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Departments of Molecular and Clinical Endocrinology and Oncology (F.O., T.C., S.S., G.L., A.C.), Obstetrics and Gynecology (A.T.), and Clinical and Experimental Medicine, Gastroenterology Unit (F.M.), and Institute of Internal Medicine and Metabolic Disease (B.D.S.), Federico II University, Naples 80131, Italy; Department of Obstetrics and Gynecology (S.P., T.R., F.Z.), University of Catanzaro Magna Graecia, Catanzaro 88100, Italy; and Department of Obstetrics and Gynecology (R.A.), Cedars-Sinai Medical Center, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Dr. Francesco Orio, Department of Molecular, Clinical Endocrinology and Oncology, University Federico II, Via S. Pansini 5, 80131 Naples, Italy. E-mail: francescoorio{at}virgilio.it.
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Abstract |
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Objective: The objective of this study was to evaluate the effects of 6 months of metformin administration on endothelial structure and function in women with PCOS.
Design: This was a prospective, baseline-controlled, clinical study.
Setting: The study was performed at University Federico II (Naples, Italy).
Patients: Thirty young normal-weight women with PCOS without additional metabolic or cardiovascular diseases were studied.
Interventions: Metformin (850 mg daily) was administered for 6 months.
Mean Outcome Measures: The main outcome measures were complete hormonal profile, including total testosterone, SHBG, dehydroepiandrosterone sulfate, prolactin, and gonadotropin levels; serum insulin and glucose levels during a 75-g 2-h oral glucose tolerance test; plasma endothelin-1 concentrations (picomoles per liter ± SD); serum lipid profile; brachial artery baseline diameter (millimeters ± SD), diameter after reactive hyperemia (millimeters ± SD), and flow-mediated dilation (percentage ± SD); and the intima media thickness (millimeters ± SD) on both common carotid arteries.
Results: After treatment, SHBG levels and the free androgen index changed significantly (P < 0.001). High-density lipoproteins and the area under curve for glucose/area under curve for insulin ratio also significantly (P < 0.001) increased, whereas low-density lipoproteins and plasma endothelin-1 levels were significantly (P < 0.001) reduced. No other change was found in any of the biochemical parameters evaluated. A significant difference was observed in brachial artery baseline diameter (3.24 ± 0.30 vs. 3.0 ± 0.30), flow-mediated dilation (14.30 ± 1.90 vs. 15.70 ± 1.50) (P < 0.01, each), diameter after reactive hyperemia (3.70 ± 0.30 vs. 3.55 ± 0.10) (P < 0.05), and intima media thickness (0.53 ± 0.09 vs. 0.40 ± 0.07) (P < 0.001) after metformin treatment in comparison with baseline values.
Conclusions: A 6-month course of metformin improves endothelial structure and function in young, normal-weight women with PCOS.
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Introduction |
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Endothelial injury is one of the early signs of cardiovascular damage (16), and in a highly selected sample of women with PCOS without any additional risk factors for CVD, we have previously demonstrated early endothelial impairment (4). In fact, our PCOS sample, despite being composed of young, normal-weight women who were not hypertensive or dyslipidemic, had abnormal intima media thickness (IMT) of carotid arteries, flow-mediated dilation (FMD) of brachial arteries, and plasma endothelin-1 (ET-1) levels compared with healthy controls (4). Other investigators also observed evidence of endothelial injury in women with PCOS (17, 18, 19). IR may play a key role in the development of endothelial damage, potentially inducing disturbances of subcellular signaling pathways common to both insulin action and nitric oxide production, or through other links, such as increased oxidant stress, higher ET levels, overactivation of the renin-angiotensin system, and excessive secretion of hormones and cytokines by adipose tissue (20).
Several insulin-sensitizing agents have been demonstrated to improve IR and reduce circulating insulin levels in women with PCOS (21). Among these, metformin cloridrate, a biguanide class drug used in patients with type 2 diabetes mellitus, has been recognized to ameliorate IR, hyperinsulinemia, and hyperandrogenism in women with PCOS (21). However, to date, there are no data available regarding the effect of metformin administration on the endothelium in PCOS. The aim of the present pilot study was to evaluate the effectiveness of metformin treatment on endothelial structure and function in a group of women with PCOS without confounding factor for endothelial injury.
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Subjects and Methods |
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Thirty young normal-weight women with PCOS were screened from the patient population of the Department of Molecular and Clinical Endocrinology and Oncology in Naples (4). The diagnosis of PCOS was made based according to the Rotterdam criteria (22). Specifically, patients with anovulation and clinical and/or biochemical hyperandrogenism were enrolled.
Exclusion criteria included age less than 18 yr or more than 25 yr, pregnancy, hypothyroidism, hyperprolactinemia, Cushing’s syndrome, nonclassical congenital adrenal hyperplasia, and use of oral contraceptives, glucocorticoids, antiandrogens, ovulation induction agents, antidiabetic or antiobesity drugs or other hormonal drugs within the previous 6 months. Subjects with neoplastic, metabolic (including glucose intolerance), hepatic, and cardiovascular disorder or other concurrent medical illness (i.e. diabetes, renal disease, or malabsorptive disorders) were also excluded from the study. All subjects were nonsmokers and had normal physical activity, and none drank alcoholic beverages.
The institutional review board of University Federico II of Naples approved the study. The purpose of the protocol was explained to each subject, and written consent was obtained from each before beginning the study.
Protocol and treatment
At study entry, all subjects underwent blood sampling for hormonal assessment, lipid profile, serum ET-1, and fasting glucose and insulin levels. All blood samples were obtained in the morning between 0800–0900 h after an overnight fast during the early follicular phase (second to fourth day) of a spontaneous or progesterone-induced menstrual cycle. Blood samples were collected into tubes containing EDTA after a 30-min resting period in the supine position. Each subject underwent an oral glucose tolerance test for which they received 75 g glucose orally, and blood samples were obtained before and at 30-min intervals for 2 h (at 0, 30, 60, 90, and 120 min). All blood samples were immediately centrifuged at 4 C for 20 min at 1600 x g and stored at –20 C until assayed.
During the same visit, all subjects underwent transvaginal ultrasonography (TV-USG); anthropometric measurements, including height, weight, body mass index (ratio between the weight and the square of the height), and waist to hip ratio (ratio between the smallest circumference at the torso and the widest circumference at the hip); evaluation of heart rate, and diastolic and systolic blood pressures; and assessments of daily physical activity at their job and at home using a well-validated semiquantitative questionnaire (4, 7). Finally, a careful echocardiographic and color Doppler evaluation was performed by an experienced operator (B.D.S.), who was blinded to the clinical data, using a color Doppler (GE Vingmed Ultrasound, Horten, Norway) with a high resolution 10-MHz linear probe.
Briefly, each patient underwent to several longitudinal ultrasonographic scans of the carotid artery and was examined in the supine position, with the head hyperextended and turned away from the side being scanned. The sonographer scanned the right and left common carotid arteries and the carotid bifurcation bulb area from multiple planes. The IMT of the posterior (far) wall of both common carotid arteries was measured at the end diastole from the B-mode screen as the distance between the junction of the lumen and intima and that of the media and adventitia. The mean IMT for each side was calculated as the average of 10 measurements made in the right and left carotid arteries using electronic calipers. Ambient light and temperature were controlled throughout the procedure. In our studies, the intra- and interobserver coefficients of variation (CVs) for the repeated measurements of IMT were 7.0% and 12.0%, respectively. During the same visit, vascular reactivity was also assessed using brachial artery ultrasound. A 7.5-MHz linear phased array ultrasound transducer (GE Vingmed Ultrasound) was used to image the dominant brachial artery longitudinally just above the antecubital fossa. Subjects were asked to fast for at least 8–12 h and to refrain from physical activity for at least 4–6 h before the examination. All hemodynamic measurements were obtained with the subjects in a supine comfortable position in a quiet temperature-controlled room. In all studies, blood pressure in the contralateral brachial artery was recorded at regular intervals, and the electrocardiogram was monitored continuously. After baseline images of brachial arterial diameter (BAD) had been obtained, limb flow occlusion was produced by inflating a standard sphygmomanometry cuff on the upper arm to 40 mm Hg above systolic blood pressure for 4 min. This caused ischemia and consequent dilatation of downstream resistance vessels. Subsequent cuff deflation induced a brief high-flow state through the brachial artery (reactive hyperemia) for the release of endothelial nitric oxide, to accommodate the dilated resistance vessels. The BAD was measured at 30 sec, 1 min, 2 min, 3 min, and 4 min after ischemia. All images were recorded on videotape for subsequent off-line analysis. FMD of the brachial artery was expressed as the percent change in the arterial diameter from baseline to 4 min after deflation cuff, and diameter after reactive hyperemia (DARH) was expressed as the size of the diameter 4 min after deflation cuff, i.e. after ischemia. The FMD was used as a measure of endothelium-dependent vasodilatation. In our studies, the intra- and interobserver CVs for the repeated measurements of resting arterial diameter were 2.3% and 5.6%, respectively.
Then all subjects received metformin cloridrate (Glucophage, Merck, Florence, Italy) at a dosage of 850 mg, twice daily, for 6 months. The patients were instructed to take the tablets with the meals. To evaluate compliance with the treatment and the protocol, the number of tablets forgotten and the changes in diet, physical activity, and weight were also recorded. In the same daily diary, each woman recorded the frequency and severity of her menses and the onset of any adverse experiences, specifying the severity, duration, and a possible cause-effect relationship with drug administration. In addition, standard clinical evaluations and laboratory analyses, including hematological, renal function, and liver function tests, were performed at baseline and after 3 and 6 months of treatment as safety measures.
Throughout the study, no changes in lifestyle were implemented, and subjects were instructed to follow their usual diet and physical activity and to use barrier contraceptive.
During treatment, ovulatory function was evaluated in the midluteal phase (7 d before the expected menses) with the use of TV-USG and a plasma progesterone (P) assay. The presence of fluid in the cul-de-sac at TV-USG and a plasma P level greater than 32 nmol/liter (>10 ng/ml, metric units) were considered confirmatory criteria for ovulation (11).
After the treatment period, in each patient all of the above parameters were reevaluated as at baseline.
Biochemical assays.
Plasma LH, FSH, prolactin, estradiol, P, 17
-hydroxyprogesterone, T, androstenedione, and dehydroepiandrosterone sulfate levels were measured by specific RIAs, as previously described (4, 7). SHBG levels were measured using an immunoradiometric assay (4, 7), and the free androgen index was calculated [T (nmol/liter)/SHBG (nmol/liter) x 100]. Blood insulin and glucose levels were measured by a solid-phase chemiluminescent enzyme immunoassay and the glucose oxidase method, respectively (4, 7). The glucose and insulin areas under curve (AUCs) and the AUCglucose/AUCinsulin ratio (23) in response to the oral glucose tolerance test were calculated. The lipid profile consisted of serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride levels (4, 7). Serum ET-1 was measured by ELISA (Biomedica Gesellschft, Wien, Austria) with a sensitivity of 0.05 pmol/liter, and intra- and interassay CVs, respectively, of 4.5% and 6.9% (4)
Statistical analysis
Clinical and biochemical data were compared before and after treatment using the general linear model repeated measures procedure. This test was also used for FMD, IMT, and ET-1, on which the effects of covariates [
AUCglucose/AUCinsulin ratio, free androgen index (FAI), LDL, and HDL] were included. Continuous data were expressed as the mean ± SD. A value of P < 0.05 was considered statistically significant. The SPSS 13.0 (SPSS, Inc., Chicago, IL) package was used for statistical analyses.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. At study entry, the mean age (±SD) of the PCOS patients was of 22.8 ± 2.5 yr, and all subjects had polycystic ovaries at TV-USG.
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After 6 months of metformin administration, 26 patients (of 30, 86.7%) demonstrated normal ovulatory cycles. SHBG and FAI significantly (P < 0.001) increased and decreased, respectively. No other significant change in hormonal levels was observed compared with baseline values (Table 1
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Table 2 denotes the metabolic profile of subjects before and after treatment. No difference was detected in fasting glucose levels or AUCglucose, whereas fasting insulin levels and AUCinsulin were significantly (P < 0.001) reduced. With treatment, the AUCglucose/AUCinsulin ratio significantly (P < 0.001) increased, with a net change of +0.15 ± 0.18. The circulating levels of HDL-C (net change of +0.11 ± 0.18) and those of LDL-C (net change of –0.096 ± 0.029) were significantly increased and decreased, respectively. Alternatively, serum total cholesterol and triglycerides levels were not different from baseline values (Table 2).
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). After treatment, a significant decrease was observed in BAD, DARH, IMT, and plasma ET-1 levels, whereas FMD increased significantly (Table 3).
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AUCglucose/AUCinsulin ratio, FAI, LDL, and HDL as quantitative predictive variables, demonstrated only a direct relationship with metformin treatment for AUCglucose/AUCinsulin. In particular, in each model only the FMD x AUCglucose/AUCinsulin, IMT x AUCglucose/AUCinsulin, and ET-1 x AUCglucose/AUCinsulin ratios resulted significant (P < 0.001). Conversely, FAI, LDL, and HDL did not have any measurable effect on FMD, IMT, or ET-1 variations. |
Discussion |
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In our study population, endothelial impairment was strongly related to measures estimating IR, suggesting that IR and chronic hyperinsulinemia could predispose women with PCOS to an increased risk not only for diabetes, but also for CVD (4). Likewise, Diamanti-Kandarakis et al. (26) observed that a reduction in circulating ET-1 levels was associated with an improvement in IR. Metformin seems to reduce ET-1 concentrations without requiring concomitant changes in body weight. In our study and that by Diamanti-Kandarkis (26), in fact, no significant change in body mass index was reported during metformin treatment. Our data are also consistent with those reported by Morin-Papunen et al. (27), who observed that metformin treatment was associated with a significant reduction in serum C-reactive protein levels in women with PCOS.
Our data confirm that SHBG levels increase and FAI decreases with metformin treatment, demonstrating an overall improvement in androgenic profile (28, 29, 30). Whether amelioration in the hyperandrogenemic pattern in PCOS is one of the mechanisms by which metformin improves endothelial function remains unclear. However, Vryonidou et al. (31) actually suggested that hyperandrogenism could attenuate the consequences of the dysmetabolic phenotype on the vascular wall.
The significant difference in IR observed with only 6 months of metformin treatment, accompanied by the improvement in endothelial structure and function, suggests an important role of insulin excess in the precocious development of atherosclerosis in these patients. IR and hyperandrogenism have been widely accepted as risk factors for CVD in PCOS (4, 17). In this regard, insulin could promote atherogenesis by direct action on the arterial wall and the enhanced formation of superoxide anion O2, leading to an impaired endothelium-dependent arterial relaxation (32). Furthermore, hyperandrogenemia, which is often linked to IR in PCOS, and, in particular, dehydroepiandrosterone sulfate could have a protective role in early atherosclerotic arterial disease (carotid IMT) in overweight women with PCOS (33). In diabetic subjects, and in agreement with our results, IMT appears to be impaired, whereas metformin has potent antiatherogenic effects in these patients (34, 35).
The mechanisms by which metformin acts to improve endothelial function are still unclear, although suppressive effects on circulating androgens and insulin and improvements in insulin sensitivity are probably important components. In addition, it is necessary to highlight the beneficial effect of this insulin sensitizer on the dyslipidemic pattern present in PCOS. In fact, the improvement of arterial structure and function observed in our sample after metformin administration was associated with an increase in HDL-C and a reduction in LDL-C, demonstrating the beneficial effect of metformin even on lipid disturbances in PCOS. In this regard, some investigators observed improvements in carotid IMT with metformin treatment in diabetic patients, although without an associated change in the lipid profile (35). To the contrary, others have observed that metformin improves the lipid profile in diabetic patients (36, 37).
The lack of a placebo group was a limitation of the present research, although we should note that markers of endothelial function are probably not easily alterable by a placebo effect. For example, in a recent study of diabetic patients randomized to either placebo or metformin (2550 mg/d), placebo-treated patients did not experience a significant change in circulating lipids or markers of inflammation (37). In conclusion, our data indicate that endothelial structure and function in nondyslipdemic, nonhypertensive, young, normal-weight women with PCOS are significantly improved by a 6-month course of metformin administration. These findings suggest that metformin treatment could be effective in reducing the long-term CVD risk of patients with PCOS. This beneficial effect could be potentially more marked in those patients with other risk factors, such as dyslipidemia or obesity, although this remains to be confirmed. Additional long-term randomized studies are required to demonstrate these and other vascular benefits of metformin in women with PCOS.
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Acknowledgments |
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Footnotes |
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First Published Online August 23, 2005
Abbreviations: AUC, Area under curve; BAD, brachial artery baseline diameter; CV, coefficient of variation; CVD, cardiovascular disease; , change; DARH, diameter after reactive hyperemia; ET-1, endothelin-1; FAI, free androgen index; FMD, flow-mediated dilation; HDL-C, high-density lipoprotein cholesterol; IMT, intima media thickness; IR, insulin resistance; LDL-C, low-density lipoprotein cholesterol; P, progesterone; PCOS, polycystic ovary syndrome; TV-USG, transvaginal ultrasonography.
Received May 2, 2005.
Accepted August 12, 2005.
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