REGULARLY MENSTRUATING premenopausal women

0021-972X/00/$03.00/0 Vol. 85, No. 12 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2000 by The Endocrine Society Preserved Forearm Endothelial Responses with Acute Exposure to Progesterone: A Randomized Cross-Over Trial of 17- Estradiol, Progesterone, and 17- Estradiol with Progesterone in Healthy Menopausal Women* KIEREN J. MATHER, ERIC G. NORMAN, JERILYNN C. PRIOR, AND THOMAS G. ELLIOTT Division of Endocrinology (K.J.M.), Indiana University School of Medicine, Indianapolis, Indiana; and University of British Columbia and Vancouver Hospital and Health Sciences Centre (E.G.N., J.C.P., T.G.E.), Department of Medicine, Division of Endocrinology and Metabolism, Vancouver, British Columbia, Canada V5Z 1C6 ABSTRACT Regularly menstruating women are relatively protected from cardiovascular disease. Epidemiological and endothelial function studies attribute this protection to estradiol (E 2 ), but both progesterone (P) and E 2 are normally present. A range of vascular effects of added progestins have been described, from neutral to detrimental, but the effects of P per se on endothelial function in humans have not been reported. We therefore investigated the acute effects of E 2, P, and E 2 combined with P, on endothelium-dependent and -independent forearm blood flow responses. Using venous occlusion plethysmography, forearm blood flow (FBF) was measured during acute brachial artery infusions, achieving physiologic levels of 17- -E 2, P, and 17- -E 2 with P in healthy menopausal women with no cardiovascular disease risk factors. Vehicle or hormones were infused, in random order, on 4 days, 1 week REGULARLY MENSTRUATING premenopausal women are relatively protected against cardiovascular disease (1). This effect cannot simply be attributed to the younger age of premenopausal (compared with menopausal) women because young women, after ovariectomy or with premature gonadal failure, also experience higher rates of cardiovascular disease (1). Attempts to understand this vascular protection have recently turned to studies of endothelial function, with a particular focus on the endothelial effects of estrogens. In menopausal women, chronic estrogen therapy has been shown to improve endothelium-dependent control of blood flow in the coronary (2, 3) and brachial artery vascular beds (4 8). Interestingly, acute exposure to estrogen has also been found to improve endothelium-dependent responses in the coronary arteries of postmenopausal women with vascular disease (9 13) and in the brachial arteries of women with (14 17) and without (14 18) cardiovascular risk factors. Received June 27, 2000. Revision received August 11, 2000. Accepted August 18, 2000. Address all correspondence and requests for reprints to: T. G. Elliott, M.B.B.S., FRCPC, Suite 380, 575 West 8th Avenue, Vancouver, British Columbia, Canada V5Z 1C6. * Supported by the British Columbia Endocrine Research Foundation (Vancouver, British Columbia, Canada). A clinical research fellow with the Alberta Heritage Fund for Medical Research. apart. Flow responses were measured during coinfusions of hormone with the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator sodium nitroprusside. Twenty-seven healthy menopausal women were studied, and all had normal baseline endothelial responses. Small ( 15%), statistically nonsignificant increases in endothelium-dependent flow responses were seen after all acute hormone treatments. No impairment in response was seen with P alone or in combination with 17- -E 2. In healthy menopausal women without cardiovascular disease risk factors and without baseline defects in endothelial function, acute exposure to physiologic levels of 17- -E 2, P, and 17- -E 2 with P produced equivalent endothelium-dependent responses. These data suggest that P does not have detrimental vascular effects in humans. (J Clin Endocrinol Metab 85: 4644 4649, 2000) Comparatively little is known of the effects of progestagens on the vasculature (19). Menopausal declines in progesterone (P) levels precede those of estradiol (E 2 ) (20) and could play an important role in the loss of vascular protection. Across the menstrual cycle, vasodilatory influences of sex steroids are seen in both the follicular (estrogen only) and luteal (estrogen plus P) phases (21). Unfortunately, during the menstrual cycle, any vascular effects of P alone cannot be separated from the effects of E 2. Dissociation of estrogen and P secretion is, however, seen during cycles with ovulation disturbances, wherein E 2 is commonly secreted in physiologic amounts but P production is significantly diminished. In nonhuman primates (22) and in women with the polycystic ovary syndrome (23), such ovulation disturbances are associated with increased atherosclerosis, perhaps suggesting a protective role attributable to P. These studies suggest that P and E 2 both have important physiologic vascular effects. However, adding synthetic progestins to estrogen has produced (at best) neutral and (in some cases) detrimental effects on vascular responses (24 26). Important questions therefore remain regarding the role of P, as distinct from estrogen, in modulating endothelial responses. Therefore, we set out to define the acute effects of physiologic levels of P alone, and in combination with phys- 4644

PROGESTERONE AND FOREARM BLOOD FLOW 4645 iologic levels of 17 -E 2, on forearm endothelial function in healthy menopausal women. Subjects Materials and Methods We recruited healthy menopausal women without cardiovascular risk factors and without recent exposure to exogenous sex steroids. Menopausal status was defined as the absence of menstrual flow for 12 months and/or FSH 35 IU/mL. Exclusion criteria included the use of menopausal ovarian hormone therapy within the previous 6 months, diabetes mellitus (American Diabetes Association criteria), hypertension [blood pressure (BP) 140/90], hypercholesterolemia (total cholesterol 6.2 mmol/l), current smoking, and known coronary artery disease or symptoms of cardiac ischemia or failure. Aspirin and any prescribed or over-the-counter nonsteroids antiinflammatory medications were stopped at least 10 days before the first plethysmography session and avoided throughout the 4-week study. The study was approved by the University of British Columbia and the Vancouver Hospital and Health Sciences Center research ethics review boards. All participants provided written informed consent. FBF measurements Studies were performed in a quiet clinical laboratory maintained at 21 to 23 C. Subjects were asked to refrain from drinking alcohol- or caffeine-containing beverages for at least 24 h before the study. All solutions were infused at 1.0 ml/min (Harvard Apparatus, South Natick, MA) into the brachial artery of the nondominant arm via an epidural catheter (Concord Portex, Keene, NH) sealed with dental wax to a 27-guage dental needle (Sherwood Medical, St. Louis, MO). FBF was measured simultaneously in the infused arm and in the noninfused arm using venous occlusion plethysmography as reported previously (27). Both arms were supported at heart level. Before each set of measurements, circulation to the hand was prevented by inflation of a wrist cuff to 160 mm Hg. For each measurement, a cuff placed on the upper arm was inflated to 40 mm Hg to occlude venous egress. This was achieved by rapidly inflating a cuff (Model E10; Hokanson, Inc.) for 10 sec of every 20 sec. Dose-response profiles to brachial arterial infusions of the endothelium-dependent vasodilator acetylcholine (ACh, Iolab, Claremont, CA), and the endothelium-independent vasodilator sodium nitroprusside (SNP, Roche, Basel, Switzerland), were measured. On each study day, the following infusion sequence was observed: saline for 9 min; vehicle [ hormone(s)] for 20 min; SNP at 1, 3, and 10 g/min for 6 min at each concentration; vehicle [ hormone(s)] for 9 15 min to allow flow to return to baseline; and ACh at 3, 10, and 30 g/min for 4 min at each concentration. Except for the initial saline-only period, vehicle [ hormone(s)] was infused continuously. FBF was recorded during the last 3 min of each infusion. The FBF value used for statistical analysis was the mean of the last five flow measurements at a given drug dose. Data were expressed as absolute flow rates and percent increase from the immediately preceding baseline in the infused arm, and as the percent increase in the ratio of blood flow rates observed in the infused and noninfused arms (28). Because serial measurements were taken, the average of all flows for each vasodilator infusion was used as a summary measure (29, 30), and statistical tests were applied to this summary measure. Protocol Each participant was tested on four separate occasions at weekly intervals. On each occasion, FBF was measured, as above, during the constant infusion of one of four separate treatments chosen in random order: 1) vehicle (0.79% ethanol in 0.9% sodium chloride) without hormone; 2) 17- -E 2,5 10 8 mol/l (Sel-Win Chemicals, Ltd., Vancouver BC, Canada) in vehicle; 3) P, 2 10 6 mol/l (Sel-Win Chemicals, Ltd.) in vehicle; or 4) E 2 and P in the above concentrations in vehicle. Hormone infusions and measurements We performed a pilot study in nine women to ensure that the hormone levels achieved were within physiologic target ranges (E 2, 500-1450 pmol/l; P, 18 90 nmol/l). Initial 17- -E 2 dose and vehicle concentrations were taken from the literature (15), and the P dose was estimated from these values, assuming equal volumes of distribution and solubility and using the normal ratio of the two hormones during the luteal phase as a guide. These results guided an adjustment of administered concentrations of both hormones to the values reported above. Hormone levels were measured in a venous sample drawn from a dorsal hand vein or the antecubital fossa of the infused arm, at the end of the 80 90-min hormone infusions. Commercial RIA assays (Coat-a- Count, Diagnostics Products, Los Angeles, CA) were used for E 2 and P. Analyses were performed in the Vancouver Hospital and Health Sciences Center clinical chemistry laboratory. Statistical analysis In our laboratory, the plethysmography technique has good repeatability, with a measured coefficient of variation of 15% on an individual on a single day and approximately 21% on separate days. Clinically meaningful changes in flow responses require approximately a 30% change in response. Therefore, we calculated that 24 subjects would be required to demonstrate such a response between any 2 infusions (2- sided 0.05, 0.80). Participant characteristics and hormone levels are presented as mean sd with ranges. Flow and flow ratio data are expressed as mean sem. Error bars on the figures represent 1 sem. The blood flow data were reasonably normally distributed, allowing parametric testing with repeated-measures ANOVA. After significant differences were found by ANOVA, post hoc pairwise analysis was performed using the Bonferonni/Dunn adjustment for multiple comparisons. A two-tailed value of P 0.05 was considered statistically significant. Statistical analysis was performed using Statview 5.0 (SAS Institute, Inc., Carey, NC). Results We studied 27 healthy menopausal women, 56.4 4.8 yr old (mean sd; range, 45 65). None of the subjects had taken any menopausal ovarian hormone therapy previously. The duration from menopause was 6.6 4.6 yr (range, 2.0 8.5). All participants were normotensive (systolic BP, 124 2; diastolic BP, 81 1 mm Hg), nondiabetic, had normal cholesterol levels (total cholesterol, 5.0 0.4 mmol/l), and were nonsmokers. In 5 subjects, cuff artifact precluded analysis of data from 1 or 2 study days. The statistical comparisons therefore include 22 subjects for comparisons of all 4 treatments, and 23 subjects for pairwise comparisons between individual treatments. Controls and baseline Neither the vehicle used for administering the hormones nor the hormones themselves significantly altered FBF. As has been reported by other laboratories (15), the ethanol in normal saline vehicle produced a significant decrease in FBF, compared with the saline control (saline FBF, 3.8 0.4 vs. EtOH FBF, 2.8 0.2 ml/100 ml min; P 0.0045). No change in FBF was seen during infusion of vehicle plus 17- -E 2 (vehicle, 2.9 0.2 vs. E 2, 3.08 0.3; P 0.56), vehicle plus P (vehicle, 3.5 0.2 vs. P, 3.3 0.2; P 0.51), or vehicle plus both hormones (vehicle, 3.3 0.3 vs. E 2 P, 3.1 0.2; P 0.47). In the absence of hormone (control day), baseline vehicle flows were 3.3 0.7 ml/100 ml min, and dose responses to ACh and SNP were similar; mean peak flows of 24.5 and 19.1 ml/100 ml min, respectively, were observed (Tables 1 and 2). These values and the 5-fold increases in FBF observed at

4646 MATHER ET AL. JCE&M 2000 Vol. 85 No. 12 peak vasodilator doses are comparable with values found in normal premenopausal female subjects (unpublished observations) and greater than those observed in healthy male volunteers (the reference population in Ref. 31) in our laboratory. This, together with the finding that the peak ACh flows were not markedly diminished (relative to SNP flows), suggests that there was no baseline defect in endothelial function. Vasodilator flow responses with acute sex steroid exposure Acute exposure to physiologic concentrations of 17- -E 2, P, and the combination (in a randomized cross-over schema) produced small ( 15%) statistically nonsignificant enhancements in endothelium-dependent vasodilation (EDV). Peak percent increase without hormone averaged 576 67%; E 2 produced peak increases of 677 78%, P gave 658 59% increases, and the combination produced 647 85% increases (Fig. 1). Endothelium-independent vasodilation (EIV) was increased by a similar degree ( 15%, P not significant) after E 2 treatment, but no such changes were apparent with the other two treatments (Fig. 2). Importantly, there was no apparent detrimental effect of P alone or in combination with E 2. This was the case when considering either the absolute flow responses (Tables 1 and 2) or the percent increase from baseline (Figs. 1 and 2) in the treated arm. Increases in EDV responses, expressed as percent change in the ratio of flows in the infused and noninfused arms (28), were significant (Fig. 1, P 0.016 by repeatedmeasures ANOVA). Pairwise comparison for treatments vs. control achieved statistical significance only for P infusion (P 0.0018). No changes in EIV responses were apparent using this ratio (P 0.19 by repeated-measures ANOVA). Overall, we did not see statistically significant increases in EDV or EIV responses after acute exposure to any of the hormone treatments. Our data suggest, if anything, small enhancements to EDV responses with all treatments. Importantly, no detrimental effects of P on EDV or EIV were seen. Sex steroid concentrations Baseline serum 17- -E 2 levels averaged 67.9 11.9 pmol/l (range, 22.2 121.2), and P levels were 0.61 0.10 nmol/l (range, 0.16 1.38). In a random subset of 10 patients, the hormone infusions achieved 17- -E 2 levels of 420 128 (range, 357 588) pmol/l, and P levels of 20.8 6.9 (range, 10 31) nmol/l. These means fall within the luteal phase range of values for premenopausal women. Discussion In healthy menopausal women without cardiovascular disease risk factors, we observed unimpaired baseline endothelial function and found that acute infusion of 17- -E 2, P, or the combination (achieving physiologic concentrations) did not produce significant enhancements in vasodilator responses. There were no direct effects of physiologic levels of 17- -E 2 or P on flow. Importantly, endothelium-dependent vasodilator responses were preserved with acute exposure to P. Further, the change in EDV responses observed with 17- -E 2 alone was not altered by coexposure with P, and all three treatments gave similar trends to improved EDV. Endothelium-independent flow responses were not significantly altered by the hormone coinfusions. To our knowledge, these are the first data detailing blood flow responses to acute infusions of physiological levels of P, alone and with E 2,in humans. The use of ratios of blood flow rates in treated and control arms is intended to increase the sensitivity of plethysmography by correcting for minor systemic hemodynamic changes (28). Taken in this light, the suggestion of a beneficial effect of P is interesting (Fig. 1). However, this apparent effect was related principally to systemic, rather than local, changes (Table 1) and is difficult to interpret, in view of the lack of significant changes in blood flow in the treated arm. This observation does, however, further support the lack of detrimental effects of P on endothelial function. Acute effects of sex steroid hormones on the vasculature Our observations of a lack of acute effects of E 2 infusion are contrary to a body of literature reporting significant improvements in endothelium-dependent blood flow responses after acute infusions in the coronary (9 13) and brachial (15 18) arteries. Of these, relatively few studies have examined the effects of physiologic levels of sex steroids (10, 14, 15), and the majority of the subjects studied had significant coronary artery disease risk. One report included a subset of healthy postmenopausal women apparently similar to our subjects (15). However, these women demonstrated considerably diminished ACh-stimulated flows before E 2 infusion (mean peak flows, 14.4 ml/100 ml min), compared with those described in the present study (24.5 ml/100 ml min). Although direct comparisons between studies are problematic, our subjects unimpaired flows at baseline could account for the lack of further improvement. The single study in the literature with comparable methodologies had led us to anticipate impaired baseline re- TABLE 1. Endothelium-dependent flows Acute infusion Infused arm Noninfused arm Flow ratio ACh 3 ACh 10 ACh 30 ACh 3 ACh 10 ACh 30 ACh 3 ACh 10 ACh 30 Control 11.8 (1.3) 16.6 (1.8) 24.5 (3.3) 3.1 (0.3) 3.1 (0.3) 3.2 (0.3) 4.1 (0.5) 6.0 (0.7) 9.3 (1.4) 17- E 2 10.5 (1.3) 15.7 (1.7) 23.2 (2.2) 2.8 (0.2) 2.8 (0.2) 3.0 (0.3) 3.9 (0.6) 5.9 (0.8) 8.3 (0.9) P 11.9 (1.1) 17.9 (1.7) 25.1 (2.0) 2.8 (0.3) 2.7 (0.2) 2.6 (0.2) 4.7 (0.5) 7.3 (0.7) 10.7 (1.1) 17- E 2 & P 10.9 (1.1) 16.9 (2.1) 24.4 (2.6) 2.8 (0.3) 2.9 (0.3) 3.1 (0.3) 4.3 (0.5) 5.9 (0.5) 8.2 (0.7) Forearm blood flow (ml/100 ml min) in the infused and noninfused arms, and ratio of the infused divided by the noninfused forearm blood flow (unitless). Response to acetylcholine (doses in g/min) during acute infusions of vehicle (control), 17- -E 2 (5 10 8 mol/min), P (2 10 6 mol/min), and both 17- -E 2 and P at the same rates. Values are mean SEM.

PROGESTERONE AND FOREARM BLOOD FLOW 4647 TABLE 2. Endothelium-independent flows Acute infusion Infused arm Noninfused arm Flow ratio SNP 1 SNP 3 SNP 10 SNP 1 SNP 3 SNP 10 SNP 1 SNP 3 SNP 10 Control 8.5 (0.7) 13.8 (1.6) 19.1 (2.5) 2.6 (0.2) 2.8 (0.3) 2.5 (0.2) 3.5 (0.3) 5.3 (0.6) 7.9 (0.8) 17- E 2 9.0 (0.8) 14.1 (1.5) 20.4 (1.7) 3.0 (0.2) 3.1 (0.3) 3.0 (0.3) 3.3 (0.2) 4.5 (0.3) 7.3 (0.5) P 10.4 (1.0) 15.1 (1.3) 21.4 (1.4) 2.9 (0.2) 2.8 (0.2) 2.8 (0.2) 3.6 (0.3) 5.6 (0.4) 8.2 (0.6) 17- E 2 & P 9.9 (0.8) 14.7 (1.3) 19.9 (1.2) 3.1 (0.2) 3.2 (0.3) 2.8 (0.2) 3.3 (0.2) 5.0 (0.3) 7.5 (0.5) Forearm blood flow (ml/100 ml min) in the infused and noninfused arms, and ratio of the infused divided by the noninfused forearm blood flow (unitless). Flow response to sodium nitroprusside (doses in g/min) during acute infusions of vehicle (control), 17- -E 2 (5 10 8 mol/min), P(2 10 6 mol/min), and both E 2 and P at the same rates. Values are mean SEM. FIG. 1. Endothelium-dependent responses to ACh during hormone coinfusion. Top, Percent change in blood flow rates in the treated arm; bottom, percent changes in blood flow ratio (infused to noninfused arm). All data are mean SEM. *,P 0.016. sponses to EDV (15), and our sample sizes were estimated with the assumption that such dysfunction would be evident. The number of subjects studied did provide sufficient power to demonstrate a relevant change of approximately 30% in flow responses. The observation of approximately 15% increases in EDV responses above the robust baseline response is of uncertain significance, but we were simply not powered for this difference to be statistically significant. Therefore, although our data do not document beneficial effects of E 2, P, or the combination on endothelial function, in the setting of normal baseline function we cannot firmly exclude Type 2 statistical errors (i.e. concluding that no effect exists when, in fact, such an effect does exist). We can be more definitive that no harmful effects on endothelial function were evident with any of the infusions. FIG. 2. Endothelium-independent responses to SNP during hormone coinfusion. Top, Percent change in blood flow rates in the treated arm; bottom, percent changes in blood flow ratio (infused to noninfused arm). All data are mean SEM. Another potentially important difference between these studies is the concentration of 17- -E 2 achieved in the forearm. After adjustments to the administered doses, we achieved mid-normal luteal phase E 2 concentrations in our subjects, whereas the study by Gilligan et al. reported highnormal values. Whether this difference has a bearing on vascular function is an open question. P and endothelial function We did not find a detrimental effect of acute exposure to P on vascular endothelial function. This is an important addition to the described effects of progestins on the vasculature. Data from animal models of atherosclerosis suggest that medroxyprogesterone acetate alone is not beneficial and, in combination therapy, mitigates the beneficial effects of es-

4648 MATHER ET AL. JCE&M 2000 Vol. 85 No. 12 trogen therapy (32 35). P, however, has been shown to have less detrimental, or even neutral, vascular effects (34, 36 39). Studies in humans fall into two categories: studies of vascular changes across the normal menstrual cycle; and studies of the effects of menopausal ovarian hormone therapy on vascular responses. The physiologic effects of the normal menstrual cycle on endothelial function have been examined in three recent studies of healthy young women (21, 40, 41). Hashimoto and co-workers (21) found that during menstruation (early in the follicular phase), when hormone levels were at their nadir, flow-mediated dilation (FMD) was similar to that seen in men. FMD during both the late follicular (estrogen-predominant) and luteal (combination estrogen and P) phases were comparable, and both were significantly elevated, relative to the menstrual phase (21). This group also found improved endothelium-independent flows during both the follicular and luteal phases. Conflicting results are reported by English and co-workers (40), who found diminished FMD in the luteal phase, compared with the follicular phase. Observational data in menopausal women taking hormone replacement therapy is available from two cohort studies (24, 42). In both studies, subjects receiving combined estrogen and progestin (including medroxyprogesterone acetate) showed improvement in FMD equivalent to that of those treated with estrogen alone. Three intervention studies have used combination hormone replacement therapy and assessed vascular responses. No detrimental effect of P was seen in mildly hypercholesterolemic women given transdermal E 2 with cyclical intravaginal micronized P (25). Norethisterone with E 2 improved FMD in gonadotropin-suppressed young women, compared with their hypogonadal counterparts (43). However, no longterm beneficial effect on FMD was apparent with combined E 2 and norethisterone (26); design issues (lack of baseline flow measures, and vascular studies not standardized to timing of the treatment cycle) limit the interpretation of these data, however. In all of these studies, no frank detrimental effect of short- or long-term treatment with progestagens was evident, and only the latter study suggests that beneficial vascular effects of estrogen might be mitigated by concurrent synthetic progestin exposure. Our finding of a lack of detrimental effects with acute exposure to P is concordant with these data. Of note, however, long-term exposures can engender broader responses, including receptor down-regulation and alterations in the transcriptional profile, which limit the comparison of acute and chronic responses. Conclusion In healthy menopausal women, we observed preserved endothelium-dependent responses with acute exposure to physiologic levels of P or the combination of 17- -E 2 and natural P, compared with 17- -E 2 alone. These findings support suggestions that P may not exert the detrimental effects on endothelial function seen with synthetic progestins. Acknowledgments We thank our study participants for their dedication and enthusiasm. References 1. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. 1987 Menopause and the risk of coronary heart disease in women. N Engl J Med. 316:1105 1110. 2. Herrington DM, Braden GA, Williams JK, Morgan TM. 1994 Endothelialdependent coronary vasomotor responsiveness in postmenopausal women with and without estrogen replacement therapy. Am J Cardiol. 73:951 952. 3. 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