Sprung, Victoria S.1; Cuthbertson, Daniel J.2; Pugh, Christopher J. A.1,3; Aziz, Nabil4; Kemp, Graham J.5; Daousi, Christina2; Green, Daniel J.1,3; Cable, Nigel Timothy1; Jones, Helen1
Polycystic ovarian syndrome (PCOS) is a highly complex heterogeneous phenotype consisting of clinical or biochemical hyperandrogenism, oligo- or amenorrhea, and polycystic appearance of the ovaries (13). Internationally, PCOS affects up to 10% of women according to the “classical” National Institutes of Health criteria (6) and up to 20% using the broader Rotterdam criteria (9) and is therefore the most common endocrinopathy in females of reproductive age. PCOS is associated with several cardiometabolic pathologies, including obesity, insulin resistance, impaired glucose tolerance or type 2 diabetes, nonalcoholic fatty liver disease, dyslipidemia, and hypertension (13). It may therefore be associated with increased prevalence of cardiovascular disease (CVD).
Several studies using surrogate markers in women with PCOS, including measures of vascular structure and function (3) and serum concentrations of CVD risk markers (38), have supported an association with CVD. The relationship between PCOS and CVD is further evidenced by a recent meta-analysis of follow-up studies in women with PCOS, which described an approximately twofold increased risk of arterial disease compared with non-PCOS women, specifically coronary heart disease and stroke (11). Although there is some disparity between published reports, our recent meta-analysis suggests that endothelial dysfunction, manifested as reduced flow-mediated dilation (FMD), is an intrinsic feature of PCOS (33), further supporting the notion that women with PCOS are at increased risk of developing CVD.
Lifestyle modification has been endorsed by the Androgen Excess and PCOS Society as a first line treatment in the prevention of CVD (43). Existing exercise intervention studies in women with PCOS have reported the beneficial effects of exercise on reproductive function (29), obesity (27), and cardiorespiratory fitness (41). Furthermore, we recently found that exercise training improves microvascular function in obese women with PCOS (34), but the effect of exercise on macrovascular function remains unknown.
Exercise training improves endothelial function in patients who exhibit similar risk factors to women with PCOS, for example, patients with type 2 diabetes (24). Indeed, exercise-induced improvements in endothelial function have been reported to occur both before and independent of changes in traditional markers of CVD risk, such as body mass index (BMI) and blood pressure, in patients with coronary artery disease (42), hypertension (20), and type 2 diabetes (15,18). Nevertheless, it is unknown whether endothelial dysfunction in women with PCOS is modified by interventions. For instance, a study assessing the effects of metformin on arterial stiffness, a surrogate measure of vascular function, reported that enhanced vascular function may be independent of changes in insulin resistance (2). Therefore, the aim of this study was to determine the effects of a 16-wk moderate-intensity aerobic exercise training program, compared with a control intervention, on endothelial function in obese women with PCOS and to elucidate potential implicated mechanisms by examining changes in adipose tissue volume and distribution and insulin resistance.
Seventeen women with PCOS age 28 yr (95% CI = 25–31), of BMI 33 kg·m−2 (95% CI = 31–35) were recruited from a gynecology clinic specifically for women with PCOS at Liverpool Women’s Foundation Trust. In line with current clinical practice in the UK, PCOS was defined according the Rotterdam criteria for diagnosing PCOS (32), based on the presence of two of the following three criteria: (i) clinical or biochemical hyperandrogenism, (ii) oligomenorrhea or amenorrhea, (iii) polycystic appearance of the ovaries upon ultrasound, having excluded other possible causes by appropriate biochemical assessment. Only sedentary individuals were recruited, defined as <2 h low-intensity activity per week based on a self-reported questionnaire. None of the PCOS women performed any structured or vigorous physical activity. All participants were older than 18 yr and had no history of type 2 diabetes, cardiovascular, liver, kidney, or respiratory disease. Current use or use within the last 3 months of insulin-sensitizing agents (metformin and/or pioglitazone), orlistat, antiandrogens, fertility treatments, glucocorticoids, or medications that could alter vascular structure and/or function such as the oral contraceptive pill resulted in exclusion. Pharmacological treatment(s), such as metformin and the oral contraceptive pill, are commonly administered as part of clinical care for women with PCOS, but importantly, only those who declined these options were recruited onto the study. Furthermore, none of the participants began any such treatment during the study. All participants were nonsmokers and drank <14 U of alcohol per week. It was not feasible to control for menstrual cycle phase in women with PCOS due to the erratic nature of their cycles. All women with PCOS experienced oligo- or anovulation, and therefore it would be problematic to track the luteal phase of their menstrual cycle. Participants were asked to fast for 12 h, to abstain from alcohol and caffeine for 24 h, and to refrain from exercise for 48 h before testing sessions. The study conformed to the Declaration of Helsinki and was approved by the local research ethics committee. All participants provided written informed consent.
Participants reported to the laboratory on two occasions. Visit 1 included anthropometric measurements, a fasting blood sample, an assessment of brachial artery endothelial function, and lastly a cardiorespiratory fitness test. Visit 2 involved whole-body magnetic resonance imaging (MRI) with proton magnetic resonance spectroscopy (1H-MRS) to quantify liver and skeletal muscle fat. All participants were studied at the same time of day to control for the impact of circadian variation.
Participation in the exercise intervention or control intervention was based on patient choice. Ten (n = 10; 27 yr, 95% CI = 23–32; 31 kg·m−2, 95% CI = 28–34) women with PCOS received a 16-wk program of supervised moderate-intensity aerobic exercise training, and seven women (n = 7; 29 yr, 95% CI = 24–35; 35 kg·m−2, 95% CI = 31–40) acted as a control group. The primary comparison was between structured, supervised exercise training, and current clinical care. The control group therefore followed current conventional care as advised by their gynecologist during a clinic consultation. All patients underwent the physiological measurements at baseline and after the 16-wk intervention period.
Anthropometric and biochemical evaluation
After a full history, a single person recorded all anthropometric measurements (weight, height, waist, and hip circumference). After an overnight fast, blood was taken for biochemical profile, including total cholesterol, triglycerides, HDL and LDL cholesterol, alanine aminotransferase (ALT), glucose, and insulin. Circulating reproductive hormone levels were measured, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, testosterone, and sex hormone–binding globulin (SHBG).
Biochemical assays and calculations
Samples were analyzed using the Olympus AU2700 analyzer (Beckman Coulter (UK) Ltd., Buckinghamshire, UK) with standard proprietary reagents as follows: glucose with hexokinase, total cholesterol, and HDL with cholesterol esterase/oxidase, triglyceride with glycerol kinase, and ALT with IFCC kinetic UV (without pyridoxal phosphate activation). The intra- and interassay coefficients of variation were ≤10%. LDL was calculated according to the Friedwald formula. LH, FSH, estradiol, progesterone, total testosterone, and SHBG concentrations were measured by a chemiluminescence method (Siemens Centaur; Siemens Healthcare, Malvern, PA). Free androgen index was calculated as 100 × (testosterone concentration [nmol·L−1] / SHBG concentration [nmol·L−1]) (normal<7%). Insulin was measured using an ELISA kit (Invitrogen, UK). The intra- and interassay coefficients of variation were 9%. Using fasting baseline glucose and insulin concentrations, insulin resistance was calculated by the homeostasis model assessment for IR (HOMA-IR) (25). All laboratory assays were performed in the clinical biochemistry laboratory at University Hospital Aintree.
All vascular function assessments were performed in a quiet, temperature-controlled laboratory. Upon arrival, participants rested in the supine position for ∼20 min to facilitate assessment of baseline mean arterial pressure and HR. After the rest period, HR and mean arterial pressure were determined from an average of three measures on the left arm. Participants were then positioned with their right arm extended and immobilized with foam supports at an angle of ∼80° from the torso.
Brachial Artery FMD
A 10-MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (Siemens Medical Solutions, Malvern, PA) was used to image the brachial artery in the distal third of the upper right arm. When an optimal image was acquired, the probe was held stable and the ultrasound parameters were set to optimize longitudinal, B-mode images of the lumen–arterial wall interface. Continuous Doppler velocity assessment was also obtained using the high-resolution ultrasound machine and was collected using a 60° isonation angle. Nitric oxide–mediated endothelial function was assessed by measuring FMD in response to a 5-min ischemic stimulus, induced by forearm cuff inflation (36). Baseline images were recorded using a specialized recording software (Camtasia; TechSmith, Okemos, MI). A rapid inflation and deflation pneumatic device (D.E. Hokanson, Bellevue, WA) was used with an inflation cuff placed immediately distal to the olecranon process of the imaged arm to provide a stimulus for forearm ischemia (36). A baseline recording lasting 1 min was acquired before the forearm cuff was inflated (∼220 mm Hg) for 5 min. Artery diameter and blood flow velocity recordings resumed 30 s before cuff deflation and continued for 3 min thereafter (36). Peak brachial artery diameter and blood flow velocity, and the time taken to reach these peaks after cuff release, were recorded.
Brachial Artery Endothelium-Independent Vasodilation
After an approximately 15-min rest period, a 1-min baseline recording of the brachial artery was again acquired. Subsequently, brachial artery endothelium-independent vasodilation was examined after the administration of sublingual glyceryl trinitrate (GTN; 400 μg), an NO donor. The brachial artery was imaged for 10 min after the administration of GTN.
Posttest analysis of brachial artery diameter was undertaken using custom-designed automated edge detection and wall-tracking software; the validity and reproducibility of this software has been previously demonstrated (36).
Peak Oxygen Consumption Test
A fitness test (V˙O2peak) was performed on a treadmill ergometer using the protocol of Bruce et al. (10). After a 2-min warm-up at 2.2 km·h−1 on a flat gradient, the initial workload was set at 2.7 km·h−1 at 5° grade. Thereafter, stepwise increments in speed and grade were made every minute. HR (Polar Electro Oy, Kempele, Finland) and rate of perceived exertion were monitored throughout (8). V˙O2peak was calculated from expired gas fractions (Oxycon Pro, Jaegar, Germany) as the highest consecutive 15-s period of data in the last minute before volitional exhaustion.
All women underwent MRI scanning in a 1.5-T Siemens Symphony scanner (Siemens Medical Solutions, Erlangen, Germany) in a prone position, being moved through the magnet to acquire full body coverage.
Volumetric analysis of adipose tissue content.
Visceral adipose tissue (VAT) and abdominal subcutaneous adipose tissue were calculated from whole-body axial T1-weighted fast spin echo scans (axial scans, 10-mm slice thickness followed by a 10-mm gap using the integral body coil). The abdominal region was defined as the image slices from the slice containing the femoral heads, to the slice containing the top of the liver/base of the lungs. All scans were analyzed centrally, and anonymized before analysis ensuring blindness to all clinical details.
Proton magnetic resonance spectroscopy (1H-MRS)
Three voxels of interest were identified in the liver at standard sites avoiding ducts and vasculature. In the skeletal muscle, a single voxel was identified in each of the tibialis anterior and soleus muscles, avoiding bone, fascia, and the neurovascular bundle. Single voxel spectroscopy was conducted at each of these five sites. Voxel size was 20 × 20 × 20 mm, echo time was 135 ms, and repetition time was 1500 ms, with 64 acquisitions. Where the muscle was too small to allow placement of a 20-mm voxel, a 15 × 15 × 20 mm voxel was placed, and the number of acquisitions was increased to 200 to maintain signal-to-noise ratio. 1H MR spectra were quantified using the AMARES algorithm in the software package jMRUI-3.0 (28,39). As previously described, liver fat is expressed as % of CH2 lipid signal amplitude relative to water signal amplitude after correcting for T1 and T2 (37), and IMCL is expressed as CH2 lipid amplitude relative to total creatine amplitude after correcting for T1 and T2 (31).
Before commencing the exercise intervention, all participants attended a thorough familiarization session. Participants were required to attend the university gymnasium on a weekly basis, during which time they wore an HR monitor throughout (Polar Fitness; Polar Electro Oy) and provided with full supervision and guidance from a trained exercise physiologist. On the baiss of individual basal fitness level, participants underwent 30 min of moderate-intensity aerobic exercise three times a week (heart rate reserve 30%), which progressed weekly based on HR responses. At week 12, participants were exercising five times a week for 45 min at 60% heart rate reserve. To facilitate maximum compliance throughout the 16-wk period, all participants were closely monitored via the Wellness Key system, a software program that enables remote and accurate tracking of exercise activity. No dietary modifications were made throughout the course of the exercise intervention, confirmed by use of a standard food diary. Three-day food diaries were collected immediately before and after the exercise intervention and subsequently analyzed for macronutrient intake (total energy, carbohydrate, fat, protein, and sugar).
Women with PCOS in the control group received typical lifestyle advice provided at clinical consultation. Participants were simply advised by their gynecologist to modify their lifestyle by losing weight and to increase their physical activity. After consent and physiological assessment, women with PCOS who opted for the conventional care group had no contact with the research team throughout the 16-wk study. The research team did not manipulate or influence dietary intake or exercise status throughout the intervention period, nor did they monitor it. This ensured that there were no external factors that had potential impact to behavior and/or lifestyle, aside from the advice provided in the clinic, which adheres to current clinical conventional care guidelines for this patient group.
The primary outcome variable for this study was FMD, and the primary comparison was the effect of exercise versus conventional care. On the basis of previously reported data between PCOS and control women (33), using the NQUERY (Statistical Solutions, Cork, Ireland) that a two-sample t-test with a 0.05 two-sided significance level would have 80% power to detect a difference in means of 4.0%, assuming that the common standard deviation of change score is 2.7%, when the sample sizes in the exercise and control groups are 10 and 7, respectively. First, all data were analyzed for distribution and transformed appropriately. For the comparison of the exercise versus conventional care intervention, delta (Δ) change from preintervention was calculated (40) and analyzed using generalized estimating equations (GEE), with preexercise data as a covariate. The analysis approach based on the generalized estimating equation is considered a powerful and robust approach to the analysis of repeated-measures data (7). We also controlled the covariate for the baseline diameter measured before the introduction of hyperemia in each test of FMD. This allometric approach is more accurate for scaling changes in diameter than simple percentage change, which makes implicit assumptions about the relationship between baseline diameter and peak diameter (4). Data were analyzed using the Statistical Package for the Social Sciences (Version 17.0; SPSS Inc., Chicago, IL) software. Data are presented in the text as mean (95% CI), unless otherwise indicated, and exact P values are cited (values of P = 0.000 provided by the statistics package are reported as <0.001).
Women with PCOS who completed 16 wk of exercise training demonstrated 91% compliance to exercise sessions. Dietary intake, assessed during a 3-d period, was not significantly different after the exercise intervention in terms of energy intake (0.4 MJ, 95% CI = −5.3 to 6.1, P = 0.52), protein (2 g, 95% CI = −36 to 40, P = 0.63), carbohydrate (22 g, 95% CI = −492 to 448, P = 0.66), fat (8.5 g, 95% CI = −91 to 74, P = 0.42), and sugar (3 g, 95% CI = −98 to 104, P = 0.77).
There was a greater improvement in cardiorespiratory fitness after exercise training compared with conventional care (4.7 mL·kg−1·min−1, 95% CI = 1.4–7.9, P < 0.001; Fig. 1).
Exercise training and conventional care did not affect fasting glucose (−0.02 mmol·L−1, 95% CI = −0.09 to 0.13, P = 0.78; Table 1) or insulin (−4.4 μL U·mL−1, 95% CI = −11.3 to 2.5, P = 0.21; Table 1) differently; therefore, there was no difference in HOMA-IR after the respective interventions (−0.6, 95% CI = −2.2 to 1.0, P = 0.47; Fig. 1). Cholesterol reduced to a greater degree after exercise training compared with the conventional care group (−0.20 mmol·L−1, 95% CI = −0.28 to 0.04, P = 0.01). LDL also decreased after exercise training (−0.7 mmol·L−1, 95% CI = −1.1 to −0.3, P = 0.001; Table 1).
Mean FMD demonstrated a greater improvement after exercise training compared with conventional care (Table 2, Fig. 1). When baseline diameter and preintervention measurements of FMD were covariate controlled in our statistical model, the adjusted mean difference in FMD change scores was 3.6% (95% CI = 0.5 to 6.7, P = 0.03). Time to reach peak diameter was faster after exercise training (−16.8 s, 95% CI = −32.1 to 1.5, P = 0.09). There were negligible and nonstatistically significant differences in baseline or peak arterial diameter or shear rate between interventions (P > 0.05). All vascular data are summarized in Table 2.
Hepatic and Abdominal Fat Deposition
There were negligible differences in the change in liver fat between exercise-trained and conventional care (−0.5% CH2/H2O, 95% CI = −1.2 to 0.6, P = 0.56; Table 1). Similarly, there was no significant difference in weight, BMI, waist circumference muscle fat, VAT, or abdominal subcutaneous adipose tissue between interventions (P > 0.05; Table 1).
The novel finding of the current study was that supervised, moderate-intensity exercise training induced a greater improvement in brachial artery endothelial function in women with PCOS when compared with a conventional care group who did not undertake supervised exercise training. Importantly, this improvement in endothelial function occurred in the absence of changes in body weight, liver fat, VAT, or insulin resistance. These data suggest that improvements in vascular function, which reflect cardiovascular risk, are achievable in the absence of changes in common cardiovascular risk factors, a finding consistent with some previous observations (15). These results suggest that an improvement in fitness in the absence of weight loss reduces cardiovascular risk in women with PCOS. This supports exercise training as a management strategy capable of improving NO-mediated endothelial function even if such training does not produce changes in traditional cardiovascular risk factors (17).
Endothelial dysfunction is the earliest manifestation of atherosclerotic disease. It precedes morphological signs and clinical symptoms and is evident in women with PCOS (33). To our knowledge, this is the first study to assess the effect of exercise training on endothelial function in women with PCOS. Previously, other vascular markers, such as carotid intima-media thickness, have been used to evaluate CVD risk in women with PCOS. A recent study investigated the effect of a 1-yr lifestyle intervention, consisting of nutritional advice, exercise training, and behavioral therapy, in obese adolescent girls with PCOS and reported a reduced carotid intima-media thickness in those girls who achieved significant weight loss (23). Nevertheless, the authors could not distinguish between the effects of exercise, diet, behavioral therapy, and weight loss, nor did they include a control group for comparison. Importantly, the present findings indicate that moderate-intensity aerobic exercise, which meets the current recommended physical activity guidelines of the American College of Sports Medicine (14) and the American Heart Association (30), can induce significant improvements in conduit artery endothelial function, an early and prognostic marker of future cardiovascular risk, in the absence of weight loss.
There were no significant changes in parameters of obesity including BMI, waist circumference, or abdominal adipose tissue volumes after exercise training in the obese women we studied. This finding is in contrast to Hutchinson et al. (21), who reported a significant decrease in BMI, VAT, and insulin resistance after 12 wk of exercise, consisting of alternated moderate-intensity continuous exercise (∼70% V˙O2peak) and high-intensity intermittent exercise (∼90%–100% V˙O2peak) for∼3 h·wk−1, in women with PCOS. It is plausible that an exercise-training stimulus of higher intensity might have elicited changes in adipose tissue volume in this study. It is also possible that the lack of changes in these variables could be related to no changes dietary habits. Nevertheless, given that our exercise intervention enhanced FMD in women with PCOS to a level comparable to control women of similar age and BMI (33) and that body composition does not seem to mediate the difference in FMD observed in PCOS and control women (33), it is unlikely that changes in body composition within the current study would induce a greater increase in FMD.
Although the exercise intervention significantly reduced total and LDL cholesterol, neither insulin resistance nor liver fat significantly changed with exercise training. Notably both insulin resistance and liver fat (35) were not clinically elevated in these women before the intervention. One explanation for the data being within normal healthy limits may be related to the criteria used to diagnose PCOS in these women. The National Institutes of Health (44) and the Androgen Excess Society (5) definitions describe more obese and insulin-resistant phenotypes of PCOS, as hyperandrogenism is an integral criteria for this diagnosis (26). Biochemical hyperandrogenic PCOS represents a distinct metabolic phenotype characterized by an increased risk of hepatic steatosis and insulin resistance (22). Hyperandrogenism is not an essential feature of the Rotterdam diagnosis criteria (32), as indicated within the current study population, as only 11 of the 17 women demonstrated biochemical hyperandrogenism. Nevertheless, it is unlikely that androgens contributed to the exercise-induced improvement in FMD observed in the present study, as testosterone and free androgen index did not change after either exercise or conventional care.
Given that the exercise-induced improvement in endothelial function occurred in the absence of concomitant changes in body composition, insulin resistance, or lipid profiles, the current data suggest that exercise has a direct effect on the endothelium in women with PCOS. It is widely accepted that the FMD response in conduit vessels is endothelium dependent and nitric oxide mediated (12). Exercise training has been shown to promote NO bioavailability by reducing the number of oxygen free radicals and up-regulating endothelial NO synthase in CAD patients (19). Moreover, it has been hypothesized that episodic increases in arterial shear stress, induced by exercise, act as a key stimulus to the functional adaptations of the endothelium, which decrease atherosclerotic risk (15). Elevated CVD (hypertension) and event (stroke) risk purported in this population are vascular in origin, and therefore a direct effect on vascular function with exercise would be highly beneficial in these women.
One limitation of this study was that we did not randomize the women to an intervention. This limitation is highlighted by the result that the conventional care group had a higher BMI compared with the exercise group. However, given that we used a robust covariate control statistical analysis technique that incorporated adjustments for differences in baseline measurements and significant changes in BMI did not occur with the intervention, we are confident that study finding are robust. Furthermore, we ran additional analyses with BMI and preintervention FMD data as covariates and found exercise training still results in a greater increase in FMD than conventional care (4.4%, 95% CI = −2.4 to 6.5, P < 0.001). Given that, it was not feasible to control for menstrual cycle phase in women with PCOS due to the erratic nature of their cycles. Although not statistically significant, estradiol was higher in the exercise group. However, with estradiol and preintervention data as covariates, exercise training still resulted in a greater increase in FMD (4.0%, 95% CI = 2.1 to 6.0, P < 0.001). A further limitation was the method used to assess insulin resistance; the use of an oral glucose tolerance test (1) or a two-stage hyperinsulinemic–euglycemic clamp, with infusion of deuterated glucose, might have provided a more in-depth assessment of insulin sensitivity.
We also acknowledge that monitoring of diet and physical activity in the conventional care group in addition to the exercise group would have provided additional information regarding lifestyle changes in the control group. It is well documented that diet-induced weight loss enhances vascular function. Nevertheless, we propose that the conventional care group did not substantially increase physical activity or alter diet given that fitness, and FMD did not increase and body composition and blood lipids did not decrease. Furthermore, it is plausible that the lack of change in HOMA-IR after the exercise intervention was due to the lack of dietary changes.
There are several noteworthy strengths in the methodology of this study. The application of 1H-MRS and whole-body MRI is considered to be the most sensitive, noninvasive method to quantify liver fat and adipose tissue volume, respectively. Finally, the study was appropriately powered to detect a difference of 4% in the primary variable (FMD) between the control and exercise groups.
In summary, the current data suggest that supervised, moderate-intensity exercise training enhances endothelial function in women with PCOS. Given that FMD provides independent prognostic information relating to CVD risk (16), these data support the use of supervised exercise training as a cardioprotective strategy for vascular dysfunction and primary prevention of CVD in women with PCOS.
The authors express their gratitude to Terry Owen, the Aintree Volunteers, and the medical research charity, Weight Matters, for funding support. Support for Ph.D. student V. S. Sprung was provided by the medical research charity, Weight Matters.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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