Metformin therapy improves the menstrual pattern with minimal endocrine and metabolic effects in women with polycystic ovary syndrome

FERTILITY AND STERILITY VOL. 69, NO. 4, APRIL 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Metformin therapy improves the menstrual pattern with minimal endocrine and metabolic effects in women with polycystic ovary syndrome Laure C. Morin-Papunen, M.D.,* Riitta M. Koivunen, M.D.,* Aimo Ruokonen, M.D., and Hannu K. Martikainen, M.D.* University Central Hospital of Oulu, Oulu, Finland Objective: To determine the clinical, hormonal, and biochemical effects of 4 6 months of metformin therapy in obese patients with polycystic ovary syndrome (PCOS). Design: Prospective study. Setting: The Gynecological Endocrine Unit of University Central Hospital, Oulu, Finland. Patient(s): Twenty obese patients with PCOS. Intervention(s): Patients were treated with 0.5 g of metformin three times daily for 4 6 months. Main Outcome Measure(s): Clinical symptoms, menstrual pattern, and hirsutism, as well as serum concentrations of sex steroids, sex hormone-binding globulin (SHBG), gonadotropins, and lipids were assessed during the treatment. Result(s): Eleven women (68.8% of the women with menstrual disturbances) experienced more regular cycles during therapy. No changes in hirsutism, body mass index, or blood pressure occurred. The mean testosterone level was decreased significantly after 2 months of treatment but returned to the starting level by 4 6 months. Free testosterone levels decreased significantly during the treatment. There was no significant change in the levels of other sex steroids or lipids measured at 4 6 months of treatment. Conclusion(s): Metformin therapy is well tolerated by the majority of patients and may be clinically useful, especially in obese patients with PCOS and menstrual disturbances. (Fertil Steril 1998;69:691 6. 1998 by American Society for Reproductive Medicine.) Key Words: PCOS, metformin, insulin resistance, hyperinsulinemia Received April 25, 1997; revised and accepted December 22, 1997. Metformin hydrochloride, Diformin, Leiras, Finland. * Department of Obstetrics and Gynecology. Department of Clinical Chemistry. Reprint requests: Laure C. Morin-Papunen, M.D., Department of Obstetrics and Gynecology, University Central Hospital of Oulu, Kajaanintie 50 90220 Oulu, Finland (FAX: 358-8-3154310). 0015-0282/98/$19.00 PII S0015-0282(98)00011-9 Polycystic ovary syndrome (PCOS) is a disorder characterized by chronic anovulation, elevated androgen levels, signs of hyperandrogenism, enlarged cystic ovaries, and obesity. Many patients with PCOS also have metabolic disturbances, which may have important implications for long-term health (1). Both lean and obese women with PCOS show evidence of decreased insulin sensitivity (2), but insulin resistance, accompanied by compensatory hyperinsulinemia, is most marked when there is an interaction between obesity and the syndrome (1, 3). There is a strong correlation between hyperinsulinemia and hyperandrogenism (4), and it has been shown recently that insulin concentrations have a strong positive correlation with adrenal steroid secretion in hyperandrogenic patients with PCOS (5). It therefore appears that insulin resistance precedes, and may even cause, hyperandrogenism, and not the reverse (3, 6, 7). Hyperinsulinemia in patients with PCOS is often accompanied by elevated levels of total and low-density lipoprotein (LDL), cholesterol, and triglyceride (8). This lipid pattern is a well-established risk factor for coronary artery disease (9). Insulin is an atherogenic hormone, and hyperinsulinemia, independent of other factors, is a risk for the development of cardiovascular disease in diabetic and nondiabetic individuals (10, 11). Furthermore, insulin resistance and hyperinsulinemia are major risk factors for the development of noninsulin-de- 691

pendent diabetes mellitus (NIDDM) at a young age (3). Many treatments have been tested to alleviate the clinical symptoms of PCOS, but most of them, such as oral contraceptives and sequential progestins, do not abolish the basic problem, that is, insulin resistance. Metformin is a biguanide antihyperglycemic drug used to treat NIDDM. It lowers blood glucose mainly by increasing the intestinal use of glucose, enhancing peripheral glucose uptake, and inhibiting hepatic glucose production. It also enhances insulin sensitivity at postreceptor levels and stimulates insulin-mediated glucose disposal but does not stimulate insulin secretion (12). Velazquez et al. (13) studied 26 women with PCOS treated with metformin. Despite the short treatment period (8 weeks), they were able to show an improvement in insulin sensitivity associated with decreases in serum LH and androgens. They postulated that most of the metabolic disturbances of PCOS can be reversed by metformin, with the additional benefit of normalization of the endocrine conditions to allow regular menstrual cycles, reversal of infertility, and spontaneous pregnancy (13). In contrast, Crave et al. (14) showed that the administration of metformin in 24 obese hirsute patients had no additional benefit over the effect of a low-calorie diet in improving hyperinsulinemia and hyperandrogenism. The aim of our study was to assess in more detail the effects of long-term metformin therapy on clinical and biochemical indices in obese patients with PCOS. MATERIALS AND METHODS Patients Thirty-one women with PCOS were recruited from the Endocrinology Outpatient Clinic at the University Hospital of (Oulu, Finland). The ages of the patients varied between 20 and 41 years (mean, 30 years). Eleven women stopped the treatment after 2 months for various reasons: three underwent IVF-ET treatment, one conceived, one moved away, three had side effects (vomiting and diarrhea lasting for 2 months), two had no apparent reason, and one because she received no help for her hirsutism. Twenty women continued the treatment for 4 6 months and were included in the analysis. Regular (normal) menstrual cycles were defined as cycles with a minimum-maximum length variation of 4 days and with an intermenstrual interval between 21 and 35 days (15). Irregular cycles were those with variations of the intermenstrual interval of more than 4 days and a period of 35 days. Oligomenorrhea was defined as menstruation with an interval of 35 days and amenorrhea as the absence of periods for 6 months. The criteria for PCOS were as defined by Homburg (16). All of the patients had polycystic ovaries shown by vaginal ultrasonography ( 8 subcapsular follicles of 3 8 mm diameter in one plane in one ovary and increased stroma) and at least one of the following symptoms: oligomenorrhea or amenorrhea (13/20, 65%), hirsutism (Ferriman-Gallwey score of 7) (17) (12 of 20, 60%), or acne (7 of 20, 35%). Seventeen (85%) of the patients were obese, with a body mass index (BMI) of 27 kg/m 2. Ten of 20 (50%) had elevated serum testosterone levels ( 0.89 ng/ml). One of the patients had hypothyroidism, had used thyroxine medication before the study, and was euthyroid. After 4 months of treatment, hypothyroidism was diagnosed in one other patient. Patients were excluded if they were diabetic, smoked, used alcohol, or had received sex hormones or drugs known to affect lipoprotein metabolism during the 2 months preceding the study. A 1-mg dexamethasone suppression test was performed in hirsute and hyperandrogenic women (testosterone level of 1.5 ng/ml to exclude Cushing s syndrome. Protocol of the Study The treatment consisted of 500 mg of metformin therapy (metformin hydrochloride, Diformin; Leiras, Finland) administered three times daily. All patients were evaluated before the treatment, 18 patients were evaluated at 2 months, and all 20 were evaluated at 4 6 months. Blood pressure was measured after the patient had been sitting for 20 minutes. Transvaginal ultrasonography with a 6-MHz probe (General Electric RT-200; Milwaukee, WI) was performed to measure ovarian volumes and the number of follicles. Volume determinations were made using the formula for the volume of an ellipsoid: 0.523 length width thickness (18). Venous blood samples were obtained after 10 12 hours of fasting within the first week of the cycle for those patients who were menstruating or at any convenient time for amenorrheic women. An oral glucose tolerance test (OGTT) and insulin response were performed before and after metformin treatment after 10 12 hours of fasting between 8 A.M. and 10 A.M. After a basal blood sample was obtained, a 75-g glucose load was administered orally. Blood samples were obtained, at 60 and 120 minutes after glucose loading for glucose and insulin determinations. Glucose tolerance was evaluated using the criteria of the World Health Organization (19). The study was approved by the Ethics Committee of the University Hospital of Oulu, Oulu, Finland, and informed written consent was obtained from each subject. Assays The concentrations of sex hormone-binding globulin (SHBG), LH, FSH, and TSH were analyzed by fluoroimmunoassays (Wallac Ltd., Turku, Finland); prolactin and free T 4 were assessed by a chemiluminescence system (Ciba Corn- 692 Morin-Papunen et al. Metformin in PCOS Vol. 69, No. 4, April 1998

TABLE 1 Menstrual pattern before and during metformin therapy. Patient no. Menstrual period before therapy Menstrual period during therapy 1 Oligomenorrhea Irregular 2 Oligomenorrhea Irregular 3 Regular Regular 4 Oligomenorrhea Irregular 5 Regular Regular 6 Oligomenorrhea Irregular 7 Irregular Regular 8 Oligomenorrhea Oligomenorrhea 9 Oligomenorrhea Oligomenorrhea 10 Oligomenorrhea Oligomenorrhea 11 Oligomenorrhea Irregular 12 Oligomenorrhea Irregular 13 Amenorrhea Amenorrhea 14 Irregular Regular 15 Irregular Regular 16 Amenorrhea Oligomenorrhea 17 Amenorrhea Oligomenorrhea 18 Regular Regular 19 Regular Regular 20 Amenorrhea Amenorrhea ing Diagnostics, Fernwald, Germany). Radioimmunoassays were used for DHEA, DHEAS, androstenedione, and free testosterone (Diagnostics Product Corporation, Los Angeles, CA); testosterone (Farmos Diagnostica, Orion, Turku, Finland); and serum insulin (Pharmacia, Uppsala, Sweden), following the instructions of the manufacturers. Levels of serum total cholesterol, triglycerides, highdensity lipoprotein cholesterol (HDL), and blood glucose were determined by standard methods. The serum LDL level was calculated with the Friedewald formula if the serum triglyceride level was 354 mg/dl; if the triglyceride level was 354 mg/dl, it was precipitated by heparin in isoelectric point (LDL-cholesterol; Merck, Darmstadt, Germany). Statistical Analysis A paired Wilcoxon s nonparametric rank sum test was used to compare the means of hormonal indices. The Mann- Whitney U-test was used to compare the clinical and hormonal indices between responders and nonresponders. P values were two-tailed, with 0.05 considered the limit of statistical significance. RESULTS Menstrual Pattern Eleven women (68.8% of those with menstrual disturbances before therapy) had a change in their menstrual pattern at 4 6 months of treatment: Six oligomenorrheic women achieved irregular cycles, three women with irregular cycles achieved regular menses, and two amenorrheic women became oligomenorrheic (Table 1). We divided the patients into two groups according to menstrual changes: responders (n 11) and nonresponders (n 5). Women with regular periods were excluded from these groups. The groups did not differ from each other according to mean age, BMI, or waist-hip ratio before the treatment. Hirsutism Score, BMI, Waist-Hip Ratio, and Blood Pressure There were no significant changes in the hirsutism score, BMI (Table 2), waist-hip ratio, or blood pressure during therapy. TABLE 2 Characteristics of women with PCOS before and during metformin treatment. Characteristic Before treatment After 2 mo (n 18) After 4 6 mo (n 20) Reference range BMI (kg/m 2 ) 31.5 5.6 31.9 5.3 31.2 5.7 27 Fasting insulin (mu/ml) 17.3 7.1 16.9 9.8 15.2 8.3* 2.4 28.2 Testosterone (ng/ml) 1.03 0.4 0.9 0.3 0.95 0.4 0.1 0.89 Free testosterone (pg/ml) 4.1 1.9 3.8 2.2 3.6 2.1 0.43 3.2 SHBG (ng/ml) 3.1 1.2 3.0 1.2 3.2 1.4 1.86 13 Free androgen index 12.3 5.9 10.8 6.8 12.4 9.1 1.4 7.3 LH (miu/ml) 6.7 3.9 6.9 3.8 6.5 2.8 2 10 FSH (miu/ml) 5.2 1.4 5.4 1.7 5.7 1.8 2 12 LH/FSH 1.4 0.9 1.4 0.8 1.2 0.6 2 17-OHP (ng/ml) 0.9 0.6 1.04 0.6 0.9 0.4 0.33 3.0 DHEA (ng/ml) 7.4 4.4 8.2 5.3 7.3 3.9 0.5 10.4 DHEAS ( g/ml) 2.4 0.8 2.5 0.9 2.5 0.9 0.37 5.16 Androstenedione (ng/ml) 3.7 1.8 3.7 1.6 3.9 1.7 0.2 4.6 Note. Values are means SD. * P 0.04 compared with values before treatment. P 0.03 compared with values before treatment. FERTILITY & STERILITY 693

TABLE 3 Serum gonadotropins, steroids, and SHBG concentrations before and during metformin treatment in the responders and nonresponders. Before treatment After 2 mo After 4 6 mo Hormone RE (n 11) NR (n 5) RE (n 10) NR (n 5) RE (n 11) NR (n 5) Reference range LH (miu/ml) 6.0 3.3 10.5 4.1 6.8 4.4 8.8 2.5 6.4 2.6 8.8 2.4 2 10 FSH (miu/ml) 5.3 1.7 5.2 1.4 5.6 2.0 5.2 1.6 6.4 1.9* 5.2 1.3 2 12 LH/FSH 1.3 0.9 2.1 0.8 1.4 0.9 1.8 0.6 1.1 0.5 1.8 0.6 2 Testosterone (ng/ml) 0.9 0.3 1.3 0.5 0.7 0.3 1.1 0.3 0.8 0.3 1.3 0.4 0.1 0.9 Free testosterone (pg/ml) 3.7 1.4 6.1 2.6 2.8 0.9 6.6 2.4 3.1 1.5 5.8 2.5 0.4 3.2 SHBG (ng/ml) 3.4 1.4 2.4 3.5 3.1 1.2 2.6 1.5 3.3 1.4 2.4 1.6 1.9 13.0 Free androgen index 10.3 4.6 18.4 5.9 8.3 3.3 17.8 9.6 9.1 5.1 22.1 12.4 1.4 7.3 Androstenedione (ng/ml) 3.1 1.3 4.7 1.5 2.9 1.0 5.3 1.3 3.4 1.6 5.5 1.3 0.2 4.6 17-OHP (ng/ml) 0.7 0.4 1.3 0.7 1.0 0.5* 1.3 0.9 0.9 0.4 1.2 0.5 0.3 3.0 DHEA (ng/ml) 4.8 1.2 12.3 4.9 5.7 2.6 14.0 6.5 6.1 2.7 11.5 4.6 0.5 10.4 DHEAS ( g/ml) 2.0 0.6 3.5 0.8 2.1 0.6 3.5 0.5 2.1 0.9 3.0 0.9 0.4 5.2 Fasting insulin (mu/ml) 16.3 4.1 21.0 11.3 14.7 6.4 13.2 5.1 13.1 4.6 21.5 11.9 2.4 28.2 Note. Values are means SD. RE responders (change in menstrual pattern), NR nonresponders (no change in menstrual pattern). Women with regular menstruation were excluded. * P 0.05 compared with values before treatment among RE. P 0.05 (NR vs. RE before treatment). P 0.05 (NR vs. RE after 6 months of treatment). P 0.05 (NR vs. RE after 2 months of treatment). Ovarian Volume The respective mean volumes of the right and left ovary were 9.3 and 9.7 cm 3 before therapy, 8.1 and 8.6 cm 3 at 2 months, and 8.1 and 7.6 cm 3 at 4 6 months of treatment. The changes were not significant. Sex Steroids and SHBG The mean testosterone level decreased significantly at 2 months but returned close to the starting level after 6 months (Table 2). The mean serum level of free testosterone was high before the treatment and decreased significantly at 4 6 months of treatment but stayed above the normal reference range (Table 2). The serum SHBG level was within normal limits at the beginning of the study and did not change significantly during metformin treatment. The mean of the free androgen index was above normal limits in the beginning of the study and did not change significantly during therapy (Table 2). The mean serum levels of 17 -hydroxyprogesterone (17-OHP), DHEA, DHEAS, and androstenedione were normal before treatment and did not change significantly during therapy. In the basal state, the serum levels of 17-OHP, DHEA, DHEAS, and free androgen index were significantly lower in the responders than in the nonresponders (Table 3). After 2 months of treatment, the serum concentrations of testosterone, free testosterone, free androgen index, DHEA, and DHEAS were significantly lower in the responders. At 4 6 months of treatment, the serum testosterone, free testosterone, free androgen index, DHEA, and androstenedione concentrations were significantly lower in the responders than in the nonresponders (Table 3). Pituitary Hormones In the basal state, the mean serum LH and FSH levels were within the normal ranges. There was no significant change in gonadotropins during the treatment in the whole study group (Table 2). However, the responders showed a significant increase in serum FSH after 4 6 months of treatment. There was no significant change in the LH level in either the responder or the nonresponder group (Table 3). The baseline level of prolactin was normal, and there was no significant change during the treatment. The serum TSH and free T 4 concentrations did not change significantly during metformin therapy. Glucose and Insulin Of the 20 patients continuing the treatment for 4 6 months, OGTT was done in 10 patients before the treatment and at 4 6 months of treatment. The results of the OGTT were normal for eight women. Two patients had impaired glucose tolerance before treatment, one of them with an elevated 60-minute value and one with an elevated 120- minute value. One of them had values within normal limits after 6 months. None of the patients had values diagnostic for NIDDM. The mean fasting insulin level obtained from all patients before treatment and at 2 and 4 6 months of treatment showed a significant decrease at 4 6 months of therapy (P 0.04) (Table 2). The mean area under the insulin curve (AUC insulin) for the OGTT decreased significantly (P 0.01) by 54% at 4 6 months of treatment. At 4 6 months, we could not find any significant correlation between the 694 Morin-Papunen et al. Metformin in PCOS Vol. 69, No. 4, April 1998

decrease in serum fasting insulin and the changes in FSH, LH, LH/FSH, 17-OHP, DHEA, DHEAS, androstenedione, free androgen index, testosterone, and free testosterone. There was no significant difference in the fasting insulin concentrations between responders and nonresponders before treatment or at 2 or 4 6 months of treatment. Lipids The serum cholesterol, HDL, LDL, and triglyceride concentrations were within normal ranges before the treatment and did not change significantly during the study. DISCUSSION To our knowledge, this is the first study in which metformin has been administered for as long as 6 months, allowing evaluation of the clinical value of this therapy in patients with PCOS. The most important change was seen in the menstrual pattern during metformin therapy. Up to 70% of the women with menstrual disturbances achieved more regular menstruation with metformin (responders). A similar effect was seen in the study by Velazquez et al. (13), in which 7 (27%) of the 26 patients resumed normal menses and 3 became pregnant. Unfortunately, we could not assess how many of the cycles were ovulatory during therapy. However, spontaneous menstruation is psychologically important for the patient because it implies better ovarian function. In addition, the more frequent occurrence of menses in these patients may alleviate the known risks of endometrial hyperplasia and carcinoma in obese patients with PCOS (20). Despite these apparently durable clinical effects, the serum testosterone level returned close to the starting value after 6 months of treatment, after being transiently decreased at 2 months of therapy. These changes were also observed in another recent study (14). Hence, the metformin effect may be to some extent transitional, and some adaptation may occur during more prolonged therapy. The hirsutism score did not change during the treatment. There was no change in the level of 17-OHP during metformin treatment. In this study, nonresponders had higher 17-OHP levels than responders did before treatment. This is in contrast to a recent study (21), in which metformin produced a significant decrease in the basal and leuprolideinduced 17-OHP and testosterone levels. The investigators postulated that obese patients with PCOS may have an abnormality of intracellular insulin signaling or of P450 c17a activity in the ovary, which renders the enzyme complex more sensitive to insulin, and that amelioration of insulin resistance by metformin may return the activity of the enzyme to normal (21). The reason for this discrepancy remains uncertain, but it may be due to the difference in the patient populations; our patients were less obese. It is possible that the hypersensitivity of ovarian cytochrome P450 to insulin in patients with PCOS may appear only in those with more severe obesity. After 4 6 months of treatment, there was a significant decrease in the levels of fasting insulin and in the AUC insulin in the OGTT, as reported previously in some (13, 21), but not in all (22, 23) studies. However, because OGTT was performed for only five women from the responder group and for two women from the nonresponder group, it was not possible to compare these groups with regard to the OGTT. In this study, no change was observed in the SHBG levels during metformin treatment. Because insulin inhibits SHBG production by Hep G2 cells (24), we could have expected an increase in SHBG levels during metformin therapy, as was reported in some previous studies (13, 21). The increase in SHBG observed in an earlier study (13) was probably due to weight loss and the simultaneous improvement of insulin resistance, rather than to any effect of metformin (14). The BMI remained constant in our study, and the change in insulin secretion was obviously too small to affect SHBG production. Metformin treatment had little effect on blood lipids or blood pressure in this study. However, abnormal lipid values in some patients normalized during the treatment, which is in accordance with the results of another recent study in obese women with PCOS (13). This is in contrast to a previous report of diabetic and nondiabetic men, in whom long-term therapy with metformin resulted in a moderate reduction in plasma triglyceride and total cholesterol levels and in a small increase in plasma HDL concentrations (25). Our results support the fact that, despite the small metabolic and hormonal changes, metformin therapy is well tolerated by the majority of patients and may be clinically useful, especially in obese patients with PCOS and menstrual disturbances and in those women with PCOS who are unable to achieve and maintain a durable, diet-induced weight loss. However, the effect may be transitory with regard to testosterone levels, and women with PCOS and hirsutism did not seem to benefit from metformin therapy. Acknowledgments: We thank Tarja Bützow, M.D., of the Family Federation of Finland, Helsinki, and Professor A. Kauppila, Department of Gynecology and Obstetrics, University Hospital of Oulu, Finland, for their critical and thoughtful readings of the manuscript. References 1. Franks S. Polycystic ovary syndrome: a changing perspective. Clin Endocrinol (Oxf). 1989;31:87 120. 2. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral insulin resistance independent of obesity in polycystic ovary syndrome. Diabetes 1989;38:1165 74. 3. Dunaif A. Molecular mechanisms of insulin resistance in the polycystic ovary syndrome. Semin Reprod Endrocrinol 1994;12:15 20. FERTILITY & STERILITY 695

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