30 mcg ethinylestradiol, alone or combined with spironolactone, on cardiovascular risk markers in women with polycystic ovary syndrome

Contraception 86 (2012) 268 – 275

Original research article

The effects of 2 mg chlormadinone acetate/30 mcg ethinylestradiol, alone or combined with spironolactone, on cardiovascular risk markers in women with polycystic ovary syndrome☆,☆☆ Carolina Sales Vieira a, b,⁎, Wellington P. Martins a, b, c , Janaína Boldrini França Fernandes a , Gustavo Mafaldo Soares d , Rosana Maria dos Reis a, b , Marcos Felipe Silva de Sá a, b , Rui Alberto Ferriani a, b a

Department of Gynecology and Obstetrics, Medical School of Ribeirão Preto, University of São Paulo, Brazil, Avenida Bandeirantes, 3900 - Campus Universitário, Monte Alegre, Ribeirão Preto, SP, CEP-14049-900, Brazil b National Institute of Hormones and Women's Health, Ribeirão Preto, SP, CEP-14049-900, Brazil c Escola de Ultrassonografia e Reciclagem Médica de Ribeirão Preto (EURP), Rua Casemiro de Abreu 660, Ribeirão Preto, SP, CEP-14020-060, Brazil d Department of Obstetrics and Gynecology, Federal University of Rio Grande do Norte, Brazil, Caixa Postal 1524, Campus Universitário Lagoa Nova, Natal, RN, CEP-59072-970, Brazil Received 18 October 2011; revised 20 December 2011; accepted 20 December 2011

Abstract Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder associated with metabolic dysfunction and changes in cardiovascular risk markers, and using oral contraceptives (OCs) may exert a further negative effect on these alterations in patients with PCOS. Thus, the primary objective of this study was to assess the effects on arterial function and structure of an OC containing chlormadinone acetate (2 mg) and ethinylestradiol (30 mcg), alone or combined with spironolactone (OC+SPL), in patients with PCOS. Study Design: This was a randomized, controlled clinical trial. Fifty women with PCOS between 18 and 35 years of age were randomized by a computer program to use OC or OC+SPL. Brachial artery flow-mediated vasodilation, carotid intima-media thickness and the carotid artery stiffness index were evaluated at baseline and after 6 and 12 months. Serum markers for cardiovascular disease were also analyzed. The intragroup data were analyzed using analysis of variance with Tukey's post hoc test. A multivariate linear regression model was used to analyze the intergroup data. Results: At 12 months, the increase in mean total cholesterol levels was greater in the OC+SPL group than in the OC group (27% vs. 13%, respectively; p=.02). The increase in mean sex hormone-binding globulin levels was greater in the OC group than in the OC+SPL group (424% vs. 364%, respectively; p=.01). No statistically significant differences between the groups were found for any of the other variables. Conclusion: The addition of spironolactone to an OC containing chlormadinone acetate and ethinylestradiol conferred no cardiovascular risk-marker advantages in young women with PCOS. © 2012 Elsevier Inc. All rights reserved. Keywords: Contraceptives; Oral; Hormonal; Cardiovascular diseases; Polycystic ovary syndrome; Spironolactone

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Disclosure: The authors have nothing to disclose. ☆☆ Funding: This study received funding from the Waldemar B. Pessoa Foundation and from the National Institute of Hormones and Women's Health, National Council for Scientific and Technological Development (CNPq). ⁎ Corresponding author. Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina de Ribeirão Preto/USP, Av. Bandeirantes 3900, Campus Universitário, Ribeirão Preto, SP, CEP-14049-900, Brazil. Tel.: +55 16 3602 2804; fax: +55 16 3633 0946. E-mail addresses: [email protected], [email protected] (C.S. Vieira). 0010-7824/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2011.12.011

1. Introduction The reconfiguration of polycystic ovary syndrome (PCOS) from an endocrinopathy with exclusively reproductive effects to a metabolic syndrome with reproductive implications [1] has altered the therapeutic management of patients with this disorder. PCOS is associated with metabolic disorders (such as hypertension, diabetes and dyslipidemia) that, in and of themselves, are risk factors for cardiovascular disease (CVD) and have clinical implications

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for women's health [2]. To reduce the risk CVD, which is elevated in these patients, lifestyle modification should always precede and/or accompany any intervention in women with PCOS [3]. Nevertheless, for women with PCOS who do not wish to conceive, oral contraceptives (OCs) are still the treatment of choice for regulating the menstrual cycle and the clinical manifestations of excess androgens [4,5]. In a previous study, our group reported increased arterial stiffness, an independent CVD risk factor, in young, nonobese women with PCOS compared to ovulating controls paired for weight and age [5]. In addition, we also reported imbalanced circulating matrix metalloproteinases in these women [6], which can contribute to the future development of atherosclerosis. These alterations are associated with the hyperandrogenism present in women with PCOS. Atherosclerosis has a long latent phase before the symptoms are manifested. Therefore, the ability to evaluate arterial function prior to developing an angiographically measurable atherosclerotic plaque is an important aspect of early detection [7,8]. Several noninvasive measures of arterial structure and function have been shown to be clinically useful, including measuring the carotid intimamedia thickness (IMT), endothelial function and arterial stiffness [9,10]. Given that OCs can reduce the hyperandrogenism associated with PCOS, there are questions about whether attempts to increase the anti-androgenic effect of OCs are associated with metabolic benefits. To our knowledge, no studies have evaluated the effects of chlormadinone acetate (2 mg) and ethinylestradiol (30 mcg), either by itself or combined with an anti-androgenic drug, on clinical and subclinical CVD markers in women with PCOS. Therefore, the primary objective of the present study was to compare the effects of this OC, alone or in combination with spironolactone (SPL), on the arterial function and structure of young women with PCOS over a 12-month period. The rationale for adding SPL to OC therapy in PCOS was to augment the antiandrogenic effects of the OC. 2. Methods 2.1. Participants An open-label, controlled, randomized clinical trial was conducted at the University Hospital of the Ribeirão Preto School of Medicine, University of São Paulo (HC-FMRPUSP), Brazil, between January 2007 and July 2009. The study was registered with ClinicalTrials.gov (http:// clinicaltrials.gov/; NCT00842140). The following inclusion criteria were used: female, 18 to 35 years of age, a PCOS diagnosis, no desire to conceive, and wishing to use an OC. The PCOS diagnosis was confirmed by the Rotterdam consensus criteria [11]. The following exclusion criteria were used at screening: patients with a body mass index (BMI) ≥40 kg/m 2; any clinical

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conditions corresponding to Categories 3 or 4 of the World Health Organization Medical Eligibility Criteria for OC use [12]; smoking; alcoholism; illicit drug use; any systemic disease (systemic arterial hypertension, diabetes mellitus, immune system diseases or thyroid diseases) except PCOS; current or previous (up to 2 months before the study) use of oral, vaginal, monthly injectable or transdermal combined hormonal contraceptives; current or previous use (up to 6 months before the study) of a long-acting hormonal contraceptive method (injectable, implant or intrauterine device); 12 or fewer weeks since childbirth; currently breastfeeding or had stopped breastfeeding within 2 months of the screening visit; chronic and/or acute inflammatory processes; and the use of medications known to interfere with inflammatory markers or CVD risk factors (hypoglycemic drugs, anti-inflammatory drugs or statins). All the volunteers gave written informed consent, and the study was approved by the institutional review board. The principal variable used to calculate the sample size was the carotid artery stiffness index because a previous study had found that this variable is higher in women with PCOS than in ovulating controls [5]. Given a standard deviation (SD) of .96 in the arterial stiffness for women with PCOS [5], at least 16 patients in each group at the end of the study were required to achieve a minimum detectable difference of 1 SD between the pre- and post-treatment periods with an alpha of .05 and 80% power. Of the 100 women with a PCOS diagnosis at the university hospital during the recruitment period of the study, 60 met the inclusion criteria. However, 10 of these women were excluded (one was being prescribed topiramate, three refused to undergo a hormonal contraceptive washout and six were smokers). Ultimately, 50 women with PCOS were enrolled in the study. Immediately following their PCOS diagnoses, these women were randomized to one of the two treatment arms in a 1:1 proportion using a randomizer program (http://www.randomizer.org). The following treatment arms were used: Group 1 received cyclic use of an OC containing 2 mg chlormadinone acetate/30 mcg ethinylestradiol (EE) (Belara ®, Janssen Cilag, Grünenthal, Germany); and Group 2 received the same OC combined with SPL (100 mg/day) (EMS, São Bernardo do Campo, SP, Brazil). Participation could be discontinued during the study for the following reasons: the subject's request, pregnancy, smoking beginning during the study, failure to comply with the protocol, and experiencing a serious adverse effect. The study subjects were followed up at 12 months, and nine subjects discontinued the study (seven had adverse gastric reactions, one moved to another city and one wanted to become pregnant) (Fig. 1). 2.2. Data collection and evaluation of variables The participants received five medical assessments during the study: prior to initiating the medication (baseline), and at 3, 6, 9 and 12 months. At these visits, patient compliance

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Fig. 1. A flowchart of the patients in the study.

was assessed, and any complaints were registered. The anthropometric, clinical, serum and ultrasonographic variables were objectively evaluated at baseline, 6 and 12 months. Except for baseline visit, the other visits (6 and 12 months) were performed 14–21 days after beginning a package of OC. As far as the objective of the study was to evaluate the chronic effect of an OC alone or in combination with SPL on the arterial function and structure of young

women with PCOS, the time of pill ingestion was not set (each woman chose the most convenient time to take the pill based on her routine). The following anthropometric variables were measured: weight, height, BMI and waist circumference (the smallest measurement between the iliac crest and the inferior margin of the last rib). The frequency of physical activity was also classified according to the American Heart Association and

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American College of Sports Medicine recommendations: moderate-intensity aerobic physical activity for a minimum of 30 min 5 days a week or vigorous-intensity aerobic physical activity for a minimum of 20 min 3 days a week [13]. Blood samples were collected in the gynecology laboratory of HC-FMRP-USP after a 12-h fast. The ultrasonography preceded the blood sample collection because the latter can influence the results of endothelial evaluation. The baseline blood samples were generally collected prior to the beginning of the treatment, during the follicular phase (Cycle Days 3 to 7); in the women with irregular cycles, however, blood was taken at any time the ultrasonography confirmed the absence of a corpus luteum or a follicle ≥10 mm. Except for baseline visit, the other visits (6 and 12 months) were performed 14–21 days after beginning a package of OC. Blood tests were not related to time of pill intake. The women were instructed not to use any medications that might alter the results of the laboratory tests. The blood samples were processed within 2 h of sampling. The serum was stored at −80°C for the simultaneous determination of the following serum variables: fasting glucose, measured by the oxidase method using a Konelab 60i analyzer (Wiener Lab ®, Rosario, Argentina); total cholesterol (TC), highdensity lipoprotein cholesterol (HDL) and triglycerides (TG), measured using an enzymatic method in a BT 3000 Plus analyzer (Wiener Lab ®); low-density lipoprotein cholesterol (LDL), calculated using the Friedewald formula, LDL=total cholesterol−(HDL+TG/5) [14]; C-reactive protein (CRP), sex hormone-binding globulin (SHBG) and fasting insulin, measured by chemiluminescence using a DPC Immulite ® 2000 immunoassay analyzer (Diagnostic Products Corporation, Los Angeles, CA, USA); and interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), measured by chemiluminescence using a DPC Immulite ® 1000 immunoassay analyzer (Diagnostic Products Corporation). The free androgen index (FAI) was calculated using the following formula: total testosterone (nmol/L)/SHBG (nmol/L)×100. The total testosterone was measured by radioimmunoassay using a Packard Tri-Carb 2100 TR scintillation counter. The homeostasis model assessment of insulin resistance (HOMAIR) index was calculated according to the following formula: {[fasting glucose (mg/dL)×0.05551]×[fasting insulin (μIU/mL)/22.5)]} [15]. Both the vascular and pelvic ultrasounds were performed between 7:00 and 9:00 a.m. after an overnight fast of at least 12 h in a room with a controlled temperature (20°C to 23°C), and the patients rested in the supine position for 15 min prior to the procedure. The ultrasound scans were performed 5 min after the blood pressure measurements using a 6.0- to 12.0MHz linear probe from Voluson 730 Expert (GE Medical Systems, Zipf, Austria) with an attached electrocardiogram. All the blood pressure measurements and ultrasound scans were performed by the same operator (WPM). The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were examined in the left upper arm using a standard mercury

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sphygmomanometer. The means of three consecutive measurements were used for the SBP and DBP values. The IMT, brachial artery flow-mediated dilation (FMD), carotid artery stiffness index (β) and mean ovarian volume (MOV) were assessed as has been previously described [5,16–19]. 2.3. Statistical analysis The data from this trial were analyzed using the intentionto-treat principle. Normality was assessed by the Shapiro– Wilk and D'Agostino–Pearson tests. The intragroup data were analyzed using analysis of variance with Tukey's post hoc test. For the intergroup analysis, a mixed-effects, multivariate linear regression model was used instead of univariate statistics. This method was appropriate because of the multiple analyses involved in the study (multiple variables, two treatment groups and three time points). The calculations were performed using the SAS program, version 9.1 (SAS Institute, Cary, NC, USA). The mean variation in each parameter was used for the intergroup comparisons, with comparisons made between baseline and 6 months and between baseline and 12 months. The significance level was defined as 5%.

3. Results 3.1. Intragroup comparisons (at 12 months of evaluation) In the OC group, the mean carotid artery stiffness index and MOV decreased by 12.5% and 28.5%, respectively, between baseline and 12 months (pb.01 for both comparisons). There was a 5% reduction in mean glucose levels and a 91.5% mean FAI reduction in the 12 months of OC use (pb.01 for all comparisons). The total testosterone also decreased by 46% in the 12 months of OC use (pb.01). The mean TC (13%), HDL (18%), TG (75%), SHBG (424%) and CRP (138%) levels increased over the 12 months of treatment (pb.01 for all comparisons). There were no significant changes in any of the other variables over the 12 months of observation (Tables 1 and 2). In the OC+SPL group, the ultrasonography showed a 9% mean increase in the IMT (p=.01) and a 36% mean MOV reduction (pb.01) after 12 months of treatment. There was a 3% reduction in mean glucose levels (p=.01) and a 93% reduction in mean FAI (pb.01). The mean total testosterone also decreased by 57% in the 12 months of OC use (pb.01). Furthermore, increases in the mean TC (27%; pb.01), LDL (21%; pb.01), HDL (26%; pb.01), TG (77%; pb.01), SHBG (364%; pb.01), CRP (118%; pb.01) and TNF-alpha (14%; p=.02) levels over the 12 months of treatment were found. No significant changes were found for any of the other variables (Tables 1 and 2). 3.2. Intergroup comparisons (at 12 months of evaluation) The frequency of mean physical activity was 16% (4/25) in the OC group and 16% (4/25) in the OC+SPL group

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Table 1 Intragroup effects of the therapeutic interventions on clinical and ultrasonographic variables over a 12-month period in women with polycystic ovary syndrome OC

Age (years) Weight (kg) BMI (kg/m 2) Waist (cm) SBP (mmHg) DBP (mmHg) FMD (%) IMT (mm) β MOV (cm 3)

OC+SPL

Baseline

6 months

12 months

Baseline

6 months

12 months

25.0 (3.8) 60.8 (10.1) 23.3 (4.3) 78.7 (10.8) 113.8 (6.2) 76.2 (6.7) 7.7 (3.2) 0.43 (0.08) 4.0 (1.1) 10.9 (2.7)

n/a 61.2 (10.1) 23.5 (4.3) 77.4 (10.4) 112.2 (8.3) 74.6 (4.6) 7.7 (3.1) 0.45 (0.05) 3.6 (0.9)⁎ 7.7 (2.6)⁎

n/a 62.0 (9.7) 23.8 (4.1) 77.2 (9.3) 115.3 (7.6) 77.6 (5.0) 7.1 (3.2) 0.45 (0.06) 3.5 (0.7) † 7.8 (3.0) †

24.4 (4.3) 66.6 (15.9) 25.4 (5.5) 82.5 (13.3) 117.2 (12.3) 78.6 (8.4) 8.2 (3.8) 0.44 (0.09) 3.9 (1.0) 12.0 (3.6)

n/a 68.7 (16.7) 26.2 (5.7) 83.7 (13.6) 115.4 (12.6) 77.8 (9.2) 8.2 (4.5) 0.46 (0.06) 3.7 (1.0) 8.4 (2.9)⁎

n/a 68.2 (16.7) 26.2 (5.7) 82.6 (13.7) 119.6 (13.1) 78.6 (10.6) 8.0 (3.0) 0.48 (0.08) † 4.0 (0.8) 7.7 (2.3) †

Values are presented as means (SD). n/a: not applicable; OC: oral contraceptive (2 mg chlormadinone acetate and 30 mcg ethinylestradiol); OC+SPL: OC and spironolactone (100 mg/day); SBP: systolic blood pressure; DBP: diastolic blood pressure; BMI: body mass index; FMD: flow-mediated dilation; IMT: common carotid intima-media thickness; β: common carotid artery stiffness index; MOV: mean ovarian volume. Italicized values mean significant difference. ⁎ pb.05 (comparison between baseline and 6 months). † pb.05 (comparison between baseline and 12 months).

(p=.91). At 12 months, the increase in TC levels was greater in the OC+SPL group than in the OC-alone group (27% vs. 13%; p=.02). The increase in mean SHBG levels was greater in the OC-alone group than in the OC+SPL group (424% vs. 364%; p=.01). No statistically significant differences between the two groups were found for any of the other variables (Tables 3 and 4).

4. Discussion Oral contraceptive use is associated with an increased risk of venous [20] and arterial [21] thrombosis and with elevated TG levels [22] and hepatic angiotensinogen synthesis [23],

which leads to poor control of blood pressure in hypertensive women [24]. OCs containing low doses of EE exert no significant effects on carbohydrate metabolism in nondiabetic women [25]; however, some studies have reported increased insulin levels and/or a reduction in insulin sensitivity in women with PCOS who use OCs [26,27]. Because of these adverse effects, some studies have attempted to add other medications (e.g., metformin) to improve the metabolic dysfunction. Our results show that using an OC containing 2 mg chlormadinone and 30 mcg EE reduces mean carotid artery stiffness by 12.5% after 12 months of follow-up. Adding SPL (100 mg/day) to the OC failed to confer any further improvement in the ultrasonographic markers of arterial

Table 2 Intragroup effects of the therapeutic interventions on metabolic variables over a 12-month period in women with polycystic ovary syndrome OC

Glucose (mg/dL) TC (mg/dL) HDL (mg/dL) LDL (mg/dL) TG (mg/dL) Insulin (μIU/mL) SHBG (nmol/L) CRP (mg/L) TNF-alpha (pg/mL) IL-6 (pg/mL) HOMA-IR Testosterone (ng/dL) FAI (%)

OC+SPL

Baseline

6 months

89.1 (10.0) 168.1 (25.3) 50.2 (8.6) 102.7 (20.7) 75.9 (58.8) 5.1 (3.4) 42.9 (17.2) 2.1 (2.5) 10.6 (6.3) 1.9 (1.6) 1.1 (0.8) 79 (37.3) 9.4 (11.5)

82.7 (9.4)⁎ 193.2 (35.6)⁎ 61.4 (9.5)⁎ 103.5 (31.6) 141.0 (90.9)⁎ 6.0 (4.8) 220.9 (111.1)⁎ 6.2 (5.1)⁎ 12.8 (3.7) 1.6 (1.1) 1.3 (1.1) 41.9 (17.8)⁎ 0.9 (0.7)⁎

12 months †

84.9 (8.7) 190.0 (33.3) † 59.1 (9.7) † 104.4 (27.3) 132.6 (67.1) † 6.1 (4.8) 224.9 (105.6) † 5.0 (3.8) † 12.0 (3.2) 1.7 (1.0) 1.3 (1.0) 42.8 (15.9) † 0.8 (0.5) †

Baseline

6 months

12 months

86.0 (9.1) 168.5 (29.0) 50.3 (10.6) 99.5 (28.0) 93.3 (57.9) 9.1 (7.3) 30.3 (16.3) 3.4 (4.9) 10.9 (6.9) 2.2 (1.8) 2.0 (1.6) 84.2 (29.1) 17.9 (32.4)

82.4 (9.7)⁎ 204.9 (34.7)⁎ 61.8 (12.9)⁎ 110.0 (29.8) 184.7 (140.9)⁎ 11.3 (7.1) 147.7 (88.9)⁎ 4.9 (4.3)⁎ 12.0 (3.9) 2.3 (1.4) 2.3 (1.5) 41.1 (9.5)⁎ 1.5 (1.4)⁎

83.2 (9.4) † 214.7 (29.8) † 63.4 (11.4) † 120.4 (27.1) † 165.3 (100.4) † 9.6 (5.9) 140.5 (82.4) † 7.4 (8.8) † 12.4 (2.7) 1.9 (1.2) 2.0 (1.2) 36.6 (16.7) † 1.3 (1.0) †

Values are presented as mean (SD). Italicized values mean significant difference. OC: oral contraceptive (2 mg chlormadinone acetate and 30 mcg ethinylestradiol); OC+SPL: OC and spironolactone (100 mg/day); TC: total cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; SHBG: sex hormone-binding globulin; CRP: C-reactive protein; TNF-alpha: tumor necrosis factor alpha; IL-6: interleukin 6; HOMA-IR: homeostasis model assessment of insulin resistance (insulin resistance index); FAI: free androgen index. ⁎ pb.05 (comparison between baseline and 6 months). † pb.05 (comparison between baseline and 12 months).

C.S. Vieira et al. / Contraception 86 (2012) 268–275 Table 3 Effects of 2 mg chlormadinone acetate and 30 mcg ethinylestradiol alone or combined with spironolactone on clinical and ultrasonographic variables over a 12-month period in women with polycystic ovary syndrome OC Weight (kg) Δ6 months Δ12 months BMI (kg/m 2) Δ6 months Δ12 months SBP (mmHg) Δ6 months Δ12 months DBP (mmHg) Δ6 months Δ12 months Waist (cm) Δ6 months Δ12 months FMD (%) Δ6 months Δ12 months IMT (mm) Δ6 months Δ12 months β Δ6 months Δ12 months MOV (cm 3) Δ6 months Δ12 months

Table 4 Effects of 2 mg chlormadinone acetate and 30 mcg ethinylestradiol alone or combined with spironolactone on metabolic variables over a 12-month period in women with polycystic ovary syndrome

OC+SPL

0.4 (1.1) 1.2 (2.2)

2.0 (2.3) 1.5 (3.1)

0.2 (0.4) 0.5 (0.9)

0.8 (0.9) 0.8 (1.2)

−1.6 (10.9) 1.5 (8.9)

−1.8 (8.3) 2.4 (9.2)

−1.6 (8.2) 1.5 (7.6)

−0.8 (8.5) 0.0 (7.0)

−1.3 (2.7) −1.5 (3.7)

1.2 (4.2) 0.1 (4.6)

0.0 (2.7) −0.6 (3.1)

0.0 (4.3) −0.2 (4.3)

0.02 (0.06) 0.02 (0.08)

0.02 (0.06) 0.04 (0.07)

−0.4 (1.2) −0.5 (1.2)

−0.2 (0.9) 0.1 (1.0)

−3.2 (3.0) −3.1 (3.3)

−3.5 (3.3) −4.2 (3.7)

The data are presented as the absolute mean variation within a time interval (±SD). Δ6 months: variation within 6 months (mean at 6 months−mean at baseline); Δ12 months: variation within 12 months (mean at 12 months− mean at baseline). OC: Oral contraceptive (2 mg chlormadinone acetate and 30 mcg ethinylestradiol); OC+SPL: OC and spironolactone (100 mg/day); SBP: systolic blood pressure; DBP: diastolic blood pressure; BMI: body mass index; FMD: flow-mediated vasodilation; IMT: common carotid intimamedia thickness; β: common carotid artery stiffness index; MOV: mean ovarian volume.

function and structure compared to the use of an OC alone. Increased arterial stiffness constitutes an independent CVD risk factor and may facilitate the onset and progression of hypertension, left ventricular hypertrophy, myocardial infarction and heart failure [28]. The FMD values of subjects in the present study were within the range expected for this group of women [29], and no significant changes, either between or within the groups, were found during the study period. With respect to the serum markers of CVD risk, adding SPL led to a greater increase in mean TC levels (27%) than did OC treatment alone. These results suggest that the OC+SPL combination produces no additional beneficial metabolic effects that would justify its routine use in women with PCOS. In a previous study of overweight women with PCOS, the combination of SPL (100 mg/day) and an OC (100 mcg levonorgestrel and 20 mcg EE) was evaluated over a 6month period and compared with using another OC alone (2

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OC Glucose (mg/dL) Δ6 months Δ12 months TC (mg/dL) Δ6 months Δ12 months HDL (mg/dL) Δ6 months Δ12 months LDL (mg/dL) Δ6 months Δ12 months TG (mg/dL) Δ6 months Δ12 months Insulin (μIU/mL) Δ6 months Δ12 months CRP (mg/L) Δ6 months Δ12 months SHBG (nmol/L) Δ6 months Δ12 months TNF-alpha (pg/mL) Δ6 months Δ12 months IL-6 (pg/mL) Δ6 months Δ12 months HOMA-IR Δ6 months Δ12 months Testosterone (ng/dL) Δ6 months Δ12 months FAI (%) Δ6 months Δ12 months

OC+SPL

−6.4 (5.2) −4.2 (9.2)

−3.5 (6.5) −2.7 (8.5)

25.1 (34.4) 21.9 (30.3)

36.5 (23.7) 46.2(27.8)

11.2 (10.7) 8.9 (10.4)

11.5 (11.4) 13.1 (10.2)

0.8 (26.5) 1.7 (21.0)

10.5 (21.1) 21.0 (22.2)

65.1 (84.7) 56.7 (50.92)

91.4 (68.7) 72.0 (84.2)

0.9 (3.8) 1.0 (3.0)

2.2 (4.1) 0.5 (5.7)

4.1 (4.2) 2.9 (2.3)

1.4 (6.8) 4.0 (5.7)

178. 0 (101.4) 182.0 (94.0)

117.4 (81.8) 110.2 (97.6)

2.2 (6.4) 1.4 (5.2)

1.1 (6.5) 1.5 (6.9)

−0.3 (1.0) −0.2 (1.5)

0.1 (1.6) −0.3 (1.2)

0.2 (0.9) 0.2 (0.7)

0.3 (0.8) 0.0 (1.2)

−37.1 (32.3) −36.1 (38)

−43.9 (26.2) −47.9 (31.6)

−8.5 (11.1) −8.6 (11.3)

−16.4 (37.1) −16.7 (38.8)

The data are presented as the absolute mean variation within a time interval (±SD). Δ6 months: Variation within 6 months (mean at 6 months−mean at baseline); Δ12 months: variation within 12 months (mean at 12 months− mean at baseline). Italicized values mean significant difference (pb.05) in the same line. OC: Oral contraceptive (2 mg chlormadinone acetate and 30 mcg ethinylestradiol; OC+SPL: OC and spironolactone (100 mg/day); TC: total cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; SHBG: sex hormone-binding globulin; CRP: C-reactive protein; TNF-alpha: tumor necrosis factor alpha; IL-6: interleukin 6; HOMA-IR: homeostasis model assessment of insulin resistance (insulin resistance index); FAI: free androgen index.

mg cyproterone acetate and 35 mcg EE) and with using metformin alone (2000 mg/day). Insulin resistance deteriorated and arterial stiffness increased by 7% in the women using the 2-mg cyproterone acetate and 35-mcg EE OC compared with those using metformin alone. By contrast, adding SPL to the OC produced no negative effects. There were no FMD changes for any of the treatments evaluated

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[30]. Spironolactone alone was evaluated in a 6-month study of 30 nonobese women with PCOS. This study showed that SPL treatment improved FMD and cholesterol levels in nonobese PCOS patients [31]. Another attempt to increase the anti-androgenic effects of OCs compared the effects of an OC containing drospirenone (3 mg) and EE (20 mcg), alone or combined with either cyproterone acetate or metformin, on the traditional cardiovascular risk markers. The OC combined with cyproterone acetate produced the worst performance of the three treatments, with lower insulin sensitivity and higher TG and TC levels [32]. The reductions in the FAI, total testosterone and MOV, and the increases in the SHBG and HDL levels were expected [26,33] and occurred in both of the groups in this study. In addition, there was an increase in CRP with no increase in IL6 levels, regardless of the treatment used. Therefore, the increase in CRP was probably not due to increased inflammatory activity induced by the OC but rather to enhanced hepatic protein synthesis related to using EE [34]. It is important to emphasize that using the OC alone or in combination with SPL led to increased TG levels of 75% and 77%, respectively, without a significant statistical difference between the groups. This information is particularly important for women with PCOS and hypertriglyceridemia. Using OCs containing anti-androgenic progestogens results in a greater increase in TG levels than does using OCs whose progestogen component has androgenic properties [35]. The patients in the present study were followed for 12 months, and this period is insufficient for any conclusions about the effects of these medications on future CVD risk in women with PCOS. While there is no metabolic benefit from increasing the anti-androgenic effect of OCs with SPL, adding SPL to an OC can be justified when clinical hyperandrogenism is important [36]. In conclusion, adding SPL to an OC containing chlormadinone acetate and EE does not decrease cardiovascular risk markers in young women with PCOS when compared with using the OC alone. Acknowledgments We wish to thank the technical laboratory and nursing team, Maria Albina V. Bortolieiro, Maria Auxiliadora Pádua Rosa, Océlia de Vasconcelos and Sandra Aparecida Cavichiollo Vianna, for contributing to this study and for ensuring the well-being of the patients.

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