tumors are benign, composed of smooth muscle cells with variable amount of collagen - rich fibrous tissue. It is well established that accumulation of extracellular matrix in leiomyoma is key feature of tissue fibrosis. However, the pathogenesis of leiomyoma is still unclear. The aim of this study was to evaluate the metabolism of collagen in cultured leiomyoma cells and in control myometrium cells. The effect of estradiol, selective modulators of estrogen receptors (raloxifene, tamoxifen) and estrogen receptor down regulator (ICI 182.780) on collagen biosynthesis (measured by 5-[3H]-proline incorporation assay and measurement of prolidase activity) and collagen degradation (measured by metalloproteinase activity assay) was studied. It was found that collagen biosynthesis is strongly stimulated by low doses of estradiol (5 nM) in leiomyoma cells while it is not changed in control myometrium cells. An increase in estradiol concentration to 10 nM results in drastic decrease of this process both in leiomyoma as well as control cells. Although raloxifene and tamoxifen only slightly affected collagen biosynthesis in control myometrium cells, they significantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosysignificantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosynthesis and prolidase activity suggesting that regulation of this process may take place at transcriptional level. Both estrogen and SERMs were found to inhibit MMP-2 in leiomyoma as well as in control myometrium cells. The data suggest that stimulatory action of estrogen on collagen biosynthesis and inhibitory effect on MMP-2 activity in uterine leiomyoma may contribute to accumulation of this protein in ECM of this tissue.

Low-dose estradiol gel 0.1% for the treatment of vasomotor symptoms associated with menopause

Estrogen therapy (ET) is the most effective treatment currently available for the relief of vasomotor symptoms such as hot flushes and night sweats, which are associated with menopause. Safety concerns associated with long-term use of standard doses of oral estrogens have led to treatment guidelines recommending the use of ET at the lowest effective dose for the shortest duration possible when indicated for treatment of menopausal symptoms. Estradiol gel 0.1% (Divigel®) is a low-dose, ‘patchless’ transdermal formulation of a bioidentical estrogen (structurally indistinguishable from the endogenous human hormone) that has been demonstrated to be effective and well tolerated in the treatment of moderate-to-severe hot flushes. Available at doses of 0.25, 0.5 and 1.0 g (containing estradiol 0.25, 0.5 and 1.0 mg, respectively), estradiol gel 0.1% is applied topically to the skin once daily. At these doses, estradiol gel 0.1% delivers estradiol 0.003–0.027 mg/day at steady-state, the lowest systemic levels of e...

Low- and standard-estrogen dosage in oral therapy: dose-dependent effects on insulin and lipid metabolism in healthy postmenopausal women

Objective To evaluate the influences of different doses of daily oral unopposed 17β-estradiol compared with placebo, both on glucose tolerance and lipid metabolism in healthy postmenopausal women.Patients and methods Forty-eight normoinsulinemic postmenopausal women were enrolled in the study. Patients were assigned to receive randomly 1 mg (group A) or 2 mg (group B) of oral micronized estradiol therapy daily or to the placebo (group C), for 12 weeks.Results The low-dose estradiol treatment determined an improvement of the peripheral insulin sensitivity, made evident by a significant increase both in the metabolic index and oral glucose insulin sensitivity index (p < 0.01 and p < 0.05, respectively) as well as a decrease in the homeostasis model assessment-estimated insulin resistance (p < 0.01). Conversely, in the standard-dose group, the metabolic index significantly decreased (p < 0.05), showing a slight deterioration in insulin sensitivity. For lipid metabolism, the 1 mg dose showed a neutral effect, while 2 mg had a beneficial effect on low density lipoprotein cholesterol, but caused an increase in triglycerides (p < 0.01 and p < 0.05, respectively).Conclusions The oral low dose of unopposed estradiol therapy had a favorable effect on glycoinsulinemic metabolism in healthy postmenopausal women; however, the standard dose caused a slight but significant deterioration in insulin sensitivity.

Effects of estrogen and estrogenic compounds on cognition in ovariectomized rats

Objectives To evaluate the effects of estrogen and estrogenic compounds on cognition in ovariectomized rats.Methods Female Sprague–Dawley rats (3–5 months old) weighing 250–300 g were randomly divided into seven groups: Sham, ovariectomized (OVX), OVX plus estradiol valerate, OVX plus ipriflavone, OVX plus raloxifene, OVX plus tibolone, OVX plus low-dose estradiol valerate and ipriflavone. All treatments were given orally for 3 months; whereas the drug groups received indicated drugs, the Sham and OVX control groups received saline. The escape latency of rats was tested by the Morris water maze test and the expression of amyloid precursor protein (APP) in hippocampus was determined by reverse transcription polymerase chain reaction. The level of serum estradiol and the diameter of the endometrial gland and the thickness of endometrium were also evaluated.Results The latency of the OVX group was noticeably longer than that of the Sham group, and the latency of all treatment groups was lower than that of OVX rats. The expression of APP mRNA in the hippocampii of OVX rats was significantly increased relative to that in Sham rats; interestingly, expression of APP in treatment groups was significantly reduced relative to OVX rats.Conclusions Our data indicate that estrogenic compounds can antagonize cognitive impairment and that all these compounds cause only mild stimulation on the endometrium compared to estrogen. Inhibition of APP expression in the hippocampus may account for, at least partially, the protective effects of these estrogenic compounds against cognitive defects. Our data suggest that estrogenic compounds (raloxifene, tibolone and ipriflavone) may be a promising approach to antagonize cognitive impairment in postmenopausal women.

Efficacy of Low-Dose Estradiol Vaginal Tablets in the Treatment of Atrophic Vaginitis

To evaluate the efficacy of two vaginal doses of estradiol (E2) compared with placebo in the treatment of atrophic vaginitis. In a multi-center, randomized, double-blind, parallel-group study, 230 postmenopausal women received treatment with 25 mcg or 10 mcg E2 or placebo for 12 weeks. Efficacy was measured through composite score of three vaginal symptoms and grading of vaginal health. Additional analyses included maturation of vaginal and urethral mucosa. Safety assessments included endometrial biopsy, adverse events, changes in laboratory tests, and physical examinations. After 12 weeks of treatment, all patients were switched to the open-label extension and received treatment with 25 mcg E2 up to week 52. Vaginal tablets with 25 mcg and 10 mcg E2 showed significant (P<.001) improvement in composite score of vaginal health. Other results with 10 mcg E2 were not entirely consistent with those for 25 mcg E2. Over 12 weeks, both active treatments resulted in greater decreases in vaginal pH than placebo. There were no significant differences between the 25 mcg and 10 mcg E2 groups in terms of improvements in maturation value or composite score of three vaginal symptoms. The efficacy was maintained to week 52 with 25 mcg E2. Vaginal tablets with 25 mcg and 10 mcg E2 provided relief of vaginal symptoms, improved urogenital atrophy, decreased vaginal pH, and increased maturation of the vaginal and urethral epithelium. Those improvements were greater with 25 mcg than with 10 mcg E2. Both doses were effective in the treatment of atrophic vaginitis. ClinicalTrials.gov, www.clinicaltrials.gov, NCT00465192 and NCT00464971 I.

Response to Windahl et al.

We have read the article by Windahl et al. (1) with interest. While we agree with the conclusion that estren may not be a suitable clinical candidate for the treatment of postmenopausal osteoporosis due to effects on reproductive tissues that we did not observe (2), we strongly disagree with the interpretation of the evidence. Postmenopausal osteoporosis is a disease of late adulthood in which bone loss occurs after peak bone mass has been established. Swiss-Webster and C57BL/6 mice do not reach peak bone mass until the age of 5 to 6 months (3). Accordingly, we (2) assessed the suitability of estren using 6- to 8-month-old animals and consider the use of 3-month-old mice inappropriate. In addition, by using 3-month-old C57BL/6 mice (20–25 g) as compared with 6- to 8-month-old Swiss-Webster mice (45–50 g), Windahl et al. have effectively doubled the original dose. Moreover, while 5 ng/g/d of estradiol is needed to prevent loss of uterine weight in 3-month-old ovariectomized C57BL/6 mice, an 8-fold higher dose, 40 ng/g/d, is needed to prevent loss of uterine weight in 6-month-old C57BL/6 mice (4). Unlike Windahl et al., others have reported that, at the dose we had used, estren was ineffective in 10- to 12-week-old mice in a 3-day uterine bioassay (5) and that it exhibited only 16% of the activity of 17β-estradiol in 11-month-old mice (6). Hence, the difference between our 2 studies could simply be accounted for by the difference in the size and age of the animals. While there is an 8-fold higher sensitivity of the uterus to estradiol in 3- versus 6-month-old mice, estradiol at 5 ng/g/d could prevent bone loss in both 3- and 6-month-old C57BL/6 mice (4), indicating a differential sensitivity of uterus and bone to estrogen replacement in adulthood. Notably, a 2-year-long administration of very low–dose estradiol to postmenopausal women decreased bone turnover without endometrial changes (7). Previously, Galien et al. (8) used an isomer of estren to maintain bone mass but, similarly to the authors’ new selective estrogen receptor modulator (SERM) PSK3471, it only “moderately increased” reproductive organ weight compared with that of controls, confirming our original finding that estren works on bone without affecting reproductive organs (2). This omission is disturbing. Worse, Supplemental Figure 3 shows that decreasing the dose of estren to 25 μg/mouse/d significantly decreased the effect of the compound on seminal vesicle weight but did not diminish its beneficiary effect on bone volume. Yet the authors chose to interpret insignificant data — characterizing values as “somewhat decreased” — to buttress their conclusion that the effects of estren on bone and seminal vesicles cannot be separated. The mice in the Windahl study had none of the expected cellular and biochemical changes that occur after loss of estrogens in mice and humans. Estradiol replacement increased rather than decreased osteoclast number, in opposition to multiple studies from the last 15 years. Also, despite the increase in osteoclast numbers, estrogen replacement increased trabecular bone volume as compared with that of the controls. Therefore, the authors concluded that estrogens (and PSK3471) are anabolic on bone. This is an erroneous and misleading conclusion, as it has been established that estrogens are anticatabolic/antiremodeling agents (9). Equally puzzling, PSK3471 had no effect on any parameter of bone turnover. That PSK3471 had the undesirable effect of stimulating uterine growth in both the myometrium and endometrium was completely ignored. We and others have advanced the idea that the development of function-specific estrogen and androgen-receptor ligands could delineate the significance of nonclassical functions of these receptors. In the process, several biologic properties of these compounds not exhibited by the natural ligands have been discovered, including the ability to induce osteoblast differentiation by stimulating Wnt and BMP signaling in a kinase-dependent manner. In dismissing estren while promoting a SERM with similar unwanted effects, Windahl et al. not only have disposed of the scholarly obligation to offer an explanation for the significant discrepancies between their findings and those of many others but have also ignored their own data.

Physiologic induction of puberty in Turner syndrome with very low-dose estradiol

Many hormone preparations are available for the induction of puberty. We present arguments and evidence that favor the use of physiologic hormone replacement regimens with estradiol and progesterone that mimic the normal changes in production of these natural hormones across puberty. Delaying estrogen therapy until 15 years of age to optimize height potential, as previously recommended, seems unwarranted. This emphasis on stature tends to undervalue the psychosocial importance of age-appropriate pubertal maturation and may be deleterious to bone and other aspects of the child's health. It is now clear that puberty induction can be initiated as early as 12 years of age with very low-dose estradiol (starting doses of one-tenth to one-eighth of the adult dose) without compromising enhancement of growth potential by growth hormone (GH). We give suggestions and recommendations for the induction of puberty in Turner syndrome on the basis of existing data from prospective clinical trials. However, such data are sparse, and there are unanswered questions about optimal treatment schedules for stature, feminization, and uterine and bone development since estradiol percutaneous preparations have only recently become available by prescription in sufficiently low dosages to initiate puberty.

Chemical Exposures: Prostate Cancer and Early BPA Exposure

In animal models, estrogens can drive carcinogenesis of the prostate and have long been suspected of playing a role in human prostate cancer. Scientists have hypothesized that prenatal exposure to estrogen-like compounds, including monomeric bisphenol A (BPA), may account for recent increases in rates of prostate cancer. Now a rat study by Gail Prins of the University of Illinois at Chicago Department of Urology, Shuk-Mei Ho of the University of Cincinnati Department of Environmental Health, and their colleagues provides the first evidence of a direct link between low-dose BPA exposure during development and later prostate cancer. BPA initially was synthesized for use in also is used as a cross-linking compound in the manufacture of polycarbonate plastics and epoxy resins. It leaches from food and beverage containers and from dental sealants, although the latter currently are not thought to be a major source of exposure. Today, this hormonally active chemical is widespread in the environment, with detectable serum levels present in approximately 90% of humans in the United States and other industrialized countries, by Prins’s estimate. BPA concentrations measured in placental and fetal tissues may be fivefold higher than maternal serum levels, with higher levels found in male fetuses compared to females, according to Prins. BPA has been in use for about 50 years in the industrialized world. Some investigators have proposed that widespread ingestion of monomeric BPA from polycarbonate food and drink containers might partially explain recent increases in prostate cancer rates. According to the American Cancer Society, rates have been on the rise since 1975. With the 1987 introduction of prostate-specific antigen testing, the newly enhanced ability to diagnose the disease caused incidence to spike to 240 age-adjusted cases per 100,000 men by 1992. After this “catch-up” period, rates dropped for three years, but are now back on the rise. In the study, described in the 1 June 2006 issue of Cancer Research, groups of newborn rats were given high or low doses of estradiol or an environmentally relevant dose of BPA. The findings provide a molecular underpinning for potential long-term effects by showing changes in methyl groups on DNA that are responsible for turning genes on or off. For example, one key prostate gene that normally fuels cell growth during development stayed turned on in the prostates of male rats exposed to BPA or elevated estradiol from birth, says Prins. Such epigenetic alterations may leave a permanent impression on genes, possibly sensitizing the animal to developing diseases later in life. One must exercise caution, however, in extrapolating the current rat findings to humans. How might one conduct an analogous program of research on men? The researchers consider such a program virtually impossible because 50 years or more typically would be required for results of early exposure to BPA to show up as prostate cancer. Indeed, Rebecca Sokol, a professor of medicine at the University of Southern California, cautions against extrapolating human effects from rat studies. She does, however, note that unlike strong carcinogens that damage DNA profoundly, BPA appears to cause subtle changes that may pass from one generation to the next. She asks whether something is happening to alter genes seemingly without changing the genetic code itself. Says Prins, “Our evidence shows that in an animal model, some genes are altered by the addition or removal of methyl groups on the DNA, which changes the ability of those genes to be transcribed and translated to proteins. It is possible that these effects may pass through generations as has been shown recently for sperm cells.” However, she adds, that analysis awaits future studies.

Phase II antioxidant enzyme activities in brain of male and female ACI rats treated chronically with estradiol

Activities of Phase II antioxidant enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), and phenol sulfotransferase 1A1 (SULT1A1) were measured in brain of August–Copenhagen Irish (ACI) rats exposed chronically to low doses of estradiol (E 2). ACI rats were selected for study because this strain is highly responsive to treatment with low doses of E 2 as indexed by a high incidence of E 2-induced mammary tumors compared to other strains. Rats were exposed chronically to 3 mg E 2 contained in cholesterol pellets implanted subcutaneously for 6 weeks. This treatment increased activities of all four enzymes in the striatum of male but not female ACI rats. Blood E 2 levels at time of sacrifice correlated closely with activities of striatal NQO1, GST, and SULT1A1, but not with striatal UGT. NQO1, GST, and SULT1A1 activities in other brain regions including the cortex, cerebellum, and hippocampus were less sensitive to chronic E 2 treatment. NQO1 was primarily localized in vascular elements and neurons and SULT1A1 primarily in neurons and neuropil of control and E 2-treated rats. Collectively, these results suggest that enhanced expression of NQO1, GST, and SULT1A1 may contribute to the antioxidant effects of E 2 in the striatum, an area of the brain that may be particularly prone to oxidative stress because of its high content of catecholamines.

Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary1

Although molecular mechanisms underlying steroid effects on GnRH and dopamine receptors are well documented in mammals, little is known in fish. Herein, we describe the expression of pituitary GnRH and dopamine receptors relative to gonadotropin expression and release. We exposed female tilapia to graded doses of estradiol or 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) in vitro, and of estradiol in vivo, and determined mRNA levels of gnrhr1, gnrhr3, drd2, lhb, and fshb by real-time PCR. We also determined gonadotropin levels using specific ELISAs. Exposure to low doses of estradiol caused increased gnrhr3 mRNA levels in vivo and in vitro, probably related to positive feedback on FSH release. Increasing concentrations of estradiol resulted in increased drd2 mRNA levels in vivo and in vitro, inhibition of LH and FSH release, and inhibition of lhb mRNA levels in vivo, possibly related to negative feedback. At high doses of estradiol, FSH release increased in preparation for a new generation of follicles. Exposure to nanomolar doses of DHP resulted in increased drd2 mRNA levels, probably related to negative feedback on LH release. A decrease in drd2 levels at the micromolar range of DHP (concomitant with increased gnrhr3 and fshb mRNA levels) may be related to the recruitment of a new generation of oocytes. Exposure to DHP also resulted in increased lhb mRNA levels toward final oocyte maturation. Salmon GnRH analog (sGnRHa) increased mRNA levels of gnrh1and gnrh3; when combined with DHP, sGnRHa synergistically increased expression of gnrh3 only. These results emphasize the role of sex steroids on positive and negative feedbacks controlling the reproductive cycle.

Aromatase inhibition and very low dose estradiol add-back: A pilot study for novel breast cancer prevention

1014 Background: Aromatase inhibitors (AIs) are important agents for the treatment of hormone receptor positive breast cancer in post menopausal women. They are being assessed as chemopreventives of breast cancer in large clinical trials, but there are known issues of tolerability notably affecting bone. It is possible that decreasing, but not abolishing, circulating estradiol levels may reduce breast cancer risk without such side effects. We have undertaken a novel pilot study to establish whether healthy volunteers on an AI could be delivered very low dose estradiol replacement to a predictable serum level of 10–20 pmol/L using a transdermal patch, and whether this would reverse changes in the bone resorption biomarker CTx. Methods: Ten healthy postmenopausal women with normal estradiol levels received letrozole 2.5mg/day for 16 weeks. From weeks 6–16 they were also given a quarter of Estraderm MX25 transdermal patch (6.25 μg of estradiol) replaced, twice weekly. Fasting blood samples were taken at baseline, 6, 10, 12, 14 and 16 weeks for estradiol, FSH, LH, SHBG and CTx. Results: Serum estradiol levels fell to &lt;3pmol/L at 6 weeks consistent with AI therapy. The quarter patch successfully replaced estradiol levels to a median of 12pmol/L although there was marked inter and intrasubject variability. FSH rose significantly with AI therapy with no appreciable change in SHBG or LH. There is encouraging evidence that CTx levels which increased with AI (p=0.076) can be corrected with low dose estradiol replacement although this did not reach statistical significance in this initial group. We continue to collect data on a further 8 subjects already recruited into the study. Conclusions: A quarter of Estraderm MX25 transdermal patch can be used to provide a serum estradiol level of 10–20 pmol/l in postmenopausal women given an AI. Further data will confirm whether this novel approach to chemoprevention can reverse the negative impact of AIs on bone metabolism and be suitable for extending to a full chemoprevention trial. [Table: see text] No significant financial relationships to disclose.

Systemic and local administration of estradiol into the prefrontal cortex or hippocampus differentially alters working memory

The influence of estradiol on learning and memory is dependent on a number of factors. The effects of physiological levels of estradiol on the acquisition of a spatial working memory task mediated by the prefrontal cortex (PFC) and the hippocampus were examined in Experiment 1. Ovariectomized Long–Evans rats received daily injections of estradiol or vehicle were tested on the win-shift version of the radial arm maze. A high dose of estradiol benzoate (5 μg) enhanced acquisition of the task, whereas a low dose of estradiol (0.3 μg) increased the number of errors committed over 17 days of testing. Experiment 2 was conducted to examine site-specific influences of estradiol on spatial working memory in well-trained rats. Saline and estradiol cyclodextrin (0.1 and 0.9 μg) were infused into the prelimbic region of the PFC or dorsal hippocampus 40 min prior to testing on the win-shift task. Infusions of estradiol into both brain areas attenuated saline-infusion disruptions in working memory. Specifically, the higher dose of estradiol facilitated working memory when infused into the PFC, whereas the lower dose of estradiol facilitated performance when infused into the dorsal hippocampus. Moreover, working memory was significantly impaired 24 h after infusions of estradiol into the dorsal hippocampus but not the PFC. These data provide further evidence for the notion that estradiol can dose-dependently alter memory processes and suggest that facilitation or disruptions of working memory by estradiol are site- and time-specific.

Effects of HRT on liver enzyme levels in women with type 2 diabetes: a randomized placebo‐controlled trial

Increasingly strong links are being recognized between diabetes, insulin resistance and liver fat accumulation [e.g. nonalcoholic fatty liver disease (NAFLD)]. Recent data indicating that hormone replacement therapy (HRT) may lessen diabetes risk is intriguing but explanatory mechanisms are unclear. Post hoc investigation of the possibility that HRT may favourably influence liver enzyme levels commonly elevated in patients with diabetes. We examined liver function test data from a 6-month trial of a low-dose continuous combined HRT (1 mg 17beta oestradiol and 0.5 mg norethisterone acetate). Double-blind, randomized placebo-controlled. Fifty women with type 2 diabetes. Liver enzyme levels (AST, ALT, gamma-glutamylytransferase [GGT], and alkaline phosphatase [ALP]). Forty-five women completed the study with 19/22 in the active group demonstrating compliance as measured by sex hormone changes. Relative to placebo recipients (n = 23), women randomized and compliant to HRT demonstrated significant reductions in ALT [-14 (-23 to -6) U/l, P = 0.002], AST [-9.2 (-14 to -5) U/l, P < 0.001] and ALP [-60.8 (-80 to -42) U/l, P < 0.001]. Circulating concentrations in GGT were also significantly reduced (P = 0.035). All changes were significant using an intention-to-treat analysis. HRT containing low-dose oestradiol and norethisterone reduces serum concentrations of liver function enzymes, potentially due to a lowering of liver fat accumulation. Better understanding of mechanisms by which this HRT improves liver function tests could help the design of new therapies to treat individuals with NAFLD.

Oral hormone therapy with 17??-estradiol and 17??-estradiol in combination with norethindrone acetate in the prevention of bone loss in early postmenopausal women: dose-dependent effects

A 2-year multicenter, double-blind, randomized, placebo-controlled study examined the efficacy and safety of different doses of 17beta-estradiol (E(2)) alone and continuous-combined oral formulations of E(2) and norethindrone acetate (NETA) versus placebo in the prevention of bone loss in newly menopausal women. Patients were randomized to one of seven groups: placebo, E(2) 0.25 mg, E2 0.5 mg, E(2) 1 mg, E(2) 1 mg/NETA 0.25 mg, E(2) 1 mg/NETA 0.5 mg, or E(2) 2 mg/NETA 1 mg. Treatment was a once-daily tablet taken for 26 months. The primary efficacy endpoint was the change in bone mineral density (BMD) at the lumbar spine, measured by dual-energy x-ray absorptiometry, at screening and at 13, 19, and 26 months. BMD changes at the femoral neck and trochanter were also assessed. Biochemical markers of bone metabolism were measured at baseline, and at 3, 6, 13, 19, and 26 months. Histological diagnoses of endometrial samples were tabulated for each treatment group. A total of 327 women were randomized and 189 women completed the 2-year trial. BMD at the lumbar spine decreased 2.3% in the placebo group. The lowest dose of unopposed E(2) prevented bone loss at the spine and hip. Significant increases in spine BMD compared with placebo occurred in all groups of treatment with E(2) and were more pronounced in the combination groups. Compared with placebo, women receiving active treatment experienced greater reductions in bone resorption markers. The effects were evident by 6 months and generally remained stable thereafter. Adverse events, primarily associated with the endometrium, were the most common reasons for discontinuation. There is a dose-dependent effect of E(2) on BMD. The addition of NETA seems to enhance the response in BMD observed with E(2). Low doses of E(2) (1 mg and lower) can be considered for the prevention of osteoporosis, while titrating the hormone dose to individual patient's needs.

Chronic Treatment with Low Doses of Estradiol Affects Pituitary and Thyroid Function in Young and Middle-aged Ovariectomized Rats

Menopause marks the end of the reproductive life span of women and is characterized by a dramatic drop of circulating estrogen. Hormone replacement therapy (HRT) has been successfully used to treat the symptoms of menopause because estrogen has strong suppressive effects on climacteric complaints. As the hypothalamo-pituitary-thyroid axis is a target for estrogen action we assessed the effect of ovariectomy (OVX) and long-term (3 months) estradiol (E2) treatment by means of subcutaneously (s.c.) implanted silastic capsules on pituitary and thyroid function in middle-aged female rats. Our results demonstrate that s.c. administration of E2 resulted in physiological serum concentrations of E2. Upon OVX, ERbeta mRNA in the pituitary was induced and amounts of transcripts decreased after E2 administration. Implanted E2 capsules led to a reduction of ERalpha mRNA in the pituitary. E2 treatment attenuated the OVX-induced increases in TSHbeta and TSHalpha mRNA. Chronic E2 treatment reduced total T4 levels in OVX animals. TSH and total T3 serum levels were not altered upon E2 treatment. Taken together, our results clearly demonstrate that both, the pituitary and the thyroid of middle-aged rats remain susceptible to the influence of OVX and E2 treatment.

Characterization of Osteocrin Expression in Human Bone

Osteocrin (Ostn), a bone-active molecule, has been shown in animals to be highly expressed in cells of the osteoblast lineage. We have characterized this protein in human cultured primary human osteoblasts, in developing human neonatal bone, and in iliac crest bone biopsies from adult women. In vivo, Ostn expression was localized in developing human neonatal rib bone, with intense immunoreactivity in osteoblasts on bone-forming surfaces, in newly incorporated osteocytes, and in some late hypertrophic chondrocytes. In adult bone, Ostn expression was specifically localized to osteoblasts and young osteocytes at bone-forming sites. In vitro, Ostn expression decreased time dependently (p<0.02) in osteoblasts cultured for 2, 3, and 6 days. Expression was further decreased in cultures containing 200 nM hydrocortisone by 1.5-, 2.3-, and 3.1-fold (p<0.05) at the same time points. In contrast, alkaline phosphatase expression increased with osteoblast differentiation (p<0.05). Low-dose estradiol decreased Ostn expression time dependently (p<0.05), whereas Ostn expression in cultures treated with high-dose estradiol was not significantly changed. These results demonstrate that Ostn is expressed in human skeletal tissue, particularly in osteoblasts in developing bone and at sites of bone remodeling, suggesting a role in bone formation. Thus, Ostn provides a marker of osteoblast lineage cells and appears to correlate with osteoblast activity.

Endometrial and vaginal effects of low-dose estradiol delivered by vaginal ring or vaginal tablet

Aims The major aims of the study were to compare the safety of a continuous low-dose estradiol-releasing vaginal ring (ESTring) to that of a vaginal estradiol tablet (Vagifem®) on the endometrium and the relief of subjective symptoms and signs of urogenital estrogen deficiency. Quality of life and acceptability of treatment delivery were also assessed.Study design A prospective, randomized study in which women were assigned in a 2 : 1 ratio to ESTring and Vagifem and followed for 12 months. The primary endpoint was endometrial safety, based on the results of ultrasound measurement of endometrial thickness and a progestogen challenge test at baseline and week 48. Efficacy was determined by subjective assessment of urogenital estrogen deficiency symptoms at baseline and weeks 3, 12, 24, 36 and 48 and assessment of signs of vaginal epithelial atrophy by the clinician at baseline, 12 and 48 weeks. In addition, pelvic floor strength, vaginal cytological evaluation and pH, bacteruria and patient acceptability were assessed. Quality of life was assessed using a menopause-specific quality-of-life questionnaire and a 2-day bladder diary at baseline and 12 and 48 weeks. The comparability of the two groups was assessed using ANOVA, χ2 or Fisher's exact tests.Results A total of 126 women were randomized to ESTring and 59 to Vagifem. There was no statistical difference between the groups in the alleviation of symptoms and signs of urogenital estrogen deficiency. Maturation indices increased in both groups, from generally atrophic at baseline to proliferative or highly proliferative at 48 weeks. After 48 weeks of treatment, there was no statistically significant difference in endometrial thickness between the two groups. A statistically smaller proportion of bleeding/spotting occurred in the ESTring group (n = 0) compared to the Vagifem users (n = 4). Estradiol and total estrone serum levels increased during treatment in both groups but remained within the normal postmenopausal range. General health status in both groups was unchanged but the urogenital component of health burden was significantly improved in both groups. Bladder diary variables showed no differences between treatment groups.Conclusion Equivalent endometrial safety and efficacy in the relief of the symptoms and signs of urogenital estrogen deficiency were demonstrated for the 12 months' use of a low-dose estradiol-releasing vaginal ring and a vaginal estradiol tablet.

A Randomized Placebo-Controlled Trial of Short-Term Graded Transdermal Estradiol in Healthy Gonadotropin-Releasing Hormone Agonist-Suppressed Pre- and Postmenopausal Women: Effects on Serum Markers of Bone Turnover, Insulin-Like Growth Factor-I, and Osteoclastogenic Mediators

The acute effects of estradiol on procollagen type 1 formation in pre- and postmenopausal women are controversial. Twenty-three premenopausal women and 13 postmenopausal women received two consecutive im injections of 3.75 mg leuprolide acetate 3 wk apart to block endogenous ovarian steroidogenesis. Transdermal estradiol therapy commenced on the night of the second leuprolide injection in all subjects, except five pre- and two postmenopausal women who were randomized to receive placebo patches. Estradiol therapy was applied incrementally, with each dose of 0.05, 0.10, 0.15, and 0.20 mg/d administered for 4 consecutive days, to mimic the estradiol changes typifying the follicular phase of the menstrual cycle. Blood aminoterminal propeptide of type I procollagen (PINP), intact osteocalcin (OC), carboxyterminal telopeptide of type I collagen (CTx), IGF-I, and estradiol were measured before and at the end of each estradiol increment. Potential mediators such as osteoprotegerin and receptor activator of nuclear factor-kappaB ligand (RANKL) were also measured. Despite comparable increases in serum estradiol, PINP increased more in postmenopausal compared with premenopausal women (between-group P = 0.03) and occurred at a time when CTx and OC did not change. CTx and IGF-I changed minimally and inconsistently, whereas OC, RANKL, and osteoprotegerin were stable. Repeated measures linear regression disclosed a significant negative association between increases in estradiol and PINP in premenopausal women (P = 0.0006) only. This suggests that lower dose estradiol should greatly increase PINP. Analogous regressions also showed significant negative relationships between changes in estradiol and RANKL in both pre- (P = 0.04) and postmenopausal (P = 0.002) women. Changes in serum markers of bone formation (PINP or OC) did not correlate with those of IGF-I. We conclude that lower dose estradiol rapidly increases osteoblastic collagen synthesis in women at a time when collagen degradation is stable and that this response differs between pre- and postmenopausal women. The effect of estradiol on bone formation does not appear to be mediated by IGF-I. In contrast, RANKL is likely to mediate the effect of estradiol on osteoclastogenesis.

Lipids and clotting factors during low dose transdermal estradiol/norethisterone use

Objective: To demonstrate the effects of 2-year transdermal continuous combined low-dose estradiol (0.025 mg/day) and norethisterone acetate (0.125 mg/day) on lipid/lipoprotein profile and coagulation/fibrinolysis. Methods: A double-blind, randomized, multicenter, parallel, 1-year trial enrolled 266 healthy women at least 2 years post menopause. Patients received either 0.025 mg estradiol and 0.125 mg norethisterone acetate daily or placebo transdermally. One hundred and thirty five women completed a second year open follow-up (96 had used Estragest TTS, 39 placebo during the first year), where all women had the estradiol/norethisterone patch. Lipid/lipoprotein profile and coagulation/fibrinolysis parameters were studied at 0, 24, 48, 72 and 96 weeks. Results: In women on estradiol/norethisterone total cholesterol, Lp(a) and VLDL cholesterol decreased significantly more than in the placebo group after 24 weeks and LDL cholesterol after 48 weeks. Women on estradiol/norethisterone had no change in HDL, triglycerides or Lp(a), an increased HDL/total cholestrol ratio and decreased LDL, VLDL and total cholesterol at 48 weeks compared to placebo. Women with active treatment also showed a significant reduction compared with the placebo group of Factor VII and antithrombin III at 24 and 48 weeks and a reduction of fibrinogen at 24 weeks. These changes persisted over the second year. Conclusions: A continuous combined low-dose transdermal patch daily delivering 0.025 mg estradiol and 0.125 mg norethisterone acetate provided beneficial effects on lipid/lipoprotein profile and coagulation/fibrinolysis. The changes were similar to those previously described after higher dose oral and transdermal estrogen/progestogen regimens.