Effects of Rosiglitazone in Obese Women with Polycystic Ovary Syndrome and Severe Insulin Resistance
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《临床内分泌与代谢杂志》
Abstract
Our objective was to evaluate the effectiveness of the insulin-sensitizing agent rosiglitazone in obese women with polycystic ovary syndrome (PCOS) and severe insulin resistance. Twelve obese women with PCOS were recruited. All were hirsute and anovulatory with acanthosis nigricans indicating severe insulin resistance. All women were treated with 4 mg of rosiglitazone daily for 6 months. A standard 75-g oral glucose tolerance test with insulin levels was performed before and after the women were treated with rosiglitazone. Glucose and insulin areas under the curve (AUC) were calculated. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate, LH, and 17-hydroxyprogesterone were also measured before and after treatment. The body mass index was determined before and after treatment. There was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during oral glucose tolerance test and basal total testosterone levels. After treatment with rosiglitazone, there were significant decreases in fasting insulin levels (46.0 ± 6.5 vs. 16.9 ± 2.0 μU/ml; P < 0.001), insulin AUC (749.3 ± 136.3 vs. 225.0 ± 15.7 μU/ml; P = 0.003), fasting glucose levels (90.8 ± 3.0 vs. 81.8 ± 1.9 mg/dl; P = 0.003), and glucose AUC (437.9 ± 25.0 vs. 322.5 ± 14.7 mg/dl; P < 0.001). Both total testosterone (96.3 ± 17.3 vs. 56.1 ± 5.8 ng/dl; P = 0.01) and free testosterone (5.8 ± 0.6 vs. 3.4 ± 0.5 pg/ml; P < 0.001) decreased significantly after treatment, although there was no significant change in LH levels. Levels of SHBG increased significantly (18.3 ± 3.4 vs. 25.8 ± 6.6 nmol/liter; P = 0.009) after treatment, and dehydroepiandrosterone sulfate levels decreased significantly (P = 0.04). There was no significant change in body mass index (40.4 ± 2.4 vs. 41.1 ± 2.7 kg/m2). Eleven of the women reverted to regular ovulatory cycles during the treatment period. We conclude that 1) rosiglitazone therapy improves insulin resistance and glucose tolerance in obese women with PCOS; 2) rosiglitazone decreases ovarian androgen production, which appears to be independent of any changes in LH levels; 3) hyperinsulinemia appears to play a key role in the overproduction of ovarian androgens in these women because attenuation of insulin levels is associated with decreased testosterone levels; and 4) short-term rosiglitazone therapy helps restore spontaneous ovulation.
Introduction
POLYCYSTIC OVARY SYNDROME (PCOS) is one of the most common endocrinopathies affecting 4–10% of women of reproductive age (1, 2). It is characterized by hyperandrogenism and chronic anovulation. Approximately 44% of women with PCOS are obese, and 60% display insulin resistance (2, 3). Hyperinsulinemia contributes to the hyperandrogenism by increasing ovarian androgen production and by suppressing hepatic production of SHBG with consequent increase in free testosterone levels (4). The hyperinsulinemia found in PCOS is more profound in obese patients, although the presence of insulin resistance appears to be independent of body weight (5). Presence of acanthosis nigricans in women with PCOS indicates severe insulin resistance and high risk for type 2 diabetes (6, 7). Women with hyperandrogenism, insulin resistance, and acanthosis nigricans syndrome have severe insulin resistance, and insulin receptor mutations have been observed in these women (8, 9).
In the past, therapeutic approaches to PCOS have focused on suppressing ovarian androgen production or ovulation induction. Recently, insulin sensitizers have been used to reduce the level of hyperinsulinemia and its negative impact on ovarian function and possibly to prevent long-term consequences of hyperinsulinemia. Women with PCOS are at higher risk for hypertension, dyslipidemia, type 2 diabetes, and cardiovascular disease (10, 11, 12). Metformin is an oral hypoglycemic agent that has been shown to improve insulin sensitivity and ovarian function in women with PCOS (13, 14, 15). Metformin is not, however, effective in obese women with PCOS (16, 17).
Rosiglitazone is a member of the thiazolidinediones (TZD) family that has been shown to be effective in the treatment of type 2 diabetes. TZDs are newer oral antidiabetic agents that exert their insulin-sensitizing actions through the peroxisome proliferator-activated receptor found in a number of tissues including the liver, skeletal muscle, and adipose tissue (18). TZDs increase insulin sensitivity without increasing insulin secretion through activation of multiple genes, including the up-regulation of glucose transporters (18). Troglitazone, a member of the TZD family, was found to have beneficial effects on insulin sensitivity and ovarian function in women with PCOS (18, 19, 20); it was taken off the market, however, over concerns of hepatotoxicity. Currently there are only limited data on the use of rosiglitazone in PCOS. The aim of our present study is to evaluate the efficacy of rosiglitazone on insulin resistance and hyperandrogenism in obese women with PCOS and severe insulin resistance.
Subjects and Methods
Subjects
We recruited 12 women with PCOS who were obese, hirsute, anovulatory, and had acanthosis nigricans, indicating severe insulin resistance to participate in the study. The National Institutes of Health/ National Institute of Child Health and Human Development criteria were used to establish PCOS. All of the women had polycystic ovaries on pelvic ultrasound examination. Menstrual history revealed either oligomenorrhea (cycle length > 45 d) or amenorrhea (cycle length > 6 months). All had normal TSH and prolactin levels. Women with possible late-onset congenital adrenal hyperplasia (17-hydroxyprogesterone levels > 2 ng/ml) and possible virilizing ovarian tumors were excluded from the study. In one patient who had testosterone levels higher than 200 ng/dl, the possibility of an ovarian tumor was excluded by wedge biopsy of the ovaries. Histological examination revealed stromal hyperthecosis. This study was approved by the University of Texas Medical Branch Institutional Review Board, and written informed consent was obtained from all subjects. After a 3-d high-carbohydrate diet, patients were instructed to fast overnight and were subsequently admitted to our clinical research center. The body mass index (BMI) of all the women was calculated, and a standard 75-g 3-h oral glucose tolerance test (OGTT) was performed. Glucose and insulin levels were measured at baseline and at 1, 2, and 3 h after the oral glucose administration. Glucose and insulin areas under the curve (AUC) were calculated using the trapezoidal method. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate (DHEA-S), LH, and 17-hydroxyprogesterone were also measured. All of the women were then treated with rosiglitazone, 4 mg once a day. They were advised to follow a low-fat, low-calorie diet, although no standardized diet was used. After 6 months of treatment, the women were readmitted to the clinical research center, the BMI was calculated again, and all of the studies, including the OGTT with insulin levels, were repeated. All the posttreatment tests were performed in the follicular phase of the cycle between cycle d 5 and 8. All of the results obtained were then analyzed, and the pre- and posttreatment values were compared.
Hormone assays
All the blood samples were centrifuged and the separated serum was kept frozen at –70 C until the time of the assay. Insulin, DHEA-S, SHBG, and 17-hydroxyprogesterone levels were measured by specific double antibody RIA using 125I-labeled hormones (Diagnostic Systems Laboratories, Webster, TX). Total and free testosterone levels were measured by coated tube RIA, and LH levels were measured by immunoradiometric assay. Plasma glucose levels were measured by the glucose oxidase technique. All the samples were run in duplicate. High and low controls were run with each assay, and the assay was accepted only if the controls were within the expected range. Intraassay variation ranged from 1.5–2.5%, and interassay variation ranged from 3.8 to 7.4%. Pre- and posttreatment samples from each patient were assayed in the same batch.
Statistical analysis
The statistical analysis was performed using SigmaStat software (SPSS Inc., Chicago, IL). Hormone levels before and after treatment were compared by paired t test. Correlation between insulin and testosterone levels was determined by Pearson correlation coefficients. Data are presented as mean ± SE, and P < 0.05 was considered statistically significant.
Results
All 12 women who had enrolled completed the study. Table 1 depicts patient characteristics and hormone studies before and after treatment with rosiglitazone. At baseline, there was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during OGTT (AUC) and basal total testosterone levels (Fig. 1). After treatment with rosiglitazone, there was a significant decrease in fasting insulin levels (46.0 ± 6.5 vs. 16.9 ± 2.0 μU/ml; P < 0.001) and insulin AUC (749.3 ± 136.3 vs. 225.0 ± 15.7 μU/ml; P = 0.003) (Fig. 2). Fasting glucose levels (90.8 ± 3.0 vs. 81.8 ± 1.9 mg/dl; P = 0.003) and glucose AUC (437.9 ± 25.0 vs. 322.5 ± 14.7 mg/dl; P < 0.001) also decreased with rosiglitazone treatment (Fig. 3). Both total testosterone (96.3 ± 17.3 vs. 56.1 ± 5.8 ng/dl; P < 0.01) and free testosterone (5.8 ± 0.6 vs. 3.4 ± 0.5 pg/ml; P < 0.001) decreased significantly after treatment, whereas SHBG levels increased (18.3 ± 3.4 vs. 25.8 ± 6.6 nmol/liter; P = 0.009) after treatment (Fig. 4). There was, however, no significant change in LH levels (10.1 ± 0.82 vs. 9.1 ± 0.84 mIU/ml). There was also a significant decrease in the levels of DHEA-S (1508.7 ± 181.9 vs. 1081.3 ± 180.8 ng/ml; P = 0.04). The decrease in 17-hydroxyprogesterone levels (1.02 ± 0.23 vs. 0.71 ± 0.11 ng/ml; P = 0.06) was not statistically significant. There was no change in BMI (40.4 ± 2.4 vs. 41.1 ± 2.7 kg/m2). At the dosage used, there were no adverse effects reported by our subjects, and there were no elevations in serum transaminases during the treatment period. None of our patients reported water retention.
Eleven of the 12 patients studied reverted to regular ovulatory cycles during the treatment period. Ovulation was confirmed using a cycle d 21 serum progesterone level that was more than 5 ng/ml after the onset of spontaneous menstruation. Length of the cycles during treatment varied from 28 to 32 d.
Discussion
Hyperinsulinemia appears to play a central role in the pathogenesis of PCOS. In vitro studies indicate that insulin stimulates androgen accumulations in the incubations of ovarian stroma obtained from women with hyperandrogenism (21). Insulin stimulates testosterone biosynthesis by human theca cells (22). Significant positive correlations have been observed between peripheral insulin levels and ovarian vein androgen levels (23). Insulin infusion acutely augments ovarian androgen production (24), and decreasing circulating insulin levels result in a reduction in ovarian androgens (25). Insulin has also been shown to suppress production of hepatic SHBG (26), resulting in an increase in the circulating levels of free testosterone.
Insulin-sensitizing agents ameliorate insulin resistance and hyperinsulinemia. The efficacy of metformin in improving ovulation in women with PCOS has been reported (14, 15). Not all women with PCOS, however, respond to metformin, and some experience significant gastrointestinal side effects with its use. Most obese women with PCOS fail to respond to metformin (16, 17).
Troglitazone, a peroxisome proliferator-activated receptor agonist that has been taken off the market because of hepatotoxicity, has been shown to be effective in improving endocrine and ovulatory performance in women with PCOS. In a large multicenter, double-blind trial, Azziz et al. (20) observed that troglitazone improves ovulatory function, hirsutism, hyperandrogenemia, and insulin resistance of PCOS in a dose-related fashion. In a group of obese women with PCOS and impaired glucose tolerance, Ehrmann et al. (19) observed significant reductions in total and free testosterone, fasting insulin and glucose, and insulin and glucose AUC after 12 wk of treatment with troglitazone. In both studies, levels of SHBG increased and levels of LH did not change, as observed in our current study.
Previous studies on the effect of rosiglitazone in women with PCOS are limited. Shobokshi and Shaarawy (27) compared women with PCOS who received either rosiglitazone and clomiphene citrate or clomiphene citrate only and observed that combined therapy with rosiglitazone and clomiphene citrate resulted in a significant decrease in insulin and serum-free testosterone levels. It is difficult to draw conclusions on the effects of rosiglitazone from this study, because there were no women who received rosiglitazone only. In a 2-month study, Ghazeeri et al. (28) treated obese clomiphene-resistant women with PCOS either with rosiglitazone and placebo or rosiglitazone and clomiphene citrate. The primary outcome of this study was ovulation, with changes in insulin sensitivity and androgens as some of the secondary outcomes. They reported ovulation in 77% of women taking rosiglitazone and clomiphene citrate and 33% of women taking rosiglitazone only. Lower ovulation rates with rosiglitazone only in this study could be due to the short treatment duration of only 2 months. Rosiglitazone therapy for 6 months restored ovulation in 11 of the 12 women (91%) we studied. Most of the women in our study had long periods of amenorrhea, and spontaneous menstruation occurred in these women within the first 3 months of initiation of therapy. Cataldo et al. (29) reported pregnancy in one patient after treatment with rosiglitazone for 5 months. Previous studies indicate that metformin, the most commonly used insulin-sensitizing drug in PCOS, is not very effective in the resumption of menstruation and ovulation in morbidly obese women (17). The mean BMI of the women of close to 40 kg/m2 in that study is very similar to the mean BMI of our patients. The high ovulation rate that was observed in our patients with rosiglitazone treatment indicates that TDZs may be the ideal treatment for obese PCOS women with severe insulin resistance who desire fertility. Since there have been no studies to evaluate use of rosiglitazone during pregnancy, rosiglitazone should be discontinued when pregnancy occurs.
In our study, we sought to study the effects of rosiglitazone in obese women with PCOS who had severe insulin resistance. Because obese women do not respond well to metformin, we wanted to study the effectiveness of rosiglitazone in these women. A standard OGTT with calculation of insulin AUC was used to assess insulin release and to establish insulin resistance, because this method has been validated as a sensitive predictor of insulin resistance (30). Furthermore, all of our patients had acanthosis nigricans, a cutaneous finding that is specific for severe insulin resistance (6, 7). We were able to demonstrate that rosiglitazone therapy improves insulin resistance and glucose tolerance in these women, because there were significant reductions in fasting insulin, fasting glucose, and insulin and glucose response to an oral glucose load. Ghazeeri et al. (28) also observed a significant decrease in fasting insulin levels and an increase in SHBG with rosiglitazone therapy for 2 months.
Rosiglitazone therapy resulted in a reduction in ovarian androgen production because levels of both total and free testosterone were significantly reduced. Levels of SHBG increased with therapy, further reducing the bioavailability of circulating androgens. These changes are most likely caused by improvements in insulin sensitivity, which resulted in amelioration of hyperinsulinemia and, thus, a reduction in ovarian androgen production. Hyperinsulinemia appears to play a key role in the pathogenesis of hyperandrogenism in these women. Interestingly, LH levels did not change with therapy. This finding is in agreement with previous studies with troglitazone, which showed reductions in circulating androgen levels without any changes in LH levels (18, 19, 20). Lack of change in LH levels could be due to the fact that our patients did not have elevated LH levels at baseline. Mor et al. (31) reported low LH levels in their group of obese women with PCOS and severe insulin resistance. The observation of low LH levels in our hyperinsulinemic patients is consistent with previous reports, which indicate two possible distinct phenotypes of PCOS, a low-LH/high-insulin group and a high-LH/low-insulin group (31, 32).
Rosiglitazone therapy also resulted in a significant decrease in circulating levels of adrenal androgen DHEA-S. Azziz et al. (33) observed similar effects on DHEA-S levels with troglitazone administration for 20 wk. It is not clear whether the decrease in DHEA-S levels is a result of decrease in insulin levels or is due to the direct effects of rosiglitazone on the steroidogenic enzymes in the adrenal gland. In a recent study, pioglitazone treatment for 6 months in women with PCOS reduced the adrenal androgen response to corticotrophin (34). These authors speculated that insulin enhances ACTH-stimulated adrenal androgen synthesis.
The reductions in ovarian androgen levels with rosiglitazone therapy could be a result of the decrease in the insulin levels, the direct effects on the ovary, or a combination of both. Previous studies indicate that TZDs may have a direct effect on ovarian steroidogenesis apart from improved insulin sensitivity. TZDs have been shown to directly affect gonadal steroidogenic cells. Mitwally et al. (35) found troglitazone to directly inhibit the production of androsterone by cultured rat theca interstitial cells and progesterone by cultured human granulosa lutein cells. Furnsinn et al. (36) showed that TZDs inhibit gonadal steroidogenesis in obese male Zucker rats, a finding that was independent of improvements in insulin sensitivity. Vierhapper et al. (37) demonstrated that 7 d of rosiglitazone therapy in a group of healthy men significantly reduced production rates of testosterone and dihydrotestosterone. Previous studies in our laboratory with porcine granulosa cells indicate that troglitazone reduces progesterone production (38). Gasic et al. (39) showed troglitazone to be a competitive inhibitor of ovarian 3?-hydroxysteroid dehydrogenase. Other in vitro studies have shown troglitazone to have a direct effect on various steroidogenic enzymes, including aromatase and 17 hydroxylase/17–20 lyase (40, 41).
It is of interest that the improvement in insulin sensitivity in our patients was independent of adiposity, because there were no changes in BMI with therapy. It is well established that obesity is associated with insulin resistance, and reductions in weight typically lead to improved insulin sensitivity. Previous studies have shown thiazolidinediones to cause some weight gain when used in the management of diabetes (42). Weight gain was not seen in our subjects, which is in agreement with most studies using thiazolidinediones in women with PCOS. Ghazeeri et al. (28) found no change in weight in their subjects with PCOS with short-term treatment of rosiglitazone. It has been suggested that thiazolidinediones cause redistribution of fat from different adipose compartments with the shift of adipose tissue from the visceral to the sc area (43, 44). Redistribution of fat remains a possibility in our subjects because BMI did not change. It is known that different adipose compartments can have varying effects on endocrine and metabolic factors in women with PCOS (45). Ehrmann et al. (19) found no changes in BMI or distribution of regional adiposity by using a dual-energy x-ray absorptiometry scan when they treated their PCOS subjects with troglitazone. Furthermore, Romualdi et al. (46) found no changes in BMI or waist-to-hip ratio in 18 obese subjects with PCOS who were treated with pioglitazone.
In summary, our results indicate that rosiglitazone improves insulin sensitivity and glucose tolerance in obese women with PCOS and severe insulin resistance. It also helps to restore ovulation and attenuate ovarian androgen production without weight gain or other side effects. Use of rosiglitazone appears to be an effective method in the management of obese PCOS women with severe insulin resistance.
Footnotes
This work was supported in part by Grant R01 CA 45181 from the National Institutes of Health (to M.N.) and Grant M01 RR00073 from the General Clinical Research Center Program.
First Published Online October 13, 2004
Abbreviations: AUC, Area under the curve; BMI, body mass index; DHEA-S, dehydroepiandrosterone sulfate; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; TZD, thiazolidinediones.
Received July 14, 2004.
Accepted September 24, 2004.
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Our objective was to evaluate the effectiveness of the insulin-sensitizing agent rosiglitazone in obese women with polycystic ovary syndrome (PCOS) and severe insulin resistance. Twelve obese women with PCOS were recruited. All were hirsute and anovulatory with acanthosis nigricans indicating severe insulin resistance. All women were treated with 4 mg of rosiglitazone daily for 6 months. A standard 75-g oral glucose tolerance test with insulin levels was performed before and after the women were treated with rosiglitazone. Glucose and insulin areas under the curve (AUC) were calculated. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate, LH, and 17-hydroxyprogesterone were also measured before and after treatment. The body mass index was determined before and after treatment. There was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during oral glucose tolerance test and basal total testosterone levels. After treatment with rosiglitazone, there were significant decreases in fasting insulin levels (46.0 ± 6.5 vs. 16.9 ± 2.0 μU/ml; P < 0.001), insulin AUC (749.3 ± 136.3 vs. 225.0 ± 15.7 μU/ml; P = 0.003), fasting glucose levels (90.8 ± 3.0 vs. 81.8 ± 1.9 mg/dl; P = 0.003), and glucose AUC (437.9 ± 25.0 vs. 322.5 ± 14.7 mg/dl; P < 0.001). Both total testosterone (96.3 ± 17.3 vs. 56.1 ± 5.8 ng/dl; P = 0.01) and free testosterone (5.8 ± 0.6 vs. 3.4 ± 0.5 pg/ml; P < 0.001) decreased significantly after treatment, although there was no significant change in LH levels. Levels of SHBG increased significantly (18.3 ± 3.4 vs. 25.8 ± 6.6 nmol/liter; P = 0.009) after treatment, and dehydroepiandrosterone sulfate levels decreased significantly (P = 0.04). There was no significant change in body mass index (40.4 ± 2.4 vs. 41.1 ± 2.7 kg/m2). Eleven of the women reverted to regular ovulatory cycles during the treatment period. We conclude that 1) rosiglitazone therapy improves insulin resistance and glucose tolerance in obese women with PCOS; 2) rosiglitazone decreases ovarian androgen production, which appears to be independent of any changes in LH levels; 3) hyperinsulinemia appears to play a key role in the overproduction of ovarian androgens in these women because attenuation of insulin levels is associated with decreased testosterone levels; and 4) short-term rosiglitazone therapy helps restore spontaneous ovulation.
Introduction
POLYCYSTIC OVARY SYNDROME (PCOS) is one of the most common endocrinopathies affecting 4–10% of women of reproductive age (1, 2). It is characterized by hyperandrogenism and chronic anovulation. Approximately 44% of women with PCOS are obese, and 60% display insulin resistance (2, 3). Hyperinsulinemia contributes to the hyperandrogenism by increasing ovarian androgen production and by suppressing hepatic production of SHBG with consequent increase in free testosterone levels (4). The hyperinsulinemia found in PCOS is more profound in obese patients, although the presence of insulin resistance appears to be independent of body weight (5). Presence of acanthosis nigricans in women with PCOS indicates severe insulin resistance and high risk for type 2 diabetes (6, 7). Women with hyperandrogenism, insulin resistance, and acanthosis nigricans syndrome have severe insulin resistance, and insulin receptor mutations have been observed in these women (8, 9).
In the past, therapeutic approaches to PCOS have focused on suppressing ovarian androgen production or ovulation induction. Recently, insulin sensitizers have been used to reduce the level of hyperinsulinemia and its negative impact on ovarian function and possibly to prevent long-term consequences of hyperinsulinemia. Women with PCOS are at higher risk for hypertension, dyslipidemia, type 2 diabetes, and cardiovascular disease (10, 11, 12). Metformin is an oral hypoglycemic agent that has been shown to improve insulin sensitivity and ovarian function in women with PCOS (13, 14, 15). Metformin is not, however, effective in obese women with PCOS (16, 17).
Rosiglitazone is a member of the thiazolidinediones (TZD) family that has been shown to be effective in the treatment of type 2 diabetes. TZDs are newer oral antidiabetic agents that exert their insulin-sensitizing actions through the peroxisome proliferator-activated receptor found in a number of tissues including the liver, skeletal muscle, and adipose tissue (18). TZDs increase insulin sensitivity without increasing insulin secretion through activation of multiple genes, including the up-regulation of glucose transporters (18). Troglitazone, a member of the TZD family, was found to have beneficial effects on insulin sensitivity and ovarian function in women with PCOS (18, 19, 20); it was taken off the market, however, over concerns of hepatotoxicity. Currently there are only limited data on the use of rosiglitazone in PCOS. The aim of our present study is to evaluate the efficacy of rosiglitazone on insulin resistance and hyperandrogenism in obese women with PCOS and severe insulin resistance.
Subjects and Methods
Subjects
We recruited 12 women with PCOS who were obese, hirsute, anovulatory, and had acanthosis nigricans, indicating severe insulin resistance to participate in the study. The National Institutes of Health/ National Institute of Child Health and Human Development criteria were used to establish PCOS. All of the women had polycystic ovaries on pelvic ultrasound examination. Menstrual history revealed either oligomenorrhea (cycle length > 45 d) or amenorrhea (cycle length > 6 months). All had normal TSH and prolactin levels. Women with possible late-onset congenital adrenal hyperplasia (17-hydroxyprogesterone levels > 2 ng/ml) and possible virilizing ovarian tumors were excluded from the study. In one patient who had testosterone levels higher than 200 ng/dl, the possibility of an ovarian tumor was excluded by wedge biopsy of the ovaries. Histological examination revealed stromal hyperthecosis. This study was approved by the University of Texas Medical Branch Institutional Review Board, and written informed consent was obtained from all subjects. After a 3-d high-carbohydrate diet, patients were instructed to fast overnight and were subsequently admitted to our clinical research center. The body mass index (BMI) of all the women was calculated, and a standard 75-g 3-h oral glucose tolerance test (OGTT) was performed. Glucose and insulin levels were measured at baseline and at 1, 2, and 3 h after the oral glucose administration. Glucose and insulin areas under the curve (AUC) were calculated using the trapezoidal method. Serum levels of total and free testosterone, dehydroepiandrosterone sulfate (DHEA-S), LH, and 17-hydroxyprogesterone were also measured. All of the women were then treated with rosiglitazone, 4 mg once a day. They were advised to follow a low-fat, low-calorie diet, although no standardized diet was used. After 6 months of treatment, the women were readmitted to the clinical research center, the BMI was calculated again, and all of the studies, including the OGTT with insulin levels, were repeated. All the posttreatment tests were performed in the follicular phase of the cycle between cycle d 5 and 8. All of the results obtained were then analyzed, and the pre- and posttreatment values were compared.
Hormone assays
All the blood samples were centrifuged and the separated serum was kept frozen at –70 C until the time of the assay. Insulin, DHEA-S, SHBG, and 17-hydroxyprogesterone levels were measured by specific double antibody RIA using 125I-labeled hormones (Diagnostic Systems Laboratories, Webster, TX). Total and free testosterone levels were measured by coated tube RIA, and LH levels were measured by immunoradiometric assay. Plasma glucose levels were measured by the glucose oxidase technique. All the samples were run in duplicate. High and low controls were run with each assay, and the assay was accepted only if the controls were within the expected range. Intraassay variation ranged from 1.5–2.5%, and interassay variation ranged from 3.8 to 7.4%. Pre- and posttreatment samples from each patient were assayed in the same batch.
Statistical analysis
The statistical analysis was performed using SigmaStat software (SPSS Inc., Chicago, IL). Hormone levels before and after treatment were compared by paired t test. Correlation between insulin and testosterone levels was determined by Pearson correlation coefficients. Data are presented as mean ± SE, and P < 0.05 was considered statistically significant.
Results
All 12 women who had enrolled completed the study. Table 1 depicts patient characteristics and hormone studies before and after treatment with rosiglitazone. At baseline, there was a highly significant (r = 0.881, P < 0.0001) positive correlation between insulin response during OGTT (AUC) and basal total testosterone levels (Fig. 1). After treatment with rosiglitazone, there was a significant decrease in fasting insulin levels (46.0 ± 6.5 vs. 16.9 ± 2.0 μU/ml; P < 0.001) and insulin AUC (749.3 ± 136.3 vs. 225.0 ± 15.7 μU/ml; P = 0.003) (Fig. 2). Fasting glucose levels (90.8 ± 3.0 vs. 81.8 ± 1.9 mg/dl; P = 0.003) and glucose AUC (437.9 ± 25.0 vs. 322.5 ± 14.7 mg/dl; P < 0.001) also decreased with rosiglitazone treatment (Fig. 3). Both total testosterone (96.3 ± 17.3 vs. 56.1 ± 5.8 ng/dl; P < 0.01) and free testosterone (5.8 ± 0.6 vs. 3.4 ± 0.5 pg/ml; P < 0.001) decreased significantly after treatment, whereas SHBG levels increased (18.3 ± 3.4 vs. 25.8 ± 6.6 nmol/liter; P = 0.009) after treatment (Fig. 4). There was, however, no significant change in LH levels (10.1 ± 0.82 vs. 9.1 ± 0.84 mIU/ml). There was also a significant decrease in the levels of DHEA-S (1508.7 ± 181.9 vs. 1081.3 ± 180.8 ng/ml; P = 0.04). The decrease in 17-hydroxyprogesterone levels (1.02 ± 0.23 vs. 0.71 ± 0.11 ng/ml; P = 0.06) was not statistically significant. There was no change in BMI (40.4 ± 2.4 vs. 41.1 ± 2.7 kg/m2). At the dosage used, there were no adverse effects reported by our subjects, and there were no elevations in serum transaminases during the treatment period. None of our patients reported water retention.
Eleven of the 12 patients studied reverted to regular ovulatory cycles during the treatment period. Ovulation was confirmed using a cycle d 21 serum progesterone level that was more than 5 ng/ml after the onset of spontaneous menstruation. Length of the cycles during treatment varied from 28 to 32 d.
Discussion
Hyperinsulinemia appears to play a central role in the pathogenesis of PCOS. In vitro studies indicate that insulin stimulates androgen accumulations in the incubations of ovarian stroma obtained from women with hyperandrogenism (21). Insulin stimulates testosterone biosynthesis by human theca cells (22). Significant positive correlations have been observed between peripheral insulin levels and ovarian vein androgen levels (23). Insulin infusion acutely augments ovarian androgen production (24), and decreasing circulating insulin levels result in a reduction in ovarian androgens (25). Insulin has also been shown to suppress production of hepatic SHBG (26), resulting in an increase in the circulating levels of free testosterone.
Insulin-sensitizing agents ameliorate insulin resistance and hyperinsulinemia. The efficacy of metformin in improving ovulation in women with PCOS has been reported (14, 15). Not all women with PCOS, however, respond to metformin, and some experience significant gastrointestinal side effects with its use. Most obese women with PCOS fail to respond to metformin (16, 17).
Troglitazone, a peroxisome proliferator-activated receptor agonist that has been taken off the market because of hepatotoxicity, has been shown to be effective in improving endocrine and ovulatory performance in women with PCOS. In a large multicenter, double-blind trial, Azziz et al. (20) observed that troglitazone improves ovulatory function, hirsutism, hyperandrogenemia, and insulin resistance of PCOS in a dose-related fashion. In a group of obese women with PCOS and impaired glucose tolerance, Ehrmann et al. (19) observed significant reductions in total and free testosterone, fasting insulin and glucose, and insulin and glucose AUC after 12 wk of treatment with troglitazone. In both studies, levels of SHBG increased and levels of LH did not change, as observed in our current study.
Previous studies on the effect of rosiglitazone in women with PCOS are limited. Shobokshi and Shaarawy (27) compared women with PCOS who received either rosiglitazone and clomiphene citrate or clomiphene citrate only and observed that combined therapy with rosiglitazone and clomiphene citrate resulted in a significant decrease in insulin and serum-free testosterone levels. It is difficult to draw conclusions on the effects of rosiglitazone from this study, because there were no women who received rosiglitazone only. In a 2-month study, Ghazeeri et al. (28) treated obese clomiphene-resistant women with PCOS either with rosiglitazone and placebo or rosiglitazone and clomiphene citrate. The primary outcome of this study was ovulation, with changes in insulin sensitivity and androgens as some of the secondary outcomes. They reported ovulation in 77% of women taking rosiglitazone and clomiphene citrate and 33% of women taking rosiglitazone only. Lower ovulation rates with rosiglitazone only in this study could be due to the short treatment duration of only 2 months. Rosiglitazone therapy for 6 months restored ovulation in 11 of the 12 women (91%) we studied. Most of the women in our study had long periods of amenorrhea, and spontaneous menstruation occurred in these women within the first 3 months of initiation of therapy. Cataldo et al. (29) reported pregnancy in one patient after treatment with rosiglitazone for 5 months. Previous studies indicate that metformin, the most commonly used insulin-sensitizing drug in PCOS, is not very effective in the resumption of menstruation and ovulation in morbidly obese women (17). The mean BMI of the women of close to 40 kg/m2 in that study is very similar to the mean BMI of our patients. The high ovulation rate that was observed in our patients with rosiglitazone treatment indicates that TDZs may be the ideal treatment for obese PCOS women with severe insulin resistance who desire fertility. Since there have been no studies to evaluate use of rosiglitazone during pregnancy, rosiglitazone should be discontinued when pregnancy occurs.
In our study, we sought to study the effects of rosiglitazone in obese women with PCOS who had severe insulin resistance. Because obese women do not respond well to metformin, we wanted to study the effectiveness of rosiglitazone in these women. A standard OGTT with calculation of insulin AUC was used to assess insulin release and to establish insulin resistance, because this method has been validated as a sensitive predictor of insulin resistance (30). Furthermore, all of our patients had acanthosis nigricans, a cutaneous finding that is specific for severe insulin resistance (6, 7). We were able to demonstrate that rosiglitazone therapy improves insulin resistance and glucose tolerance in these women, because there were significant reductions in fasting insulin, fasting glucose, and insulin and glucose response to an oral glucose load. Ghazeeri et al. (28) also observed a significant decrease in fasting insulin levels and an increase in SHBG with rosiglitazone therapy for 2 months.
Rosiglitazone therapy resulted in a reduction in ovarian androgen production because levels of both total and free testosterone were significantly reduced. Levels of SHBG increased with therapy, further reducing the bioavailability of circulating androgens. These changes are most likely caused by improvements in insulin sensitivity, which resulted in amelioration of hyperinsulinemia and, thus, a reduction in ovarian androgen production. Hyperinsulinemia appears to play a key role in the pathogenesis of hyperandrogenism in these women. Interestingly, LH levels did not change with therapy. This finding is in agreement with previous studies with troglitazone, which showed reductions in circulating androgen levels without any changes in LH levels (18, 19, 20). Lack of change in LH levels could be due to the fact that our patients did not have elevated LH levels at baseline. Mor et al. (31) reported low LH levels in their group of obese women with PCOS and severe insulin resistance. The observation of low LH levels in our hyperinsulinemic patients is consistent with previous reports, which indicate two possible distinct phenotypes of PCOS, a low-LH/high-insulin group and a high-LH/low-insulin group (31, 32).
Rosiglitazone therapy also resulted in a significant decrease in circulating levels of adrenal androgen DHEA-S. Azziz et al. (33) observed similar effects on DHEA-S levels with troglitazone administration for 20 wk. It is not clear whether the decrease in DHEA-S levels is a result of decrease in insulin levels or is due to the direct effects of rosiglitazone on the steroidogenic enzymes in the adrenal gland. In a recent study, pioglitazone treatment for 6 months in women with PCOS reduced the adrenal androgen response to corticotrophin (34). These authors speculated that insulin enhances ACTH-stimulated adrenal androgen synthesis.
The reductions in ovarian androgen levels with rosiglitazone therapy could be a result of the decrease in the insulin levels, the direct effects on the ovary, or a combination of both. Previous studies indicate that TZDs may have a direct effect on ovarian steroidogenesis apart from improved insulin sensitivity. TZDs have been shown to directly affect gonadal steroidogenic cells. Mitwally et al. (35) found troglitazone to directly inhibit the production of androsterone by cultured rat theca interstitial cells and progesterone by cultured human granulosa lutein cells. Furnsinn et al. (36) showed that TZDs inhibit gonadal steroidogenesis in obese male Zucker rats, a finding that was independent of improvements in insulin sensitivity. Vierhapper et al. (37) demonstrated that 7 d of rosiglitazone therapy in a group of healthy men significantly reduced production rates of testosterone and dihydrotestosterone. Previous studies in our laboratory with porcine granulosa cells indicate that troglitazone reduces progesterone production (38). Gasic et al. (39) showed troglitazone to be a competitive inhibitor of ovarian 3?-hydroxysteroid dehydrogenase. Other in vitro studies have shown troglitazone to have a direct effect on various steroidogenic enzymes, including aromatase and 17 hydroxylase/17–20 lyase (40, 41).
It is of interest that the improvement in insulin sensitivity in our patients was independent of adiposity, because there were no changes in BMI with therapy. It is well established that obesity is associated with insulin resistance, and reductions in weight typically lead to improved insulin sensitivity. Previous studies have shown thiazolidinediones to cause some weight gain when used in the management of diabetes (42). Weight gain was not seen in our subjects, which is in agreement with most studies using thiazolidinediones in women with PCOS. Ghazeeri et al. (28) found no change in weight in their subjects with PCOS with short-term treatment of rosiglitazone. It has been suggested that thiazolidinediones cause redistribution of fat from different adipose compartments with the shift of adipose tissue from the visceral to the sc area (43, 44). Redistribution of fat remains a possibility in our subjects because BMI did not change. It is known that different adipose compartments can have varying effects on endocrine and metabolic factors in women with PCOS (45). Ehrmann et al. (19) found no changes in BMI or distribution of regional adiposity by using a dual-energy x-ray absorptiometry scan when they treated their PCOS subjects with troglitazone. Furthermore, Romualdi et al. (46) found no changes in BMI or waist-to-hip ratio in 18 obese subjects with PCOS who were treated with pioglitazone.
In summary, our results indicate that rosiglitazone improves insulin sensitivity and glucose tolerance in obese women with PCOS and severe insulin resistance. It also helps to restore ovulation and attenuate ovarian androgen production without weight gain or other side effects. Use of rosiglitazone appears to be an effective method in the management of obese PCOS women with severe insulin resistance.
Footnotes
This work was supported in part by Grant R01 CA 45181 from the National Institutes of Health (to M.N.) and Grant M01 RR00073 from the General Clinical Research Center Program.
First Published Online October 13, 2004
Abbreviations: AUC, Area under the curve; BMI, body mass index; DHEA-S, dehydroepiandrosterone sulfate; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; TZD, thiazolidinediones.
Received July 14, 2004.
Accepted September 24, 2004.
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