Francesco Corrado

Effects of Genistein and Hormone-Replacement Therapy on Bone Loss in Early Postmenopausal Women: A Randomized Double-Blind Placebo-Controlled Study

The natural isoflavone phytoestrogen genistein has been shown to stimulate osteoblastic bone formation, inhibit osteoclastic bone resorption, and prevent bone loss in ovariectomized rats. However, no controlled clinical trial has been performed so far to evaluate the effects of the phytoestrogen on bone loss in postmenopausal women. We performed a randomized double‐blind placebo‐controlled study to evaluate and compare with hormone‐replacement therapy (HRT) the effect of the phytoestrogen genistein on bone metabolism and bone mineral density (BMD) in postmenopausal women. Participants were 90 healthy ambulatory women who were 47–57 years of age, with a BMD at the femoral neck of <0.795 g/cm2. After a 4‐week stabilization on a standard fat‐reduced diet, participants of the study were randomly assigned to receive continuous HRT for 1 year (n = 30; 1 mg of 17β‐estradiol [E2] combined with 0.5 mg of norethisterone acetate), the phytoestrogen genistein (n = 30; 54 mg/day), or placebo (n = 30). Urinary excretion of pyridinoline (PYR) and deoxypyridinoline (DPYR) was not significantly modified by placebo administration either at 6 months or at 12 months. Genistein treatment significantly reduced the excretion of pyridinium cross‐links at 6 months (PYR = −54 ± 10%; DPYR = −55 ± 13%; p < 0.001) and 12 months (PYR = −42 ± 12%; DPYR = −44 ± 16%; p < 0.001). A similar and not statistically different decrease in excretion of pyridinium cross‐links was also observed in the postmenopausal women randomized to receive HRT. Placebo administration did not change the serum levels of the bone‐specific ALP (B‐ALP) and osteocalcin (bone Gla protein [BGP]). In contrast, administration of genistein markedly increased serum B‐ALP and BGP either at 6 months (B‐ALP = 23 ± 4%; BGP = 29 ± 11%; p < 0.005) or at 12 months (B‐ALP = 25 ± 7%; BGP = 37 ± 16%; p < 0.05). Postmenopausal women treated with HRT had, in contrast, decreased serum B‐ALP and BGP levels either at 6 months (B‐ALP = −17 ± 6%; BGP = −20 ± 9%; p < 0.001) or 12 months (B‐ALP = −20 ± 5%; BGP = −22 ± 10%; p < 0.001). Furthermore, at the end of the experimental period, genistein and HRT significantly increased BMD in the femur (femoral neck: genistein = 3.6 ± 3%, HRT = 2.4 ± 2%, placebo = −0.65 ± 0.1%, and p < 0.001) and lumbar spine (genistein = 3 ± 2%, HRT = 3.8 ± 2.7%, placebo = −1.6 ± 0.3%, and p < 0.001). This study confirms the genistein‐positive effects on bone loss already observed in the experimental models of osteoporosis and indicates that the phytoestrogen reduces bone resorption and increases bone formation in postmenopausal women.

Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial

Context Women seeking alternative treatments to preserve bone often use phytoestrogens, but evidence of their effectiveness is lacking. Phytoestrogens are found in soy products. Genistein is a phytoestrogen with a structure similar to that of 17-estradiol. Contribution This randomized trial compared genistein, 54 mg/d, with placebo for 24 months in 389 osteopenic postmenopausal women. Increases in bone mineral density were greater with genistein than with placebo. Genistein also had favorable effects on markers of bone metabolism. Genistein did not increase endometrial thickness, but it did cause gastrointestinal side effects. Implications Genistein appears to have a favorable effect on markers of bone health in osteopenic postmenopausal women. Studies of its effect on fractures are needed. The Editors Postmenopausal osteoporosis is caused by a sharp decrease in estrogen levels that leads to an increased rate of bone remodeling (13). Currently available treatments for postmenopausal osteoporosis include hormone replacement therapy; calcitonin; bisphosphonates; and selective estrogen receptor modulators, such as raloxifene (4, 5). Although hormone replacement therapy is effective in reducing postmenopausal bone loss (68), it is associated with a higher risk for breast, endometrial, and ovarian cancer; cardiovascular disease; venous thromboembolism; and stroke (810). Epidemiologic data indicate that women who ingest high amounts of phytoestrogens, particularly isoflavones in soy products, have less risk for osteoporosis than do those who consume a typical Western diet (1113). Consequently, many women use phytoestrogens to maintain bone density. Genistein, an isoflavone phytoestrogen that is abundant in soybean products, structurally resembles 17-estradiol (14). As a natural selective estrogen receptor modulator, genistein may positively regulate bone cell metabolism without harmful estrogenic activity in the breast and uterus. This safe profile results from the greater affinity of genistein for estrogen receptor-, which is more abundant in bone, than for estrogen receptor-, which is abundant in reproductive tissue. Observational studies suggest that postmenopausal Asian women who consume diets high in isoflavones have a lower rate of fracture than that in other groups (15, 16). However, the mechanism of action of genistein on bone is not yet fully understood. In postmenopausal women, treatment with genistein (54 mg/d) increased bone mineral density (BMD) at the lumbar spine and femoral neck with no clinically significant adverse effects on the breast and uterus (17). In the same cohort, genistein decreased the ratio of soluble receptor activator of nuclear factor-B ligand to osteoprotegerin, which may partly account for its positive effects on BMD (18). These investigations were the first to evaluate the effects of purified, standardized genistein on bone health, but they were short in duration and included only 90 patients. One published trial assessed the effect of isoflavones on BMD in postmenopausal women, but it compared isoflavone-rich soy milk (containing only a small amount of genistein) with natural transdermal progesterone (19). Other studies of pure genistein from our research group have focused on cardiovascular outcomes or menopausal symptoms rather than on bone health (2022). We performed a randomized, placebo-controlled trial of the effects of pure genistein on bone density and bone metabolism over 24 months in a larger cohort of osteopenic postmenopausal women. Methods Design and Setting The study protocol is consistent with the principles of the Declaration of Helsinki, and participants gave written informed consent. Participants were recruited from women reporting to the Center for Osteoporosis in the Department of Internal Medicine and the Center for Menopause in the Department of Obstetrical and Gynecological Sciences, University of Messina (Messina, Italy), and to the Department of Medical Physiopathology, University La Sapienza (Rome, Italy). Three hundred eighty-nine women met the inclusion criteria and agreed to participate (Figure 1). Figure 1. Study flow diagram. Participants Participants were women 49 to 67 years of age who had been postmenopausal for at least 12 months at baseline, were in good general health, had not had a menstrual period in the preceding year, had not undergone surgically induced menopause, and had a follicle-stimulating hormone level greater than 50 IU/L and a serum 17-estradiol level of 100 pmol/L or less (27 pg/mL). At the start of the study, a complete family history was obtained, physical examination and laboratory evaluation (chemical analytes and hematologic measurements) were performed, and BMD was measured at the lumbar spine and femoral neck. Exclusion criteria were clinical or laboratory evidence of confounding systemic diseases, such as cardiovascular, hepatic, or renal disorders; coagulopathy; use of oral or transdermal estrogen, progestin, androgens, selective estrogen receptor modulators, or other steroids; use of biphosphonates, cholesterol-lowering therapy, or cardiovascular medications (including antihypertensive drugs) in the preceding 6 months; smoking habit of more than 2 cigarettes daily; treatment in the preceding year with any drug that could have affected the skeleton; family history of estrogen-dependent cancer; and BMD at the femoral neck greater than 0.795 g/cm2 (which corresponds to a T-score of 1.0 SD). Randomization and Intervention We assigned patients to groups by using a computer-generated randomization sequence with a permuted block size of 4, stratified by center. After a 4-week stabilization period during which participants received a standard low-soy, reduced-fat diet, participants were assigned to receive genistein (n= 198), 54 mg/d in 2 tablets (Laboratori Plants, Messina, Italy), or placebo (n= 191) (Figure 1). The biological effects of phytoestrogen intake are described elsewhere (23). No patient withdrew from the study during the stabilization period. The purity of genistein was 98%. Placebo and genistein tablets were identical in appearance and taste. Both genistein and placebo tablets contained calcium carbonate (500 mg) and vitamin D (400 IU). All participants were counseled on an isocaloric, reduced-fat diet composed of 25% to 30% energy from fat, less than 10% energy from saturated fatty acids, 55% to 60% energy from carbohydrates, and 15% energy from protein, with a cholesterol intake less than 300 mg/d and fiber intake of 35 g/d or greater. Recommended daily caloric intake was based on body size and was calculated by using the HarrisBenedict equation. We used this diet during the stabilization period to ensure that all participants had the same energy intake and to avoid interference with the lipid profile. The intake of soy products, legumes, or other nutrient supplements was prohibited. The isoflavone intake before randomization, as assessed by using a food-frequency questionnaire, was 1 to 2 mg/d. This intake has been shown to be typical in Western populations (24). Participants used this diet throughout the study, and adherence was reinforced by a nutritionist. Diet and body mass index were evaluated in all participants during follow-up. Primary Outcome The BMD at the anteroposterior lumbar spine and femoral neck was measured by using dual-energy x-ray absorptiometry (Hologic QDR 4500 W, Technologic, Turin, Italy) at baseline and after 12 and 24 months of treatment. The instrument was calibrated daily according to the manufacturers instructions. Reproducibility was calculated as a coefficient of variation obtained by weekly measurements of a standard phantom on the instrument and by repeated measurements obtained in 3 patients of different ages. The coefficient of variation of our instrument is 0.5% with the standard phantom; in vivo, we calculated a coefficient of variation of 1.1% for the lumbar spine and 1.5% for the femoral neck. Secondary Outcomes Bone Resorption Markers At baseline, 12 months, and 24 months, a 2-hour fasting morning urine sample was collected at the same time of day to assess urinary excretion of pyridinium crosslinks (pyridinoline and deoxypyridinoline). Pyridinoline (normal range, 26 to 91 pmol/mol creatinine) and deoxypyridinoline (normal range, 3 to 21 pmol/mol creatinine) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories, Hercules, California). Bone Formation and Bone Growth Markers and Other Variables After an overnight fast, venous blood samples were collected between 8 a.m. and 9 a.m. through a polyethylene catheter inserted in a forearm vein. The serum was separated from the blood corpuscles by centrifugation and kept frozen at 70C until analysis for bone formation and bone growth markers, calcium, intact parathyroid hormone, 25-hydroxyvitamin D3, 17-estradiol, follicle-stimulating hormone, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Serum bone-specific alkaline phosphatase (normal range, 8.5 to 17.9 g/L) and insulin-like growth factor I (normal range, 9.6 to 21.2 nmol/L) were measured by using an immunoenzymatic assay (Pantec, Turin, Italy). Serum calcium (normal range, 2.25 to 2.75 mmol/L [9 to 11 mg/dL]), serum phosphorus (normal range, 1.13 to 1.45 mmol/L [3.5 to 4.5 mg/dL]), and urinary creatinine (130 to 220 molkg1d1 [14.71 to 24.89 mg/kg of body weight per day]) were measured by using automated routine procedures. Parathyroid hormone (normal range, 12 to 100 pg/dL), 25-hydroxyvitamin D3 (normal range, 1.25 to 7.5 nmol/L), and follicle-stimulating hormone (normal range, 21 to 153 IU/L in the postmenopausal phase) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories). 17-Estradiol (normal range, 37 to 110 pmol/L in the postmenopausal phase) was evaluated by using a solid-phase immunoassay (Roche Diagnostics, Monza, Italy). Total cholesterol,

The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women

The phytoestrogen genistein improves endothelial dysfunction in ovariectomized rats through a nitric oxide-dependent mechanism. We investigated whether genistein alters the balance between the nitric oxide products and endothelin-1 and influences endothelium-dependent vasodilation in postmenopausal women. Sixty healthy postmenopausal women were enrolled in the study. A double-blind, placebo controlled, randomized design was employed. After a 4-week stabilization on a standard fat-reduced diet, participants to the study were randomly assigned to receive either genistein (n=30; 54 mg/day) or placebo (n=30). Flow-mediated, endothelium-dependent vasodilation of the brachial artery, plasma nitric oxide breakdown products and endothelin-1 levels were measured at baseline and after 6 months of genistein therapy. The mean baseline level of nitrites/nitrates was 22+/-10 micromol/l and increased to 41+/-10 micromol/ml after 6 months of treatment. The mean baseline plasma endothelin-1 level was 14+/-4 pg/ml and decreased to 7+/-1 pg/ml following 6 months of treatment with genistein. The mean baseline ratio of nitric oxide to endothelin also significantly increased at the end of treatment. Flow-mediated, endothelium-dependent vasodilation of the brachial artery was 3.9+/-0.8 mm at baseline and increased to 4.4+/-0.7 mm after 6 months of treatment. Placebo-treated women showed no changes in plasma nitrites/nitrates, endothelin-1 levels and flow-mediated vasodilation. Genistein therapy improves flow-mediated endothelium dependent vasodilation in healthy postmenopausal women. This improvement may be mediated by a direct effect of genistein on the vascular function and could be the result of an increased ratio of nitric oxide to endothelin.

Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study

PURPOSE Genistein, a phytoestrogen found in soybeans, corrects endothelial dysfunction induced by oophorectomy in animals. Using a double-blind, controlled, randomized design, we evaluated its effects on endothelial function in women. METHODS We enrolled 79 healthy postmenopausal women (mean [+/- SD] age, 56 +/- 4 years) and randomly assigned them to receive continuous estrogen/progestin therapy (n = 26; 17beta-estradiol [1 mg/d] combined with norethisterone acetate [0.5 mg/d]), genistein (n = 27; 54 mg/d), or placebo (n = 26). Brachial artery flow-mediated, endothelium-dependent vasodilation and plasma levels of nitrites/nitrates (a marker of nitric oxide metabolism) and endothelin-1 were measured at baseline and after 1 year of therapy. RESULTS Treatment with genistein increased levels of nitrites/nitrates (mean increase, 21 micromol/L; 95% confidence interval [CI]: 15 to 26 micromol/L; P <0.001 vs. placebo); estrogen/progestin therapy caused similar changes (P <0.001 vs. placebo). Plasma endothelin-1 levels decreased following 12 months of genistein (mean decrease, 7 pg/mL; 95% CI: 3 to 10 pg/mL; P <0.001 vs. placebo) and after 12 months of estrogen/progestin (P <0.001 vs. placebo). When compared with placebo, brachial artery flow-mediated dilation was improved by genistein (mean increase, 5.5%; 95% CI: 3.9% to 7.0%; P <0.001) and by estrogen/progestin (P <0.001). There were no significant differences between estrogen and genistein for any of these parameters (all P >0.4). CONCLUSION One year of genistein therapy improves endothelium function in postmenopausal women to a similar extent as does an estrogen/progestin regimen.

Endoglin, PlGF and sFlt-1 as markers for predicting pre-eclampsia

Objective. To evaluate the ability of endoglin, placental growth factor (PlGF) and the soluble form of vascular endothelial growth factor receptor (sFlt‐1) measurements in gestational weeks 24–28 were used to predict pre‐eclampsia. Design. Observational, prospective study. Setting. Department of Gynecological, Obstetrical Sciences and Reproductive Medicine, University of Messina. Sample. Fifty‐two pre‐eclamptic and 52 healthy pregnant women. Methods. A maternal serum sample was frozen and stored at 1‐h 50‐g glucose challenge test between 24 and 28 weeks’ gestation. A second maternal serum sample was collected at admission for the onset of the disease in the pre‐eclamptic group and at admission for delivery in the control group. Levels of endoglin, sFlt‐1 and the PlGF were measured in the stored serum. Pre‐eclamptic subjects were also divided into women with early‐onset (<37 weeks) and women with late‐onset pre‐eclampsia (≥37 weeks). Results. Levels of endoglin, sFlt‐1, and sFlt‐1: PlGF ratio were found to be higher in the pre‐eclamptic group in both trimesters. No differences were found between early‐ and late‐onset pre‐eclamptic. The Receiver Operating Characteristics curve, applied to the second trimester marker values, showed the best diagnostic profile for sFlt‐1: PlGF (area under the curve, AUC =0.92) followed by endoglin (AUC =0.88), sFlt‐1 (AUC =0.87) and PlGF (AUC = 0.83). This finding was confirmed by Bayesian analysis which highlighted a specificity, a sensitivity, a diagnostic accuracy, a positive predictive value and a negative predictive value of 88.5% for sFlt‐1: PlGF using a cut‐off of 38.47. Conclusions. Endoglin, PlGF and sFlt‐1 might be used as markers for predicting pre‐eclampsia, but sFlt‐1: PlGF seems to be more accurate.

Adiponectin and insulin resistance in early- and late-onset pre-eclampsia

Objective  To evaluate the importance of adiponectin and insulin resistance in early‐ and late‐onset pre‐eclampsia.

Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study.

Objective: To evaluate in a 24-month, prospective, randomized, double-blind, placebo-controlled study whether pure administration of the phytoestrogen genistein (54 mg/d) might reduce the number and severity of hot flushes in postmenopausal women, with no adverse effect on the endometrium and vagina. Methods: A total of 389 participants met the parent study criteria and were randomly assigned to receive the phytoestrogen genistein (n = 198) or placebo (n = 191). About 40% of participants in both groups did not experience hot flushes, and the evaluation was performed in a subgroup of 236 participants (genistein, n = 119; placebo, n = 117). Reductions from the baseline in the frequency and severity of hot flushes were the principal criteria of efficacy. Endometrial thickness was evaluated by ultrasonography. The maturation value was also used to determine hormonal action on the vaginal cells. Results: There were no significant differences in vasomotor symptoms between groups at the baseline (4.4 ± 0.33 hot flushes per day in the genistein group and 4.2 ± 0.35 hot flushes per day in the control group). After 12 months of genistein therapy, there was a significant reduction (−56.4%) in the mean number of hot flushes, with a significant difference compared with the control group. After 24 months, there was no further decrease in the number of hot flushes in both groups. No significant difference was found in mean endometrial thickness and the maturation value score between the two groups, either at the baseline or after 24 months. Conclusions: The phytoestrogen genistein has been shown to be effective on vasomotor symptoms without an adverse effect on the endometrium and vagina, but after the first year, there was no further improvement in the decrease in hot flushes.

Breast Safety and Efficacy of Genistein Aglycone for Postmenopausal Bone Loss: A Follow-Up Study

CONTEXT Genistein aglycone improves bone metabolism in women. However, questions about the long-term safety of genistein on breast as well as its continued efficacy still remain. OBJECTIVE We assessed the continued safety profile of genistein aglycone on breast and endometrium and its effects on bone after 3 yr of therapy. DESIGN The parent study was a randomized, double-blind, placebo-controlled trial involving 389 osteopenic, postmenopausal women for 24-months. Subsequently, a subcohort (138 patients) continued therapy for an additional year. PATIENTS AND INTERVENTIONS Participants received 54 mg of genistein aglycone daily (n = 71) or placebo (n = 67). Both treatment arms received calcium and vitamin D(3) in therapeutic doses. MAIN OUTCOMES Mammographic density was assessed at baseline, 24 and 36 months by visual classification scale and digitized quantification. BRCA1 and BRCA2, sister chromatid exchange, and endometrial thickness were also evaluated. Lumbar spine and femoral neck bone mineral density were also assessed. Secondary outcomes were biochemical levels of bone markers. RESULTS After 36 months, genistein did not significantly change mammographic breast density or endometrial thickness, BRCA1 and BRCA2 expression was preserved, whereas sister chromatid exchange was reduced compared with placebo. Bone mineral density increases were greater with genistein for both femoral neck and lumbar spine compared to placebo. Genistein also significantly reduced pyridinoline, as well as serum carboxy-terminal cross-linking telopeptide and soluble receptor activator of NF-kappaB ligand while increasing bone-specific alkaline phosphatase, IGF-I, and osteoprotegerin levels. There were no differences in discomfort or adverse events between groups. CONCLUSIONS After 3 yr of treatment, genistein exhibited a promising safety profile with positive effects on bone formation in a cohort of osteopenic, postmenopausal women.

Inflammatory markers in women with a recent history of gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a risk factor for both Type 2 diabetes (DM2) and insulin-resistance syndrome (IRS). C-reactive protein (CRP), fibrinogen and leukocyte count are increased in the IRS and predict DM2 and cardiovascular disease (CVD). The chemochine monocyte chemoattractant protein-1 (MCP-1/CCL2) is also elevated in DM2 and CVD. Recent evidence suggests a relation between chronic inflammation and GDM, but post-delivery information on inflammatory markers in these high-risk women is lacking. Serum levels of CRP, fibrinogen, MCP-1/CCL2, and leukocyte blood count have been assessed in 26 women with and 26 women without a recent history of GDM, matched for age, body mass index (BMI), post-partum duration and parity. DM2 was excluded in all the participants by an oral glucose tolerance test (OGTT). Women with previous GDM showed significantly higher CRP (p=0.007) and fibrinogen (p=0.02) serum concentrations, whereas MCP-1/CCL2 serum levels and leukocyte blood count were comparable in the two groups. Overall, CRP levels significantly correlated with BMI (r=0.40, p=0.03), waist-to-hip ratio (WHR) (r=0.44, p=0.001), fasting insulin (r=0.27, p=0.04), insulin-resistance assessed by means of the homeostatic model (HOMA) (r=0.28, p=0.04), and fibrinogen concentration (r=0.49, p=0.0001). At linear regression analysis, only WHR and fibrinogen were independently associated with CRP levels. In conclusion, the increase of inflammatory markers may be one of the first detectable disorders in healthy women at high risk of DM2 and IRS, like those with a GDM history.

myo-Inositol Supplementation and Onset of Gestational Diabetes Mellitus in Pregnant Women With a Family History of Type 2 Diabetes: A prospective, randomized, placebo-controlled study

OBJECTIVE To check the hypothesis that myo-inositol supplementation may reduce gestational diabetes mellitus (GDM) onset in pregnant women with a family history of type 2 diabetes. RESEARCH DESIGN AND METHODS A 2-year, prospective, randomized, open-label, placebo-controlled study was carried out in pregnant outpatients with a parent with type 2 diabetes who were treated from the end of the first trimester with 2 g myo-inositol plus 200 µg folic acid twice a day (n = 110) and in the placebo group (n = 110), who were only treated with 200 µg folic acid twice a day. The main outcome measure was the incidence of GDM in both groups. Secondary outcome measures were as follows: the incidence of fetal macrosomia (>4,000 g), gestational hypertension, preterm delivery, caesarean section, shoulder dystocia, neonatal hypoglycemia, and neonatal distress respiratory syndrome. GDM diagnosis was performed according to the International Association of the Diabetes and Pregnancy Study Groups (IADPSG) recommendations. RESULTS Incidence of GDM was significantly reduced in the myo-inositol group compared with the placebo group: 6 vs. 15.3%, respectively (P = 0.04). In the myo-inositol group, a reduction of GDM risk occurrence was highlighted (odds ratio 0.35). A statistically significant reduction of fetal macrosomia in the myo-inositol group was also highlighted together with a significant reduction in mean fetal weight at delivery. In the other secondary outcome measures, there were no differences between groups. CONCLUSIONS myo-Inositol supplementation in pregnant women with a family history of type 2 diabetes may reduce GDM incidence and the delivery of macrosomia fetuses.