Cytokine xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome Mirelle O. Sóter a, Cláudia N. Ferreira b, Mariana F. Sales c, Ana L. Candido d, Fernando M. Reis d, Kátia S. Milagres e, Carla Ronda e, Ieda O. Silva a, Marinez O. Sousa a, Karina B. Gomes a,c,⇑ a
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Colégio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil d Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil e Simile, Laboratório de Imunologia, Belo Horizonte, Minas Gerais, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 February 2015 Received in revised form 1 June 2015 Accepted 15 June 2015 Available online xxxx Keywords: Polycystic ovary syndrome Cytokine Gene Polymorphisms Metabolism
a b s t r a c t Background: The imbalance between proinflammatory and anti-inflammatory pathways plays a role in polycystic ovary syndrome (PCOS) etiology. We aimed to investigate the relationship between polymorphisms of genes encoding inflammation-associated cytokines and the metabolic profile of Brazilian women with PCOS. Design: Case-control study. Methods: The study included 196 women – 97 with PCOS (diagnosed based on Rotterdam criteria, 2003) and 99 age-matched, healthy women (controls). It was investigated polymorphisms in cytokines genes from peripheral blood-derived DNA by using PCR. Results: The frequencies of alleles, genotypes, and phenotypes were similar between women with PCOS and controls. The GG genotype of the 179C/G polymorphism (IL6) was associated with higher glucose levels, while the GA and AA genotypes of the 1082A/G polymorphism (IL10), CT and TT genotypes of the 819A/T polymorphism (IL10), CA and AA genotypes of the 522A/G (IL10) polymorphism, and TA genotype of the +874T/A polymorphism (IFN-c) were associated with lower total cholesterol and triglycerides levels. The GA genotype of the 1082A/G polymorphism (IL10) and the CC genotype of the 10T/C polymorphism (TGF-b1) were associated with lower and higher Ferriman indices, respectively, in women with PCOS. The AA genotype of the 1082A/G polymorphism (IL10) was associated with lower glucose levels, while the TC genotype of the 10T/C polymorphism (TGF-b1) was associated with a lower lipid accumulation product index and higher high-density lipoprotein cholesterol levels in the PCOS group. Conclusions: The genetic polymorphisms of cytokines are not associated with PCOS development, but may contribute to common metabolic disorders associated with PCOS. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder in reproductive age with a prevalence rate of 5– 10% [1–4]. This syndrome is characterized by clinical and/or biochemical hyperandrogenism and menstrual irregularities associated with infertility [1,5]. The etiology of PCOS remains unknown [1,6–8]. However, several biochemical abnormalities have been described, including the primary defect of insulin resistance in ⇑ Corresponding author at: Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Avenida Antonio Carlos, Belo Horizonte, Minas Gerais, Brazil. E-mail address: [email protected] (K.B. Gomes).
muscle and adipose tissues with compensatory hyperinsulinemia associated with intrinsic b-cell dysfunction [7,9,10]. In PCOS, oxidative stress is involved on the development of b-cell dysfunction, which is linked to hyperglycemia-induced mononuclear cell (MNC) activation of NFjB pathway and systemic inflammation. This inflammation may play a role in impairing insulin secretion prior to developing overt hyperglycemia [11]. A relationship among inflammation, hormonal, and metabolic alterations in women with PCOS has been reported. Increased concentrations of C-reactive protein (CRP), a marker of low-grade inflammation and a predictor of coronary heart disease, as well as an increase white cell count are commonly observed among women with PCOS [12–14]. Moreover, the systemic low-grade inflammation observed in patients with PCOS is also characterized
http://dx.doi.org/10.1016/j.cyto.2015.06.008 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sóter MO et al. Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.008
2
M.O. Sóter et al. / Cytokine xxx (2015) xxx–xxx
by elevated interleukin-18 levels, plasminogen activator inhibitor-1 levels, endothelial dysfunction, and oxidative stress [11,15,16]. New evidence suggests that the immune system exacerbates clinical features and can be associated with other important aspects of PCOS, including insulin resistance and cardiovascular disease [17,18]. Indeed, inflammation is considered a hallmark of endothelial dysfunction and atherosclerosis [19], which contributes to an increased risk of heart diseases in PCOS [15]. PCOS is often associated with excess abdominal adiposity (AA) with or without obesity. Circulating leukocytes of normal weight women with PCOS are pre activated during the fasting state and glucose ingestion in this population promotes an increased leukocytic reactive oxygen species generation and altered TNF-a release [20–22]. The association of AA with markers of oxidative stress and CRP suggest that the AA excess is an additional source of oxidative stress in normal-weight women with PCOS. Women with PCOS fail to suppress mononuclear cell-derived cytokine release in response to glucose ingestion, and this response is independent of excess adiposity. Nevertheless, circulating mononuclear cell and adipose tissue excess are separate in addition distinct sources of inflammation in this population [23–25]. It is not clear whether inflammatory cytokines are elevated in women with PCOS due to an intrinsic perturbation of the inflammatory pathway or if the systemic proinflammatory state is resulted from obesity and/or insulin resistance. An approach to differentiate between parameters that are intercorrelated and which are related to causality ought to be investigated whether genetic variations in these pathways are associated with the outcome of interest. Given the potentially confound impact that obesity and insulin resistance may have on the inflammatory response in PCOS, genetic tests are necessary in order to determine whether the increased inflammatory state observed in PCOS is an intrinsic feature of the syndrome or simply a reflection of the systemic proinflammatory state due to obesity and insulin resistance [26]. Studies have shown that genetic polymorphisms, especially single nucleotide polymorphisms (SNPs), are associated with clinical features [27] and the development of PCOS [28–30]. However, despite progress in elucidating the genetic mechanisms associated with this endocrine disorder, there are still many challenges posed by the multigenic nature of the syndrome, the complex network of molecular mediators of systemic inflammation, and the different distribution of genetic polymorphisms according to the background of each population. The purpose of this study was to investigate the relationship between polymorphisms of genes encoding cytokines associated with inflammation and the metabolic profile of Brazilian women with PCOS. In particular, we investigated the frequencies of SNPs in the genes for tumor necrosis factor a (TNF-a), interleukin-6 (IL6), interleukin-10 (IL10), interferon c (IFN-c) and transforming growth factor b (TGF-b1) in this women group.
2. Subjects and methods 2.1. Subjects Written informed consent was obtained from all participants prior to inclusion in the study. The local Ethics Committee (COEP) of the Federal University of Minas Gerais approved the study (CAAE 0379.0.203.000-11). This case-control study evaluated 97 women with PCOS (aged from 20 to 44 years) and 99 healthy women without the syndrome (18–45 years). The PCOS group was recruited at Hospital Borges da Costa, Universidade Federal de Minas Gerais (UFMG), Brazil, in the
period 2011–2013. The control group was recruited among employees and students from UFMG in the same period. The PCOS diagnosis was performed according to European Society of Human Reproduction/Embryology and the American Society for Reproductive Medicine criteria (ESHRE/ASRM) [2], considering the presence of at least two of three criteria: (1) oligo/amenorrhea and anovulation (oligomenorrhea – the absence of menstruation for 3 menstrual periods and amenorrhea – the absence at least 6 menstrual periods); (2) clinical or laboratory hyperandrogenism (considering hirsutism and testosterone levels) and (3) ultrasonography showing micropolycystic ovaries. The presence of 12 or more follicles in the ovary each measuring 9– 2 mm in diameter and/or increased ovarian volume (>10 mL) were considered micropolycystic ovarian. All control subjects presented ovulatory cycles evidenced by regular menses lasting 25–35 days and a luteal phase serum progesterone level greater than 5 ng/mL. All control subjects exhibited normal circulating androgen levels, absence of skin manifestation caused by the androgen excess and polycystic ovaries on ultrasound. No especial diet was recommended for patients or controls. None of them exercised during the 24 h before the study participation. Exclusion criteria for both groups included the presence of diabetes mellitus, autoimmune disease, adrenal disease, kidney and liver disease, thyroid disorders, cancer or tumors, acute inflammatory disease, hyperprolactinemia, hypogonadism, and pregnancy. People treated with the following medications were also excluded: steroidal and non-steroidal anti-inflammatory medications, isotretinoin, cyclosporine, antiretroviral, insulin, metformin, and oral contraceptives. 2.2. Blood sampling Venous blood samples were obtained after fasting for 12 h using tubes with and without EDTA/heparin (VacuetteÒ) anticoagulants. In PCOS patients, a second blood sample was collected 2 h after 75 g oral glucose load. The samples were centrifuged at 2500g for 20 min at 4 °C to obtain the plasma or serum. Aliquots were stored at 80 °C until the use. Whole blood samples were used for genotyping. 2.3. Measurements Insulin, total testosterone, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and 17-OH-progesterone were measured by using ARCHITECTÒ immunoassays (Abbott, Chicago, IL, USA) according to the manufacturer’s instructions. Glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), very low-density lipoprotein cholesterol (VLDLc), triglycerides (TG) and CRP were determined using Vitros system (Johnson & Johnson, New Brunswick, NJ, USA). Height was measured using a rigid stadiometer. Weight was measured using a calibrated balance scale. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. The lipid accumulation product (LAP) index was calculated by using the following formula: LAP = [waist circumference (cm) 58] TG (mmol/L) [31]. Hypertension was defined as systolic blood pressure P140 mmHg and/or diastolic blood pressure P90 mmHg at the time of interview or a documented history of high blood pressure in individuals that were on regular use of antihypertensive medication. Those individuals who did not practice physical exercises regularly for at least 40 min, three times a week were considered sedentary. Smokers were individuals with regular consumption of any quantity of cigarettes, for a period exceeding 6 months until the month prior to the completion of the clinical record, and
Please cite this article in press as: Sóter MO et al. Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.008
3
M.O. Sóter et al. / Cytokine xxx (2015) xxx–xxx
alcoholism was defined by the consumption of more than 30 mL of ethanol per day. The Ferriman–Gallwey index was used in order to assess hirsutism. The density of terminal hair on the upper lip, chin, chest, upper back, lower back, upper abdomen, lower abdomen, arms and thighs were visually rated on a scale of zero to four, where zero indicates no growth of terminal hair and four indicates strong growth in male pattern hair. A total score was calculated. A single observer scored all subjects to avoid bias in rating. HOMA-IR (homeostatic model assessment for insulin resistance) was calculated using the formula: HOMA-IR = [insulin (mU/L) glucose (mM/L)]/22.5 [32]. The molecular analyses using specific primers were performed using the multiplex PCR System kit (Cytokine genotyping Tray – One LambdaÒ), followed by agarose gel electrophoresis. The cytokine genes polymorphisms evaluated were 308A/G (rs 1800629) for TNF-a; 174C/G (rs 1800795) for IL6; 1082A/G (rs 1800896), 819T/C (rs 1800871), 592C/A (rs 1800872) for IL10; +874T/A (rs 2430561) for IFN-c; and 10T/C (rs 1800470), 25C/G (rs 1800471) for TGF-b1. Phenotype was defined as the relationship between the polymorphism and the genetic expression level [33–36]. 2.4. Statistics Statistical analyses were performed using SPSS version 13.0. The Kolmogorov–Smirnov test was used to determine the normality of the variables. For normal continuous variables, the mean and standard deviation were calculated, and those that did not follow normality were expressed as median values and interquartile ranges. For data that were normally distributed, we used the Student t-test or ANOVA (with LSD post-hoc test) and for non-parametric variables, we used the Mann–Whitney test or Kruskall Wallis test to compare two or three groups, respectively. Categorical variables are presented as absolute and relative frequencies, and the chi-square test was used to compare the frequencies and the Hardy–Weinberg equilibrium. Spearman or Pearson correlation test was applied to normally or non-normally distributed variables. P < 0.05 was considered significant. The sample size was estimated to allow the detection of differences of at least 20% in the proportion of each genotype between the case and the control groups, with statistical power of 80% and confidence interval (CI) of 95%. 3. Results The PCOS and control groups were matched for age. Hypertension frequency, smoking, physical inactivity and alcohol consumption did not differ between the groups (Table 1). Fasting glucose and CRP were not different between the groups; however, BMI, abdominal circumference, LAP index, HOMA-IR index, insulin, lipids, testosterone, and FSH levels were higher, and HDL-C was lower in PCOS when compared to controls (Table 1). The genotype distribution of the polymorphisms studied was in Hardy–Weinberg equilibrium (P > 0.05) for both groups. The frequencies of alleles, genotypes, and phenotypes were similar between PCOS and controls (Supplemental Tables 1–3). A multivariate logistic regression analysis was conducted and the cytokine gene polymorphisms frequency was independent of BMI in both groups (P > 0.05 for all the polymorphisms, data not shown). In the PCOS group, the TNF-a 308A/G polymorphism and the TGF-b1 25C/G polymorphism did not influence the biochemical parameters (all P > 0.05, data not shown). In women with PCOS, glucose levels were higher in those with the GG genotype of the IL6 174C/G polymorphism (Table 2). The categorized genotypes showed correlation with glucose levels (r = 0.313; P = 0.002). No
Table 1 Characterization of women with PCOS and control group considering anthropometric, biochemical and risk factors for cardiovascular diseases.
Age (years) BMI (kg/m2) Abdominal circumference (cm) Smoker Sedentarism Alcohol use Hypertension Glucose (mmol/L) HOMA-IR Insulin (uUI/mL) TC (mmol/L) LAP HDLc (mmol/L) LDLc (mmol/L) VLDLc (mmol/L) TG (mmol/L) Testosterone (ng/dL) FSH (mUI/mL) CRP (mg/dL)
PCOS (N = 97)
Control (N = 99)
P
30.6 ± 4.9 28.7 (7.8) 97.0 (17.5) 9 (9%) 63 (65%) 17 (18%) 4 (4%) 4.8 ± 9.1 2.7 (3.4) 12.3 (17.4) 4.9 ± 35.1 46.8 (51.2) 1.2 (19) 3.5 ± 45 0.24 (18) 1.2 (89) 53.3 (47.2) 5.5 (2.1) 5.0 (9)
29.4 ± 7.7 23.1 (5.4) 80.5 (19.5) 5 (5%) 65 (66%) 29 (29%) 1 (1%) 4.7 ± 8.2 1.6 (0.8) 7.7 (3.4) 4.5 ± 33.1 20.25 (18.3) 1.4 (19) 2.9 ± 38 0.18 (8) 0.9 (39) 27.7 (19.5) 4.5 (3.3) 4.0 (2)
0.214
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome Mirelle O. Sóter a, Cláudia N. Ferreira b, Mariana F. Sales c, Ana L. Candido d, Fernando M. Reis d, Kátia S. Milagres e, Carla Ronda e, Ieda O. Silva a, Marinez O. Sousa a, Karina B. Gomes a,c,⇑ a
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Colégio Técnico, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil d Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil e Simile, Laboratório de Imunologia, Belo Horizonte, Minas Gerais, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 February 2015 Received in revised form 1 June 2015 Accepted 15 June 2015 Available online xxxx Keywords: Polycystic ovary syndrome Cytokine Gene Polymorphisms Metabolism
a b s t r a c t Background: The imbalance between proinflammatory and anti-inflammatory pathways plays a role in polycystic ovary syndrome (PCOS) etiology. We aimed to investigate the relationship between polymorphisms of genes encoding inflammation-associated cytokines and the metabolic profile of Brazilian women with PCOS. Design: Case-control study. Methods: The study included 196 women – 97 with PCOS (diagnosed based on Rotterdam criteria, 2003) and 99 age-matched, healthy women (controls). It was investigated polymorphisms in cytokines genes from peripheral blood-derived DNA by using PCR. Results: The frequencies of alleles, genotypes, and phenotypes were similar between women with PCOS and controls. The GG genotype of the 179C/G polymorphism (IL6) was associated with higher glucose levels, while the GA and AA genotypes of the 1082A/G polymorphism (IL10), CT and TT genotypes of the 819A/T polymorphism (IL10), CA and AA genotypes of the 522A/G (IL10) polymorphism, and TA genotype of the +874T/A polymorphism (IFN-c) were associated with lower total cholesterol and triglycerides levels. The GA genotype of the 1082A/G polymorphism (IL10) and the CC genotype of the 10T/C polymorphism (TGF-b1) were associated with lower and higher Ferriman indices, respectively, in women with PCOS. The AA genotype of the 1082A/G polymorphism (IL10) was associated with lower glucose levels, while the TC genotype of the 10T/C polymorphism (TGF-b1) was associated with a lower lipid accumulation product index and higher high-density lipoprotein cholesterol levels in the PCOS group. Conclusions: The genetic polymorphisms of cytokines are not associated with PCOS development, but may contribute to common metabolic disorders associated with PCOS. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder in reproductive age with a prevalence rate of 5– 10% [1–4]. This syndrome is characterized by clinical and/or biochemical hyperandrogenism and menstrual irregularities associated with infertility [1,5]. The etiology of PCOS remains unknown [1,6–8]. However, several biochemical abnormalities have been described, including the primary defect of insulin resistance in ⇑ Corresponding author at: Department of Clinical and Toxicological Analysis, Faculty of Pharmacy, Federal University of Minas Gerais, Avenida Antonio Carlos, Belo Horizonte, Minas Gerais, Brazil. E-mail address: [email protected] (K.B. Gomes).
muscle and adipose tissues with compensatory hyperinsulinemia associated with intrinsic b-cell dysfunction [7,9,10]. In PCOS, oxidative stress is involved on the development of b-cell dysfunction, which is linked to hyperglycemia-induced mononuclear cell (MNC) activation of NFjB pathway and systemic inflammation. This inflammation may play a role in impairing insulin secretion prior to developing overt hyperglycemia [11]. A relationship among inflammation, hormonal, and metabolic alterations in women with PCOS has been reported. Increased concentrations of C-reactive protein (CRP), a marker of low-grade inflammation and a predictor of coronary heart disease, as well as an increase white cell count are commonly observed among women with PCOS [12–14]. Moreover, the systemic low-grade inflammation observed in patients with PCOS is also characterized
http://dx.doi.org/10.1016/j.cyto.2015.06.008 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sóter MO et al. Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.008
2
M.O. Sóter et al. / Cytokine xxx (2015) xxx–xxx
by elevated interleukin-18 levels, plasminogen activator inhibitor-1 levels, endothelial dysfunction, and oxidative stress [11,15,16]. New evidence suggests that the immune system exacerbates clinical features and can be associated with other important aspects of PCOS, including insulin resistance and cardiovascular disease [17,18]. Indeed, inflammation is considered a hallmark of endothelial dysfunction and atherosclerosis [19], which contributes to an increased risk of heart diseases in PCOS [15]. PCOS is often associated with excess abdominal adiposity (AA) with or without obesity. Circulating leukocytes of normal weight women with PCOS are pre activated during the fasting state and glucose ingestion in this population promotes an increased leukocytic reactive oxygen species generation and altered TNF-a release [20–22]. The association of AA with markers of oxidative stress and CRP suggest that the AA excess is an additional source of oxidative stress in normal-weight women with PCOS. Women with PCOS fail to suppress mononuclear cell-derived cytokine release in response to glucose ingestion, and this response is independent of excess adiposity. Nevertheless, circulating mononuclear cell and adipose tissue excess are separate in addition distinct sources of inflammation in this population [23–25]. It is not clear whether inflammatory cytokines are elevated in women with PCOS due to an intrinsic perturbation of the inflammatory pathway or if the systemic proinflammatory state is resulted from obesity and/or insulin resistance. An approach to differentiate between parameters that are intercorrelated and which are related to causality ought to be investigated whether genetic variations in these pathways are associated with the outcome of interest. Given the potentially confound impact that obesity and insulin resistance may have on the inflammatory response in PCOS, genetic tests are necessary in order to determine whether the increased inflammatory state observed in PCOS is an intrinsic feature of the syndrome or simply a reflection of the systemic proinflammatory state due to obesity and insulin resistance [26]. Studies have shown that genetic polymorphisms, especially single nucleotide polymorphisms (SNPs), are associated with clinical features [27] and the development of PCOS [28–30]. However, despite progress in elucidating the genetic mechanisms associated with this endocrine disorder, there are still many challenges posed by the multigenic nature of the syndrome, the complex network of molecular mediators of systemic inflammation, and the different distribution of genetic polymorphisms according to the background of each population. The purpose of this study was to investigate the relationship between polymorphisms of genes encoding cytokines associated with inflammation and the metabolic profile of Brazilian women with PCOS. In particular, we investigated the frequencies of SNPs in the genes for tumor necrosis factor a (TNF-a), interleukin-6 (IL6), interleukin-10 (IL10), interferon c (IFN-c) and transforming growth factor b (TGF-b1) in this women group.
2. Subjects and methods 2.1. Subjects Written informed consent was obtained from all participants prior to inclusion in the study. The local Ethics Committee (COEP) of the Federal University of Minas Gerais approved the study (CAAE 0379.0.203.000-11). This case-control study evaluated 97 women with PCOS (aged from 20 to 44 years) and 99 healthy women without the syndrome (18–45 years). The PCOS group was recruited at Hospital Borges da Costa, Universidade Federal de Minas Gerais (UFMG), Brazil, in the
period 2011–2013. The control group was recruited among employees and students from UFMG in the same period. The PCOS diagnosis was performed according to European Society of Human Reproduction/Embryology and the American Society for Reproductive Medicine criteria (ESHRE/ASRM) [2], considering the presence of at least two of three criteria: (1) oligo/amenorrhea and anovulation (oligomenorrhea – the absence of menstruation for 3 menstrual periods and amenorrhea – the absence at least 6 menstrual periods); (2) clinical or laboratory hyperandrogenism (considering hirsutism and testosterone levels) and (3) ultrasonography showing micropolycystic ovaries. The presence of 12 or more follicles in the ovary each measuring 9– 2 mm in diameter and/or increased ovarian volume (>10 mL) were considered micropolycystic ovarian. All control subjects presented ovulatory cycles evidenced by regular menses lasting 25–35 days and a luteal phase serum progesterone level greater than 5 ng/mL. All control subjects exhibited normal circulating androgen levels, absence of skin manifestation caused by the androgen excess and polycystic ovaries on ultrasound. No especial diet was recommended for patients or controls. None of them exercised during the 24 h before the study participation. Exclusion criteria for both groups included the presence of diabetes mellitus, autoimmune disease, adrenal disease, kidney and liver disease, thyroid disorders, cancer or tumors, acute inflammatory disease, hyperprolactinemia, hypogonadism, and pregnancy. People treated with the following medications were also excluded: steroidal and non-steroidal anti-inflammatory medications, isotretinoin, cyclosporine, antiretroviral, insulin, metformin, and oral contraceptives. 2.2. Blood sampling Venous blood samples were obtained after fasting for 12 h using tubes with and without EDTA/heparin (VacuetteÒ) anticoagulants. In PCOS patients, a second blood sample was collected 2 h after 75 g oral glucose load. The samples were centrifuged at 2500g for 20 min at 4 °C to obtain the plasma or serum. Aliquots were stored at 80 °C until the use. Whole blood samples were used for genotyping. 2.3. Measurements Insulin, total testosterone, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and 17-OH-progesterone were measured by using ARCHITECTÒ immunoassays (Abbott, Chicago, IL, USA) according to the manufacturer’s instructions. Glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), very low-density lipoprotein cholesterol (VLDLc), triglycerides (TG) and CRP were determined using Vitros system (Johnson & Johnson, New Brunswick, NJ, USA). Height was measured using a rigid stadiometer. Weight was measured using a calibrated balance scale. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. The lipid accumulation product (LAP) index was calculated by using the following formula: LAP = [waist circumference (cm) 58] TG (mmol/L) [31]. Hypertension was defined as systolic blood pressure P140 mmHg and/or diastolic blood pressure P90 mmHg at the time of interview or a documented history of high blood pressure in individuals that were on regular use of antihypertensive medication. Those individuals who did not practice physical exercises regularly for at least 40 min, three times a week were considered sedentary. Smokers were individuals with regular consumption of any quantity of cigarettes, for a period exceeding 6 months until the month prior to the completion of the clinical record, and
Please cite this article in press as: Sóter MO et al. Peripheral blood-derived cytokine gene polymorphisms and metabolic profile in women with polycystic ovary syndrome. Cytokine (2015), http://dx.doi.org/10.1016/j.cyto.2015.06.008
3
M.O. Sóter et al. / Cytokine xxx (2015) xxx–xxx
alcoholism was defined by the consumption of more than 30 mL of ethanol per day. The Ferriman–Gallwey index was used in order to assess hirsutism. The density of terminal hair on the upper lip, chin, chest, upper back, lower back, upper abdomen, lower abdomen, arms and thighs were visually rated on a scale of zero to four, where zero indicates no growth of terminal hair and four indicates strong growth in male pattern hair. A total score was calculated. A single observer scored all subjects to avoid bias in rating. HOMA-IR (homeostatic model assessment for insulin resistance) was calculated using the formula: HOMA-IR = [insulin (mU/L) glucose (mM/L)]/22.5 [32]. The molecular analyses using specific primers were performed using the multiplex PCR System kit (Cytokine genotyping Tray – One LambdaÒ), followed by agarose gel electrophoresis. The cytokine genes polymorphisms evaluated were 308A/G (rs 1800629) for TNF-a; 174C/G (rs 1800795) for IL6; 1082A/G (rs 1800896), 819T/C (rs 1800871), 592C/A (rs 1800872) for IL10; +874T/A (rs 2430561) for IFN-c; and 10T/C (rs 1800470), 25C/G (rs 1800471) for TGF-b1. Phenotype was defined as the relationship between the polymorphism and the genetic expression level [33–36]. 2.4. Statistics Statistical analyses were performed using SPSS version 13.0. The Kolmogorov–Smirnov test was used to determine the normality of the variables. For normal continuous variables, the mean and standard deviation were calculated, and those that did not follow normality were expressed as median values and interquartile ranges. For data that were normally distributed, we used the Student t-test or ANOVA (with LSD post-hoc test) and for non-parametric variables, we used the Mann–Whitney test or Kruskall Wallis test to compare two or three groups, respectively. Categorical variables are presented as absolute and relative frequencies, and the chi-square test was used to compare the frequencies and the Hardy–Weinberg equilibrium. Spearman or Pearson correlation test was applied to normally or non-normally distributed variables. P < 0.05 was considered significant. The sample size was estimated to allow the detection of differences of at least 20% in the proportion of each genotype between the case and the control groups, with statistical power of 80% and confidence interval (CI) of 95%. 3. Results The PCOS and control groups were matched for age. Hypertension frequency, smoking, physical inactivity and alcohol consumption did not differ between the groups (Table 1). Fasting glucose and CRP were not different between the groups; however, BMI, abdominal circumference, LAP index, HOMA-IR index, insulin, lipids, testosterone, and FSH levels were higher, and HDL-C was lower in PCOS when compared to controls (Table 1). The genotype distribution of the polymorphisms studied was in Hardy–Weinberg equilibrium (P > 0.05) for both groups. The frequencies of alleles, genotypes, and phenotypes were similar between PCOS and controls (Supplemental Tables 1–3). A multivariate logistic regression analysis was conducted and the cytokine gene polymorphisms frequency was independent of BMI in both groups (P > 0.05 for all the polymorphisms, data not shown). In the PCOS group, the TNF-a 308A/G polymorphism and the TGF-b1 25C/G polymorphism did not influence the biochemical parameters (all P > 0.05, data not shown). In women with PCOS, glucose levels were higher in those with the GG genotype of the IL6 174C/G polymorphism (Table 2). The categorized genotypes showed correlation with glucose levels (r = 0.313; P = 0.002). No
Table 1 Characterization of women with PCOS and control group considering anthropometric, biochemical and risk factors for cardiovascular diseases.
Age (years) BMI (kg/m2) Abdominal circumference (cm) Smoker Sedentarism Alcohol use Hypertension Glucose (mmol/L) HOMA-IR Insulin (uUI/mL) TC (mmol/L) LAP HDLc (mmol/L) LDLc (mmol/L) VLDLc (mmol/L) TG (mmol/L) Testosterone (ng/dL) FSH (mUI/mL) CRP (mg/dL)
PCOS (N = 97)
Control (N = 99)
P
30.6 ± 4.9 28.7 (7.8) 97.0 (17.5) 9 (9%) 63 (65%) 17 (18%) 4 (4%) 4.8 ± 9.1 2.7 (3.4) 12.3 (17.4) 4.9 ± 35.1 46.8 (51.2) 1.2 (19) 3.5 ± 45 0.24 (18) 1.2 (89) 53.3 (47.2) 5.5 (2.1) 5.0 (9)
29.4 ± 7.7 23.1 (5.4) 80.5 (19.5) 5 (5%) 65 (66%) 29 (29%) 1 (1%) 4.7 ± 8.2 1.6 (0.8) 7.7 (3.4) 4.5 ± 33.1 20.25 (18.3) 1.4 (19) 2.9 ± 38 0.18 (8) 0.9 (39) 27.7 (19.5) 4.5 (3.3) 4.0 (2)
0.214
