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Department of Clinical Biochemistry (A.V.,A.D.-A.), Athens University Medical School, 11527 Athens, Greece; Departments of Endocrinology, Diabetes, and Metabolism (A.V., T.T., V.L., I.-A.V., C.P.) and Radiology (A.P., N.B.), Red Cross Hospital, 15121 Athens, Greece; and Laboratory of Pharmacology (A.T.), Democritus University of Thrace Medical School, 68100 Alexandroupolis, Greece
Address all correspondence and requests for reprints to: Andromachi Vryonidou, Voriou Hepirou 37, Athens 15235, Greece. E-mail: mahi_vr{at}hotmail.com.
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Abstract |
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In the studied population (n = 130), PCOS status, age, body mass index, and parental history of coronary heart disease were strong positive predictors of carotid IMT, whereas dehydroepiandrosterone sulfate was a strong negative predictor. In PCOS patients lower 
4-androstenedione and high-density lipoprotein-cholesterol levels were additionally strong positive predictors of carotid IMT, whereas in control women only total cholesterol was the additional positive predictor of carotid IMT.
In conclusion, young women with PCOS have an early increase of cardiovascular risk factors and greater carotid IMT, both of which may be responsible for subclinical atherosclerosis. The hyperandrogenemic phenotype of the syndrome may attenuate the consequences of the dysmetabolic phenotype on the vascular wall.
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Introduction |
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Measurement of carotid IMT is considered an easy and reliable marker for the prediction and progression of CVD in middle-aged and older people in large epidemiological studies. Carotid IMT has been found to correlate well with traditional cardiovascular risk factors such as increasing age, obesity, and adverse lipid profiles (16, 17, 18), as commonly observed in PCOS. An inverse correlation of carotid IMT with androgen levels in women has been demonstrated in limited studies. Although middle-aged women with a history of PCOS were found to be at increased risk for coronary artery disease, compared with normal cycling women (19, 20, 21), mortality from CVD was not reported to be higher in two follow-up studies (22, 23).
Limited data are available concerning the prevalence of metabolic disorders in young women with PCOS, and the question of whether chronic PCOS status leads over time to greater impairment of cardiovascular function is still open (24, 25). The present study was set up to evaluate the presence of premature atherosclerosis by measurement of carotid IMT in young women with PCOS (<35 yr) and the possible contribution of the hyperandrogenemic and metabolic phenotype that exists in these patients, in the variability of carotid IMT.
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Subjects and Methods |
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We prospectively studied 130 Caucasian women aged 17–35 yr (75 with PCOS and 55 healthy controls). All studied subjects were citizens of Athens and of similar socioeconomic status. Patients with PCOS (75) were selected from our outpatient clinic. A clinical diagnosis of PCOS was made if there was a history of chronic anovulation (less than eight menses per year) in association with a free androgen index (FAI) greater than 5 (26). Nonclassical adrenal 21-hydroxylase deficiency, Cushing’s syndrome, thyroid dysfunction, hyperprolactinemia, and androgen-secreting tumors were excluded by appropriate tests before the diagnosis of PCOS was made. No PCOS patient had diagnosed DM. No women had been taking any medication for the last 3 months. Control subjects (55) were again recruited from our outpatient clinic to which they came for evaluation of thyroid function or obesity problems. After a complete clinical, biochemical, and hormonal examination that excluded all pathological conditions, subjects who were normal were selected to participate in the study. Control women had normal cycles (menses every 27–32 d) with ovulation confirmed by progesterone levels greater than 6 ng/ml in the luteal phase. They were not hirsute, nor did they have a personal history of diabetes or hypertension or a family history of PCOS. From every woman (patient or control), a complete family history was obtained regarding parental DM, dyslipidemia (DSL), and coronary heart disease (CHD). A positive CHD history was defined as a myocardial infarction event or a positive coronary angiography before the age of 60 yr in either of the parents.
A physical examination was performed by the same doctor on all women. Obesity was assessed by estimating body mass index (BMI), with normal ranging 18.5–25, overweight 26–30, and obese over 30 (27). Body fat distribution was assessed by measurement of the waist to hip ratio (WHR). A Ferriman and Gallwey score of 6 or greater (F-G > 6) was considered hirsutism (28). Systolic blood pressure (SBP), diastolic blood pressure, and heart rate were measured by standard methods. SBP and diastolic blood pressure were measured in the right arm, with the subjects in a seated position. The average of two measurements taken by the same examiner with a mercury sphygmomanometer was used.
The study was performed according to the guidelines of the Helsinki Declaration on human experimentation and was approved by the ethical committee of the Medical School of Athens and the Red Cross Hospital. All subjects gave their written informed consent before the study.
Study protocol
Blood samples were collected between 0800 and 0900 h, after a 3-d normal carbohydrate diet and an overnight fast for at least 12 h. Levels of glucose (GLU), insulin (INS), total testosterone (TT), SHBG, dehydroepiandrosterone sulfate (DHEAS), 4-androstendione (4A), and lipids were determined during the early follicular phase of a spontaneous or progesterone-induced withdrawal bleeding. Fasting glucose to insulin ratio (FGIR) and glucose x insulin/22.5 [homeostasis model assessment (HOMA)] were estimated and used as indicators of insulin sensitivity. In cases of impaired fasting GLU (110–125 mg/dl), a 75-g oral glucose tolerance test was performed and subjects with impaired GLU tolerance or overt DM were excluded from the study.
Biochemical study
Plasma GLU levels, total and high-density lipoprotein (HDL)-cholesterol and triglycerides were measured by an enzymatic, colorimetric method in a Cobas Integra/400/700/800 autoanalyzer (Roche Laboratory Systems, Mannheim, Germany). Low-density lipoprotein (LDL) cholesterol was calculated according to the Friedewald formula (total cholesterol – HDL cholesterol – triglycerides/5).
Assays
Serum INS was measured by an immunoradiometric assay (DiaSorin, Vercelli, Italy) with a sensitivity of 0.3 µIU/ml and intra- and interassay coefficients of variation of 2.3 and 4.1%, respectively. Serum SHBG levels were also measured by an immunoradiometric assay (Orion Diagnostica, Espoo, Finland) with a sensitivity of 0.5 nmol/liter and intra- and interassay coefficients of variation of 4.8 and 5.3%, respectively. Serum TT and 4A levels were measured by a RIA (Diagnostic Systems Laboratories, Webster, TX) as well as DHEAS levels (Diagnostic Products Corp., Los Angeles, CA). The assays had sensitivity of 0.05 ng/ml, 0.02 ng/ml, and 1.1 µg/dl and intra- and interassay coefficients of variation of 7.5, 4.3, 4.6, 8.5, 6, and 8.2%, respectively.
Carotid measurement technique
Carotid artery ultrasound imaging was performed by the same experienced radiologist (P.A.), who was blinded to PCOS status. Two properly maintained and calibrated ultrasound systems were used with excellent resolution [ATL HDI 5000 with a 12-MHz high-resolution linear transducer (Philips/ATL) and a LOGIC 700 with a 5- to 12-MHz high-resolution linear probe; General Electric (Fairfield, CT)]. Depth, gain, and focus were optimized to obtain the best IMT image (29). Longitudinal images of both common carotid arteries were acquired just proximal to the carotid bifurcation (bulb) (30). Five measurements for IMT at each carotid artery were taken at 1.5 cm from the bifurcation, averaging all values (31). Carotid IMT was defined as the distance between the lumen-intima and the media interfaces. Only the posterior carotid wall measurements for IMT were taken (32). Inferior-quality images were excluded from the study. The patient was in a supine position with the head facing away from the side of examination. If there was plaque in the carotid artery, care was taken not to include it in the IMT measurement. Reproducibility of carotid IMT measurement was assessed in 20 women who underwent two ultrasound examinations with the same machine within 1 month. The between-visit coefficient of variation was 5% (0.04 mm).
Statistical analysis
Statistical analyses were performed using the Statistical Package for Social Sciences (version 10.0, SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant. Descriptive statistics were computed for PCOS cases and controls and compared by the unpaired t test for continuous data and a 
2 test for categorical data. Skewed variables were logarithmically transformed before performing statistical comparisons. Spearman correlation coefficients between hormone and lipid levels were used to measure the correlations.
Models of multiple regression analysis were built using the stepwise command of SPPS. Variables were included in or removed from the model if the significance of the F value was less than 0.05 or less than 0.1, respectively. Univariate regression analysis of carotid IMT with various hormonal and traditional cardiovascular risk factors was conducted on an exploratory basis to determine possible predictors of carotid IMT.
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Results |
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. Subjects and control women were similar in age and BMI; however, PCOS subjects had more pronounced central adiposity than control women (0.79 vs. 0.75, P = 0.001), as estimated by WHR. Significant differences were noted as expected in all measured androgens including TT, DHEAS, and 4A, which were higher in PCOS subjects than control women. SHBG levels were significantly lower in PCOS women than control subjects (P < 0.001). From the measured lipids, HDL-cholesterol was significantly lower (P < 0.001), whereas total and LDL-cholesterol levels were significantly higher in PCOS subjects, compared with control women (P = 0.02 and P = 0.01, respectively). Indices of insulin resistance, HOMA, and FGIR were significantly higher in PCOS women than control women (P < 0.001), whereas fasting GLU levels tended to be higher in PCOS women (P = 0.05). History of parental DM was more prevalent in PCOS subjects than controls (30.6% vs. 14%, P = 0.04). No difference was observed between the two groups in smoking status, arterial blood pressure, or history of parental CHD and DSL.
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, 0.58 vs. 0.47 mm, P < 0.001). Also, the maximum IMT was significantly higher in PCOS women than control women (0.60 vs. 0.50 mm, P < 0.001). Univariate linear regressions of carotid IMT with traditional cardiovascular risk factors are shown in Table 3.
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4A, HOMA, LDL-cholesterol, and triglycerides. A significantly negative association was found between IMT and levels of DHEAS, SHBG, and HDL-cholesterol. Parental history of DM, CHD, and DSL were also significant predictors of IMT (P < 0.001, P = 0.001, and P < 0.02, respectively).
Multivariate linear regression analysis (Table 4) was carried out with carotid IMT as the dependent variable. Hormonal and traditional cardiovascular risk factors found to be associated with IMT in the univariate analysis were the independent variables (model 1). In a second model, the effect of disease histories was also examined (model 2). In the studied subjects (n = 130) (model 1), PCOS status was the strongest predictor of carotid IMT (P < 0.001), explaining 30.6% of its variability. Age and BMI were also independent positive predictors of IMT (P < 0.001 for both). DHEAS was a significant negative independent predictor of IMT (P = 0.002). PCOS status, age, BMI, and DHEAS explained 65.2% of the variability of IMT (model 1). When disease histories were included in the analysis (model 2), the above predictors as well as the parental history of CHD (P = 0.003) were still significant. Using this model, a total of 68% of the variability of IMT was explained.
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). In control women, only total cholesterol was a significant positive predictor of IMT, explaining 24.8% of its variability (results not shown).
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Discussion |
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In our study we found that women with PCOS had thicker carotid intima-media, compared with matched-for-age controls. The risk factors for premature atherosclerosis and CVD, such as increased central adiposity, higher total and LDL-cholesterol levels, lower HDL-cholesterol levels, and insulin resistance, were more prevalent in PCOS subjects, compared with matched-for-age and BMI control women, as have been shown in previous studies (33, 34, 35, 36). The difference in carotid IMT still existed when comparison was made between lean and obese women with PCOS and controls of similar weight (results not shown), suggesting that the pathogenesis of this abnormality in PCOS is not dependent on BMI alone. The above-mentioned metabolic risk factors may predict the development of atherosclerotic lesions and the consequent increased mortality from CVD events in young women, as has been demonstrated from epidemiological studies in middle-aged and older subjects. However, as yet no firm conclusions have been drawn in women with PCOS, and the risk of the syndrome for CVD remains uncertain (25). Data from cross-sectional studies are converging in support of increased disposition for atherosclerosis in late premenopausal women with PCOS (14, 15), but a similar trend has not been demonstrated in retrospective studies (22, 23).
Carotid IMT determined by B-mode ultrasound provides an index of generalized atherosclerosis (37, 38, 39, 40) and has been associated, in numerous studies, with the incidence of myocardial infarction and stroke (41, 42, 43). Thus, it has been proposed as an index of premature atherosclerosis or an independent risk factor for CVD in adults (17). Atherosclerosis is thought to begin in childhood and develop silently for decades before clinical events such as myocardial infarction and stroke occur in later life. Autopsy studies have disproved the assumption that time lapse in young women does not suffice for atherosclerotic lesions to manifest. These studies confirmed the presence of preclinical atherosclerotic lesions in children and adolescents and established their associations with antemortem vascular risk factors (44).
Limited data exist concerning the relationship of carotid IMT with various cardiovascular risk factors or future development of CVD in healthy young people (45). Case-control studies in children and young adults demonstrated that apart from age and BMI, which are strong predictors of carotid IMT, hypercholesterolemia (46), hypertension (47), and hyperglycemia (48) but not insulin resistance are associated with greater carotid IMT. To date, an estimation of carotid IMT in women with PCOS has been made in two studies. In the first, Talbott et al. (14) have shown that in young women with PCOS, carotid IMT was not different, compared with matched controls, whereas in women older than 45 yr, a significant difference was found. By contrast, in the second study Orio et al. (12) found an early impairment of endothelial structure and function and greater carotid IMT in 30 normal-weight women with PCOS as we did. These women had a mean age of 22 yr and were compared with 30 matched-for-age and weight controls. In addition, it should be noted that the IMT in the control subjects of the first study, aged 30–44 yr, was found significantly higher, compared with the studies of Orio et al. and our own (0.66 vs. 0.39 mm vs. 0.47 mm, respectively). This is somewhat puzzling, taking into account that these women had significantly lower BMI than women with PCOS (26.5 vs. 30.1).
Multiple regression analysis in our patients and controls taken as a group demonstrated a positive association of carotid IMT with age, BMI, PCOS status, and a positive CHD parental history and a negative association with DHEAS levels. No association was found between IMT and the degree of insulin resistance expressed by HOMA, SBP, or serum lipid levels (Table 4
). Epidemiological prospective and cross-sectional studies have documented that age, BMI, and parental CHD history are strong positive predictors of carotid IMT with variable contribution depending on other coexisting risk factors (40, 49). These correlations were also corroborated in our study. The lack of association between carotid IMT and insulin resistance and lipids could be explained by their cosegregation with BMI or PCOS status. When PCOS cases were studied separately, carotid IMT was predicted positively by age and BMI and negatively by low HDL-cholesterol, DHEAS, and 4A levels, explaining 19.7, 24.8, and 5.7% of its variability, respectively (Table 5). In control women total cholesterol alone was the additional positive predictor of IMT (results not shown). The antiatherogenic role of HDL-cholesterol and the atherogenic role of total cholesterol, especially in women, are well documented (50). The negative relationship among carotid IMT, blood DHEAS, and 4A levels is an interesting and stimulating subject for further observation.
The role of androgens in cardiovascular physiology and disease remains unclear. Nevertheless, considerable evidence suggests that androgens have beneficial or neutral cardiovascular effects at early and late stages of atherosclerosis (51). The prolonged sustained exposure of women with PCOS to higher androgens was not associated with excess of CVD or mortality despite increases in cardiovascular risk factors (22, 23). Because it is highly unlikely that these studies could have overlooked the greatly increased risk (7-fold) predicted by factor modeling (21), this striking discrepancy raises the possibility that androgens may have beneficial effects, at least in part, on atherogenesis in women.
The hyperandrogenemia in PCOS patients is linked to insulin resistance (4). Therapeutic interventions that increase insulin sensitivity are followed by dropping of androgens levels. In girls with a precocious pubarche history, who are at high risk for progression to PCOS, and in adult patients with PCOS, the administration of INS sensitizers resulted, along with the decrease of insulin resistance, in a significant decrease in DHEAS levels and all androgens levels, respectively (52, 53, 54). DHEAS and 4A are precursors of testosterone and estradiol, and it is possible that their effects on the cardiovascular system may be direct or indirect via enzymatic conversion to active metabolites. A favorable effect of the above androgens on lipid levels and lipoprotein metabolism has been reported in humans (55, 56). The progressive decline of these androgens and the parallel increased incidence of atherosclerosis with aging may indicate an atheroprotective effect (57). In vitro and in vivo animal studies have shown that DHEAS improves endothelial function and has characteristics of a peroxisome proliferator with antiproliferative and chemoprotective effects as well as favorable effects on obesity and diabetes (58, 59, 60). This protective effect seems to be abolished in women with CHD, who have lower 4A, DHEAS, and TT, compared with healthy controls (61, 62). Moreover, cross-sectional studies in pre- and postmenopausal women demonstrated that greater IMT was associated with lower levels of androgens, irrespective of other cardiovascular risk factors (63, 64, 65).
A genetic component in PCOS is indicated by the observations that, in a substantial proportion of women with PCOS and their first-degree relatives, greater BMI, increased testosterone and DHEAS levels, and lower insulin sensitivity were demonstrated (66). Additionally, women with PCOS exhibit increased prevalence and a strong family history for type 2 DM (67).
On the basis of our data, we can speculate that perhaps some factors present in PCOS counteract the deleterious effect of other risk factors on vasculature (68). Among these factors may be DHEAS and/or 4A, which counterbalance insulin resistance and its consequences. More research as well as longitudinal follow-up of PCOS cohorts beyond menopause is required to determine whether these women are at increased risk for CVD.
Our study has both its strengths and limitations. To our knowledge, this is the largest study examining the effect of the metabolic environment of PCOS on the carotid intima media of young women. The well-defined PCOS cohort and the control group were of comparable age, BMI, smoking status, and parental CHD history as well as presenting normal blood pressure and normal glucose levels. The significantly higher proportion of PCOS patients with a positive parental DM history, which could participate in IMT variability as was recently reported (69), and the fact that physical activity was not assessed were limitations of the study.
In conclusion, this study shows that women with PCOS demonstrate an early appearance of increased cardiovascular risk factors and indexes of preclinical atherosclerosis, compared with women of the same age without PCOS. Our data also suggest that androgens and especially DHEAS may counterbalance in part the adverse effect of the dyslipidemic profile and insulin resistance. Whether this protective effect attenuates or eliminates clinical CVD in women with PCOS until or after menopause remains to be clarified.
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Footnotes |
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Abbreviations: 4A, 4-Androstenedione; BMI, body mass index; CHD, coronary heart disease; CVD, cardiovascular disease; DHEAS, dehydroepiandrosterone sulfate; DM, diabetes mellitus; DSL, dyslipidemia; FGIR, fasting glucose to insulin ratio; GLU, glucose; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; IMT, intima-media thickness; INS, insulin; LDL, low-density lipoprotein; PCOS, polycystic ovary syndrome; SBP, systolic blood pressure; TT, total testosterone; WHR, waist to hip ratio.
Received December 3, 2004.
Accepted February 18, 2005.
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