Arch Gynecol Obstet DOI 10.1007/s00404-014-3499-7
GENERAL GYNECOLOGY
No relationship between osteoprotegerin concentrations and endothelial dysfunction in non-obese women with and without polycystic ovary syndrome Remzi Abali • Nicel Tasdemir • Seref Alpsoy • Ufuk Go¨ker Tasdemir • Savas Guzel • Mehmet Aytac Yuksel • Ilkbal Temel Yuksel • Murat Yilmaz
Received: 4 May 2013 / Accepted: 25 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose To investigate the relationships of osteoprotegerin (OPG) concentrations to brachial artery flow-mediated vasodilation (FMD) and the carotid artery intima media thickness (CIMT) in polycystic ovary syndrome (PCOS). Methods Thirty-seven women with PCOS and 41 controls matched for body mass index (BMI) and age were included in study. The serum OPG concentrations, hormonal and metabolic profiles were measured in women
R. Abali (&) N. Tasdemir Department of Gynecology and Obstetrics, Faculty of Medicine, Namik Kemal University, 100. YilMah. Barbaros Cad, No: 132, Tekirdag, Turkey e-mail: [email protected]; [email protected] S. Alpsoy Department of Cardiology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey U. G. Tasdemir Department of Gynecology and Obstetrics, Tekirdag Hayrabolu State Hospital, Tekirdag, Turkey S. Guzel Department of Biochemistry, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. A. Yuksel Department of Gynecology and Obstetrics, Cerrahpasa Faculty of Medicine, ˙Istanbul University, Istanbul, Turkey I. Temel Yuksel Department of Obstetrics and Gynecology, Istanbul Okmeydani Research and Training Hospital, Istanbul, Turkey M. Yilmaz Department of Endocrinology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey
with PCOS and in control group. The CIMT and brachial artery FMD were evaluated in both groups. Results The mean serum concentrations of all hormones were comparable, except LH, which was higher in women with PCOS. Lipid parameters were similar between groups. There were no differences between groups with respect to fasting glucose, 2-h glucose, fasting insulin, HbA1c and HOMA-IR. The mean osteoprotogerin concentrations were higher in PCOS group (11.39 ± 2.29 vs. 10.22 ± 2.25 pmol/L, P = 0.026). The mean CIMT was higher in PCOS group than control group (0.52 ± 0.058 vs. 0.45 ± 0.059 mm, P \ 0.01). The mean brachial artery FMD was lower in PCOS group (0.068 ± 0.022 vs. 0.055 ± 0.029, P = 0.017). Conclusions We found high osteoprotogerin concentrations, increased CIMT and decreased FMD, in women with PCOS. However, there was no correlation between osteoprotegerin and cardiovascular risk markers. Keywords Polycystic Ovary syndrome Osteoprotegerin Carotid intima-media thickness FLOWmediated dilatation Cardiovascular disease
Introduction Polycystic ovary syndrome (PCOS) affects nearly 5–8 % of women of reproductive age [1]. It is characterized by chronic ovulatory dysfunction, clinical and/or biochemical hyperandrogenism and polycystic ovarian morphology. PCOS is associated with major risk factors for cardiovascular events, such as insulin resistance, hypertension and dyslipidemia [2]. However, an increase in cardiovascular morbidity and mortality has not been shown by population studies [3].
123
Arch Gynecol Obstet
Carotid artery intima-media thickness (CIMT) is a marker of early atherosclerosis. It can be measured relatively simply and noninvasively. CIMT has been reported as a predictor of subsequent cardiovascular events independent of traditional risk factors and is being used for risk stratification with other established risk factors for future cardiovascular diseases (CVDs) [4]. Endothelial dysfunction is associated with various forms of CVDs, such as hypertension, coronary artery disease, chronic heart failure and peripheral artery disease. It is characterized by reduced vasodilator responses of the endothelium due to reduced nitric oxide generation, oxidative stress and reduced production of hyperpolarizing factor (endothelium-derived hyperpolarizing factor). The endothelial dysfunction is related with poor prognosis for cardiovascular events, and improvement of endothelial dysfunction may reduce cardiovascular risk [5]. Postischemic flow-mediated dilatation (FMD) of the brachial artery is one of the classic techniques used to evaluate endothelial function. Age is a major determinant of arterial parameters; with increasing age, arterial compliance decreases [6]. Various genetic and environmental factors, including food intake, may influence FMD [7]. Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily and its expression has been demonstrated in human normal vascular smooth muscle, endothelial cells and atherosclerotic lesions [8, 9]. OPG inhibits osteoclastic bone resorption, and older studies have shown that OPG concentration increases with advancing age and reflects the severity of osteoporosis [10]. Increased OPG levels have also been found in women who were using hormone replacement therapy [11]. Several clinical studies in various populations have reported that elevated OPG levels are associated with subclinical atherosclerosis [12] and overall cardiovascular morbidity and mortality [11]. OPG may promote atherogenesis by inducing endothelial inflammation [13] and stimulating proliferation of endothelial and vascular smooth muscle cells [14]. We designed a cross-sectional study to investigate OPG levels, endothelial dysfunction and carotid artery intima media thickness in women with PCOS.
Materials and methods Thirty-seven women with PCOS and 41 controls matched for body mass index (BMI) and age were included in this study. The study was conducted in the gynecology clinic of Namik Kemal University School of Medicine (Tekirdag, Turkey) between November 2010 and June 2012. The study was approved by the local Ethics Committee of
123
Namik Kemal University. Written informed consent was obtained from all participants. The control group included 41 individuals selected from women who did not show any clinical and ultrasonography signs of PCOS. Diagnosis of PCOS was made in the presence of at least two of the following criteria in accordance with the Rotterdam Consensus [15]: oligoovulation and/or anovulation, clinical and/or biochemical signs of hyperandrogenism and polycystic ovaries on ultrasound examination (the presence of 12 or more follicles measuring 2–9 mm in diameter and/or ovarian volume [10 cm3). Oligoanovulation was defined as the presence of oligomenorrhea (menstrual cycles of [35 days) or amenorrhea (lack of the menstrual period for 6 months or more). Patients who had diabetes, hyperprolactinemia, congenital adrenal hyperplasia, Cushing’s disease, androgen-secreting tumors, thyroid disorders, hypercholesterolemia or hypertension were not included in the study. Smokers and patients who had received medication (e.g., insulin-sensitizing drugs, oral contraceptives, anti-androgens, statins, and aspirin) within 3 months prior to enrollment were excluded. For each patient, height and weight were measured to calculate BMI. The BMI was calculated as the weight in kilograms divided by the square height in meters. Waist circumference was measured at the narrowest level between the costal margin and the iliac crest, and the hip circumference was measured at the level of the major trochanter with the subject standing and breathing normally. The waist/hip ratio was calculated as the waist circumference divided by the hip circumference. Biochemical analyses and hormone assays For hormonal and biochemical analyses, serum and plasma samples were collected between 8 and 10 am after an overnight fast of at least 12 h during the early follicular phase (2nd to 5th days of the cycle) in subjects with regular menses or on random days in subjects with amenorrhea. Serum levels of follicle stimulating hormone, luteinizing hormone (LH), estradiol, total testosterone, dehydroepiandrosterone sulfate, and insulin were determined by enzyme-linked immunosorbent assay (Cobas 411; Roche Diagnostics, Mannheim, Germany). Glucose, total cholesterol, low-density lipoprotein cholesterol (LDL-C), highdensity lipoprotein cholesterol (HDL-C) and triglycerides were measured by an autoanalyzer using standard enzymatic methods (Cobas 311; Roche Diagnostics). All participants underwent a 75-g, 2-h oral glucose tolerance test after 3 days on a carbohydrate-rich diet. Insulin resistance, defined by the homeostasis model assessment insulin index (HOMA-IR), was calculated with
Arch Gynecol Obstet
the use of the following equation: HOMA-IR = fasting insulin (lU/L) 9 fasting glucose (mmol/L)/22.5 [16]. OPG levels in the serum were quantified by Biovendor enzyme-linked immunosorbent assays. The assay detects both monomer and dimeric forms of OPG, including OPG bound to its ligand. Briefly, mouse anti-human OPG was used as a capture antibody, and a biotinylated polyclonal anti-human OPG antibody was used for detection. The detection limit of this assay was 0.03 pmol/L. Intra- and interassay variabilities for this test were 3.5 and 5.8 % respectively.
Statistical analysis The data were analyzed with SPSS for Windows 11.5. The Kolmogorov–Smirnov test was used to test the normality of distribution for continuous variables, and data were expressed as mean ± standard deviation or median and interquartile range as appropriate. The differences between groups were assessed by using unpaired t tests for parametric data and the Mann–Whitney U test for nonparametric data. Correlations between variables were evaluated with the use of Spearman’s correlation coefficient. Statistical significance was defined as P \ 0.05.
Arterial parameters Carotid intima-media thickness (CIMT) This parameter was derived from noninvasive ultrasonography scans of the carotid arteries with the use of a high-resolution ultrasound machine (Esaote, mylab50CV, 2008, Italy) with a 13.4MHz linear transducer. All women were examined in the supine position, with the head hyperextended and turned away from the side being scanned. The CIMT was defined as the distance between the blood-intima and mediaadventitia boundaries on B-mode imaging [6]. Images for intima-media thickness at each carotid artery were obtained from the distal portion of common carotid arteries, 1 and 5 cm proximal to the carotid bulb and immediately proximal to the origin of the bifurcation. The mean intimamedia thickness for each side was calculated as the average of five measurements obtained in the right and left carotid arteries, and subsequently, the mean value of the right- and left-side intima-media thickness was calculated. Intimamedia thickness measurement was performed by one experienced cardiologist, who was blinded to PCOS status and serum assays, for each patient to avoid inter-observer variability. The results are reported as mean common CIMT. Brachial artery flow-mediated dilatation Brachial artery diameter was measured from B-mode ultrasound images captured on an Esaote mylab50CV machine using a 13.4-MHz linear transducer; an electrocardiogram trace was simultaneously recorded. Longitudinal scanning identified the clearest image of the brachial artery above the elbow. With continuous scanning held for 30 s prior and 4 min after ischemia, induced via a pneumatic tourniquet inflated around the upper arm to 40 mmHg above systolic pressure for 4 min. Vessel diameter was measured during systole and diastole and averaged over five cardiac cycles. FMD was determined from the point of maximal dilation, which can be described as the percentage change from the baseline to 60 s after ischemia [16].
Results The clinical characteristics and biochemical data of the women diagnosed with PCOS and control subjects are shown in Table 1. As expected from appropriate matching, no differences were found between study groups with respect to BMI and age (P = 0.616, P = 0.638, respectively). The mean serum levels of all hormones were comparable between the two groups, except LH, which was significantly higher in the women with PCOS (P = 0.011). Levels of total cholesterol, triglyceride, LDL-C and HDL-C were similar between the two groups (P = 0.664, P = 0.337, P = 0.669, P = 0.321, respectively) (Table 1). There were no differences between the groups with respect to fasting glucose, 2-h glucose, fasting insulin, HbA1c and HOMA-IR (P = 0.499, P = 0.734, P = 0.178, P = 0.440, P = 0.234, respectively). The mean OPG levels were higher in the PCOS group compared with control subjects (11.39 ± 2.29 vs. 10.22 ± 2.25 pmol/L, P = 0.026). The mean carotid intima-media thickness was higher in the PCOS group than the control group (0.52 ± 0.058 vs. 0.45 ± 0.059 mm, P \ 0.01). The mean brachial artery FMD was lower in the PCOS group (0.068 ± 0.022 vs. 0.055 ± 0.029, P = 0.017) (Table 1). Correlations between OPG levels and biochemical and arterial CVD risk factors were investigated. No correlation was found between OPG levels and biochemical parameters in either group (Table 2). OPG levels were not correlated with CIMT or brachial artery FMD in either group (Table 2). Moreover, there was no correlation between OPG levels and determinants of insulin resistance in women with PCOS or controls (Table 2).
Discussion Polycystic ovary syndrome is related to insulin resistance, obesity and dyslipidemia, which are features of metabolic
123
Arch Gynecol Obstet Table 1 Clinical and laboratory characteristics of the subjects
Bold values indicate statistical significance (p \ 0.05) BMI body mass index, WHR waist hip ratio, FSH follicle stimulating hormone, LH luteinizing hormone, E2 estradiol, DHEAS dehydroepiandrosterone sulfate, HDL-C high density lipoprotein cholesterol, LDL-C low density lipoprotein cholesterol, OGTT oral glucose tolerance test, HOMA-IR insulin resistance by homeostatic model assessment, CIMT carotis intima media thickness, FMD flow-mediated dilatation
Age (years)
26.4 ± 4.9
25.8 ± 5.4
23.8 ± 3.5
24.2 ± 3.7
0.616
WHR
0.77 (0.74–0.81)
0.82 (0.77–0.87)
0.005
Fasting glucose (mg/dl)
91.6 ± 8.2
90.2 ± 10.8
0.499
96.4 ± 20.7
98.4 ± 30.0
0.734
HbA1C
5.41 ± 0.36
5.48 ± 0.42
0.440
HOMA-IR
1.49 (0.77–2.75)
1.67 (1.01–1.67)
0.234
HDL-C (mg/dL)
57.34 ± 12.47
54.47 ± 12.71
0.321
LDL-C (mg/dL)
103.99 ± 28.36
106.67 ± 26.59
0.669
Total cholesterol (mg/dL)
180.13 ± 39.7
184.07 ± 40.11
0.664
Triglycerides (mg/dL)
78.7 (64.75–97.31)
88.7 (64.35–126)
0.337
Fasting insulin (lU/mL)
6.18 (3.62–11.02)
7.57 4.67–9.28
0.178
FSH (IU/L)
6.37 ± 1.82
5.99 ± 1.74
0.360
LH (IU/L)
5.28 (3.80–6.97)
7.47 (4.79–13)
0.011
E2 (pmol/L) DHEAS (lmol/L)
4.32 (3.36–5.96) 216.22 ± 9.57
4.37 (3.04–6.85) 213.92 ± 9.95
0.856 0.921
Total testosterone (ng/dL)
0.31 ± 0.12
0.36 ± 0.17
0.581
Prolactin (mIU/L)
229.6 (147–307.1)
348.5 (88–463.4)
0.089
TSH (mIU/ml)
1.94 (1.26–2.69)
1.82 (1.05–2.69)
CIMT (mm)
0.45 ± 0.059
0.52 ± 0.058
FMD (%)
6.8 ± 2.2
5.5 ± 2.9
0.017
Osteoprotogerin (pmol/L)
10.22 ± 2.25
11.39 ± 2.29
0.026
Control P value
r
P value
0.040
0.814
-0.144
0.369
-0.104
0.541
0.026
0.874
HDL-C (mg/dL) LDL-C (mg/dL)
0.111 -0.166
0.512 0.327
0.220 0.200
0.174 0.209
Triglycerides (mg/dL)
-0.013
0.941
-0.038
0.814
0.091
0.592
0.238
0.134
Fasting glucose (mg/dL)
-0.061
0.718
0.139
0.386
HOMA-IR
-0.143
0.407
0.111
0.509
HbA1C
-0.093
0.601
0.096
0.566
Fasting insulin (lU/mL)
-0.145
0.398
0.066
0.695
CIMT (mm)
-0.055
0.746
-0.112
0.487
FMD
-0.080
0.636
-0.005
0.975
BMI body mass index, WHR waist hip ratio, FSH follicle stimulating hormone, LH luteinizing hormone, E2 estradiol, DHEAS dehydroepiandrosterone sulfate, HDL-C high density lipoprotein cholesterol, LDL-C low density lipoprotein cholesterol, OGTT oral glucose tolerance test, HOMA-IR insulin resistance by homeostatic model assessment, CIMT carotis intima media thickness, FMD flow-mediated dilatation
syndrome. However, an increase in the incidence of CVD in women with PCOS remains controversial. Lifelong metabolic dysfunction in women with PCOS makes it
123
0.638
OGTT 120th min glucose (mg/dL)
BMI (kg/m2)
Total cholesterol (mg/dL)
P value
Age (years)
PCOS r
PCOS (n = 37)
BMI (kg/m2)
Table 2 Correlation analysis of measured parameters and osteoprotogerin in subjects with PCOS and controls Osteoprotogerin
Control (n = 41)
0.438
GENERAL GYNECOLOGY
No relationship between osteoprotegerin concentrations and endothelial dysfunction in non-obese women with and without polycystic ovary syndrome Remzi Abali • Nicel Tasdemir • Seref Alpsoy • Ufuk Go¨ker Tasdemir • Savas Guzel • Mehmet Aytac Yuksel • Ilkbal Temel Yuksel • Murat Yilmaz
Received: 4 May 2013 / Accepted: 25 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose To investigate the relationships of osteoprotegerin (OPG) concentrations to brachial artery flow-mediated vasodilation (FMD) and the carotid artery intima media thickness (CIMT) in polycystic ovary syndrome (PCOS). Methods Thirty-seven women with PCOS and 41 controls matched for body mass index (BMI) and age were included in study. The serum OPG concentrations, hormonal and metabolic profiles were measured in women
R. Abali (&) N. Tasdemir Department of Gynecology and Obstetrics, Faculty of Medicine, Namik Kemal University, 100. YilMah. Barbaros Cad, No: 132, Tekirdag, Turkey e-mail: [email protected]; [email protected] S. Alpsoy Department of Cardiology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey U. G. Tasdemir Department of Gynecology and Obstetrics, Tekirdag Hayrabolu State Hospital, Tekirdag, Turkey S. Guzel Department of Biochemistry, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey M. A. Yuksel Department of Gynecology and Obstetrics, Cerrahpasa Faculty of Medicine, ˙Istanbul University, Istanbul, Turkey I. Temel Yuksel Department of Obstetrics and Gynecology, Istanbul Okmeydani Research and Training Hospital, Istanbul, Turkey M. Yilmaz Department of Endocrinology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey
with PCOS and in control group. The CIMT and brachial artery FMD were evaluated in both groups. Results The mean serum concentrations of all hormones were comparable, except LH, which was higher in women with PCOS. Lipid parameters were similar between groups. There were no differences between groups with respect to fasting glucose, 2-h glucose, fasting insulin, HbA1c and HOMA-IR. The mean osteoprotogerin concentrations were higher in PCOS group (11.39 ± 2.29 vs. 10.22 ± 2.25 pmol/L, P = 0.026). The mean CIMT was higher in PCOS group than control group (0.52 ± 0.058 vs. 0.45 ± 0.059 mm, P \ 0.01). The mean brachial artery FMD was lower in PCOS group (0.068 ± 0.022 vs. 0.055 ± 0.029, P = 0.017). Conclusions We found high osteoprotogerin concentrations, increased CIMT and decreased FMD, in women with PCOS. However, there was no correlation between osteoprotegerin and cardiovascular risk markers. Keywords Polycystic Ovary syndrome Osteoprotegerin Carotid intima-media thickness FLOWmediated dilatation Cardiovascular disease
Introduction Polycystic ovary syndrome (PCOS) affects nearly 5–8 % of women of reproductive age [1]. It is characterized by chronic ovulatory dysfunction, clinical and/or biochemical hyperandrogenism and polycystic ovarian morphology. PCOS is associated with major risk factors for cardiovascular events, such as insulin resistance, hypertension and dyslipidemia [2]. However, an increase in cardiovascular morbidity and mortality has not been shown by population studies [3].
123
Arch Gynecol Obstet
Carotid artery intima-media thickness (CIMT) is a marker of early atherosclerosis. It can be measured relatively simply and noninvasively. CIMT has been reported as a predictor of subsequent cardiovascular events independent of traditional risk factors and is being used for risk stratification with other established risk factors for future cardiovascular diseases (CVDs) [4]. Endothelial dysfunction is associated with various forms of CVDs, such as hypertension, coronary artery disease, chronic heart failure and peripheral artery disease. It is characterized by reduced vasodilator responses of the endothelium due to reduced nitric oxide generation, oxidative stress and reduced production of hyperpolarizing factor (endothelium-derived hyperpolarizing factor). The endothelial dysfunction is related with poor prognosis for cardiovascular events, and improvement of endothelial dysfunction may reduce cardiovascular risk [5]. Postischemic flow-mediated dilatation (FMD) of the brachial artery is one of the classic techniques used to evaluate endothelial function. Age is a major determinant of arterial parameters; with increasing age, arterial compliance decreases [6]. Various genetic and environmental factors, including food intake, may influence FMD [7]. Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily and its expression has been demonstrated in human normal vascular smooth muscle, endothelial cells and atherosclerotic lesions [8, 9]. OPG inhibits osteoclastic bone resorption, and older studies have shown that OPG concentration increases with advancing age and reflects the severity of osteoporosis [10]. Increased OPG levels have also been found in women who were using hormone replacement therapy [11]. Several clinical studies in various populations have reported that elevated OPG levels are associated with subclinical atherosclerosis [12] and overall cardiovascular morbidity and mortality [11]. OPG may promote atherogenesis by inducing endothelial inflammation [13] and stimulating proliferation of endothelial and vascular smooth muscle cells [14]. We designed a cross-sectional study to investigate OPG levels, endothelial dysfunction and carotid artery intima media thickness in women with PCOS.
Materials and methods Thirty-seven women with PCOS and 41 controls matched for body mass index (BMI) and age were included in this study. The study was conducted in the gynecology clinic of Namik Kemal University School of Medicine (Tekirdag, Turkey) between November 2010 and June 2012. The study was approved by the local Ethics Committee of
123
Namik Kemal University. Written informed consent was obtained from all participants. The control group included 41 individuals selected from women who did not show any clinical and ultrasonography signs of PCOS. Diagnosis of PCOS was made in the presence of at least two of the following criteria in accordance with the Rotterdam Consensus [15]: oligoovulation and/or anovulation, clinical and/or biochemical signs of hyperandrogenism and polycystic ovaries on ultrasound examination (the presence of 12 or more follicles measuring 2–9 mm in diameter and/or ovarian volume [10 cm3). Oligoanovulation was defined as the presence of oligomenorrhea (menstrual cycles of [35 days) or amenorrhea (lack of the menstrual period for 6 months or more). Patients who had diabetes, hyperprolactinemia, congenital adrenal hyperplasia, Cushing’s disease, androgen-secreting tumors, thyroid disorders, hypercholesterolemia or hypertension were not included in the study. Smokers and patients who had received medication (e.g., insulin-sensitizing drugs, oral contraceptives, anti-androgens, statins, and aspirin) within 3 months prior to enrollment were excluded. For each patient, height and weight were measured to calculate BMI. The BMI was calculated as the weight in kilograms divided by the square height in meters. Waist circumference was measured at the narrowest level between the costal margin and the iliac crest, and the hip circumference was measured at the level of the major trochanter with the subject standing and breathing normally. The waist/hip ratio was calculated as the waist circumference divided by the hip circumference. Biochemical analyses and hormone assays For hormonal and biochemical analyses, serum and plasma samples were collected between 8 and 10 am after an overnight fast of at least 12 h during the early follicular phase (2nd to 5th days of the cycle) in subjects with regular menses or on random days in subjects with amenorrhea. Serum levels of follicle stimulating hormone, luteinizing hormone (LH), estradiol, total testosterone, dehydroepiandrosterone sulfate, and insulin were determined by enzyme-linked immunosorbent assay (Cobas 411; Roche Diagnostics, Mannheim, Germany). Glucose, total cholesterol, low-density lipoprotein cholesterol (LDL-C), highdensity lipoprotein cholesterol (HDL-C) and triglycerides were measured by an autoanalyzer using standard enzymatic methods (Cobas 311; Roche Diagnostics). All participants underwent a 75-g, 2-h oral glucose tolerance test after 3 days on a carbohydrate-rich diet. Insulin resistance, defined by the homeostasis model assessment insulin index (HOMA-IR), was calculated with
Arch Gynecol Obstet
the use of the following equation: HOMA-IR = fasting insulin (lU/L) 9 fasting glucose (mmol/L)/22.5 [16]. OPG levels in the serum were quantified by Biovendor enzyme-linked immunosorbent assays. The assay detects both monomer and dimeric forms of OPG, including OPG bound to its ligand. Briefly, mouse anti-human OPG was used as a capture antibody, and a biotinylated polyclonal anti-human OPG antibody was used for detection. The detection limit of this assay was 0.03 pmol/L. Intra- and interassay variabilities for this test were 3.5 and 5.8 % respectively.
Statistical analysis The data were analyzed with SPSS for Windows 11.5. The Kolmogorov–Smirnov test was used to test the normality of distribution for continuous variables, and data were expressed as mean ± standard deviation or median and interquartile range as appropriate. The differences between groups were assessed by using unpaired t tests for parametric data and the Mann–Whitney U test for nonparametric data. Correlations between variables were evaluated with the use of Spearman’s correlation coefficient. Statistical significance was defined as P \ 0.05.
Arterial parameters Carotid intima-media thickness (CIMT) This parameter was derived from noninvasive ultrasonography scans of the carotid arteries with the use of a high-resolution ultrasound machine (Esaote, mylab50CV, 2008, Italy) with a 13.4MHz linear transducer. All women were examined in the supine position, with the head hyperextended and turned away from the side being scanned. The CIMT was defined as the distance between the blood-intima and mediaadventitia boundaries on B-mode imaging [6]. Images for intima-media thickness at each carotid artery were obtained from the distal portion of common carotid arteries, 1 and 5 cm proximal to the carotid bulb and immediately proximal to the origin of the bifurcation. The mean intimamedia thickness for each side was calculated as the average of five measurements obtained in the right and left carotid arteries, and subsequently, the mean value of the right- and left-side intima-media thickness was calculated. Intimamedia thickness measurement was performed by one experienced cardiologist, who was blinded to PCOS status and serum assays, for each patient to avoid inter-observer variability. The results are reported as mean common CIMT. Brachial artery flow-mediated dilatation Brachial artery diameter was measured from B-mode ultrasound images captured on an Esaote mylab50CV machine using a 13.4-MHz linear transducer; an electrocardiogram trace was simultaneously recorded. Longitudinal scanning identified the clearest image of the brachial artery above the elbow. With continuous scanning held for 30 s prior and 4 min after ischemia, induced via a pneumatic tourniquet inflated around the upper arm to 40 mmHg above systolic pressure for 4 min. Vessel diameter was measured during systole and diastole and averaged over five cardiac cycles. FMD was determined from the point of maximal dilation, which can be described as the percentage change from the baseline to 60 s after ischemia [16].
Results The clinical characteristics and biochemical data of the women diagnosed with PCOS and control subjects are shown in Table 1. As expected from appropriate matching, no differences were found between study groups with respect to BMI and age (P = 0.616, P = 0.638, respectively). The mean serum levels of all hormones were comparable between the two groups, except LH, which was significantly higher in the women with PCOS (P = 0.011). Levels of total cholesterol, triglyceride, LDL-C and HDL-C were similar between the two groups (P = 0.664, P = 0.337, P = 0.669, P = 0.321, respectively) (Table 1). There were no differences between the groups with respect to fasting glucose, 2-h glucose, fasting insulin, HbA1c and HOMA-IR (P = 0.499, P = 0.734, P = 0.178, P = 0.440, P = 0.234, respectively). The mean OPG levels were higher in the PCOS group compared with control subjects (11.39 ± 2.29 vs. 10.22 ± 2.25 pmol/L, P = 0.026). The mean carotid intima-media thickness was higher in the PCOS group than the control group (0.52 ± 0.058 vs. 0.45 ± 0.059 mm, P \ 0.01). The mean brachial artery FMD was lower in the PCOS group (0.068 ± 0.022 vs. 0.055 ± 0.029, P = 0.017) (Table 1). Correlations between OPG levels and biochemical and arterial CVD risk factors were investigated. No correlation was found between OPG levels and biochemical parameters in either group (Table 2). OPG levels were not correlated with CIMT or brachial artery FMD in either group (Table 2). Moreover, there was no correlation between OPG levels and determinants of insulin resistance in women with PCOS or controls (Table 2).
Discussion Polycystic ovary syndrome is related to insulin resistance, obesity and dyslipidemia, which are features of metabolic
123
Arch Gynecol Obstet Table 1 Clinical and laboratory characteristics of the subjects
Bold values indicate statistical significance (p \ 0.05) BMI body mass index, WHR waist hip ratio, FSH follicle stimulating hormone, LH luteinizing hormone, E2 estradiol, DHEAS dehydroepiandrosterone sulfate, HDL-C high density lipoprotein cholesterol, LDL-C low density lipoprotein cholesterol, OGTT oral glucose tolerance test, HOMA-IR insulin resistance by homeostatic model assessment, CIMT carotis intima media thickness, FMD flow-mediated dilatation
Age (years)
26.4 ± 4.9
25.8 ± 5.4
23.8 ± 3.5
24.2 ± 3.7
0.616
WHR
0.77 (0.74–0.81)
0.82 (0.77–0.87)
0.005
Fasting glucose (mg/dl)
91.6 ± 8.2
90.2 ± 10.8
0.499
96.4 ± 20.7
98.4 ± 30.0
0.734
HbA1C
5.41 ± 0.36
5.48 ± 0.42
0.440
HOMA-IR
1.49 (0.77–2.75)
1.67 (1.01–1.67)
0.234
HDL-C (mg/dL)
57.34 ± 12.47
54.47 ± 12.71
0.321
LDL-C (mg/dL)
103.99 ± 28.36
106.67 ± 26.59
0.669
Total cholesterol (mg/dL)
180.13 ± 39.7
184.07 ± 40.11
0.664
Triglycerides (mg/dL)
78.7 (64.75–97.31)
88.7 (64.35–126)
0.337
Fasting insulin (lU/mL)
6.18 (3.62–11.02)
7.57 4.67–9.28
0.178
FSH (IU/L)
6.37 ± 1.82
5.99 ± 1.74
0.360
LH (IU/L)
5.28 (3.80–6.97)
7.47 (4.79–13)
0.011
E2 (pmol/L) DHEAS (lmol/L)
4.32 (3.36–5.96) 216.22 ± 9.57
4.37 (3.04–6.85) 213.92 ± 9.95
0.856 0.921
Total testosterone (ng/dL)
0.31 ± 0.12
0.36 ± 0.17
0.581
Prolactin (mIU/L)
229.6 (147–307.1)
348.5 (88–463.4)
0.089
TSH (mIU/ml)
1.94 (1.26–2.69)
1.82 (1.05–2.69)
CIMT (mm)
0.45 ± 0.059
0.52 ± 0.058
FMD (%)
6.8 ± 2.2
5.5 ± 2.9
0.017
Osteoprotogerin (pmol/L)
10.22 ± 2.25
11.39 ± 2.29
0.026
Control P value
r
P value
0.040
0.814
-0.144
0.369
-0.104
0.541
0.026
0.874
HDL-C (mg/dL) LDL-C (mg/dL)
0.111 -0.166
0.512 0.327
0.220 0.200
0.174 0.209
Triglycerides (mg/dL)
-0.013
0.941
-0.038
0.814
0.091
0.592
0.238
0.134
Fasting glucose (mg/dL)
-0.061
0.718
0.139
0.386
HOMA-IR
-0.143
0.407
0.111
0.509
HbA1C
-0.093
0.601
0.096
0.566
Fasting insulin (lU/mL)
-0.145
0.398
0.066
0.695
CIMT (mm)
-0.055
0.746
-0.112
0.487
FMD
-0.080
0.636
-0.005
0.975
BMI body mass index, WHR waist hip ratio, FSH follicle stimulating hormone, LH luteinizing hormone, E2 estradiol, DHEAS dehydroepiandrosterone sulfate, HDL-C high density lipoprotein cholesterol, LDL-C low density lipoprotein cholesterol, OGTT oral glucose tolerance test, HOMA-IR insulin resistance by homeostatic model assessment, CIMT carotis intima media thickness, FMD flow-mediated dilatation
syndrome. However, an increase in the incidence of CVD in women with PCOS remains controversial. Lifelong metabolic dysfunction in women with PCOS makes it
123
0.638
OGTT 120th min glucose (mg/dL)
BMI (kg/m2)
Total cholesterol (mg/dL)
P value
Age (years)
PCOS r
PCOS (n = 37)
BMI (kg/m2)
Table 2 Correlation analysis of measured parameters and osteoprotogerin in subjects with PCOS and controls Osteoprotogerin
Control (n = 41)
0.438
