Susan E. Appt

Effects of High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys

Abstract Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.

MPA and postmenopausal coronary artery atherosclerosis revisited

Whether progestins, particularly medroxyprogesterone acetate (MPA), attenuate the cardiovascular benefits of postmenopausal estrogen replacement therapy (ERT) has been controversial for over a decade. Concerns related first to findings that MPA attenuated increases of high density lipoprotein cholesterol (HDLC) concentrations of postmenopausal women compared to conjugated equine estrogen (CEE) alone. That observation was followed by early cynomolgus monkey studies that suggested MPA decreased estrogens cardiovascular benefits (vascular reactivity and coronary artery atherosclerosis inhibition). In a more recent and larger trial with cynomolgus monkeys, no differences were seen in the coronary artery atherosclerosis protective effect of CEE when MPA was co-administered (HRT). The lack of attenuation of ERTs benefits by progestins has also been seen in at least three studies of carotid artery intima-media thickness (IMT) of postmenopausal women. Additionally, the majority of studies of vascular reactivity of postmenopausal women have not found differences when CEE is given alone or with MPA. Seven observational studies of cardiovascular outcomes of postmenopausal women permit separate consideration of ERT versus HRT use; there is no evidence of attenuation of ERTs benefits by progestin use. In conclusion, it is evident that the current experimental, clinical, and observational data do not provide evidence that progestins attenuate estrogens cardiovascular benefits.

Impairment of ovarian function and associated health-related abnormalities are attributable to low social status in premenopausal monkeys and not mitigated by a high-isoflavone soy diet

BACKGROUND Psychological stress may impair premenopausal ovarian function and contribute to risk for chronic disease. Soy isoflavones may also influence ovarian function and affect health. Here, we report the effects of a psychological stressor (subordinate social status) and dietary soy on reproductive function and related health indices in female monkeys. We hypothesized that reproductive compromise and adverse health outcomes would be induced in subordinate when compared with dominant monkeys and be mitigated by exposure to soy. METHODS Subjects were 95 adult cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six. Animals consumed a soy-free, animal protein-based diet during an 8-month Baseline phase and then, during a 32-month Treatment phase, consumed either the baseline diet or an identical diet that substituted high-isoflavone soy protein for animal protein. RESULTS Across more than 1200 menstrual cycles, subordinate monkeys consistently exhibited ovarian impairment [increased cycle length (P < 0.02) and variability (P < 0.02) and reduced levels of progesterone (P < 0.04) and estradiol (P < 0.04)]. Subordinate status was confirmed behaviorally and was associated with elevated cortisol (P < 0.04) and relative osteopenia (P < 0.05). Consumption of the soy diet had no significant effects. CONCLUSIONS (i) Psychological stress adversely affects ovarian function and related health indices in a well-accepted animal model of womens health; (ii) Similar effects may extend to women experiencing reproductive impairment of psychogenic origin; (iii) soy protein and isoflavones neither exacerbate nor mitigate the effects of an adverse psychosocial environment; and (iv) this study was limited by an inability to investigate the genetic and developmental determinants of social status.

Premenopausal Antimullerian Hormone Concentration is Associated with Subsequent Atherosclerosis

Objective The aim of this study was to determine if premenopausal ovarian reserve is associated with susceptibility for atherosclerosis. Methods Female cynomolgus macaques (n = 66, women’s equivalent age = 45 y) consumed an atherogenic diet for ∼5 months before the measurement of a marker of ovarian reserve (antimüllerian hormone [AMH]), plasma lipids, follicular phase estradiol, and body weight (BW). Monkeys were then ovariectomized (OVX; n = 17), remained premenopausal (n = 20), or were induced to have reduced ovarian reserve (ROR, n = 29). After 26 additional months consuming the diet, atherosclerosis measurements and risk variables were reassessed. Results No differences in baseline AMH, plasma lipids, BW, and estradiol or postdiet lipids and BW were observed among the groups subsequently assigned to the OVX, premenopausal control, or reduced ovarian reserve conditions. Postdiet measurements of atherosclerosis extent did not differ among the groups. However, analysis of plaque size by tertile of baseline AMH revealed that plaques were largest in monkeys that began the experiment with the lowest baseline AMH, followed by those in the middle and high tertiles (plaque extent: low AMH, 0.76 ± 0.12 mm2; mid AMH, 0.46 ± 0.1 mm2; high AMH, 0.34 ± 0.08 mm2; P = 0.02). Baseline AMH and plaque size were also correlated negatively (r = −0.31, P = 0.01). Plasma lipids were also correlated significantly with plaque extent (all P < 0.01) but not with AMH. Conclusions We report for the first time an inverse relationship between a marker of ovarian reserve (AMH) and subsequent atherosclerosis risk.

Effects of Soybean Glyceollins and Estradiol in Postmenopausal Female Monkeys

Abstract: Glyceollins are a novel class of soybean phytoalexins with potential cancer-protective antiestrogenic effects. The purpose of this study was to evaluate the estrogen-antagonist effects of glyceollin-enriched soy protein on biomarkers for breast cancer risk. Thirty female postmenopausal cynomolgus macaques were randomized to one of three dietary treatments for 3 wk: 1) estradiol (E2, 1 mg/day) + casein/lactalbumin (control); 2) E2 + soy protein isolate (SPI) containing 194 mg/day isoflavonoids; and 3) E2 + glyceollin-enriched soy protein (GLY) containing 189 mg/day isoflavonoids + 134 mg/day glyceollins. Doses are expressed in calorically scaled human equivalents. Mean serum glyceollin concentrations at 4 h postfeeding were 134.2 ± 34.6 nmol/L in the GLY group and negligible in the SPI group (P = 0.0007). Breast proliferation was significantly increased in the control group (+237%, P = 0.01) but not in the SPI group (+198%, P = 0.08) or GLY group (+36%, P = 0.18). Gene expression of trefoil factor 1 and progesterone receptor, two markers of estrogen receptor activity in breast epithelium, were also significantly higher in the control (P < 0.05 for both) but not in the GLY group. These preliminary findings suggest that soybean glyceollins are natural compounds with potential estrogen-modulating properties in the breast.

Endometrial profile of bazedoxifene acetate alone and in combination with conjugated equine estrogens in a primate model.

OBJECTIVE Concerns of breast cancer risk in postmenopausal women taking combined estrogen + progestin therapy have generated interest in the use of selective estrogen receptor modulators (SERMs) as potential progestin alternatives. Endometrial proliferation and cancer risk are major concerns, however, for estrogens and certain types of SERMs when given alone. The primary aim of this study was to evaluate the endometrial profile of bazedoxifene acetate (BZA), a third-generation SERM, alone and in combination with conjugated equine estrogens (CEE) in a postmenopausal primate model. METHODS Ninety-eight ovariectomized cynomolgus monkeys (Macaca fascicularis) were randomized to receive no hormone treatment (controls), BZA 20 mg, CEE 0.45 mg, or the combination of BZA 20 mg + CEE 0.45 mg once daily for 20 months in a parallel-arm study design. The primary outcome measure was endometrial epithelial proliferation. RESULTS BZA + CEE and BZA treatment resulted in significantly less endometrial epithelial area and Ki67 expression compared with CEE (P < 0.001 for all). The prevalence of endometrial hyperplasia and other estrogen-induced morphologic changes in the BZA + CEE and BZA groups was not significantly different from controls. The addition of BZA to CEE completely inhibited the expression of estrogen receptor-α-regulated genes (TFF1 and PGR), whereas BZA alone had no effect. BZA + CEE and BZA treatment also resulted in lower estrogen receptor-α protein expression in the endometrium compared with the control and CEE groups (P < 0.05 for all). CONCLUSIONS BZA given at a clinically relevant dose inhibits estrogen effects on the endometrium and lacks uterotropic effects when given alone.ObjectiveConcerns of breast cancer risk in postmenopausal women taking combined estrogen + progestin therapy have generated interest in the use of selective estrogen receptor modulators (SERMs) as potential progestin alternatives. Endometrial proliferation and cancer risk are major concerns, however, for estrogens and certain types of SERMs when given alone. The primary aim of this study was to evaluate the endometrial profile of bazedoxifene acetate (BZA), a third-generation SERM, alone and in combination with conjugated equine estrogens (CEE) in a postmenopausal primate model. MethodsNinety-eight ovariectomized cynomolgus monkeys (Macaca fascicularis) were randomized to receive no hormone treatment (controls), BZA 20 mg, CEE 0.45 mg, or the combination of BZA 20 mg + CEE 0.45 mg once daily for 20 months in a parallel-arm study design. The primary outcome measure was endometrial epithelial proliferation. ResultsBZA + CEE and BZA treatment resulted in significantly less endometrial epithelial area and Ki67 expression compared with CEE (P < 0.001 for all). The prevalence of endometrial hyperplasia and other estrogen-induced morphologic changes in the BZA + CEE and BZA groups was not significantly different from controls. The addition of BZA to CEE completely inhibited the expression of estrogen receptor-&agr;–regulated genes (TFF1 and PGR), whereas BZA alone had no effect. BZA + CEE and BZA treatment also resulted in lower estrogen receptor-&agr; protein expression in the endometrium compared with the control and CEE groups (P < 0.05 for all). ConclusionsBZA given at a clinically relevant dose inhibits estrogen effects on the endometrium and lacks uterotropic effects when given alone.

Plasma lipid-dependent and -independent effects of dietary soy protein and social status on atherogenesis in premenopausal monkeys : implications for postmenopausal atherosclerosis burden

Objective: Atherosclerosis developed during premenopausal years predicts postmenopausal atherosclerosis burden. The objective of this study was to determine the effects of dietary soy protein isolate (SPI) and social status on atherogenesis and arterial gene expression in a premenopausal monkey model. Design: Socially housed premenopausal cynomolgus macaques (n = 84) were fed an atherogenic diet deriving protein from casein/lactalbumin or SPI (containing 1.88 mg isoflavones/g). After 36 months of diet consumption, iliac artery biopsies were assessed for atherosclerosis and expression of mRNA transcripts related to inflammation, macrophage and T-cell content, and estrogen receptors (ERs). Results: SPI reduced plaque size (P < 0.05), total plasma cholesterol, non-high-density lipoprotein cholesterol (HDLc), and the total plasma cholesterol/HDLc ratio (all P < 0.003), while increasing triglycerides (P < 0.006) and HDLc (P < 0.0001). Arterial mRNA for CD68 (P < 0.001), CD3 (P < 0.02), and CD4 (P < 0.001) and inflammatory markers monocyte chemotactic protein-1, intercellular adhesion molecule-1, and interleukin-6 (all P < 0.0001) were also lower in the group receiving SPI. For most outcomes, this effect remained even after adjustments for plaque size and plasma lipid concentrations. Arterial ER-&agr; was inversely associated with atherosclerosis (P < 0.02) and increased with SPI (P < 0.001). Subordinate monkeys had lower ER-&bgr; (P < 0.02) and higher interleukin-6 (P < 0.05) transcripts but did not differ from dominant monkeys in extent of atherosclerosis (P > 0.9). Conclusions: Premenopausal consumption of SPI had plasma lipid-independent beneficial effects on the pathobiological processes involved in atherosclerotic plaque development, thus potentially establishing the basis for reduced postmenopausal complications. Dominant social status provided similar, albeit less extensive, benefits in risk markers.

Individual differences in equol production capability modulate blood pressure in tibolone-treated postmenopausal women: lack of effect of soy supplementation

Objectives Equol, a gut bacterial metabolite of the isoflavone daidzein, has been associated with beneficial health effects. Recent studies indicate that women with intestinal capacity to convert daidzein to equol also have the capacity to alter steroid metabolism and bioavailability of estrogens. Methods We evaluated whether individual equol production capability, while not consuming soy supplement, was associated with lower blood pressure in postmenopausal women using tibolone. In addition, in a randomized, placebo-controlled, cross-over trial we assessed the effect of soy supplementation on blood pressure in both equol-producing (n = 20) and non-equol-producing (n = 20) women using tibolone. Blood pressure was recorded with a validated oscillometric technique. Results The circulating equol levels rose 20-fold in the equol producers and 1.9-fold in the non-equol producers. At baseline, systolic blood pressure (129.9 ± 2.6 vs. 138.5 ± 3.1 mmHg, p = 0.02), diastolic blood pressure (72.2 ± 1.5 vs. 76.6 ± 1.3 mmHg, p = 0.01) and mean arterial blood pressure (93.5 ± 1.7 vs. 99.9 ± 1.8 mmHg, p = 0.007) were lower in equol producers compared to non-equol producers. Soy supplementation had no effect on blood pressure in either group, whereas the baseline differences persisted. Conclusions Postmenopausal women using tibolone characterized as equol producers had lower blood pressure compared to non-equol producers. Soy supplementation for 2 months had no blood pressure-lowering effect.

Serum antimüllerian hormone predicts ovarian reserve in a monkey model.

Objective: A monkey model of the menopausal transition (perimenopause) would facilitate efforts to understand better the effect of hormonal fluctuations during this life phase on the initiation of chronic diseases associated with the postmenopausal years. Antimüllerian hormone (AMH) is a promising marker of ovarian reserve (primordial follicle number) in women. Here, we describe the relationship between AMH and ovarian reserve in cynomolgus monkeys (Macaca fascicularis) estimated to be 12 to 15 years of age (∼36-45 y in women). Methods and Results: The results of daily vaginal swabbing (to detect menses) and thrice weekly blood sampling for 12 weeks indicate that AMH is relatively stable across the menstrual cycle (intraclass correlation, ∼0.80), with a slight although significant (P < 0.02) reduction (∼1.4-fold) on days 2 to 5 postovulation. Substantial interindividual variation in AMH concentrations were observed between monkeys, with values ranging from 4.46 ± 0.17 to 18.80 ± 0.71 ng/mL (mean ± SE). Antimüllerian hormone concentrations were reduced by ∼63% after the removal of one ovary (7.6 ± 0.77 vs 2.75 ± 0.37 ng/mL; P < 0.001; n = 19) and were below the level of detection after the removal of both ovaries (5.8 ± 0.42 to <0.05 ng/mL; P < 0.001; n = 84), suggesting that the ovary is likely to be either the major or the sole source of AMH in the monkey. Finally, we examined the association between AMH and primordial, primary, and secondary follicles in 29 monkeys and found significant associations with all follicle types (r = 0.78, r = 0.66, and r = 0.80, respectively; P < 0.01). Conclusions: The relationship between AMH and ovarian reserve in the monkey is similar to that in women, suggesting that monkeys may be a useful model for studying hormonal fluctuations across the menopausal transition.

Effects of Long Term Sertraline Treatment and Depression on Coronary Artery Atherosclerosis in Premenopausal Female Primates

Objectives Major depressive disorder and coronary heart disease often co-occur in the same individuals. Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed for depression and other disorders, but their effects on coronary heart disease risk remain unclear. We determined the effects of an SSRI on coronary artery atherosclerosis (CAA) in an established nonhuman primate model used to clarify the association between depression and CAA. Methods Forty-two adult female cynomolgus macaques consuming a Western diet were characterized during an 18-month pretreatment phase and assigned to SSRI (sertraline hydrochloride 20 mg/kg, per os, once a day) or placebo balanced on pretreatment depression, body weight (BW), and iliac artery atherosclerosis extent measured via biopsy. After 18 months, CAA extent was measured using histomorphometry. Results Before and during treatment, depressed monkeys had lower BW, body mass index, and plasma high-density lipoprotein cholesterol, and higher heart rates during the pretreatment (p < .01) but not the treatment phase (p = .17). There were no pretreatment differences between the sertraline and placebo groups. Sertraline reduced anxious behavior but had no effect on BW, body mass index, heart rate, plasma lipids, or depression. CAA, analyzed by a 2 (depressed, nondepressed) × 2 (placebo, sertraline) × 3 (coronary arteries) analysis of covariance adjusted for pretreatment iliac atherosclerosis, was greater in depressed than in nondepressed monkeys (p < .036), and in sertraline than in placebo-treated monkeys (p = .040). The observed CAA extent in depressed monkeys treated with sertraline was 4.9 times higher than that in untreated depressed monkeys, and 6.5 times higher than that in nondepressed monkeys, on average. Conclusions Depressed animals developed more CAA, and long-term treatment with sertraline resulted in more extensive CAA.