Effects of two forms of combined oral contraceptives on carbohydrate metabolism in adolescents with polycystic ovary syndrome

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1 POLYCYSTIC OVARY SYNDROME Effects of two forms of combined oral contraceptives on carbohydrate metabolism in adolescents with polycystic ovary syndrome George Mastorakos, M.D., a,b Carolina Koliopoulos, M.D., b Efthymios Deligeoroglou, M.D., b Evanthia Diamanti-Kandarakis, M.D., c and George Creatsas, M.D. b a Endocrine Unit, b 2nd Department of Obstetrics and Gynecology, Athens University School of Medicine, Aretaieion Hospital, and c Endocrine Section, 1st Department of Internal Medicine, Athens University School of Medicine, Laiko General Hospital, Athens, Greece Objective: To compare the effects of combined oral contraceptives (OCs) containing cyproterone acetate or desogestrel on insulin sensitivity in adolescents with polycystic ovary syndrome (PCOS). Design: A prospective randomized clinical trial. Setting: Outpatient gynecological clinic of Aretaieion University Hospital. Patient(s): Thirty-six adolescent girls with hyperandrogenism and six or less menses in the preceding 12 months. Intervention(s): Patients were separated in two groups: group A received 0.15 mg of desogestrel plus mg of ethinyl E 2 daily; and group B received 2 mg of cyproterone acetate plus mg of ethinyl E 2 daily, for 21 days followed by a 7-day rest, for 12 months. Main Outcome Measure(s): Hirsutism score, lipid, androgen, and sex hormone-binding globulin (SHBG) levels were evaluated at baseline. An oral glucose tolerance test (OGTT) was performed and metabolism indices, based on previously studied mathematical formulas, were assessed at baseline and at 12 months. Result(s): After 12 months of treatment, the homeostasis model assessment index of insulin resistance increased significantly in both groups. The fasting glucose-to-insulin ratio and predicted insulin sensitivity index decreased in group B. The delta of the area under the OGTT curve for insulin and predicted first and second phase insulin secretion indices increased significantly only in group B. Conclusion(s): We conclude that treatment of adolescent girls with PCOS with the two combined OCs administered, results in unfavorable changes of insulin sensitivity. In addition, cyproterone acetate is associated with an increase of insulin secretion and hyperinsulinemia. (Fertil Steril 2006;85: by American Society for Reproductive Medicine.) Key Words: Polycystic ovary syndrome, cyproterone acetate, combined oral contraceptives, desogestrel, insulin resistance, HOMA, OGTT, adolescence Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, chronic anovulation, and insulin resistance, which results in hirsutism, irregular menses, infertility, dyslipidemia, as well as a higher risk for diabetes mellitus and cardiovascular disease. In a cross-sectional study of women of reproductive age from the Greek island of Lesbos, the incidence of PCOS was 6.77% (1) whereas among white and African American women it was 4.7% and 3.4%, respectively (2). There are studies suggesting a strong familial component in PCOS (3). It is accepted that PCOS is an oligogenic disorder in which a small number of key genes interact with environmental factors (notably dietary) resulting in the abnormalities associated to this syndrome (4). The Received November 24, 2004; revised and accepted July 19, Reprints requests: George Mastorakos, M.D., 3, Neofytou Vamva str., Athens, Greece (FAX: ; mastorak@mail. kapatel.gr). syndrome has a pubertal onset (5). Adolescent girls affected by PCOS present with menstrual irregularity, signs of hyperandrogenism, and often gain weight immediately or shortly after menarche (5). These patients may also present with hyperinsulinemia, reflecting insulin resistance as well as increased androgen levels and decreased sex hormonebinding globulin (SHBG) levels (6). A genetic origin for the decreased SHBG levels in women with PCOS has recently been suggested (7). Avvad et al. (8) did not find any difference in the glucose and insulin parameters among normal adolescents or adolescents with PCOS and they suggested that the level of insulin resistance may be less intense in these girls because they are at an early stage of PCOS. However, by using hyperinsulinemic euglycemic and hyperglycemic clamp techniques, Lewy et al. (9) have shown that adolescent girls with PCOS present approximately 50% reduction in peripheral tissue insulin sensitivity, evidence of hepatic insu- 420 Fertility and Sterility Vol. 85, No. 2, February /06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 lin resistance, and compensatory hyperinsulinemia early in the course of the syndrome. When adolescents and young women with PCOS were followed-up for 10 years, a trend to deterioration of hyperinsulinism and insulin resistance was observed without accentuation of hyperandrogenism (10). Different therapeutic approaches have been proposed for the treatment of PCOS-related hyperandrogenism. The antiandrogenic progestogen cyproterone acetate in combined oral contraceptives (OCs) is commonly administered in some parts of the world such as Europe (11). On the other hand, combined OCs containing progestogens with very low androgenic activity, such as desogestrel, may prove to be effective in reducing hyperandrogenism (12). Due to both their estrogenic and progestogenic component, OCs reduce glucose tolerance and increase insulin levels (13). To compare the effects on carbohydrate metabolism of two combined OCs containing cyproterone acetate or desogestrel as progestogenic compounds, we studied 36 adolescent girls with PCOS during a 12-month period of treatment with either of these two combined formulations in a prospective randomized study. These two combined OC pills, with slightly different content in ethinyl E 2, commonly used in Europe, have been selected to draw some practical conclusions on their usefulness for the treatment of PCOS. In addition, they have been selected because in clinical practice the question of treating adolescent girls with moderate PCOS with a pill containing an antiandrogen is most often raised. MATERIALS AND METHODS Subjects Thirty-six (14 19 years old) adolescent girls with clinically evoked and biologically confirmed hyperandrogenism and having six or less menses during the past 12 months, demonstrating chronic anovulation, were consecutively recruited for this study in the outpatient adolescent gynecology clinic of our university hospital. The diagnosis of PCOS in these adolescent girls was based on the criteria established at the 1990 PCOS conference organized at the National Institutes of Health (Bethesda, MD) by the National Institute of Child Health and Development (14). Before initiation of the protocol, PRL, TSH, free triiodothyronine plasma levels, as well as a 250- g iv ACTH stimulation test were performed to rule out, respectively, hyperprolactinemia, functional thyroidopathy, and 21-hydroxylase deficiency associated with nonclassic congenital adrenal hyperplasia. Thus, the exclusion criteria included adenoma-associated hyperprolactinemia, functional thyroidopathy, and 21-hydroxylase deficiency associated with nonclassic congenital adrenal hyperplasia, androgen-secreting neoplasms, and diabetes mellitus. None of the patients suffered from diabetes mellitus. No subject had used any hormonal medication, including combined OCs, for at least 6 months before study entry. Body mass index (BMI) and waist/hip ratio (WHR) were calculated for each patient. Hirsutism was evaluated according to Ferriman-Gallway criteria (15). Only 6 of the 36 adolescent girls who participated in this study had a Ferriman-Gallway score 10. The investigators who determined the Ferriman-Gallway scores were blinded to the treatment arms. The patients and their parents were informed about the study and gave their consent. The Ethical Committee of our institution approved the study and an Institutional Review Board approval was obtained. Protocol Baseline blood samples were obtained on days 3 7 for cycling patients or on a random day for amenorrheic patients for measurement of testosterone (T), free T, 4 -androstenedione (A), dehydroepiandrosterone sulfate (DHEA-S), 17OH-P and SHBG, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol subfractions, triglycerides, apolipoproteins (A-I, A-II, B), and Lipoprotein (Lp[a]). Fasting glucose and insulin serum levels were measured and an oral glucose tolerance test (OGTT) with 75 g of glucose was performed and blood samples for the measurement of glucose and insulin levels were obtained at 15, 30, 60, 90, and 120 minutes. On the same day, an abdominal ultrasound with a 5-mHz transducer was performed to assess the morphology and the volume of the uterus and ovaries. Subsequently, patients were randomly assigned to one of the following treatments: group A, 18 patients received 0.15 mg of desogestrel plus mg ethinyl E 2 daily; or group B, 18 patients received 2 mg of cyproterone acetate plus mg of ethinyl E 2 daily. Treatment was administered daily for 21 days followed by a 7-day rest for a total of 1 year. Clinical characteristics, androgen and lipids levels did not differ between treatment groups at baseline (Table 1). Randomization was based on computer-generated random numbers. Glucose and insulin serum levels during fasting as well as during the OGTT test were reevaluated after 1 year of treatment. To avoid a factor of confusion in the interpretation of data, patients were not advised to lose weight during the study period. As a result, patients complained about not losing weight during the study period, although they were satisfied not to gain weight as they had assumed because of the treatment with the OCs. There were no dropout during the study period. Hormone Assays Testosterone was measured by RIA (SORIN, Saluggia, Italia), with sensitivity of 0.05 ng/ml, and coefficient of 9.4% for intra-assay and of 10.0% for interassay variability. Free T was measured by an RIA method (DPC, Los Angeles, CA), with a sensitivity of 0.1 pg/ml, and coefficient of 4.3% for intra-assay and of 6.5% for interassay variability. The SHBG was measured by an Immunoradiometric assay (IRMA) method (DSL, Webster, TX), with a sensitivity of 3 nmol/l, and coefficient of 3.7% for intraassay and of 11.5% for interassay variability. Androsten- Fertility and Sterility 421

3 TABLE 1 Clinical, hormonal, and lipid profile characteristics of adolescent girls with PCOS before initiation of treatment with desogestrel/ethinyl E 2 (group A) or cyproterone acetate/ethinyl E 2 (group B) (mean SE). Characteristics Group A Desogestrel/ethinyl E 2 Group B Cyproterone acetate/ethinyl E 2 Age (y) Ferriman-Gallway score WHR BMI Testosterone (ng/ml) Free testosterone (pg/ml) androstenedione (ng/ml) DHEAS (ng/ml) 2, , SHBG (nmol/l) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Apolipoprotein A-I (mg/dl) Apolipoprotein A-II (mg/dl) Apolipoprotein B (mg/dl) Lp(a) (mg/dl) Total/HDL cholesterol LDL/HDL cholesterol Note: There is no statistical significance of any value between the two groups. dione was measured by RIA (DSL), with sensitivity of 0.02 ng/ml, and coefficient of 5.9% for intra-assay and of 7.0% for interassay variability. Insulin was measured by an electrochemiluminescence method (Roche Diagnostics GmbH, Mannheim, Germany), with sensitivity of 0.20 IU/mL (1.39 pmol/l), and coefficient of 1.2% for intraassay and of 2.7% for interassay variability. Lipid and Glucose Assays Serum total cholesterol and triglycerides were measured by enzymatic procedures. Precipitation was used to measure HDL cholesterol, whereas LDL cholesterol was calculated according to the equation: LDL cholesterol Total cholesterol Triglycerides/5 HDL cholesterol. Apolipoproteins A-I, A-II, and B were measured by tholosimetria (Biodesign, Kennebunkport, ME), with coefficient of 7.9% for intra-assay and of 10.1% for interassay variability for A1, 7% and 9% for A2, and 6.5% and 8.2% for B, respectively. An ELISA method with coefficient of intra-assay and interassay variability of 6.0% and 12.7% was used to measure Lp(a) (Innotest, Innogenetics, Gent, Belgium). Glucose was measured by an enzymatic method (BioSystems, Barcelona, Spain). Indices of Carbohydrate Metabolism Carbohydrate Metabolism Indices Derived From Fasting Values. Insulin sensitivity was estimated by the following index: Glucose-to-insulin ratio (16) Glucose at baseline (mg/dl)/insulin at baseline ( IU/mL). Insulin resistance was estimated by the following index: the homeostasis model assessment (17) [Insulin at baseline (pmol/l) Glucose at baseline (mmol/l)]/135. Carbohydrate Metabolism Indices Derived From OGTT Results. Insulin sensitivity was estimated by the following index (18): Insulin sensitivity index [ BMI] [ insulin at 120 minutes (pmol/l)] [ glucose at 90 minutes (mmol/l)]. Beta-cell secretion of insulin was estimated by the following indices (18): Predicted index of first phase of insulin secretion (1st PHIS) 1283 [1.289 insulin at 30 minutes (pmol/l)] [138.7 glucose at 30 minutes (mmol/l)] [3.772 insulin at baseline (pmol/l)] and Predicted index of second phase of insulin secretion (2nd PHIS) 287 [ insulin at 30 minutes (pmol/l)] [26.07 glucose at 30 minutes (mmol/l)] [ insulin at baseline (pmol/l)]. 422 Mastorakos et al. Insulin resistance in adolescent PCOS Vol. 85, No. 2, February 2006

4 TABLE 2 Carbohydrate metabolism indices of adolescent girls with PCOS before (baseline) and 12 months after initiation of treatment with desogestrel/ethinyl E 2 (group A) or cyproterone acetate/ethinyl E 2 (group B) (mean SE). Carbohydrate metabolism indices Group A baseline Group A at 12 months Group B baseline Group B at 12 months Fasting glucose (mg/dl) Fasting insulin ( lu/ml) Fasting G/I a HOMA a a Predicted ISI a Predicted 1st PHIS 1, , b 1, , a Predicted 2nd PHIS b a AUCI 7, , , , , a Note: G/I Glucose-to-Insulin ratio, HOMA homeostasi model of assessment, ISI Insulin sensitivity index. a Statistically significant difference at P.05 in the same group from baseline. b Statistically significant difference at P.05 between the two groups at the same time point. Hyperinsulinemia was estimated by the of the area under the curve of insulin ( AUCI). The AUCI during OGTT was calculated after having subtracted from each of the 15-, 30-, 60-, 90-, and 120-minute insulin values that of 0 minutes and applied the trapezoidal rule of area calculation. Statistical Analysis Basal parameters of the two groups were compared by the Mann-Whitney U nonparametric test. Statistical analysis was performed by multiple regression analysis and ANOVA repeated measures. For multiple regression, analysis of direct and indirect measurements of carbohydrate metabolism (glucose-to-insulin ratio, homeostasis model assessment, insulin sensitivity index, 1st PHIS, 2nd PHIS, AUCI) were considered as independent variables. Anthropometric measurements (BMI, WHR), clinical expression (Ferriman-Gallway score), and hormone values (T, free T, SHBG) of androgen secretion, and measurements of lipid profile (total cholesterol, HDL, total cholesterol-to-hdl ratio, LDL, LDL/HDL) were considered as dependent variables. However, this assumption does not necessarily ascertain causality. The LSD post-hoc test was applied and statistical significance was set at P.05. A post-hoc power analysis of the study has been done considering insulin sensitivity index as the primary outcome of the study (19). shown in Tables 1 and 2, respectively. Insulin and glucose values during OGTTs performed before the initiation of treatment are presented in Figures 1, 2, 3, and 4 (solid lines). Before treatment, four patients presented with impaired fasting glucose and none presented with impaired glucose tolerance. Twenty-one (58.33%), 10 (27.77%), and 5 (13.88%) patients presented with a WHR, respectively, 0.80, between FIGURE 1 Glucose levels (mean SE) during oral glucose tolerance test before initiation (solid line) and after 12 months (dotted line) of treatment with desogestrel/ethinyl E 2 (group A). RESULTS Initial Values and Correlations The initial values of clinical, hormonal, and lipid profile characteristics and of carbohydrate metabolism indices are Fertility and Sterility 423

5 FIGURE 2 Glucose levels (mean SE) during oral glucose tolerance test before initiation (solid line) and after 12 months (dotted line) of treatment with cyproterone acetate/ethinyl E 2 (group B). FIGURE 4 Insulin levels (mean SE) during oral glucose tolerance test before initiation (solid line) and after 12 months (dotted line) of treatment with cyproterone acetate/ethinyl E 2 (group B) and 0.85, and Fasting glucose-to-insulin ratio ratio correlates negatively to WHR. Homeostasis model assessment and AUCI correlate positively to WHR and BMI. Insulin sensitivity index show a negative correlation to WHR and BMI. First and 2nd PHIS correlate positively to WHR. Homeostasis model assessment and AUCI correlate positively to the hirsutism score. Regarding androgens, the insulin sensitivity index shows a negative correlation to T and free T, although the glucose-to-insulin ratio has a positive correlation to SHBG. Fasting glucose-to-insulin ratio correlates negatively to total cholesterol, LDL cholesterol, total cholesterol/hdl FIGURE 3 Insulin levels (mean SE) during oral glucose tolerance test before initiation (solid line) and after 12 months (dotted line) of treatment with desogestrel/ethinyl E 2 (group A). cholesterol, and LDL cholesterol/hdl cholesterol ratios and positively with HDL cholesterol. The insulin resistance index, homeostasis model assessment, 1st PHIS, and 2nd PHIS correlate positively to total cholesterol, LDL cholesterol, total cholesterol/hdl cholesterol, and LDL cholesterol/ HDL cholesterol ratios. The AUCI correlates positively with total cholesterol/hdl cholesterol and LDL cholesterol/ HDL cholesterol ratios. No correlation was found between any carbohydrate metabolism index and apolipoproteins AI, AII, B, Lp(a), and triglycerides. Carbohydrate Metabolism Indices During Treatment During treatment BMI and WHR remained statistically unchanged in both groups of adolescent girls with PCOS (group A, WHR , BMI ; group B, WHR , BMI ). Insulin and glucose values during OGTTs performed at the end of the treatment period are presented in Figures 1, 2, 3, and 4 (dotted lines). After 12 months of treatment three patients presented with impaired fasting glucose and another three presented with impaired glucose tolerance. Insulin Resistance During Treatment A decline of fasting glucose-to-insulin ratio after 12 months of treatment was observed in both groups, although it was statistically significant only in the cyproterone acetate combined OC-group. The homeostasis model assessment increased significantly in both groups. The insulin sensitivity index decreased significantly only in the cyproterone acetate combined OC-treated group. However, no difference was found between the insulin sensitivity index of the two groups at 12 months of treatment. A post-hoc power analysis revealed that to establish such a significant difference, the 424 Mastorakos et al. Insulin resistance in adolescent PCOS Vol. 85, No. 2, February 2006

6 number of the studied subjects should be more than 1,500 in each group. Insulin Secretion During Treatment First and 2nd PHIS increased significantly only in the cyproterone acetate combined OC-treated group. When the 1st and 2nd PHIS were compared between the two groups at the 12th month of treatment, a significant difference was found. A statistically significant increase in AUCI was observed only in the cyproterone acetate combined OC-treated group. DISCUSSION In this study we found that the administration of two formulations of combined OCs (desogestrel/ethinyl E 2 and cyproterone acetate/ethinyl E 2 ) to adolescent girls with PCOS increases insulin resistance. In addition, the administration of cyproterone acetate combined OC was associated with hyperinsulinemia. We have previously reported that in adolescent girls with PCOS both formulations were comparably effective at ensuring normal menstrual cycles and at decreasing hirsutism and androgens levels (12). Before initiation of treatment, the fasting glucose-toinsulin ratio, an index of insulin sensitivity, correlated negatively to WHR. Insulin sensitivity index, on one hand, and homeostasis model assessment and AUCI, indices of insulin resistance and hyperinsulinemia, on the other hand, correlated negatively and positively with WHR and BMI, respectively. The 1st and 2nd PHIS, indices of insulin secretion, correlated positively to WHR. By using euglycemic hyperinsulinemic clamps, hyperinsulinemia and impairment of insulin sensitivity were found in obese adult patients with PCOS (20). Interestingly, the responses of insulin during OGTT are significantly greater in both obese and nonobese adult PCOS women compared with weight-matched controls, whereas blood glucose levels are increased only in adult obese PCOS women (21). Android fat distribution, often observed in patients with PCOS, contributes independently of obesity to the risk for insulin resistance and diabetes mellitus type 2 (22). In this study, the hirsutism score correlated positively with the insulin resistance index, homeostasis model assessment, hyperinsulinemia index, and AUCI. The insulin sensitivity index correlated negatively to T and free T. The glucose-toinsulin ratio correlated positively with SHBG. These data are in accordance with data indicating a positive correlation between insulinemia and androgen levels in adolescent and adult PCOS women (6, 23, 24). Insulin stimulates, in vitro, ovarian production of androgens (25). Insulin receptors are found in the ovary, but insulin might also act through ovarian Insulin-like growth factor 1 receptors resulting in hyperandrogenism (26). Hyperandrogenism is reduced when women with PCOS are given pharmaceutical agents that reduce insulin levels or facilitate its action, such as diazoxide, somatostatin analog, or metformin (27, 28). Administration of antiandrogens has been reported to lead to significant improvement of insulin resistance in women with PCOS (29). Regarding the relationship of insulin and lipids, we found that insulin sensitivity expressed by a fasting glucose-toinsulin ratio correlates negatively to total cholesterol, LDL cholesterol, total cholesterol/hdl cholesterol, and LDL cholesterol/ HDL cholesterol ratios and positively to HDL cholesterol. Furthermore, insulin resistance expressed by the homeostasis model assessment, and insulin hypersecretion, expressed by 1st and 2nd PHIS correlate positively with total cholesterol, LDL cholesterol, total cholesterol/hdl cholesterol, and LDL cholesterol/hdl cholesterol ratios. Hyperinsulinemia also expressed by AUCI correlated positively to total cholesterol/hdl cholesterol and LDL cholesterol/hdl cholesterol ratios. It is known that insulin resistance is associated to a characteristic atherogenic lipid profile (increased levels of triglycerides and LDL cholesterol, decreased levels of HDL cholesterol, postprandial increase of the large triglycerides lipiprotein particles) (30). The suppression of lipolysis is reduced in insulin resistance, leading to an increased flux in nonesterified fatty acids from central sites to the portal circulation, which augments triglyceride release. In turn, LDL cholesterol concentrations increase and HDL cholesterol concentrations decrease through the mediation of cholesterol ester transfer protein. The decreased action of lipoprotein lipase in insulin resistant states may also contribute to this complex of lipoprotein disturbance (31). After 12 months of treatment we found that fasting baseline insulin resistance expressed by the homeostasis model assessment increased significantly in both groups. In our study, a fasting glucose-to-insulin ratio decreased significantly only in the cyproterone acetate-treated group. However, it has been shown that indices derived from the OGTT glucose and insulin values predict insulin sensitivity and insulin secretion with better accuracy than those indices where only fasting baseline values are used (18). It has been suggested that OGTT is the best simple office-based method to assess women with PCOS because it provides information about both insulin resistance and glucose intolerance (32). In this study, we found that the insulin sensitivity index decreased significantly only in the cyproterone acetate combined OC-treated group. Furthermore, 1st and 2nd PHIS, indices of insulin secretion, and AUCI, index of hyperinsulinemia, increased significantly only in this group. In the past, adult patients treated with cyproterone acetate combined with OC and studied by glucose clamps or OGTT presented a reduction in insulin sensitivity (33, 34). However, there are observations reporting no significant changes in AUCI and AUC glucose during OGTT before and after treatment of adult PCOS women with a cyproterone acetatecombined OC (35, 36). Also, by using hyperinsulinemic clamps, Armstrong et al. (37) reported that the glucose infusion rate required to maintain euglycemia did not change in adult women with PCOS before and after treatment with this OC. Treatment with a desogestrel-combined OC of Fertility and Sterility 425

7 obese nondiabetic hyperandrogenic adult women as well as of healthy women resulted in deterioration of glucose tolerance during OGTT (38). This intolerance is often reversible within 6 months of discontinuation of treatment (39). However, Escobar-Morreale et al. (40) found that the administration of desogestrel-combined OC to hirsute adult women for six cycles significantly reduces fasting insulin levels and insulin resistance assessed by the homeostasis model assessment. It has been reported that the desogestrel OC increases insulin half-life by 28% without affecting insulin secretion (41). Thus, it seems that the insulin profile results from a combination of estrogen-induced insulin resistance and insulin half-life changes (41). It must be noted that the measurable but generally not clinically relevant negative impact of OCs on metabolic measures is known from previous studies. 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