Folic acid supplementation in postmenopausal women with hot flushes: phase III randomised double-blind placebo-controlled trial.

OBJECTIVE: To assess whether folic acid supplementation ameliorates hot flushes. DESIGN: Double-blind, placebo-controlled randomised trial. SETTING: Nine hospitals in England. POPULATION: Postmenopausal women experiencing ≥50 hot flushes weekly. METHODS: Women (n = 164) were randomly assigned in a 1:1 ratio to receive folic acid 5 mg tablet or placebo daily for 12 weeks. Participants recorded frequency and severity of hot flushes in a Sloan Diary daily and completed Greene Climacteric and Utian Quality of Life (UQoL) Scales at 4-week intervals. MAIN OUTCOME MEASURES: The change in daily Hot Flush Score at week 12 from randomisation based on Sloan Diary Composite Score B calculation. RESULTS: Data of 143 (87%) women were available for the primary outcome. The mean change (SD) in Hot Flush Score at week 12 was -6.98 (10.30) and -4.57 (9.46) for folic acid and placebo group, respectively. The difference between groups in the mean change was -2.41 (95% CI -5.68 to 0.87) (P = 0.149) and in the adjusted mean change -2.61 (95% CI -5.72 to 0.49) (P = 0.098). Analysis of secondary outcomes indicated an increased benefit in the folic acid group regarding changes in total and emotional UQoL scores at week 8 when compared with placebo. The difference in the mean change from baseline was 5.22 (95% CI 1.16-9.28) and 1.88 (95% CI 0.23-3.52) for total and emotional score, respectively. CONCLUSIONS: The study was not able to demonstrate that folic acid had a statistically significant greater benefit in reducing Hot Flush Score over 12 weeks in postmenopausal women when compared with placebo. TWEETABLE ABSTRACT: Folic acid may ameliorate hot flushes in postmenopausal women but confirmation is required from a larger study.

Microsatellite variation of ESR1, ESR2, and AR in Serbian women with primary ovarian insufficiency.

OBJECTIVE: This study aimed to investigate the potential role of microsatellite polymorphisms of the estrogen receptor alpha gene (ESR1) TA repeat, estrogen receptor beta gene (ESR2) CA repeat, and androgen receptor gene (AR) CAG and GGN repeats among Serbian women with primary ovarian insufficiency (POI). These microsatellites have been reported to be associated with POI in different racial/ethnic populations. METHODS: A cohort of 196 POI cases matched with 544 fertile controls was recruited by the Institute for Endocrinology, Diabetes and Metabolic Disorders of Serbia between 2007 and 2010. DNA was extracted from saliva. The four microsatellites were genotyped using a PCR-based assay to determine the repeat lengths. RESULTS: POI patients carried shorter repeat lengths of ESR2 (CA)n than controls (P = 0.034), but the difference was small. ESR1 (TA)n was on the borderline of statistical differences between groups (P = 0.059). AR (CAG)n and (GGN)n showed no association with POI. CONCLUSIONS: We cautiously conclude that microsatellite polymorphisms of gonadal steroid receptor genes might contribute to the genetic basis of POI in Serbian women, but a larger-scale study and family-based studies are warranted to validate our findings even though the sample size in this study is larger than any previously published in this field.

Lack of association between ESR1 gene polymorphisms and premature ovarian failure in Serbian women.

OBJECTIVE: It has previously been reported that estrogen receptor-alpha (ERα) gene (ESR1: estrogen receptor 1) polymorphisms are associated with premature ovarian failure (POF). The aim of this study was to investigate whether these genetic polymorphisms of ESR1 are associated with POF in Serbian women. METHODS: A series of 197 POF cases matched with 547 fertile controls was recruited by the Institute for Endocrinology, Diabetes and Metabolic Disorders of Serbia between 2007 and 2010. Genomic DNA was extracted from saliva using Oragene® DNA sample collection kits. Two single-nucleotide polymorphisms (SNPs), PvuII and XbaI, in ESR1 were genotyped by dynamic allele-specific hybridization. Haplotype analyses were performed with the restriction fragment length polymorphism method. SNP and haplotype effects were analyzed by logistic regression models. RESULTS: No significant difference was found in the distribution of ESR1 PvuII and XbaI polymorphisms or haplotypes between the POF and control groups. CONCLUSION: The two ESR1 SNPs, PvuII and XbaI, are not commonly associated with POF in Serbian women and may not contribute to the genetic basis of the condition.

Testosterone treatment of HSDD in naturally menopausal women: the ADORE study.

OBJECTIVE: To evaluate the efficacy and safety of a transdermal testosterone patch (TTP, 300 microg/day) in naturally menopausal women with hypoactive sexual desire disorder (HSDD). METHODS: A total of 272 naturally menopausal women, predominantly not using hormone therapy, were randomized in this 6-month, placebo-controlled, double-blind, multicenter study to receive twice weekly either TTP or an identical placebo. Efficacy endpoints measured were the 4-week frequency of satisfying sexual episodes (SSE) using the Sexual Activity Log, the sexual desire domain of the Profile of Female Sexual Function and distress by the Personal Distress Scale. Safety was assessed by adverse events, laboratory parameters and hormone levels. RESULTS: The TTP group demonstrated significant improvements in SSE (p = 0.0089) as well as in sexual desire (p = 0.0007) and reduced personal distress (p = 0.0024) versus placebo at 6 months (intent-to-treat analysis, n = 247). The results were significant for all three endpoints in the subgroup (n = 199) not using hormone therapy. Similar numbers of women treated with placebo and TTP discontinued (n = 39, 27.5% vs. n = 26, 20%), reported adverse events (including application site reactions) (n = 101, 71.1% vs. n = 81, 62.3%) and withdrew due to adverse events (n = 20, 14.1% vs. n = 9, 6.9%). No clinically relevant changes were noted in laboratory parameters. Serum free and total testosterone levels increased from baseline in the TTP group (geometric means 5.65 pg/ml and 67.8 ng/dl, respectively, at week 24) within the physiological range; no changes were seen in estradiol and sex hormone binding globulin levels. CONCLUSIONS: TTP was effective in treating HSDD and improving sexual function in this study of naturally menopausal women with and without concurrent hormone therapy.

Therapeutic options for postmenopausal female sexual dysfunction.

BACKGROUND: Female sexual dysfunction (FSD) is a multidimensional problem combining biological, psychological and interpersonal elements of multiple etiologies. Menopause-related sexual dysfunction may not be reversible without therapy. Hormonal deficiency does not usually decrease in severity over time. Many options are available for the successful treatment of postmenopausal FSD. OBJECTIVE: To review the pharmacological and non-pharmacological therapies available for postmenopausal FSD, focusing on practical recommendations for managing postmenopausal women with sexual complaints, through a literature review of the most relevant publications in this field. PSYCHOSOCIAL THERAPY: This type of therapy (basic counselling, physiotherapy and psychosexual intervention) is considered an adaptable step-by-step approach for diagnostic and therapeutic strategies, normally combined with biomedical interventions to provide optimal outcomes. PHARMACOLOGICAL THERAPY: For postmenopausal FSD, many interventional options are now available, including hormonal therapies such as estrogens, testosterone, combined estrogen/testosterone, tibolone and dehydroepiandrosterone. CONCLUSIONS: Menopause and its transition represent significant risk factors for the development of sexual dysfunction. FSD impacts greatly on a patient's quality of life. Consequently, it is receiving more attention thanks to the development of effective treatments. Non-pharmacological approaches should be used first, focusing on lifestyle and psychosexual therapy. If required, proven effective hormonal and non-hormonal therapeutic options are available.

Differential expression of oestrogen receptor isoforms and androgen receptor in the normal vulva and vagina compared with vulval lichen sclerosus and chronic vaginitis.

BACKGROUND: Although the expression of the oestrogen receptor (ER) alpha isoform and androgen receptor (AR) has been examined in vulval lichen sclerosus (VLS), the distribution pattern of ERalpha, ERbeta and AR has not been described in chronic atrophic vaginitis nor correlated with markers of proliferation (Ki-67) in either of these diseased tissues. OBJECTIVES: To measure the levels and distribution of ERalpha, ERbeta and AR immunoreactivity in relation to Ki-67 in normal and diseased vulva and vagina. METHODS: The expression of ERalpha, ERbeta and AR in relation to the proliferation marker Ki-67 in VLS, squamous hyperplasia of the vulva and chronic atrophic vaginitis was determined by immunohistomorphometric analysis and compared with that in normal vulva and vagina. RESULTS: VLS showed similar ERalpha and ERbeta expression in the 'epidermal' and 'dermal' tissue layers to that of normal vulvae, whereas AR expression appeared to be absent in most cases. ERbeta and Ki-67 expression was correlated with ERalpha expression but only in the 'fibrovascular' layer of the vulva. ERalpha expression was absent from the 'fibromuscular' layer of diseased vulvae, while ERbeta expression was absent in normal tissues but was highly expressed in diseased vulvae. ERalpha expression was significantly correlated with AR expression in the fibrovascular layer of the vagina and inversely correlated with Ki-67 staining in the parabasal cells of the epidermis in patients with chronic atrophic vaginitis. CONCLUSIONS: These data suggest that ER expression and levels may be implicated in the aetiopathology of VLS and chronic atrophic vaginitis.

Trimegestone differentially modulates the expression of matrix metalloproteinases in the endometrial stromal cell.

Matrix metalloproteinases (MMP) are considered to be of critical importance in the initiation of menstruation where MMP protein levels are reciprocally modulated by the actions of the gonadal steroid hormones, estradiol (E(2)) and progesterone (P4), with P4 being considered the principal suppressor of endometrial MMP expression. Trimegestone (T) is a novel progestagen that tightly controls menstruation timing and duration through mechanisms that might involve MMP suppression. Endometrial stromal cells treated with 10(-6) M E(2), P4 or T in the presence and absence of 10(-6)M RU486 showed that both T and P4 suppressed the expression of MMP-1 and MMP-3 transcripts and secreted protein, whereas MMP-9 was not produced in culture. The suppressive effect of T or P4 on MMP-1 and MMP-3 transcript levels was enhanced in the presence of E(2) and attenuated in the presence of RU486, although MMP-1 proteins were unaffected by the presence of RU486, which alone acted as a partial progesterone agonist in these cultures. Immunohistochemistry with MMP-1, MMP-3 and MMP-9-specific antibodies performed on endometrial biopsies obtained from non-treated, LH-dated, normally cycling women and endometrial biopsies obtained from postmenopausal women treated with T-based HRT showed that immunoreactive MMP-1 and MMP-3 was higher in the menstrual phase, whilst MMP-9 expression was higher in the late luteal phase (P = 0.03) and T significantly inhibited the presence of MMP-9(+) cells. These data suggest that T acts in a similar manner to P4, but causes subtle differences in expression patterns of MMPs that may explain the different clinical effect that this progestagen has on endometrial behaviour compared to P4.

Nasal physiological changes during pregnancy.

Rhinitis in pregnancy has been previously investigated with variable results. This study examines all the variables of the nasal airway simultaneously for the first time. Eighteen women were recruited in the first trimester of pregnancy and followed through to the postpartum period to monitor the changes that occurred. Measurements of the nasal airway included anterior rhinoscopy (AnR), peak inspiratory nasal flow, acoustic rhinometry, anterior rhinomanometry (ARM), and the saccharin test with rhinitis questionnaire scores providing a symptomatic measurement. All the tests showed a trend consistent with decreasing nasal patency when expressed as an average for the group as a whole, although only AnR, ARM, mucociliary clearance time and rhinitis questionnaire scores were statistically significant (P < or = 0.05). This confirms the effect of pregnancy on the nasal mucosa and coincides with the rise in the serum concentration of the female sex hormones with gestational age, returning to normal postpartum. Pharmacological antagonism of oestrogens may therefore relieve nasal congestion and is currently under further research.

Randomized trial of effects of estradiol in combination with either norethisterone acetate or trimegestone on lipids and lipoproteins in postmenopausal women.

OBJECTIVE: This double-blind, randomized, multicenter study was designed to compare the blood lipid profiles in postmenopausal women after treatment with either a combined formulation containing estradiol (2 mg) and trimegestone (TMG 0.25 or 0.5 mg) or a standard hormone therapy (HT) containing estradiol and norethisterone acetate. METHOD: The serum concentrations of several lipids and lipoproteins were measured in this study, which was conducted over 13 cycles, each of 28 days. A total of 487 subjects were included, 349 of whom completed the study. RESULTS: The circulating concentrations of high density lipoprotein (HDL) cholesterol, HDL2 cholesterol and apolipoprotein (apo) AI increased from baseline in both estradiol/trimegestone groups, whilst levels of HDL3 cholesterol were unchanged. In contrast, in the estradiol/norethisterone acetate group, HDL cholesterol, HDL3 cholesterol and apo AI concentrations were reduced from baseline, while HDL2 cholesterol remained unchanged. Total cholesterol, low density lipoprotein (LDL) cholesterol, lipoprotein(a) and apo-B concentrations were reduced in all treatment groups. The concentration of triglycerides was elevated after treatment with the estradiol/trimegestone combinations but was unchanged after treatment with the estradiol/norethisterone acetate combination. The differences in the lipid pattern between the groups may be explained by the different pharmacological properties of the two progestogens: norethisterone exerts an androgenic effect and opposes the estrogen-induced increase in HDL cholesterol, whilst trimegestone has no androgenic effect and does not oppose the estrogenic effect. CONCLUSION: Overall, the results of this study suggest that the use of trimegestone in combination with estradiol may be preferable to norethisterone acetate because of the more favorable HDL and apo AI profile.

Comparison of a novel vaginal ring delivering estradiol acetate versus oral estradiol for relief of vasomotor menopausal symptoms.

OBJECTIVE: To compare a novel vaginal ring releasing estradiol acetate (Menoring; Galen Holdings) with oral estradiol for relief of moderate to severe vasomotor symptoms in healthy postmenopausal women. DESIGN: This was a prospective, double-blind, multicenter, randomized, parallel-group study. METHOD: Women (n = 159) aged < 65 years experiencing >or= 20 hot flushes/night sweats per week received either a vaginal ring releasing estradiol acetate at a rate equivalent to 50 microg/day estradiol plus placebo tablets or oral estradiol 1 mg/day plus a placebo vaginal ring for 24 weeks. For patients with inadequate control of symptoms, the dosage was doubled at 12 weeks. A 24-week, open-label extension of the vaginal ring treatment followed double-blind treatment. RESULTS: The frequency of hot flushes/night sweats was significantly reduced (p < 0.001) in both groups at 12 and 24 weeks from baseline, by 84% and 94% for the vaginal ring group and by 73% and 83% for the oral group, respectively. The mean intensity of urogenital symptoms decreased from screening to the end of treatment in both groups. The incidence of adverse events was similar for both groups. No clinically relevant local effects of the vaginal ring were observed. CONCLUSIONS: The vaginal ring relieved both systemic and urogenital symptoms and was well tolerated and accepted. Overall, the efficacy, safety and acceptability of the vaginal ring were comparable with those of oral estradiol therapy.

Oestrogen receptor beta is the predominant oestrogen receptor in human scalp skin.

Oestrogens play a major role in non-classic target tissues in both sexes, yet there have been few studies on estrogens and skin. Recently a second oestrogen receptor (ERbeta) has been discovered. Therefore, we have compared the expression of oestrogen receptor alpha (ERalpha), beta (ERbeta), the androgen receptor (AR) and a cell proliferation marker in male and female non-balding scalp skin. ERbeta was the major steroid receptor expressed in human skin. It was highly expressed in epidermis, blood vessels and dermal fibroblasts, in contrast to ERalpha and AR. In the hair follicle, ERbeta expression was localized to nuclei of outer root sheath, epithelial matrix and dermal papilla cells, in contrast to ERalpha, and the AR, which was only expressed in dermal papilla cells. Serial sections also showed strong nuclear expression of ERbeta in the cells of the bulge, while neither ERalpha nor AR was expressed. In the sebaceous gland, ERbeta was expressed in both basal and partially differentiated sebocytes. ERalpha exhibited a similar pattern of expression, while the AR was expressed in the basal and very early differentiated sebocytes. There was no obvious difference in the expression of either oestrogen receptor in male or female skin. The wide distribution of ERbeta in human skin suggests that oestrogens may play an important role in the maintenance of skin and in the regulation of the pilosebaceous unit, and provides further evidence for oestrogen action in non-classic target tissues. The differential expression of ERalpha, ERbeta and AR in human skin suggests that the mechanisms by which steroid hormones mediate their effects may be more complex than previously thought.

Meningioma and hormonal influences.

Meningiomas are slow-growing benign brain tumors. The etiology of meningioma is largely unknown, and exposure to high-dose ionizing radiation and coexistence with certain rare genetic conditions explain only a small fraction of the incidence of the disease. The evidence that implicates gender-specific hormones in the pathogenesis of meningioma emanates from data showing increased growth of meningiomas during pregnancy and change in size during menses. Observational data have identified the menopause and oophorectomy as conferring protection against the risk of developing meningiomas, while adiposity is positively associated with the disease. These tumors are also positively associated with breast cancer, although they express a different gonadal steroid receptor repertoire. About 70% of meningiomas express progesterone receptors, while fewer than 31% express estrogen receptors. These observations suggest that progesterone influences tumor growth. A progesterone antagonist such as mifepristone therefore may inhibit tumor growth. The use of hormone replacement therapy in symptomatic postmenopausal women either with previously treated disease or with dormant tumors is discussed, but remains controversial.

Pulsed estrogen therapy: from cellular mode of action to tissue effects.

Failure to achieve a uniform response to estrogen replacement therapy may be related to the dose or route of administration of estrogen. The established doctrines of estrogen replacement therapy are under scrutiny. Recent discoveries have prompted a complete re-evaluation of estrogen action at the biochemical, physiological, and molecular level. Further questions remain as regards the correct dose of estrogen. It is becoming clear that individual cell types respond differently to different doses of estrogen and, even within a single experimental system, a considerable variation in response can be seen. Furthermore, there is an evident link between the duration of exposure to estradiol (continuous versus pulsed) and cellular behavior. In addition to this, it appears that ligand binding has a significant effect on estrogen receptor dynamics. The concept of pulsed estrogen therapy has recently been exploited by the introduction of a nasal spray delivery system. The administration of estradiol via the nasal mucosa is made possible by the use of randomly methylated alpha-cyclodextrin (RAMEB), a beta-cyclodextrin which increases the solubility of estradiol. The original pulsed plasma concentration time profile of E2 following transnasal administration was studied and the results were compared with orally or transdermally administered E2. Being dose-proportional, estradiol transnasally administered at a dose of 300 microg gave an estimated 24-h systemic exposure to exogenous estradiol (area under the curve) close to that of reference treatments. It would appear that pulsed estrogen therapy has a different cellular mechanism to that of traditional forms of administration. This new concept provides reliable dose-dependent exposure to estradiol, avoids the hepatic first-pass effect and demonstrates good biological and clinical efficacy. This development, linked to our improved understanding of the cellular effects of estrogen, ushers in a new era in postmenopausal hormone replacement therapy.

Estrogen and colon cancer: current issues.

Carcinoma of the colon is common and its incidence varies according to the geographical location and dietary habits. The aims of this paper are, first, to review the current epidemiological data on the incidence and mortality of colon cancer in postmenopausal women using hormone replacement therapy (HRT); second, to review the published data on the prevalence of estrogen receptors in healthy and malignant colonic tissue; and third, to examine the available evidence of gene silencing as applicable to this and other neoplastic conditions. Estrogen use confers overall protection, with a reduction in the incidence of colon adenoma and carcinoma of about 30%. Estrogen use reduces the colon cancer-related mortality. The risk of colon cancer is decreased among current and recent users of postmenopausal HRT but the molecular mechanisms involved remain unclear. Estrogen acts either on a single major transformation step in the oncogenetic process, or is involved in multiple events that avert the course of this transformation. Aberrant methylation of the CpG islands in the promoter regions of the estrogen receptor gene, as well as of other genes, is equivalent to the silencing of that gene, with the consequence of inactivation, or reduced expression, of a number of genes downstream, including tumor suppressor genes. This epigenetic mechanism, when reversed, suppresses the growth of cancer cells in vitro and in vivo. The ubiquitous distribution of the estrogen receptor genes and their isoforms, in a tissue-specific manner, opens new avenues for the understanding of cellular behavior in health and disease.

Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides.

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.

Changes in serum vascular endothelial growth factor following initiation of estrogen replacement after hysterectomy and oophorectomy.

BACKGROUND: Vascular endothelial growth factor has been shown to play an important role in preservation and restoration of endothelial integrity. Similar effects on endothelial function have been described with estrogen replacement. This poses the question whether some of the mechanisms ascribed to estrogen are in fact vascular endothelial growth factor mediated. The objective of this study was to examine the effect of continuous transdermal estrogen replacement on serum levels of secreted vascular endothelial growth factor in women following hysterectomy and oophorectomy. METHODS: In a nonrandomized, prospective study, 40 hysterectomized and oophorectomized women were treated with transdermal estrogen. Their serum vascular endothelial growth factor levels were measured before and six months following estrogen treatment, using a commercially available ELISA kit. RESULTS: The mean serum vascular endothelial growth factor level fell from 328 ng/ml (s.d. 164) before treatment to 285 ng/ml (s.d. 124) following six months of estrogen replacement. This decrease was statistically significant (p<0.03). CONCLUSION: Transdermal estrogen replacement in hysterectomized and oophorectomized women appears to be associated with a significant reduction in secreted serum vascular endothelial growth factor. However, the effect of hysterectomy and oophorectomy per se needs to be explained in appropriately designed trials.