¹Endocrinlogy Unit, ²Gynecology Unit, University of Sao Paulo, School of Medicine, Brazil

OBJECTIVE: The aim of this study was to evaluate the influence of polycystic ovary syndrome (PCOS) and obesity on circulating markers of low-grade inflammation—tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and high sensitive C-reactive protein (hsCRP)—in young women without major cardiovascular (CV) risk factors (diabetes, dyslipidemia and arterial hypertension).
DESIGN: Twenty-five young women with PCOS and 23 eumenorrheic women without major CV risk factors and matched for body mass index (BMI) were studied. They were subdivided according to BMI and PCOS status and comparisons were made between the PCOS and Control groups, regardless of BMI, and between the Obese and Lean groups, regardless of the presence of PCOS.
RESULTS: Levels of TNF-α, IL-6 and hsCRP were similar between the PCOS group and the Control group (2.1 vs 1.9 pg/ml, p=0.397, 3.8 vs 5.7 pg/ml, p=0.805 and 0.9 vs 0.5 ng/ml, p=0.361, respectively). Levels of TNF-α were similar between the obese group and the lean group (2.1 vs 1.9 pg/ml, p=0.444). Levels of IL-6 and hsCRP were higher in the obese group than in the lean group (8.7 vs 2.0, p <0.001 and 1.4 vs 0.2 ng/ml, p <0.001, respectively).
CONCLUSION: Obesity, but not polycystic ovary syndrome, affects circulating markers of low-grade inflammation in young women without majorCV risk factors.

CV risk, Circulating markers of low-grade inflammation, Obesity, PCOS

---

---

INTRODUCTION

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic anovulation, androgen excess and infertility, increased prevalence of obesity and increased risk for type 2 diabetes mellitus (DM) and cardiovascular (CV) disease.¹ There is evidence that PCOS is also a proinflammatory disorder, characterized by the presence of chronic low-grade inflammation,^2,3 as variants in genes encoding several inflammatory cytokines and their receptors associated with insulin resistance (IR), obesity and/or DM have also been found to be associated with the syndrome.^4-8

Cytokines are soluble molecules that are involved in intercellular communication, these produced by a wide variety of cells in the body including adipocytes,⁹ and being involved in several biological processes, including atherosclerosis.^10,11 There are several subfamilies of cytokines, among them interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α).

Interleukin 6 can predict CV disease and, experimentally, contributes to the development of early atherosclerotic lesions.^12-14 Another effect of IL-6 is the induction of hepatic C-reactive protein (CRP) production, which is known to be an independent major risk marker of CV complications,¹⁴ even in individuals without known CV disease.¹⁵ Women with the highest baseline high sensitive-CRP (hs-CRP) levels had a five times greater risk of suffering a vascular event and seven times the risk of myocardial infarction or stroke than did control subjects in a prospective Women’s Health Study.¹⁶

Tumor necrosis factor-alpha is an important mediator of insulin resistance (IR)¹⁷ and is related to components of metabolic syndrome,¹⁸ such as impaired glucose tolerance (IGT) and type 2 DM,¹⁹ higher blood pressure^20-22 and dyslipidemia.²³

In recent years, several studies have been published concerning cytokines in PCOS patients, albeit with contradictory results. An important bias is the diagnostic criterion employed in the diagnosis of the syndrome. However, a question that remains unresolved is whether circulating cytokines are useful markers of the proinflammatory state and if this state could be due to PCOS itself or to the concomitant presence of obesity and/or CV risk factors, as obesity is a proinflammatory state per se²⁴ and CV risk factors could themselves trigger an inflammatory response.

Bearing all the above in mind, the aim of this study was to ascertain whether CRP is a better marker than TNF-α and IL-6 as inflammatory markers and if the low-grade inflammation described in women with PCOS could be attributed to the syndrome itself or to the concomitant presence of obesity and/or CV risk factors. With this objective, we adopted diagnostic criteria to restrict the number of PCOS phenotypes and employed a study design associated with a statistical methodology capable of determining the influence of PCOS and obesity, separately, on circulating levels of TNF-α, IL-6 and CRP in women with minor CV risk factors. As far we know, there is no study in the literature with this primary endpoint and involving the exclusion of CV risk factors.

PATIENTS AND METHODS

The procedures used were in accordance with the guidelines of the Helsinki Declaration on human experimentation. The study protocol was approved by the Ethical Committee of the Hospital das Clínicas da Universidade de São Paulo, and written informed consent was obtained from all women before beginning the study.

Twenty-five young Caucasian women with established PCOS (aged 19.0 to 33.0 yr) and 23 eumenorrheic women without evidence of hyperandrogenism (aged 18.0 to 35.0 yr) matched for body mass index (BMI) were studied. The data regarding these patients were already published.²⁵ All women were non-smokers, had a normal physical activity level, had no inflammatory or other diseases and had not taken chronic medications (hormones in general, hormonal contraceptives, glucocorticoids, nonsteroidal anti-inflammatory drugs, and drugs with an influence on body weight, glucose metabolism, lipid profile and on the CV system) for at least six months prior to the study.

The subjects were divided into two groups according to BMI: lean (18-25 kg/m²) and obese (30-40 kg/m²). Exclusion criteria for all subjects were age <18 or >35 years, pregnancy, arterial hypertension [systolic blood pressure (SBP) ≥140mmHg and/or diastolic blood pressure (DBP) ≥90mmHg], impaired fasting glucose, IGT on the oral glucose tolerance test (OGTT) or DM^26,27 and severe abnormalities of lipid profile (LDL-cholesterol >160 mg/dl or triglycerides >250 mg/dl).

Polycystic ovary syndrome diagnosis was based on the presence of menstrual dysfunction, hyperandrogenism, hyperandrogenemia and polycystic ovaries morphology, after ruling out related disorders through appropriate tests. The presence of polycystic ovaries was established by the presence of 12 or more follicles in each ovary measuring 2-9 mm in diameter, and/or increased ovarian volume (>10 ml).²⁸

Hirsutism was defined by a Ferriman and Gallwey score of ≥8.²⁹ Eumenorrhea was defined by the presence of menstrual cycles of 25-34 days for at least 12 months. Menstrual disturbance was defined by a cycle length ≥35 days and 8 or fewer menstrual cycles in the last year.

Body mass index was calculated by the formula: weight (kg)/height (m²). The minimum waist measurements between the pelvic brim and the costal margin were used to determine waist circumference (WC).³⁰

Blood pressure (BP) was measured twice in a mercury sphygmomanometer with cuffs that were adequate for the circumference of the patient’s arm. The patients were in the supine position after a 20-minute rest period. The blood samples were collected at 08:00 AM after an overnight fast, up to the 7th day of the menstrual cycle for the control group, and was aleatory for the women with PCOS. Serum progesterone was determined to confirm the absence of ovulation (<1.0 ng/ml). For the OGTT, a 75 gr glucose load was given and a blood sample was collected before to determine glucose, insulin, total (TC), LDL (LDL-C) and HDL-cholesterol (HDL-C), triglycerides (TG), steroid hormone-binding globulin (SHBG), total testosterone (TT), progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), TNF-a, IL-6, high-sensitive C-reactive protein (hs-CRP) and then, at 30, 60, 90 and 120 minutes after, to measure glucose and insulin levels.

Standard methodologies for lipids, lipoproteins and glucose determination and for LDL-C calculation were used.³¹

Tumor necrosis factor-alpha and IL-6 were determined by Multiplex-Based immunoassays-HSCYTO-60SK (Millipore Corp., Missouri 63304, USA). The intra- and interassay coefficients of variation for these two cytokines were 3.1% and 5.8%, respectively. The lower limit detection for TNF-α and IL-6 were 0.05 pg/ml and 0.1 pg/ml, respectively. High-sensitive C-reactive protein was measured by ELISA-KAPDB4360 (Diasource ImmunoAssays S.A., Louvain-la-Neuve, Belgium). The intra- and interassay coefficients of variation for hs-CRP were <15.2% and <9.9%, respectively, and the lower limit detection was 0.08 ng/ml.

For hormone assays, blood samples were processed by centrifuging and serum was stored at -20° C until assayed. Progesterone and total testosterone were measured by a fluoroimmunometric assay (Wallac, Finland). Insulin, SHBG, LH and FSH were measured by an immunofluorometric assay (Wallac, Finland). All the assays were performed in duplicate and the intra-assay and interassay coefficients of variation did not exceed 10% and 15%, respectively.

Insulin resistance was estimated by HOMA-IR (insulin in μU/mL x glucose in mg/dL/22.5×18) and the area under the curve for insulin (AUC_insulin) was calculated using the trapezoidal rule.

Statistical analysis was performed using the software InStat (GraphPad Software Inc., San Diego, CA, USA). Data were presented as median and minimum-maximum values. Comparisons between the PCOS and Control groups, regardless of BMI, and between the Obese and Lean groups, regardless of PCOS, were performed using the Mann-Whitney U test followed by Bonferroni adjustment, with p values adjusted according to the number of comparisons; p <0.005 was considered significant (p corrected). Spearman’s correlation coefficient analyses were performed and p-value <0.05 was considered statistically significant.

RESULTS

Four groups were initially formed (Obese-PCOS, Lean-PCOS, Obese-Control and Lean-Control). The clinical and chemical characteristics of these groups have been previously described.²⁵ There was no difference between the medians of TNF-α among the four groups (p=0.296), while the medians of IL-6 levels were significantly higher in the Obese-Control group when compared with the Lean-Control group (p=0.009) and with the Lean-PCOS group (p=0.001). For hs-CRP, the median was higher in the Obese-Control and Obese-PCOS groups than in the Lean-Control group and in the Lean-PCOS group (Obese-Control vs Lean-Control, p=0.006 and Obese-Control vs Lean-PCOS, p=0.004 and Obese-PCOS vs Lean-Control, p <0.001 and Obese-PCOS vs Lean-PCOS, p=0.001).

The data regarding the comparisons between the PCOS and Control groups, regardless of BMI, and between the Obese and Lean groups, irrespective of the presence of PCOS, are depicted in Table 1. For the comparison of the PCOS and Control groups, significant differences were seen only for HOMA-IR (p <0.001) and AUCi (p <0.001), these being higher in the PCOS group than in the Control group. Regarding the comparison between the Obese and Lean groups, significant differences were seen for BMI (p <0.001), HOMA-IR (p <0.001), AUCi (p=0.001), IL-6 (p <0.001) and hs-CRP (p <0.001), these being higher in the Obese than in the Lean group.

In the Obese group, Spearman’s correlation coefficient showed a positive and a negative statistically significant correlation between hs-CRP and SBP (r=0.53; p=0.012) and between hs-CRP and HDL-C (r= -0.63; p=0.001), respectively. In the PCOS group, hs-CRP was positively and statistically significant correlated with BMI (r=0.62; p=0.001).

DISCUSSION

Although there is evidence of a state of a chronic low-grade inflammation in PCOS, studies of cytokines profiles in women with this syndrome have yielded controversial results.^32-37 We attribute these differences to several factors, such as the diagnostic criterion for the syndrome, the presence of CV risk factors and the presence of obesity itself. In this study we sought to select such a group of women that would avoid such bias and, through the employment of a select statistical method, to separate the confounding effect of PCOS and obesity.

It should be noted here that the early diagnostic criteria recognized only one phenotype of PCOS.³⁸ The Rotterdam²⁸ and the Androgen Excess and PCOS society diagnostic criteria for PCOS³⁹ identified several phenotypes of the syndrome each exhibiting a distinct phenotype, hormone profile, insulin resistance and metabolic disturbances.⁴⁰ We have used strict diagnostic criteria in order to restrict the number of phenotypes and our women with PCOS fulfilled all relevant criteria.

We did not observe significant differences in IL-6, TNF-α and hs-CRP circulating levels in our women with PCOS when compared to normal women, just as Ciaraldi et al did not observe significant differences in circulating cytokines, including TNF-α.⁴¹ In our subjects, we attributed these results and the divergent results in other studies of the literature to a more homogeneous phenotype, but also to the exclusion of major CV risk factors (glucose metabolism disturbances, arterial hypertension and severe abnormalities of lipid profile), since CV risk factors by themselves could trigger an inflammatory response. The only CV risk factor that was not excluded was a low HDL-C, as this is so frequent in PCOS women that it would be impossible to have an adequate sample size.⁴² In any case, there is no evidence that a low HDL-C could trigger an inflammatory response.

The presence of classical CV risk factors, such as chronic hyperglycemia, can influence the inflammatory response in vitro and lead to misinterpretation of CV risk markers.⁴³ Moreover, it is established that the effect of TNF-α, through activation of the soluble receptors TNFR1 and sTNFR2, is related to components of the metabolic syndrome,¹⁸ such as IGT and type 2 DM(19), higher blood pressure²⁰ and dyslipidemia,²³ which are common in women with PCOS, even in those with normal BMI.^42,44

As for the inflammatory molecules, data on the relationship between PCOS and carotid intima-media wall thickness (cIMT), a marker of preclinical atherosclerosis, are not conclusive. In a previous study,²⁵ in this same cohort of women, we have demonstrated that by adopting stricter diagnostic criteria and excluding major CV risk factors^25,45 no differences were evident in vascular parameters related to early atherosclerosis in women with PCOS. This was also shown by Kahal et al⁴⁶ by studying obese women with PCOS diagnosed by strict diagnostic criteria and matched not only for BMI but also for abdominal obesity, blood pressure, lipid profile and smoking history. Although the main objective of this study was to examine cIMT and platelet function, our results also did not reveal any difference between normal and PCOS women with regard to hsCRP. As expected, we have found that the circulating levels of IL-6 and hs-CRP in obese women were higher compared to the lean women, regardless of their PCOS status, suggesting that obesity was the only major determinant of the circulating inflammatory markers.

It is well known that adipose tissue releases large amounts of IL-6 in vivo and this cytokine appears able to mediate several weight regulating processes. It is estimated that 15 to 30% of total circulating concentrations of IL-6 originate in adipose tissue in healthy subjects in the absence of acute inflammation, due to the fact that IL-6 adipose tissue production and systemic concentrations increase with adiposity.¹³ As CRP synthesis is induced by IL-6, it was no surprise that hs-CRP concentration was also elevated in obese women when compared with lean women, regardless of PCOS status, and that hs-CRP was positively correlated with BMI in PCOS women. Similarly to IL-6, CRP is related to body fat mass and this association was seen to be somewhat stronger in women and in non-diabetic subjects.¹³ It was also not surprising to observe in the obese women positive and negative correlations between hs-CRP and SBP and between hs-CRP and HDL-C, respectively. There is evidence that hs-CRP correlates with SBP, HDL-C and other components of the metabolic syndrome.⁴⁷

On the other hand, TNF-α were not different between PCOS and control women, as related to the presence of obesity, and between obese and lean women, as related to PCOS status. These results are in accordance with other reports which did not display differences of TNF-α in PCOS women when compared with controls despite the presence of more IR in PCOS women.³⁴ This could be explained by the fact that the effect of TNF-α is a paracrine one, as adipose tissue did not release large amounts of this cytokine in vivo.⁴⁸

The presence of overweight (BMI between 25 and 30 kg/m²) is associated with the increase of some CV risk factors, which has been demonstrated in patients with PCOS.^41,49 For this reason, we considered it highly appropriate to compare obese subjects with women with normal BMI (BMI between 18-25 kg/m²) and not with overweight (BMI between 25-30 kg/m²).

CONCLUSION

The circulating levels of IL-6, TNF-α and hs-CRP were similar between women with and without PCOS, but the levels of IL-6 and hs-CRP were higher in obese women when compared with lean women, regardless of the presence of PCOS. We attributed these findings to a better homogenization of our sample of PCOS women due to stricter criteria adopted for the diagnosis of the syndrome and to the exclusion of CV risk factors, as the latter can trigger by themselves an inflammatory response. We suggest that these factors must be considered in designing future studies.

GRANTS SUPPORTS

This study was supported by grants from CNPQ (142119/2005) and FAPESP (07100661-6).

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

REFERENCES

1. Toulis KA, Goulis DG, Mintziori G, et al, 2011 Meta-analysis of cardiovascular disease risk markers in women with polycystic ovary syndrome. Hum Reprod Update 17: 741-760.
2. Escobar-Morreale HF, Luque-Ramírez M, González F, 2011 Circulating inflammatory markers in polycystic ovary syndrome: a systematic review and metaanalysis. Fertil Steril 95: 1048-1058.
3. Diamanti-Kandarakis E, Paterakis T, Kandarakis HA, 2006 Indices of low-grade inflammation in polycystic ovary syndrome. Ann N Y Acad Sci 1092: 175-186.
4. Escobar-Morreale HF, Calvo RM, Sancho J, San Millan JL, 2001 TNF-alpha and hyperandrogenism: a clinical, biochemical, and molecular genetic study. J Clin Endocrinol Metab 86: 3761-3767.
5. Peral B, San Millan JL, Castello R, Moghetti P, Escobar-Morreale HF, 2002 The methionine 196 arginine polymorphism in exon 6 of the TNF receptor 2 gene (TNFRSF1B) is associated with the polycystic ovary syndrome and hyperandrogenism. J Clin Endocrinol Metab 87: 3977-3983.
6. Villuendas G, San Millan JL, Sancho J, Escobar-Morreale HF, 2002 The -597 G->A and -174 G->C polymorphisms in the promoter of the IL-6 gene are associated with hyperandrogenism. J Clin Endocrinol Metab 87: 1134-1141.
7. Erdogan M, Karadeniz M, Berdeli A, Alper G, Caglayan O, Yilmaz C, 2008 The relationship of the interleukin-6 -174 G>C gene polymorphism with oxidative stress markers in Turkish polycystic ovary syndrome patients. J Endocrinol Invest 31: 624-629.
8. Escobar-Morreale HF, Calvo RM, Villuendas G, Sancho J, San Millan JL, 2003 Association of polymorphisms in the interleukin 6 receptor complex with obesity and hyperandrogenism. Obes Res 11: 987-996.
9. Tracey KJ, Cerami A, 1993 Tumor necrosis factor, other cytokines and disease, 1993 Annu Rev Cell Biol 9: 317-343.
10. Miller AM, McInnes IB, 2011 Cytokines as therapeutic targets to reduce cardiovascular risk in chronic inflammation. Curr Pharm Des 17: 1-8.
11. Tedgui A, Mallat Z, 2006 Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiol Rev 86: 515-581.
12. Ridker PM, Rifai N, Stampfer MJ, Hennekens CH, 2000 Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 101: 1767-1772.
13. Festa A, D’Agostino R Jr, Howard G, Mykkänen L, Tracy RP, Haffner S, 2000 Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS). Circulation 102: 42-47.
14. Sarwar N, Thompson AJ, Di Angelantonio E, 2009 Markers of inflammation and risk of coronary heart disease. Dis Markers 26: 217-225.
15. Clearfield MB, 2005 C-reactive protein: a new risk assessment tooll for cardiovascular disease. J Am Osteopath Assoc 105: 409-416.
16. Ridker PM, Buring JE, Shih J, Matias M, Hennekens CH, 1998 Prospective study of C-reactive protein and the risk of future cardiovascular events among apparently healthy women. Circulation 98: 731-733.
17. Hotamisligil GS, Murray DL, Choy LN, Spielgeman BM, 1994 Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 91: 4854-4858.
18. Indulekha K, Surendar J, Mohan V, 2011 High sensitivity C-reactive protein, tumor necrosis factor-a, interleukin-6, and vascular cell adhesion molecule-1 levels in Asian Indians with metabolic syndrome and insulin resistance (CURES-105). J Diabetes Sci Technol 5: 982- 988.
19. Olson NC, Callas PW, Hanley AJ, et al, 2012 Circulating levels of TNF-a are associated with impaired glucose tolerance, increased insulin resistance, and ethnicity: the Insulin Resistance Atherosclerosis Study. J Clin Endocrinol Metab 97: 1032-1040.
20. Musialik K, 2012 The influence of chosen adipocytokines on blood pressure values in patients with metabolic syndrome. Kardiol Pol 70: 1237-1242.
21. Mathieu P, Poirier P, Pibarot P, Lemieux I, Després JP, 2009 Visceral obesity: the link among inflammation, hypertension, and cardiovascular disease. Hypertension 53: 577-584.
22. Bautista LE, Vera LM, Arenas IA, Gamarra G, 2005 Independent association between inflammatory markers (C-reactive protein, interleukin-6, and TNF-alpha) and essential hypertension. J Hum Hypertens 19: 149-154.
23. Popa C, Netea MG, van Riel PL, van der Meer JW, Stalenhoef AF, 2007 The role of TNF-alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 48: 751-762.
24. Balistreri CR, Caruso C, Candore G, 2010 The role of adipose tissue and adipokines in obesity-related inflammatory diseases. Mediators Inflamm 2010: 802078.
25. Barcellos CR, Lage SH, Rocha MP, et al, 2013 Polycystic ovary syndrome and obesity do not affect vascular parameters related to early atherosclerosis in young women without glucose metabolism disturbances, arterial hypertension and severe abnormalities of lipid profile. Gynecol Endocrinol 29: 370-374.
26. Genuth S, Alberti KG, Bennett P, et al, 2003 The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus: follow-up report on the diagnosis of diabetes mellitus. Diabetes Care 26: 3160-3167.
27. World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications: report of a WHO consultation, 1999 Part I. Diagnosis and classification of diabetes mellitus. WHO: Geneva ADA.
28. Rotterdam ESHRE/ASRM – Sponsored PCOS Consensus Workshop Group, 2004 Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 81: 19-25.
29. Ferriman DM, Gallwey JD, 1961 Clinical assessment of body hair growth in women. J Clin Endocrinol 21: 1440-1447.
30. Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III) final report, 2002 Circulation 106: 3143-3421.
31. Rocha MP, Maranhão RC, Seydell TM, et al, 2010 Metabolism of trygliceride-rich lipoproteins and lipid transfer to high-density lipoprotein in young obese and normal-weight patients with polycystic ovary syndrome. Fertil Steril 93: 1948-1956.
32. Möhlig M, Spranger J, Osterhoff M, et al, 2004 The polycystic ovary syndrome per se is not associated with increased chronic inflammation. Eur J Endocrinol 150: 525-532.
33. Olszanecka-Glinianowicz M, Banás M, Zahorska-Markiewicz B, et al, 2007 Is the polycystic ovary syndrome associated with chronic inflammation per se? Eur J Obstet Gynecol Reprod Biol 133: 197-202.
34. Soares GM, Vieira CS, Martins WP, et al, 2009 Increased arterial stiffness in nonobese women with polycystic ovary syndrome (PCOS) without comorbidities: one more characteristic inherent to the syndrome? Clin Endocrinol (Oxf) 71: 406-411.
35. Jakubowska J, Bohdanowicz-Pawlak A, Milewicz A, Szymczak J, Bednarek-Tupikowska G, Demissie M, 2008 Plasma cytokines in obese women with polycystic ovary syndrome, before and after metformin treatment. Gynecol Endocrinol 24: 378-384.
36. Gonzalez F, Thusu K, Abdel-Rahman E, Prabhala A, Tomani M, Dandona P, 1999 Elevated serum levels of tumor necrosis factor alpha in normal-weight women with polycystic ovary syndrome. Metabolism 48: 437-441.
37. Vgontzas AN, Trakada G, Bixler EO, et al, 2006 Plasma interleukin 6 levels are elevated in polycystic ovary syndrome independently of obesity or sleep apnea. Metabolism 55: 1076-1082.
38. Zawadeski JK, Dunaif A 1992 Diagnostic criteria for PCOS: towards a more rational approach. In: Dunaif A, Givens JR, Haseltine FP, Merriam MR (eds) PCOS. Boston: Blackwell Scientific.
39. Azziz R, Carmina E, Dewailly D, et al, 2006 Androgen Excess Society. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab 91: 4237-4245.
40. Wild RA, Carmina E, Diamanti-Kandarakis E, et al, 2010 Assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: a consensus statement by the Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society. J Clin Endocrinol Metab 95: 2038-2049.
41. Ciaraldi TP, Aroda V, Mudaliar SR, Henry RR, 2013 Inflammatory cytokines and chemokines, skeletal muscle and polycystic ovary syndrome: Effects of pioglitazone and metformin treatment. Metabolism 62: 1587-1596.
42. Rocha MP, Marcondes JA, Barcellos CR, et al, 2011 Dyslipidemia in women with polycystic ovary syndrome: incidence, pattern and predictors. Gynecol Endocrinol 27: 814-819.
43. González F, Rote NS, Minium J, Kirwan JP, 2006 In vitro evidence that hyperglycemia stimulates tumor necrosis factor-alpha release in obese women with polycystic ovary syndrome. J Endocrinol 188: 521-529.
44. Barcellos CR, Rocha MP, Hayashida SA, Nery M, Marcondes JA, 2007 Prevalence of abnormalities of glucose metabolism in patients with polycystic ovary syndrome. Arq Bras Endocrinol Metabol 51: 601-605.
45. Meyer ML, Malek AM, Wild RA, Korytkowski MT, Talbott EO, 2012 Carotid artery intima-media thickness in polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod Update 18: 112-126.
46. Kahal H, Aburima A, Ungvari T, et al, 2013 Polycystic ovary syndrome has no independent effect on vascular, inflammatory or thrombotic markers when matched for obesity. Clin Endocrinol (Oxf) 79: 252-258.
47. Ridker PM, Buring JE, Cook NR, Rifai N, 2003 C-Reactive Protein, the metabolic syndrome, and risk of incident cardiovascular events: an 8-year follow-up of 14 719 initially healthy american women. Circulation 107: 391-397.
48. Bastard JP, Maachi M, Lagathu C, et al, 2006 Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw 17: 4-12.
49. Barcellos CR, Rocha MP, Hayashida SA, Mion Junior D, Lage SG, Marcondes JÁ, 2007 Impact of body mass index on blood pressure levels in patients with polycystic ovary syndrome. Arq Bras Endocrinol Metabol 51: 1104-1109.

---

Address for correspondence:
Cristiano Roberto Grimaldi Barcellos, 220 Ap 92-A Belchior de Azevedo Str., Zip Code: 05089-030, Sao Paulo, Sao Paulo, Brazil, Tel.: +55 11 9-8389-8074 / +55 11 3645-1410, Fax: +55 11 3832-7501, E-mail: crbarcel@bol.com.br / endocrinologia.barcellos@gmail.com

Received: 22-04-2014, Accepted:13-01-2015