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Departments of Obstetrics and Gynecology (R.S., A.K., M.M., A.D.), Radiology (E.J.R.V.B.), and Internal Medicine (D.J.), University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242
Address all correspondence and requests for reprints to: Anuja Dokras, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Iowa, 200 Hawkins Drive, Iowa City, Iowa 52242. E-mail: ADokras{at}obgyn.upenn.edu.
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Abstract |
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Objective: The objectives of the study were to detect early-onset subclinical coronary atherosclerosis (using coronary artery calcium as a marker) in young women with PCOS, compared with age- and body mass index-matched controls, and to compare traditional CV risk factors and inflammatory markers in the two groups.
Design: This was a prospective case-control study.
Setting: The study was conducted at a university hospital.
Subjects: Twenty-four obese (body mass index 
30 kg/m2) PCOS subjects and 24 obese controls participated.
Outcome Measures: Coronary artery calcium, inflammatory markers (high-sensitivity C-reactive protein, IL-6, TNF
, adiponectin, leptin), fasting blood tests (glucose, lipids, insulin), and dual-energy x-ray absorptiometry scan for body fat distribution were measured.
Results: Coronary artery calcium was detected in eight of 24 PCOS subjects (33%) and two of 24 controls (8%) (odds ratio 5.5, 95% confidence interval 1.03, 29.45, P < 0.03). Traditional CV risk factors did not differ significantly between the two groups, nor did markers of inflammation or adiposity, body fat distribution, or metabolic parameters with the exception of significantly lower quantitative insulin sensitivity check index (marker for insulin resistance) in the PCOS group (P < 0.05).
Conclusions: Young, obese women with PCOS have a high prevalence of early asymptomatic coronary atherosclerosis, compared with obese controls. This increased risk is independent of traditional CV risk factors and novel markers of inflammation. These findings underscore the need to screen and aggressively counsel and treat these women to prevent symptomatic CV disease.
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Introduction |
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CVD is one of the leading causes of female morbidity and mortality in the United States (8). Given the high prevalence of PCOS, this condition may potentially account for a significant proportion of atherosclerotic heart disease observed in women. The precise CVD risk in women with PCOS remains unclear, in part because there are no longitudinal studies examining CV events and/or mortality. A few studies suggest an increased risk of CAD in women with presumed features of PCOS (9, 10, 11). Several studies examined the prevalence of markers of subclinical CVD in women with PCOS. There is evidence for impaired endothelial function, an early marker of atherosclerosis, in young women with PCOS (6). Carotid intima-media thickness, another marker associated with increased prevalence of stroke and myocardial infarction, is also increased in premenopausal women with PCOS, compared with age-matched controls (12). The detection of both traditional CV risk factors and surrogate markers of atherosclerosis at a young age in these women puts them at a significantly increased risk for development of symptomatic CVD.
In the past decade, markers of low-grade chronic inflammation have been associated with CVD. Large-scale studies demonstrated that high-sensitivity C-reactive protein (hs-CRP) is a strong independent risk factor of future CVD and/or stroke (13). Some researchers have speculated that PCOS may also be a state of chronic low-grade inflammation (14). There are data suggesting serum levels of hs-CRP (15) and adiponectin, an adipose tissue-specific cytokine, may be altered in PCOS (16). The role of hs-CRP in predicting increased carotid intima-media thickness is, however, not independent of body mass index (BMI) in PCOS (17). The clinical and prognostic implications of chronic inflammation are currently uncertain in PCOS (18).
Although there are no prospective studies demonstrating the increased risk of CAD in women with PCOS, coronary artery calcium (CAC) scores (19) have been shown to be increased in a limited number of studies (20, 21). The detection of CAC confirms the presence of coronary atherosclerosis independently of symptoms or risk factors, and the quantity of CAC is directly related to the risk of myocardial infarction and sudden cardiac death in asymptomatic and symptomatic subjects (22). In one study, PCOS was found to be a significant predictor of CAC after adjustment for age and BMI (21). Another study failed to show the higher prevalence of CAC in women with PCOS after correction for BMI (20). Neither of these studies measured markers of chronic inflammation as predictors of early atherosclerosis in women with PCOS.
Our study was designed to detect early-onset CAC as a marker for subclinical atherosclerosis in young obese women with PCOS, compared with age- and weight-matched controls, and compare traditional CVD risk factors and others variables such as obesity, insulin resistance, and novel markers of inflammation between these two groups.
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Subjects and Methods |
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The University of Iowa Institutional Review Board approved this study, and all participants gave written, informed consent. Obese subjects (BMI 30 kg/m2) between the ages of 21 and 50 yr attending the PCOS clinic were asked to participate. These women had less than six menstrual cycles per year, evidence of clinical or biochemical hyperandrogenism, and normal thyroid and adrenal function (2). Sixteen of the 24 PCOS subjects had a transvaginal ultrasound and all had polycystic ovaries. Obese controls were simultaneously recruited from patients seen for an annual exam at the Gynecology Clinic and by advertisement in the hospital newspaper. All controls had regular menstrual cycles (at least 12/yr) and no history of hirsutism or infertility. Other exclusion criteria for all subjects included pregnancy, lactation, chronic illnesses such as asthma or inflammatory bowel disease, hysterectomy, menopause, known CVD, and any illnesses in the month preceding and during study participation. Smoking status was defined on the basis of self-reported current or past smoking. Drugs affecting inflammation (aspirin, nonsteroidal antiinflammatory drugs) and oral contraceptive pills were withheld for 2 wk and 1 month before the study, respectively. All subjects were on an unrestricted diet and were asked to refrain from excessive physical activity.
Methods
On the day of the study, subjects reported to the General Clinical Research Center. BMI was calculated as kilograms per square meter. Waist and hip circumferences were measured in duplicate at the level of the umbilicus (waist) and greater trochanter (hip). Mean systolic and diastolic blood pressure was assessed with two readings after 5 min of seated rest.
Laboratory analysis
A urine pregnancy test was performed on all subjects before the onset of the study. Subjects had fasting (12 h overnight) blood samples collected between 0730 and 0900 h. The samples were immediately centrifuged and serum was aliquoted and stored at –70 C until batch analyzed. All PCOS patients had TSH, prolactin, dehydroepiandrosterone, and 17-hydroxy progesterone levels checked to confirm the diagnosis of PCOS (2). TSH, prolactin, total testosterone, and insulin were measured by electrochemiluminescence immunoassay. SHBG was performed by chemiluminescent immunoassay. Dehydroepiandrosterone level was determined using immunometric chemiluminescence. 17-Hydroxyprogesterone, total cholesterol, triglycerides, HDL cholesterol, and glucose were measured using enzymatic methods. Low-density lipoprotein (LDL) cholesterol and free testosterone levels were mathematically derived. Estradiol values were also checked on the day of the study because serum levels of some inflammatory markers vary with the menstrual cycle (23).
Adiponectin and leptin were measured using a RIA kit (Linco Research, Inc., St. Charles, MO) with a sensitivity of 1 ng/ml and intra- and interassay coefficients of variation averaging 6.2 and 6.9% for adiponectin and 6.0 and 6.7% for leptin, respectively. Both human IL-6 and TNF were assayed using a sandwich enzyme immunoassay technique using a high-sensitivity Quantikine kit (R&D Systems Inc., Minneapolis, MN) with a sensitivity of approximately 0.1 pg/ml. The intra- and interassay coefficients of variation averaged 7.4 and 7.8% for IL-6 and 8.8 and 16.7%, respectively, for TNF. C-reactive protein was measured with a high-sensitivity sandwich enzyme immunoassay (ALPCO Diagnostics, Windham, NH) with a sensitivity of approximately 0.05 mg/liter. The intra- and interassay coefficients of variation averaged 6.0 and 11.6%, respectively.
Multislice computed tomography (CT) protocol
On the day of the study, electrocardiogram-gated noncontrast cardiac CT using a 16-row multislice CT scanner (Sensation-16; Siemens, Erlangen, Germany) was used to determine the CAC scores. Subjects were positioned supine within the gantry of the multislice CT (MSCT) scanner, and a 0.33-sec rotation time was used to obtain 1.0-mm-thick sections. During a single breath hold, images of the heart, from the level of the tracheal bifurcation to below the base of the heart, were acquired using prospective electrocardiogram triggering at 50–80% of the RR-interval, depending on the heart rate. Scan duration was approximately 10 sec, depending on heart rate and patient size. All regions with a density greater than 130 Hounsfield units and a 3-pixel-area threshold minimum were identified as potential foci of calcifications. A calcium score was calculated by the Agatston method from calcium area and signal intensity and represents the total amount of intracoronary calcium (24) (ScImage, version 2.0.01, Los Altos, CA). The radiologist and technicians were blinded to the case/control status of the subject. Total radiation was estimated to be between 5 and 10 mGy (1.0 rad).
Dual-energy x-ray absorptiometry (DEXA) scan protocol
On the study day, body fat distribution was measured with DEXA (GE Lunar Prodigy, Madison, WI) for fat mass quantification (25). The scanner uses an x-ray source to calibrate the bone mineral content and an external luciate and aluminum phantom to determine the percentage of fat of each soft tissue sample scanned. Regions of interest (including arms, legs, and trunk) were standardized. Percent body fat and percent lean body mass were calculated for each region. Percent trunk fat was calculated as the ratio of trunk fat to total fat x 100. Percent extremity fat was calculated as the ratio of total extremity fat (right and left arm fat and right and left leg fat) to total fat x 100. Trunk to extremity (T/E) fat ratio was determined by dividing percent trunk fat by percent extremity fat. All subjects were scanned in light clothing while lying flat on their backs with arms at their sides. One experienced technician who was blinded to the study status performed all readings. The BMI was restricted to less than 40 kg/m2 due to limitations with the DEXA equipment.
CV risk factors, metabolic syndrome, and Framingham risk score
Based on National Cholesterol Education Program guidelines CV risk factors included age older than 55 yr, current cigarette smoking, diabetes, history of premature CAD in first-degree relative (men < 55 yr, women < 65 yr), hypertension, and dyslipidemia. Participants were classified as having metabolic syndrome if they had a waist circumference greater than 85 cm and two of the following criteria: serum triglycerides 150 mg/dl or greater, serum HDL cholesterol less than 50 mg/dl, blood pressure 130/85 mm Hg or greater or on antihypertensive medication, and fasting blood glucose 100 mg/dl or greater or the presence of non-insulin-dependent diabetes mellitus (26). The Framingham risk score predicts the 10-yr risk of CAD with points assigned for blood pressure, total cholesterol, HDL cholesterol, age category, and smoking and diabetes history (27).
Statistical analysis
For the primary analysis reported here, only the presence/absence of CAC was evaluated because of the relatively low prevalence of CAC in this age group. Continuous variables were analyzed using a two-tailed t test or a Mann-Whitney U test. A 
2 or Fisher’s exact test was used to evaluate categorical variables. Variables with a skewed distribution were log transformed for all analyses. We had 85% power to detect a 30% difference in hs-CRP levels between the two groups at P < 0.05. A Spearman’s correlation was used to examine the relationship between markers of inflammation and other known variables. Statistical significance was defined as P < 0.05.
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Results |
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We studied a total of 24 PCOS and 24 control subjects who were primarily white non-Hispanic (Table 1
). Control subjects were slightly older than the PCOS group (P = 0.049). As expected, the PCOS group had significantly elevated free and total testosterone levels and fewer menses per year, compared with the controls (P < 0.001). Only obese subjects were selected to participate in this study, and the mean BMI of the PCOS group (36 ± 5.4 kg/m2) was comparable with the controls (35 ± 3.2 kg/m2). Other parameters of body fat distribution were also compared (Table 1). Although the waist circumference, percent trunk fat, and T/E fat ratio were greater in the PCOS group, they were not significantly different.
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The prevalence of CAC (CAC score > 0) was significantly higher in the PCOS group, 33% (eight of 24), compared with the controls, 8% (two of 24) (P < 0.03, odds ratio 5.5, 95% confidence interval 1.03, 29.45, Fig. 1). By comparing a subject’s calcium score with others of the same age and sex through the use of large databases, a calcium percentile rank for any given individual patient can be determined (28). The ranges of calcium scores were 0–9.3 in the PCOS group and 0–2.1 in the controls. Although low, all positive CAC scores in this study were in the 95th percentile for age and gender, given the subjects’ young age. The presence of traditional CV risk factors was found to be similar in both groups of subjects (Table 2). Two controls and four PCOS subjects had hypertension. The mean systolic blood pressure in the PCOS group was 125 ± 15 mm Hg, compared with 117 ± 14 mm Hg in controls (P < 0.03), indicating evidence of prehypertension in the PCOS group. Only one subject (PCOS) had a fasting glucose value of 126 mg/dl or greater and also had a positive CAC score. The mean fasting glucose levels in the PCOS group were 101 ± 31 mg/dl, compared with 95 ± 5.8 mg/dl in the controls. None of the three subjects with HDL less than 40 mg/dl had a positive CAC score. Of the five subjects with a significant family history of CAD, none had a positive CAC score. The Framingham risk score, which is generated using most of these risk factors, was also not significantly different between the two groups. These data suggest that women with PCOS are at an increased risk for CAC independent of the presence of traditional risk factors for CAD.
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). Quantitative insulin sensitivity check index (QUICKI), a surrogate marker of insulin resistance, was significantly lower in the PCOS group (P < 0.05). HDL, LDL, and triglycerides were not significantly different in the two groups. Although the prevalence of metabolic syndrome was higher in women with PCOS (six of 24, 25%), compared with controls (four of 24, 16%), it was not statistically significant. Only one of these PCOS women had positive CAC.
Novel markers of chronic inflammation and adiposity also help stratify women at an increased risk for CVD (13, 14). The median levels of hs-CRP, a biomarker that has proven to be a strong, independent predictor of both diabetes and CVD, were not significantly different in the two groups (Table 3). The percentage of women with an elevated hs-CRP (>3 mg/dl) in the PCOS group (14 of 24) were similar to the controls (16 of 24). Median IL-6 and TNF levels were also similar in both groups. We did not detect any differences in the median levels of two adipose tissue-specific cytokines adiponectin and leptin in PCOS and control subjects.
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(P = 0.05). Total fat mass correlated with IL-6 (P < 0.03) and TNF (P < 0.02).
Next we compared variables in all women with presence of CAC (n = 10) with those without CAC (n = 38, Table 4). The only variable that was significantly different in the two groups was a higher BMI in women with CAC (P = 0.027). Although percent trunk fat was higher in the CAC-positive group, it was not significantly different from the CAC-negative group. We also compared the variables in PCOS women with and without CAC (Table 5). Although women with PCOS who had CAC detected were older and had higher mean BMI than PCOS women without CAC, these differences were not statistically significant. The androgen levels in the two groups were also similar.
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Discussion |
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In our study we found the prevalence of CV risk factors to be low in both groups of premenopausal women. The prevalence of metabolic syndrome was higher in women with PCOS, compared with controls, but not significantly different. The Framingham risk scores were similar and low in both groups due to the large negative points assigned for young age. Recently a large study of asymptomatic subjects (n = 25,253) showed that CAC is an independent predictor of all cause mortality after controlling for age, gender, ethnicity, and cardiac risk factors (32). Our findings also suggest that asymptomatic women with PCOS may have CAC without the presence of other traditional CV risk factors.
CAC is virtually always associated with mural atheromatous plaque (33) and is a radiographic marker for atherosclerosis. CAC correlates with the extent of coronary atherosclerotic plaque and can be quantified by modalities such as electron-beam CT and MSCT. MSCT uses a rotating gantry with a special x-ray tube and variable number of detectors (from 4 to 64), with images acquired over 165–420 msec at 0.5- to 3.0-mm intervals, and correlates well with CAC scores obtained by electron-beam CT (34). The sensitivity of detection of CAC at lower scores is high and reproducible, and we used a calcium board to continuously ensure the quality of our images (35). Noncontrast cardiac CT detects only calcified coronary artery plaques. Hence, we probably underestimated the burden of CAD by not detecting noncalcified plaques, which may be visualized by intravascular ultrasound or CT coronary angiography.
Two studies have examined the prevalence of CAC in women with PCOS. Christian et al. (20) reported a higher prevalence of CAC in women with PCOS (39 vs. 21% in controls, P = 0.05). The mean age of PCOS subjects was slightly higher in that study (38.5 yr), compared with our study (31.8 yr). Traditional CV risk factors contributed to the risk of CAC, but PCOS alone did not predict the presence of CAC (odds ratio 1.99, P = 0.21). The second study reported the prevalence of CAC in older PCOS women (40–61 yr) to be 46 vs. 30% in controls (P = 0.059), with the mean score being significantly higher in the PCOS group (21). After controlling for BMI and age, PCOS was found to be a significant predictor of CAC in the total sample (P = 0.049). Neither of these studies measured markers of chronic inflammation.
Plasma levels of hs-CRP, an inflammatory marker predictive of the risk of myocardial infarction greater than that estimated by traditional risk factors alone (13), are increased in patients with visceral obesity. Increased levels of hs-CRP have been reported in women with PCOS, compared with age- and weight-matched controls (14). However, limited numbers of studies have examined obese women separately and some have found no significant difference between these groups (15). Low-grade chronic inflammation may be associated with increased central fat excess rather than PCOS status per se (36). We did not find a significant difference in hs-CRP levels between women with PCOS and controls. Our inclusion of subjects well matched for truncal fat distribution may explain our findings. Also, we did not have sufficient power to detect less than 30% difference in hs-CRP levels between the two groups. Because serum hs-CRP levels vary significantly during the menstrual cycle (23), we measured estradiol levels on the study day and found them to be comparable between the two groups. Although the hs-CRP levels were similar, mean hs-CRP levels were elevated (>3 mg/liter) in both groups, indicating that majority of subjects in our study are at a higher risk for subsequent CVD.
In addition to the storage and mobilization of lipids, the adipose tissue is also a remarkable endocrine organ, releasing cytokines and proinflammatory molecules such as IL-6 and TNF. In patients with truncal obesity, macrophage infiltration in adipose tissue (37) contributes to the inflammatory profile. In our study, we found no significant difference in TNF and IL-6 levels when compared between the PCOS and control groups as well as between CAC-positive and CAC-negative subjects. The plasma concentrations of TNF have been variable in women with PCOS (36, 38). We may not have had adequate power to detect a difference in these markers. However, levels of IL6 and TNF correlated with total fat mass, suggesting that levels of these markers may be related to obesity rather than PCOS per se (18, 39). Clinical studies have yet to demonstrate an increased risk of CVD in PCOS, particularly in the context of elevated levels of inflammatory cytokines.
Leptin and adiponectin are two adipokines more closely related to obesity than an inflammatory process (16). Adiponectin is inversely related to insulin resistance, obesity, metabolic syndrome, and diabetes. It is controversial whether adiponectin levels in women with PCOS are lower than controls after matching for BMI (16, 40). In contrast to adiponectin, elevated leptin levels are implicated in the pathogenesis of obesity and associated with insulin resistance (41). Recent studies have linked leptin levels to the degree of adiposity and insulin resistance in obese PCOS and control subjects (42, 43). We did not find a significant difference in serum adiponectin and leptin levels in the two groups.
The strength of our study is the inclusion of young women, providing evidence for the early detection of subclinical coronary atherosclerosis in PCOS. The subjects were well matched for weight, and we used multiple modalities to assess body fat distribution in an attempt to control for adiposity, a major variable that plays a role in insulin resistance and contributes to the pool of markers of inflammation. However, we did not differentiate between visceral and sc central fat.
In conclusion, our study demonstrates the presence of early coronary atherosclerosis in young women with PCOS. In our study, the majority of subjects with detectable CAC did not have traditional CV risk factors, and the presence of PCOS status per se appeared to contribute to this increased risk of CAC. The American College of Obstetricians and Gynecologists currently recommends screening women with PCOS for glucose intolerance and lipid abnormalities. In addition all obese women with PCOS should be counseled and treated for CV risk factor modification. Should one target the highest risk PCOS woman with documented subclinical atherosclerosis for more aggressive treatment of CVD including antiplatelet and cholesterol reduction pharmacotherapy? Longitudinal studies to document the progression of CAC in this population are urgently needed to address these issues.
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Footnotes |
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This work was presented at the 62nd Annual American Society of Reproductive Medicine meeting, New Orleans, LA, October 21–25, 2006.
Disclosure Statement: R.S., A.K., M.M, D.J., and A.D. have nothing to declare. E.J.R.V.B. consults for Edda Technologies, Vital Images, and Schering Plough Corp.
First Published Online September 11, 2007
Abbreviations: BMI, Body mass index; CAC, coronary artery calcium; CAD, coronary artery disease; CT, computed tomography; CV, cardiovascular; CVD, CV disease; DEXA, dual-energy x-ray absorptiometry; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; MSCT, multislice CT; PCOS, polycystic ovary syndrome; QUICKI, quantitative insulin sensitivity check index; T/E, trunk to extremity (ratio).
Received June 18, 2007.
Accepted September 5, 2007.
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